Mitochondria transfer transiently rescues cerebellar neurodegeneration at early stage by alleviating mitophagy

Mitochondria transfer transiently rescues cerebellar neurodegeneration at early stage by alleviating mitophagy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondria transfer transiently rescues cerebellar neurodegeneration at early stage by alleviating mitophagy Yayun Wang, Shu-Jiao Li, Qian-Wen Zheng, Jie Zheng, Jin-Bao Zhang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4612386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Cerebellar ataxia is the main manifestation of cerebellar degenerative diseases, and the mitochondrial function of Purkinje cells (PCs) plays a key role in the disease. And treatments targeting the cerebellum need further research. We constructed a model of cerebellar PCs degeneration, characterized by ataxia, through conditional knockout of Drp1 in PCs (PCKO mice). And we further explored the pathogenesis and possible effective treatment of cerebellar degenerative diseases. Drp1 knockout results in pervasive and progressive apoptosis of PCs, accompanied by severe glial cell activation surrounding them. Mitochondrial dysfunction, as a cause of mitophagy, is a key pathogenic factor of PCs morphological damage and dysfunction. Transfer of liver-derived mitochondria into the cerebellum of PCKO mice at 1 month improved mitochondrial function and reduced mitophagy, resulting in a delay of PCs apoptosis and cerebellar ataxia for 3 weeks. This study demonstrates that mitochondria transfer may be an potential treatment for cerebellar degenerative diseases. Health sciences/Neurology/Neurological disorders/Neurodegeneration Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration Cerebellar neurodegeneration Mitochondrial transfer mitochondrial dysfunction Purkinje cells ataxia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Neurodegenerative diseases (ND) are characterized by a progressive loss of neuronal function. Cerebellar neurodegenerative diseases are characterized by cerebellar ataxia, including ataxia telangiectasia 1 , spinocerebellar ataxia 2 , etc. The main mechanism of cerebellar neurodegenerative diseases is still unknown, thus preventing the development of its drugs. There are no existing treatments to totally correct the underlying neurodegenerative disease process. Current treatments can only relieve symptoms. As the underlying disease progresses, the effectiveness of drug treatment decreases despite increased drug doses. As a result, the side-effect to benefit ratio increases 3 . Purkinje cells, as the only efferent neurons in the cerebellar cortex, play an important role in cerebellar neurodegenerative diseases. Purkinje cell degeneration is an inherited or sporadic disease characterized by progressive gait and coordination disorders 4 , 5 , 6 . Although immunotherapy 7 , electrical stimulation 8 , gene therapy 9 , 10 , stem cell transplantation 11 , 12 , 13 and other means have been studied to treat animal models of neurodegenerative diseases, the curative effect is not ideal. Chronic mitochondrial dysfunction accompanied by biological energy depletion is a pathological marker of major neurodegenerative diseases. Brain injury triggers acute mitochondrial damage and a local energy crisis that accelerates neuronal death. Defective mitochondrial maintenance and insufficient axon energy have become central problems in neurodegenerative diseases and brain injuries 13 . Alterations in mitochondrial DNA have been described in diseases, including deletions, point mutations, depletion, and maintenance alterations. In Alzheimer’s disease or Parkinson’s disease, mutations in genes directly related to mitochondrial function, such as Parkin and PINK1, are responsible for rare inherited forms of the disease 14 , 15 . And PINK1 regulates PARKIN translocation in damaged mitochondria and is involved in neurodegenerative diseases by driving its removal through selective autophagy, a process called mitochondrial autophagy 16 . Gasdermin protein, a hangman pore-forming molecule that mediates cell death, plays a role in mitochondrial damage and axon loss, and GSDME knockout prevents neurite loss in iPSC-derived motor neurons in ALS patients 17 . Current studies have shown that the main mechanism is closely related to mitochondrial dysfunction, and the mutation of multiple mitochondria-related genes will lead to the dysfunction and death of Purkinje cells 18 . Therefore, targeting Purkinje cell mitochondria for the treatment of degenerative changes may be a reliable treatment 19 , 20 , 21 . Mitochondrial transplantation is a therapeutic approach developed by McCully and colleagues that entails the injection of normal mitochondria harvested from unaffected tissue into an ischemic organ of the same subject 22 . It has recently been applied to human pediatric patients with myocardial ischemia, receiving widespread media attention 23 . In the central nervous system, an increasing number of reports in animal models or cultured cell lines have suggested the effectiveness of the mitochondrial transplant. Direct injection through veins 18 , 23 , 24 , 25 , 26 or arteries 27 , 28 , or other channels 29 , 30 , 31 , or local region 32 , 33 , 34 , or indirect injections of modified and packaged mitochondrial complexes 35 , for various neurological diseases, including Parkinson's disease 26 , 32 , brain trauma 36 , schizophrenia 37 , spinal cord injury 38 , nerve injury 39 , and stroke 24 . We have noticed the same disadvantage in these studies: mitochondrial dysfunction is not the only factor contributing to neurological defects. Thus, the subsequent recovery of neurological function due to mitochondrial transplantation is helpful but raises questions since it is difficult to assess the effectiveness of the treatment among complex factors. Our study reveals that Purkinje cell degeneration caused by Drp1 knockout results in ataxia through increased mitochondrial autophagy and apoptosis, as well as induced activation of astrocytes and microglia around Purkinje cells. However, upregulation of Drp1 in cerebellar Purkinje cells by the transgenic technique did not alleviate progressive Purkinje cell loss and motor dysfunction. Interestingly, we showed that in-situ exogenous healthy mitochondria injection into the cerebellum at an early stage of cerebellar neurodegeneration in mice significantly inhibited progressive Purkinje cell loss and behavioral impairment. We identified Purkinje cell and mitochondrial function status as key indicators for ameliorating cerebellar neurodegeneration, and mitochondrial transfer as an effective treatment for early-stage cerebellar neurodegeneration. 2. Results 2.1 Generation of cerebellar neurodegeneration model (CBND) by conditional knock-out of Drp1 in Purkinje cells (PCKO) Based on previous studies 40 , we generated Drp1 flox/flox ::Pcp2-Cre mice ( Fig. 1 a and 1 b ) whose Drp1 gene was specifically knocked out in cerebellar Purkinje cells (PCs) (PCKO), also named CBDN general mice. Their wild-type (WT) littermates were used as controls. PCKO and control mice were viable, fertile, and normal in size and weight ( Fig. 1 c ) . The cerebellum of 3-month-old (3M) PCKO mice showed atrophy ( Fig. 1 d and 1 e ) . Sagittal plane of mouse cerebellum (1M-4M) were subjected to immunofluorescence with Calbindin, and the PC number quantified ( Fig. 1 f ) . The number of PCs ( Fig. 1 g ) was decreased at 2M to 22.55%, 3M to 6.19%, and 4M to 3.55%. And the thickness of molecular layer (ML) also decreased sharply since their age of 2 months (Fig. 1 H). Next, immunofluorescent staining and RNAscope experiment showed that DRP1 protein and Drp1 mRNA both reduced obviously in PC of PCKO mice at 3M ( Fig. 1 i-k ) . Furthermore, we performed behavioral testes to detect the motor function of PCKO mice, including gait analysis, rotarod and balance beam. Compared to WT-3M mice, the PCKO mice appeared swaying gait at the age of 3-month ( Fig. 1 l ) . The results of rotarod and balance beam tests indicated that PCKO mice developed impaired coordination and inbalanced motion since their age of 4 weeks and the symptoms worsened over time ( Fig. 1 m and 1 n ) . These results demonstrated that PCKO mice presented cerebellum atrophy, Purkinje cells neurodegeneration and progressive ataxia. 2.2 Generation of TdTomato-expressing CBND (CBND TdTomato ) by TdTomato knock-out mice (PC TdTomato KO) In order to ensure the stability of the experiment and exclude the non-characteristic interference of immunofluorescence staining, we constructed PC TdTomato KO mice whose cerebellar Purkinje cells genetically express TdTomato protein ( Fig. 2 a and 2 b ) , also named TdTomato-expressing CBND (CBND TdTomato ) mice. The middle sagittal planes were detected and the PCs also degenerated at 2M ( Fig. 2 c and 2 d ) . similarly, the cerebellum area on middle sagittal plane had atrophied at 2M ( Fig. 2 e ) . Based on the behavioral results of PCKO mice, we performed rotarod and balance beam tests on PC TdTomato KO mice at the age of 1 month, who similarly exhibited ataxia ( Fig. 2 f and 2 g ) . Pearson correlation Analysis was used to explore the relationship between PCs and motor behavior, showing that the number of PCs and the number of hindfoot slips were negatively correlated ( Fig. 2 h ) . Based on these results, it can be inferred that motor dysfunction can be reversibly alleviated before PC significantly dies. In other words, therapeutic interventions at an early stage of mouse development, such as 1 month of age, may be able to delay the progression of cerebellar neurodegeneration. 2.3 Targeted overexpression of DRP1 in PCs failed to rescue ataxia in adolescent Drp1-PCKO mice We designed gain-of function experiments (Fig S1 a) to explore whether Drp1 supplement to PCs could ameliorate ataxia. The PCKO mice at 1-month-old were injected with Drp1-DIO virus at the cerebellum. After 4 weeks, the virus were widely expressed on the cerebellum (Fig S1 b) , and Drp1 mRNA obviously upregulated successfully in the cerebellum of PCKO mice (Fig S1 c and S1d) . The rotarod test showed that the coordination activity of PCKO mice had no improvement after Drp1-targeted OE treatment (Fig S1 e) . Unfortunately, there also was no improvement of balance ability of PCKO mice following Drp1 upregulation compared with Drp1 NC treatment (Fig S1 f) . 2.4 Strategy of liver-originated free mitochondria transfer into degenerated cerebellum of CBND mice (Mito-transfer) and mitochondrial quality control In consideration of the mitochondrial deficit of PCKO mice and the favorable therapeutic effect of mitochondria transfer on neuronal disease, we explored the potential of mitochondria transfer on cerebellar neurodegeneration. We injected hepatic-derived mitochondria from WT mouse into the cerebellum of PCKO mice ( Fig. 3 ) . Healthy mitochondria isolated from 100 mg of liver tissues were stained with Mito-tracker to elucidate their bioactive functions ( Fig. 3 a-d ) . The VDAC protein results further testified the mitochondria compound ( Fig. 3 e ) . Considering the vulnerability of mitochondria, we controlled the process of mitochondria transfer within 1 hour. And we proved that the mitochondrial function will not change within 1 hour after mitochondria extracting ( Fig. 3 f and 3 g ) . Thus, the mitochondria were transferred at a concentration of 50 mg/ml by direct injection into 4 sites of cerebellar cortex coordinates, as described in Fig. 3 h. 2.5 Mito-transfer, at early stage, significantly alleviates neuron loss and ataxia disorder of both CBND general mice and CBND TdTomato mice At 1, 2, 3 and 4-week after mitochondira transfer, the PCKO mice were assessed by behavioral tests ( Fig. 4 a ) . Fortunately, the mice receiving mitochondria transfer for 3 weeks at the age of 1M (CBDN general +Mito) could walk quickly and well crossing the beam, while the hindfoot of PCKO mice showed sustained continuous slipping down on the balance beam ( Fig. 4 b and Video S1) . The balance ability tested by balance beam of CBDN general +Mito mice improved significantly at first week after mitochondria transfer. And this effectiveness maintained 3 weeks and faded away in the 4th week ( Fig. 4 c and 4 d ) . We further identified GFAP-positive astroglia and Iba1-positive microglia in cerebellum ( Fig. 4 e ) . Compared to the PCKO group, the CBDN general +Mito mice showed less GFAP- and Iba1-positive cells as well as luxuriant Purkinje cells after 3 weeks of mito-transfer ( Fig. 4 e-h ) . Moreover, we injected sparse virus into cerebellum and found that CBDN general +Mito can delay the progression of PCs degeneration ( Fig. 4 i and 4 j ) . Basides, to explore the effects of mito-transfer on whole cerebllum, the CBND TdTomato mice were also treated with mitochondria and performed CUBIC brain clearing experiment. The number and morphology of PCs showed obviously improvement at 3 weeks after mito-transfer ( Fig. 5 a and 5 b ) . For the CUBIC experiment, after fixation, delipidation and refractive index matching, the brain from CBND TdTomato mice become transparent totally ( Fig. 5 c ) . The whole brain was imaged using light-sheet microscope and we found the number of PCs significantly increased in CBND TdTomato +Mito group compared with CBND TdTomato mice (Fig S2 and S3 and Video S2-4) . The cerebellum were magnified locally, of which the results showed the number of PCs of CBND TdTomato mice increased significantly and the dendritic tree were still flourish after mito-transfer ( Fig. 5 d and Video S5-7) . Though the imaris-processed analysis, the size of PCs also recovered ( Fig. 5 e and 5 f ) . 2.6 Mito-transfer, at middle and late stages, can not improve neuron loss and ataxia disorder in CBND general mice To investigate whether mitochondria transfer contributes to CBDN general mice at middle and late stages of neurodegeneration. We performed mitochondria transfer to CBDN general mice at 2 or 3-month-old and detected behavioral test at 3 weeks after that (Fig S4a and S4f ) . Calbindin staining showed that Purkinje cells of CBDN general mice lost severely and the results of cerebellum atrophy had no difference after mitochondria transfer (Fig S4b, S4c, S4g and S4h ) . The astroglial and microglial activation of cerebellum from CBDN general mice did not present any remission after mitochondria therapy (Fig S4b and S4g ) . Similarly, there was no significant difference in coordination or balance ability between CBDN general mice and mito-treated CBDN general mice at 2 (Fig S4d and S4e ) or 3-month-old (Fig S4i and S4j ) . These results indicated that the necessary condition for the effectiveness of mitochondria transfer to delay the progression of Purkinje cells degeneration is that the number of Purkinje cells is enough. 2.7 Enter and residence of exogenous mitochondria into Purkinje cells To make sure that exogenous mitochondria were capable of entering Purkinje cells, we generated GFP-Mito tagfloxed mice and crossed them with ObRb-Cre mice to generate ObRb-Mito-GFP mice ( Fig. 6 a ) , whose ObRb + cells in liver exhibited bright mito-GFP fluorescence localized specifically to the mitochondrial compartment ( Fig. 6 b ) . We transplanted the isolated green mitochondria from the liver of ObRb-Mito-GFP mice to the cerebellum of CBDN general mice according to the same protocol, and the results confirmed that the green exogenous mitochondria entered the soma of PCs after 24 hours ( Fig. 6 c and 6 d ) . To identify whether the exogenous mitochondria communicated with the endogenous mitochondria of Purkinje cells, we injected the cerebellum of Pcp2-Cre mice with mitochondria-driving virus. And 3 weeks later, the mitochondria were transferred to that mouse ( Fig. 6 e ) . The results manifested that the exogenous green mitochondria were able to enter and interacted with Purkinje cells ( Fig. 6 f ) . 2.8 Transferred mitochondria support degenerated Purkinje cells by inhibiting caspase 3 - related cell apoptosis and parkin - related mitophagy We next investigated whether the well effectiveness of mitochondria transfer on CBDN general mice was attributed to DRP1 recovery in the Purkinje cells. Calbindin and DRP1 co-staining showed that mitochondria transfer could not up-regulate DRP1 of Purkinje cells (Fig S5) . To explore the possible mechanism of mitochondria transfer, we detected the molecule-related experiment in Purkinje cells of CBDN general mice. Firstly, the Drp1 mRNA decreased in the cerebellum of CBDN general mice at the age of 1 month when the ataxia-related molecule, like SCA1 (ATXN1), SCA6 (CACNA1A), SCA7 (ATXN7) and SCA17 (TBP), also reduced in the cerebellum ( Fig. 7 a ) . Next, we found that DRP1, FIS1 and MFF protein in the cerebellum of CBDN general mice showed prominent reduction, while there was no difference in phosphated DRP1, like DRP1 S616 and DRP1 S637 ( Fig. 7 b ) . And SOD level reduced in CBDN general mice ( Fig. 7 b ) , which showed the increased level of oxidative stress in mitochondria. These results indicated that the mitochondrial dysfunction led to Purkinje cells degeneration. We further used CBDN TdTomato mice to investigate the mitochondria function in Purkinje cells. We found that the level of caspase3 on Purkinje cells increased markedly in CBDN TdTomato mice since their age of 1 month ( Fig. 7 c and 7 d ) . And mitochondria transfer were capable of slowing the progression of apoptosis of Purkinje cells ( Fig. 7 c and 7 e ) . COX4, a marker presenting the mitochondrial function, decreased significantly in Purkinje cells of CBDN TdTomato mice from 2-month-old ( Fig. 7 c and 7 f ) , which can be reversed by mitochondria transfer at the age of 1 month ( Fig. 7 c and 7 g ) . Similarly, the key molecule of mitophagy, PARKIN, showed remarkable increment in Purkinje cells of CBDN TdTomato mice at their 2-month-old ( Fig. 7 c and 7 h ) and noteworthy decrease after mitochondria therapy ( Fig. 7 c and 7 i ) . These results demonstrated that exogenous mitochondria are able to slow the progression of neurodegeneration by reducing apoptosis and increasing the mitochondrial function. Besides, Calbindin, GFAP and Iba1 tri-staining were used to estimate the intensity of astrocytes and microglia in cerebellum of PCKO mice. In the cerebellum of WT mice, little GFAP and Iba1 positive cells was found ( Fig. 7 j ) . Accompanying the progression of PC loss ( Fig. 7 k ) , GFAP intensity ( Fig. 7 l ) and Iba1 intensity ( Fig. 7 m ) gradually and significantly increased since the age of 1 month. These results stated that motor dysfunction and glial activation tend to occur earlier than PCs loss. 3. Discussion Mitochondrial damage plays a pivotal role in maintaining and promoting neurodegeneration, such as the impairment of respiratory chain, mitochondrial DNA and mitophagy 14 . Therefore, the therapy targeted to mitochondria may play a key role in neurodegenerative process. In this study, we described that mitochondrial transfer technology can reduce the level of mitophagy of Purkinje cells by improving mitochondrial function, thereby controlling apoptosis of Purkinje cells and delaying the development of cerebellar neurodegenerative diseases. In 2012, Yusuke Kageyama et al. 40 constructed the mice with DRP1-specific knockout in cerebellar Purkinje cells, in which the loss of Purkinje cells occurred over time. We have selected this model as the neurodegeneration of the cerebellum. And we demonstrated a behavioral correlation between the degeneration of cerebellar Purkinje cells and ataxia. In addition, with the progressive loss of Purkinje cells, a large number of astrocytes and microglia in the cerebellum were activated. The molecules related to apoptosis or mitophagy in Purkinje cells were significantly increased and mitochondrial function was significantly impaired. Although we up-regulated DRP1 in Purkinje cells of PCKO mice, this operation did not improve the degeneration of Purkinje cells and the ataxia of mice. Fortunately, when liver-derived mitochondria of healthy mice were injected into the cerebellum, the apoptosis process and mitochondrial dysfunction in Purkinje cells were alleviated. Due to the recovery of Purkinje cells, the motor impairment of the PCKO mouse was significantly alleviated. However, our current study found that the effects of mitochondrial transfer on neurodegeneration only lasted for three weeks and the apoptosis process of Purkinje cells was restarted in the forth week after operation. Consistent with the results of previous studies, Mitochondria can enter neuron in free form and improve the state of mitochondria in the brain 41 . Our results demonstrated that exogenous healthy mitochondria entered into Purkinje cells, but whether they can improve the physiological function of Purkinje cells and alleviate the motor dysfunction depends on the state of Purkinje cells and their mitochondria. We used Cox4 and Parkin to reflect the the level of mitochondrial respiratory chain and mitophagy, respectively 42 , 43 , 44 . The state of Purkinje cells was reflected by Calbindin and caspase3. Even though cerebellar glial cells proliferated and the caspase3 of Purkinje cells increased, the mitochondrial function was compensatory and Purkinje cells were still there. Therefore, the premise that mitochondria transfer can play a role was that the number of Purkinje cells and the function of mitochondria have not been seriously damaged. This was the most critical stage. Exogenous mitochondria can be acquired by host neurons, and then played a function to support the development and growth of host neurons. Therefore, using the Cox4 and Parkin to reflect the mitochondrial state, as well as Calbindin and caspase3 to reflect the neuron state, may gain a valuable time window and win an opportunity to treat neuronal degenerative diseases with natural loss of mitochondrial genes ( Fig. 7 n ) . Mitophagy can selectively wrap and degrade damaged mitochondria through autophagy mechanisms, thereby maintaining mitochondrial and cellular homeostasis. So dysfunction of mitophagy can lead to accumulation of damaged mitochondria and cellular dysfunction, leading to aging and age-prone neurodegeneration 45 , 46 , 47 . Our study found increased mitophagy in the Purkinje cells of PCKO mice compared to normal mice, and decreased mitophagy in Purkinje cells was found after mitochondrial transplantation. We think this is the result of mitochondrial transplantation improving mitochondrial function. As exogenous healthy mitochondria entered into dysfunctional Purkinje cells, the proportion of damaged mitochondria in Purkinje cells was reduced contributing to the decline of overall mitohagy level. Therefore, the reduction of mitophagy indirectly proved that mitochondrial transplantation can improve the mitochondrial function for recipient cells. Existing studies have shown that the survival cycle of mitochondria is about 28 days, which is consistent with our findings. After 3 weeks, the metabolism capability of exogenous mitochondria was exhausted leading to a rebound of ataxia. Whether exogenous mitochondria were able to communicate with endogenous mitochondria during mitochondrial transplantation was unclear. The results so far supported two possibilities. One is to integrate, but not replace. The other is not to blend in, do their own thing. However, the existing results showed that mitochondrial transplantation could not make up for the loss of Drp1 in the original nuclear gene. Finally, these mitochondria could not effectively divide 48 , and the damaged mitochondria in the cell could not be effectively removed 49 , 50 . In the end, neither exogenous healthy mitochondria nor endogenous defective mitochondria could survive for one month. However, the future of mitochondrial transplantation is still exciting, after all, for neurodegenerative diseases, the precious window of time still let people see the light. Therefore, correcting the lack of Drp1 has become our biggest challenge. Previous studies have shown that gene therapy is a potential protocol for the treatment of neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease 3 , 51 . In 2014, Michael S Rafii et al. conducted stereotactic gene delivery of AAV2-NGF on a patient with early and middle stage of Alzheimer’s disease. The results showed that AAV2-NGF was safe and well-tolerated for 2 years 52 . In 2018, Michael S Rafii et al. performed AAV2-NGF or sham surgery on 49 patients with early and middle stage of Alzheimer’s disease. There was no significant difference in efficacy between the gene therapy group and the placebo group 53 . In addition, for congenital genetic defects, the time condition of gene therapy are very demanding as patients grow older 54 . These studies showed that the efficacy of gene therapy has varied greatly between individuals. Our laboratory also attempted to deliver Drp1 overexpressing AAV to the cerebellum. Although the delivery was satisfactory, the overexpression of Drp1 failed to correct the fate of neuronal degeneration. We hypothesized that this may attribute to late genetic intervention or low doses. Glial cells also play an important role in neurodegenerative diseases. Consistent with previous research, this study also verified that the activation of surrounding astrocytes and microglia was manifested before mitochondrial damage and neuronal function damage 55 . It showed that the cells around neurons and neurons themselves formed a community-like overall structure, which is highly related to each other. However, it is important to note that the activation of these glial cells is the result, not the cause, of the denatalized neurons. When the mitochondria transfer corrected the neuronal state, these activated glial cells disappeared and returned to their original resting state. Therefore, we believe that rather than overestimating the potential of support cells in the community, it may be more reliable to use their status as a measure of treatment effectiveness. There are some limitations in this study. Ataxia symptoms appeared in PCKO mice at the age of one month. Although the number of Purkinje cells did not decrease significantly at this time, we speculated that the function of Purkinje cells had been damaged at this time. However, we did not conduct relevant experiments on the function of Purkinje cells in our study. Since the purity of mitochondrial extract was not verified in our study, it cannot be ruled out that other substances also play a role like mitochondria. The validity period of mitochondrial transplantation in this study is only 3 weeks, and we will try to extend the validity period of mitochondria in the next experiment, hoping that mitochondria will become a long-term effective stage for the treatment of cerebellar neurodegeneration. 4. Materials and methods 4.1 Animals We purchase Pcp2 cre mice from the Jackson Laboratory (America, Stock No: 004146). Drp1 Cre-triggered conditional knockout mice were designed by inserting a loxP site after the sequence of Drp1, and obtained from Cyagen (serial number: CKOAIS191230RT5, China). ObRb-Cre mice were purchased from Cyagen (CKOCMP-74011-Slc25a27-B6N-VA, China). Rosa26-CAG-LSL-GFP-Mito tag mice (Mito-GFP mice) were constructed by Gempharmatech (cas9-ki(Rosa26), China), of which outer mitochondria membrane carries green fluorescent protein. B6/JGpt-H11 em1Cin(CAG−LoxP−ZsGreen−Stop−LoxP−tdTomato) /Gpt (B6-G/R) mice were purchased from GemPharmatech (T006163, China). And C 57BL/6J Gpt mice were also gotten from GemPharmatech. Drp1 f/f mice were produced by crossing Drp1 f/+ mice with Drp1 fl/+ mice to generate homozygous control. Drp1 fl/+ mice were crossed with Pcp2 cre mice for two generations to generate Pcp2 cre ; Drp1 f/f mice (PCKO mice) as knockout mice. PC tdTomato KO mice were constructed by PCKO mice and B6-G/R mice. ObRb-Mito-GFP mice were generated by hybridizing ObRb-Cre mice and Mito-GFP mice. All mice were housed (maximum 5 mice in a cage) with food and water freely available under a 12-h day/night cycle (8 am to 8 pm, day). Roughly equal numbers of male and female mice were used for the experiments. All of the animal protocols complied with the Animal Care and Use Committee of the Air Force Military Medical University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (permit number IACUC-20190107). 4.2 Genotyping and confirmation The mouse genotype was identified by mouse tail PCR with genomic DNA. The primers used to identify the Pcp2 Cre sequence with 576 bp were shown as: F1: 5’-ATTCTCGTGGAACTGGATGG-3’ and R1: 5’-GGACAGGTAATGGTTGTCTGG-3’. The primers used to identify the Drp1 loxP sequence with 292 bp for mutant and 231 bp for wild type were shown as: F2: 5’-CCACTTTAGGTACTCATAACACA-3’ and R2: 5’-ATTGTCATACATTCAGATAGGG-3’. The offspring with concurrence of 576 bp and 292 bp was regarded as the Drp1 conditional knockout mouse. The primers used to identify the B6-G/R sequence with 1465 bp were shown as: F3: 5’-ATGCCCACCAAAGTCATCAGTGTAG-3’ and R3: 5’-AGGCGGGCCATTTACCGTAAGTTA-3’. The primers used to identify the ObRb Cre sequence were shown as: F4: 5’-GCTGGAAGATGGCGATTAGC-3’ and R4: 5’-TCTTCTTTCCAGAGTTCAGATGT-3’. The primers used to identify the Mito-GFP sequence were shown as: P1: 5’-CCCAAAGTCGCTCTGAGTTGTTA-3’, P2: 5’-TGGCGTTACTATGGGAACATACGTC-3’ and P3: 5’-TCGGGTGAGCATGTCTTTAATCT-3’. ObRb-Mito-GFP mice were generated by hybridizing ObRb-Cre mice and Mito-GFP mice. And their offspring, ObRb-Mito-GFP mice, show PCR result of one band of 436 bp and 232 bp. The cycling condition of PCR reaction was as follows: 94°C for 3 min, 35 cycles of 94°C for 30 s for denaturation, 60°C for 35 s for annealing, and 72°C for 35 s for extension, and after the 35 cycles with 72°C for additional extension for 5 min. The primers and PCR conditions were designed by Tsingke Biotechnology Co., Ltd. 4.3 Tissue preparation Firstly, mice were anesthetized by isoflurane. And then the mice were perfused transcardially with 0.01 M phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS for fixation. The brain was removed carefully and submerged in 4% PFA overnight at 4°C for postfixation. Subsequently, we immersed the brain in gradient dehydration with sucrose solution (10%, 20%, and 30% in order) for more than 48 hours. The brain was sectioned at 20-µm thicknesses for Immunofluorescent staining, 10-µm for RNAscope in situ hybridization on a cryostat (Leica CM1850, Germany). Finally, we mounted these sections onto the glass slides directly and returned them into − 20°C cryostat chamber immediately. 4.4 Immunofluorescent staining Immunofluorescent staining was complied with the previous instruction. Firstly, the tissues were washed in the PBS, blocked with 3% fetal bovine serum and permeabilized with 0.3% TritonX-100 at room temperature for 30 minutes. Then, we incubated these tissues with the primary antibodies at 4°C for up to 18 hours (anti-PARKIN was incubated at 4°C for up to 48 hours). And the working concentrations of primary antibodies were as follows: anti-Drp1 (Abcam, AB184247, Rabbit, 1: 50), Anti-Calbindin (Alexa Fluor® 594 Conjugate) (Cell Signaling Technology, #88831, Rabbit, 1: 50); Anti-GFAP (Alexa Fluor® 488 Conjugate) (Cell Signaling Technology, 3655S, Mouse, 1:50); anti-IBA1 (Woka, 011-27991, Goat, 1:400), anti-caspase3 (Absin, abs13825, Rabbit, 1:200), anti-PSD95 (Cell Signaling Technology, #34050S, Rabbit, 1:200), anti-COX4 (Cell Signaling Technology, #4850, Rabbit, 1: 500), anti-VDAC (Abcam, ab15895, Rabbit, 1:500), anti-PARKIN (Invitrogen, PA5-13399, Rabbit, 1:50), anti-LAMP1 (Abcam, AB208943, Rabbit, 1: 100), anti-IP3R (Invitrogen, PA1-901, Rabbit, 1: 250) and anti-Albumin (Abcam, AB207327, Rabbit, 1:500). Alexa Fluor® 647 AffiniPure Donkey Anti-Goat IgG (H + L) (Jackson Immunoresearch, 705-605-147) and Dylight 649, Goat Anti-Rabbit IgG (A23620, Abbkine, USA) were used as the secondary antibodies, which were diluted in PBS and incubated for 2 hours at room temperature. Staining 49,69-diamidino-2-phenylindole dihydrochloride (DAPI) (C1005, Beyotime, China) for 15 minutes was the last procedure of immunofluorescence to stain the nucleus. High-resolution images of section from PCKO mice were performed using confocal microscopy (FV3000, Olympus, Japen). And Leica Stellaris 5 (German) were used to imaging for PC tdTomato KO mice. The results were quantified by ImageJ. For colocalization, we used JACop plugin to get Manders’ Colocalization Coefficients (MCC). The total fluorescence intensity of protein in Purkinje cells was presented as PC i,colocal . And PC i replaced the total fluorescence intensity of Purkinje cells. So the quantification of each protein in Purkinje cells could be regarded as MCC PC . The formula of MCC PC is 4.5 RNAscope in situ hybridization The staining protocol of RNAScope was referred to previous methods. We baked the tissues at 37°C for 6 h and then washed them with 0.01 M PBS for 5 min. Each tissue was covered by hydrogen peroxide (322281, ACD, USA) at room temperature for 10 min following washing twice in distilled water. The tissues were immersed in the boiled Target Retrieval reagents (322000, ACD, USA) at 97°C for 10 min. Immediately, the tissues were transferred to the room-temperature distilled water and then to the absolute ethanol for an additional 3 min. Next, when the tissues were air-dried, we outline them with a hydrophobic pen (CIRISC PAP pen, I.S. CIRCLE WRITER, Japan). After hydrophobic boundaries completely dried, protease III reagent (322281, ACD, USA) was added to slides to cover the whole tissue. Subsequently, the slides were incubated in a preheated HybEZ oven (ACD, USA) at 40°C for 30 min, followed by washing twice in distilled water. These tissues were used for hybridization. A mixture of three probes was then added to each slide until the tissue was fully covered. The present RNAscope in situ hybridizations were divided into three types of combinations of probes. In the first combination, channel 1 probe was defined as Drp1 (434671, ACD, USA), channel 2 as Pcp2 (509991-C2, ACD, USA), and channel 3 as Nclx (1066901-C3, ACD, USA). In the second combination, channel 1 probe was defined as Mfn1 (578731, ACD, USA), channel 2 as Pcp2, and channel 3 as Mfn2 (581891-C3, ACD, USA). In the third combination, channel 1 probe was defined as Ucp2 (443781, ACD, USA), channel 2 as Pcp2, and channel 3 as Ucp4 (1073441-C3, ACD, USA). After hybridizing in a HybEZ oven at 40°C for 2 h, the slides were washed twice in 1× RNAscope® washing buffer (310091, ACD, USA) for 2 min each time. The slides were returned to the oven for 30 min following submersion in AMP-1 reagent. This step was repeated with AMP-2 and AMP-3 reagents for 30 min and 15 min, respectively. The HRP-C1, HRP-C2, and HRP-C3 signals were processed in order. Opal 520 (ASOP520, ASbio, USA) was applied to mark the channel 1 probe, Opal 570 (ASOP570, ASbio, USA) was applied to mark the channel 2 probe, and Opal 690 (ASOP690, ASbio, USA) was applied to mark the channel 3 probe. Finally, the tissue was submerged with Prolong Gold Antifade Mountant with DAPI. High-resolution imaging was performed using confocal microscopy (Zeiss, Germany). The results were quantified by ImageJ. 4.6 Behavior Mice were used to conduct all of the below behavioral tests. Each mouse was grouped and numbered by ground of genotype, which was completed by a third person. The experimenters were ignorant of the mouse genotype. All the behavioral experiments were conducted between 6 p.m. and 10 p.m. 4.7 Rotarod test. To study balance and coordination affected on mice by Drp1 of Purkinje cells, we placed the transgenic mice on an accelerated rotating rod (BZY007, Jiliang, China) with a diameter of 3 cm, which started rolling at 4 rpm and increased to 40 rpm within 180 seconds. Each trial lasted 10 minutes, with at least a 15-minutes interval. We trained the mice for 3 consecutive days, 3 times a day, and collected data on the fourth day. The latency to fall from the rod were recorded for analysis. 4.8 Balance beam To further measure the balance influenced by Drp1, the mice were placed on a horizontal beam (1-meter length and 1.2-centimeter width). We performed the experiment following the protocol of previous research. We trained the mice for 3 consecutive days, 3 times per day, and collected data on the fourth day. The latency to cross the beam and the number of hind foot slips were recorded for analysis. The hindfoot slips were counted if either the left or right hind paw slipped off the beam 18 . 4.9 Foot print The feet of the mice were stained with blue ink. Each mouse was placed on a white sheet and made to walk from the beginning to the end of the sheet. If the mouse leaves the paper from the median position without reaching the end, the round is removed. 4.10 Mitochondria isolation The procedure of mitochondria isolation was performed according to the instruction (SM0020, Solarbio, China). The tissue was rinsed with saline and the mitochondrial lysis buffer was added for grinding and homogenizing. The homogenization was centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was transferred to a new tube and centrifuged at the same condition. The secondary supernatant was then centrifuged at 12,000 g for 10 min at 4 ℃. The precipitate was resuspended with wash buffer and centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was centrifuged at 12,000 g for another 10 min at 4 ℃. Finally, mitochondria were resuspended with Store Buffer. 4.11 Mitochondria membrane potential detection The functional mitochondria were evaluated by JC-1 detection using isolated mitochondria from cerebellum, which strictly comply with the manufacturer’s instruction (C2006, Beyotime, China). In simple, 900 µl JC-1 working solution was added to 100 µl isolated mitochondria suspension and the concentration of total protein ranged from 10 µg to 100 µg. Then added 100 µl of them in a 96-well plate for test. The fluorescence measured in a Microplate reader (Spark, Tecan, Switzerland) was set as below: excitation wavelength, 485 nm; emission wavelength, 590 nm. The fluorescence (F) produced per sample was recorded, and total fluorescence per microgram of proteins (mg) was calculated and analyzed. The F/mg is an indication of the relative amount of J-aggregate formation from isolated mitochondria 56 , which reflects the potential of active mitochondria. In one control mouse, this was set to 1. The amount of relative fluorescence production of each sample was indicated as a percentage of the F/mg in control mice. 4.12 ROS level assay Reactive Oxygen Species Assay Kit (S0033S, Beyotime, China) was used to detect the ROS level. Mitochondria were extracted according to the above method. DCFH-DA was diluted in serum-free medium at 10 µM. After collection, the mitochondria were suspended in diluted DCFH-DA at a concentration of 10 to 100 µg and incubated in a cell incubator at 37ºC for 20 minutes. Inverted and mixed every 3–5 minutes and then washed three times with serum-free cell culture solution. The fluorescence measured in a Microplate reader (Spark, Tecan, Switzerland) was set as below: excitation wavelength, 488 nm; emission wavelength, 525 nm. The fluorescence (F) produced per sample was recorded, and total fluorescence per microgram of proteins (mg) was calculated and analyzed. The F/mg is an indication of the relative amount of ROS produced from isolated mitochondria. In one control mouse, this was set to 1. The amount of relative fluorescence production of each sample was indicated as a percentage of the F/mg in control mice. 4.13 Viral injection The mice were anesthetized with pentobarbital and fixed on a stereoscopic brain locator (RWD 69100, Shenzhen, China). Cut the skin on the top of the head of the mouse and expose the skull. As previously described, the RAAV-ef1α-DIO-Mito-mCherry (BC-1577, Braincase, China) was injected into the cerebellum of mice to label mitochondria of Purkinje cells. The concentrations of AAVs used in our study were all diluted to 1*10 12 v.g./mL. The injection site was determined from brain mapping and shown as below: (±0.50, -6.25, -2.25) and (±0.50, -7.20, -3.00). AAV was injected using a microsyringe (RWD 79013, Shenzhen, China) with a target injection volume of 1 µL and an injection speed of 150 nL/min. The needle remains in the cerebellum for 8 minutes before removal. The skin was sutured intermittently, and iodine disinfectant was applied to the closed wound. And 4 weeks later, the mice were used to perform behavior test, including rotarod test and balance beam test. 4.14 Mitochondria transfer C57BL/6J mice were sacrificed by neck dissection, of which about 100 mg liver was removed immediately to obtain isolated mitochondria. Nanodrop (701-058112, Thermo Scientific, USA) was used to detect the protein concentration in extracted mitochondria, which were diluted to 50 mg/ml. The Drp1 knockout mice were anesthetized with pentobarbital and fixed on a stereoscopic brain locator (RWD 69100, Shenzhen, China). Cut the skin on the top of the head of the mouse and expose the skull. The injection site was determined from brain mapping and shown as below: (±0.50, -6.25, -2.25) and (±0.50, -7.20, -3.00). Mitochondria were injected using a microsyringe (RWD 79013, Shenzhen, China) with a target injection volume of 200 nL and an injection speed of 100 nl/min. The needle remains in the cerebellum for 8 minutes before removal. And the control mice were treated with the store buffer in the same volume. The skin was sutured intermittently, and iodine disinfectant was applied to the closed wound. The entire transfusion experiment was finished within 1 hour. To testing the bioactivity of mitochondria, we stained it with MitoTacker Red (1:1000, M22426, Invitrogen, USA) under 37 ℃ for 30 minutes. Then, the mitochondrial solution was dropped onto the slide and observed under confocal microscope (FV3000, Olympus, Japen). 4.15 Sparse virus injection AAV virus were injected into cerebellum of mice at the age of 1-month-old. In the mean time, PCKO mice were transferred with mitochondria, and the control were transferred with store buffer. The injection sites were shown as: (±0.50, -6.25, -2.25; 1 µL)) and (±0.50, -7.20, -3.00; 1 µL). The concentrations of AAVs used in our study were all diluted to 1*10 12 v.g./mL. The detailed steps were similar as viral injection. The mice brain was removed after 3 weeks later and sectioned at 100-µm thicknesses and then floated them in PBS. DAPI was stained for 15 minutes. Lastly, confocal microscopy (FV3000, Olympus, Japen) was used to view images. 4.16 CUBIC tissue clearing The thorough steps of tissue clearing were complied with the hydrophilic tissue clearing reagent kit (#210701, Nuohai Life Science, China). After sacrificing, the mice were perfused with PBS, fixed with 4% paraformaldehyde, and post-fixed with 4% paraformaldehyde again at 4 ℃ for 24 hours. Next, washed with PBS for 2 hours and repeated 2 times. Then, immersed the brain in the delipidated solution (solution A : solution B = 1:1) and shook it at 37 ℃ for 6 days at a shaking speed of 60 rpm. Consequently, the refractive index was matched with solution C and the brain was shaken at the speed of 60 rpm at 25 ℃ for 2 days. Finally, the brain was immobilized with AGAR gel and photographed with Nuohai LS 18 Tiling Light Sheet Microscope (Nuohai Life Science, China). The corresponding videos were produced with Aivia (version 12.1.0, Leica, Germany). The raw data for 3D image analysis was captured by LiTScan (Light Innovation Technology Limited) and subsequently converted into an am format readable by Amira software (Thermo Fisher Scientific, USA). Subsampling of the 3D image samples was conducted to adjust resolution, utilizing the Resample module to set appropriate voxel sizes. In the preprocessing stage, the background detection and correction module of filters was employed to eliminate background noise. Structural enhancement filters, particularly 3D filters, were then applied to enhance structural features, followed by segmentation of regions of interest using the Interactive Thresholding module based on specified intensity ranges. Subsequently, manual labeling of segmented regions was performed, followed by removal of small spots to eliminate defects. It is noteworthy that the PC cell and cerebellar regions are treated independently, with segmentation of the cerebellar region accomplished through manual labeling based on atlases. Measurement and analysis modules, especially the label analysis function, were employed for data quantification, with the selection of spreadsheet and spatial map options for result export. Finally, selected data was imported into Imaris software (Oxford Instruments, UK) for visualization representation. 4.17 Real time-quantitative polymerase chain reaction (RT–qPCR) Mice were sacrificed by neck dissection, whose cerebellar cortex was removed to tube and rinsed with saline immediately. All following procedure were complied with the industrial instruction. The cerebellar cortex were dissected and homogenized. Trizol reagent (DP419, TIANGEN, China) was used to extracted the total RNA. The total RNA was then reversely transcripted to cDNA according to cDNA synthesis kit (TSK314S, TSINGKE, China). Finally, the cDNA was used to perform PCR test (TSE201, TSINGKE, China), including the gene of Drp1 , ATXN1, ATXN6, ATXN7 and ATXN17. GAPDH was used as the reference gene. All these primer sequences were exhibited in the Table S1 . We followed the 3-steps reaction procedure, shown as follow: the phase of pre-denaturation was performed under 95 ℃ for 1 min, the reaction phase firstly denaturated at 95℃ for 10 s, then annealed at 60 ℃ for 10 s, and finally extended at 72 ℃ for 10 s, and the reaction phase was repeated for 40 cycles. After the phases of amplification, the DNA products were detected using lysis curves. The quantitative was statistically calculated using the 2 −△△ Ct. 4.18 Western Blotting The cerebellums were removed instantly after neck dissection. The protein concentration was detected using BCA assay (E-BC-K318-M, Elabscience, China). Then the samples were electrophoreted on 7.5% SDS-polyacrylamide gel (PG111, Epizyme, China) or 12.5% SDS-polyacrylamide gel (PG113, Epizyme, China) and transferred to PVDF membrane (IPVH00010, Millipore, USA). Primary antibodies were diluted in 1:1000 and incubated at 4℃ over 16 hours. The information of primary antibodies were shown as: anti-DRP1 (8570, Cell Signaling Technology, USA), anti-DRP1 S616 (ab314755, Abcam, USA), anti-DRP1 S637 (ab193216, Abcam, USA), anti-FIS1 (ab156865, Abcam, USA), anti-MFF (84580s, Cell Signaling Technology, USA), anti-SOD (A12537, Abclonal, China) and anti-β-Actin (AT0001, engibody, USA). The secondary antibody was incubated at room temperature for 2 hours. Their details were presented as: ECL kit (SQ201, EpiZyme, China) and FUSION FX.EDGE (Vilber, French) were used for observing the bands. The results were quantified by Image J software. 4.19 Statistical analysis Statistical comparison of each experiment was presented in the figure legend. The data was shown by mean ± SD. All data was firstly analyzed for the Normality test and Homogeneity of variance test. For three samples comparison, one-way ANOVA was used to assess differences between groups and the Dunnett’s t test was used for multiple comparisons between groups. And for two samples comparison, Unpaired two-tailed student’s t -test was used for analysis. Kruskal-Wallis test or Mann-Whitney U test were used to analyze the data which did not conform to the Normalit test or the Homogeneity of variance test. Statistical analyses were performed using GraphPad Prism 8.0.2. P < 0.05 was considered a statistically significant difference. Abbreviation A Anterior AAV Adeno-Associated Virus ATXN1 Ataxin 1 ATXN7 Ataxin 7 CACNA1A subunit-α of the Cav2.1 voltage-gated calcium channel CBND Cerebellar neurodegeneration Cb Cerebellar lobe Cox4 Cytochrome c oxidase 4 Drp1 Dynamin-related protein 1 FIS1 Mitochondrial fission 1 protein GFAP Glial fibrillary acidic protein I Inferior Iba1 Ionized calcium binding adapter molecule 1 iPSC Induced pluripotent stem cells MFF Mitochondrial fission factor Mito Mitochondria ML Molecular layer NC Normal control ND Neurodegenerative diseases NGF Nerve growth factor KO Knock out ObRb Leptin receptor OE Over expression P posterior PCL Purkinje cells layer PCs Purkinje cells Pcp2 Purkinje cell protein 2 PINK1 PTEN-induced putative kinase 1 S Superior SCA Spinocerebellar ataxia TBP TATA-box-binding protein VDAC Voltage-dependent anion-selective channel WT Wild type Declarations Author imformation These authors contributed equally: Shujiao Li, Qian-wen Zheng, Jie Zheng, and Jinbao Zhang. Contributions Yayun Wang, Yanling Yang, and Xin Sun conceptualized the project. Yayun Wang and Shujiao Li designed experiments and wrote the manuscript. Shujiao Li carried out behavior tests, mitochondrial transfer experiments and virus regulation experiment. Qianwen Zheng and Jie Zheng conducted behavior tests and data analysis, mice genotype test and draw the cartoon images; Hui Liu performed the RNAscope experiment. Jinbao Zhang analyzed the data. Kunlong Zhang and Feifei Wu conducted virus regulation experiment. Xiaodong Li and Shuai Zhang performed images analysis. All of the authors read and approved the final manuscript. Corresponding authors Correspondence to Yayun Wang, Yanling Yang and Xin Sun. Acknowledgments We thank all members of Ya-Yun Wang’s lab for their insightful and helpful discussions during the course of the study. This project was supported by the Military Medicine Upgrade Program of Air Force Military Medical University (2020SWAQ04) from Ya-Yun Wang, the Shaanxi Innovation Capability Support Plan (2023-CX-PT-33) from Ya-Yun Wang, the Shaanxi Basic Research Program of Natural Sciences (2024JC-ZDXM-60) from Yan-Ling Yang, the Xijing Hospital Clinical New technology (2023XJSY27) from Yan-Ling Yang, the Shaanxi Basic Research Program of Natural Science (2022JQ-820) from Fei-fei Wu, and the National Natural Science Foundation of China (82201627) from Fei-fei Wu. Competing interests The authors declare that they do not have any competing interests. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Takahashi T , et al. LINE-1 activation in the cerebellum drives ataxia. Neuron 110 , 3278-3287.e3278 (2022). Switonski PM , et al. 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Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice. Sci Transl Med 10 , (2018). Rana A , et al. Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration. Proc Natl Acad Sci U S A 107 , 6988-6993 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files VideoS1balancebeam.mp4 Supplementary video 1 VideoS2.PC4XCB.1Movie.avi Supplementary video 2 VideoS3.KO4XCBMovie.avi Supplementary video 3 VideoS4.KOMITOCB.aivia.tifMovie.avi Supplementary video 4 PC.1.avi Supplementary video 5 KO.avi Supplementary video 6 KOMITO.avi Supplementary video 7 2024.6.20SupplementarymaterialsNC.doc Graphicalabstract.docx Cite Share Download PDF Status: Published Journal Publication published 22 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4612386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":321856885,"identity":"1af5c9ce-2342-40a5-8c1e-9c83e7cb3a75","order_by":0,"name":"Yayun 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14:20:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4612386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4612386/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58189-4","type":"published","date":"2025-03-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59576520,"identity":"b9930d97-a7d4-4175-94b3-ccd95f099348","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1031232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of cerebellar neurodegeneration model (CBND) by conditional knock-out of Drp1 in Purkinje cells (PCKO)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The strategy of constructing cerebellar neurodegeneration (CBND) model by knock-out of \u003cem\u003eDrp1\u003c/em\u003ein Purkinje cells (PCKO).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b) \u003c/strong\u003eGenotyping PCKO mice from mouse tail PCR assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) \u003c/strong\u003eLeft, appearance of WT and PCKO mice at 3-month-old (3M). Right, quantification of the weight (g) of WT-3M and PCKO-3M mice. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 8animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d) \u003c/strong\u003eLeft, appearance of whole brains of WT-3M and PCKO-3M mice. Bar = 1 cm. Right, quantification of weight of cerebellum from WT and Drp1-PCKO at 3-month-old. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 6 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Left, the sketch showing section position of sagittal plane from top view. Middle,the cerebellums of WT and PCKO mice were stained with Calbindin at 3-month-old. Bar = 500 mm. Right, the cerebellum area of PCKO mice counted at 3-month-old have atrophied observably.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f) \u003c/strong\u003eLeft, the sketch showing the location of the magnification. Right, representative images of Calbindin-stained Purkinje cells within the Cb6. Bar = 50 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e Quantification of the number of Purkinje cells (PCs) per mm demonstrating that PCs have significantly lost from 2 months of age. One-way ANOVA and dunnett’s t test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(h) \u003c/strong\u003eQuantification of thickness of ML (molecular layer) stating that ML have significantly atrophied from 2 months of age. One-way ANOVA and dunnett’s \u003cem\u003et\u003c/em\u003etest, n = 3animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i) \u003c/strong\u003eCerebellar DRP1 protein (left) and Drp1 mRNA (right) from WT and PCKO mice at 3-month-old were stained using immunofluoresce staining or RNAscope respectively. Bar = 20 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(j) \u003c/strong\u003eThe level of DRP1 protein in PCs soma from the left images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(k)\u003c/strong\u003e The level of \u003cem\u003eDrp1\u003c/em\u003emRNA in PCs soma from the left images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(l) \u003c/strong\u003eThe foot print of WT and PCKO mice tested from 3 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(m)\u003c/strong\u003e The latency to fall detected by open filed test showing that PCKO mice have defected coordination from 1 month old.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(n) \u003c/strong\u003eThe number of hind foot slips detected by balance beam showing that balance dysfunction was developed in PCKO mice from 1 month old.\u003c/p\u003e\n\u003cp\u003eThe data was shown by mean ± SD.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/3c31a78d5e5839bf72f6a40d.png"},{"id":59576521,"identity":"32afb5bb-80d8-4dd7-8198-a4428b376867","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1178180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of TdTomato-expressing CBND (CBND\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTdTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) by TdTomato knock-out mice (PC\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTdTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eKO)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The strategy for constructing Drp1-PC\u003csup\u003eTdTomato\u003c/sup\u003eKO (PC\u003csup\u003eTdTomato\u003c/sup\u003eKO) mice by knocking Drp1 in PC\u003csup\u003eTdTomato\u003c/sup\u003e mice, whose cerebellar PCs express TdTomato protein specifically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Genotyping of PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice identified by mouse tail PCR assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e The sketch showing the section position (red dotted line) of confocal images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e DAPI-stained cerebllum of PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice, n = 3 animals, Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Cerebellum area analyzed from PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice showing that cerebellum of PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice decreased markedly from their age of 2 months. One-way ANOVA and dunnett’s \u003cem\u003et\u003c/em\u003e test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e The latency to fall of PC\u003csup\u003eTdTomato\u003c/sup\u003eKO1M mice tested by open filed test showing that PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice developed ataxia from 1 month of age. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 6 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e The number of hind foot slips of PC\u003csup\u003eTdTomato\u003c/sup\u003eKO1M mice tested by balance beam showing that PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice developed balance deficit from 1 month of age. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 6 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(h)\u003c/strong\u003e The correlation between the number of hind foot slips and the number of PCs per mm. Pearson correlation analysis, n = 8 animals.\u003c/p\u003e\n\u003cp\u003eThe data was shown by mean ± SD.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/8d003b48f0a042df1a172347.png"},{"id":59576523,"identity":"9fa8534b-6b11-48eb-a7f9-0294d37c44ab","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":928934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStrategy of liver-originated free mitochondria transfer into degenerated cerebellum of CBND mice (Mito-transfer) and mitochondrial quality control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRapid liver removed from male C56BL/6J mouse.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Rapid mitochondrial isolated from liver of C56BL/6J mouse according to the specification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) \u003c/strong\u003eMitochondria staining using Mitotracker-red in the cell incubator at 37℃ for 30 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Mitotracker imaging under confocal microscope, Bar = 10 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e VDAC protein of liver mitochondria from C56BL/6J detected by western blot. n=3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e Relative active mitochondria stained by JC-1 showing that the mitochondria maintain a normal mitochondrial membrane potential after 1 hour since isolation. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 4 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e ROS level stained by DCFH-DA showing that the mitochondrial oxidative stress keep normal level after 1 hour since isolation. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(h)\u003c/strong\u003e The sketch showing the process of mitochondria allotransplantation into cerebellum of CBND\u003csup\u003egeneral\u003c/sup\u003e mice (PCKO mice).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; The data was shown by mean ± SD.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/4312de73c23c23c47cc173e1.png"},{"id":59577037,"identity":"1b92047d-827a-4830-b35d-1c2578d22bb5","added_by":"auto","created_at":"2024-07-03 11:33:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1433330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMito-transfer, at early stage, significantly alleviates neuron loss and ataxia disorder of CBND\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003egeneral\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eThe timeline showing Mito-transfer to CBND\u003csup\u003egeneral\u003c/sup\u003e mice at 1-month-old.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b) \u003c/strong\u003eThe photos of the CBDN\u003csup\u003egeneral\u003c/sup\u003e and Mito-treated CBDN\u003csup\u003egeneral\u003c/sup\u003e mice when crossing the balance beam.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) \u003c/strong\u003eThe crossing time spending of CBDN\u003csup\u003egeneral\u003c/sup\u003e tested by balance beam at 1, 2, 3 and 4 weeks after Mitochondria treatment decreases obviously. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 10 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d) \u003c/strong\u003eThe number of hindfoot slips tested by balance beam at 1, 2, 3 and 4 weeks after Mitochondria treatment induces significantly. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 10 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e) \u003c/strong\u003eCalbindin, GFAP and IBa1 were co-stained in the cerebellum at 3 weeks post-mito-treatment, which presented increased PCs and decreased gliosis with mitochondria therapy. n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e The number of PCs per millimeters counted from the 4th - 6th lobe of cerebellum in Mito-treated CBDN\u003csup\u003egeneral\u003c/sup\u003e mice recovered markedly after 3 weeks. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f and g)\u003c/strong\u003e GFAP (\u003cstrong\u003eF\u003c/strong\u003e) and IBa1 (\u003cstrong\u003eG\u003c/strong\u003e) intensity were quantified from D, which showing the decline of glosis. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(h) \u003c/strong\u003eThe sketch of the time line for Sparse imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i) \u003c/strong\u003eThe PCs obtained from Sparse experiment at 3 weeks after Mitochondria therapy. n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; The data was shown by mean ± SD.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/5d527ebce191f9542997c256.png"},{"id":59576526,"identity":"7de4a05f-2150-43c5-a599-9b43ea4b0c98","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2239833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMito-transfer, at early stage, significantly delay the progression of neuron degeneration in CBND\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTdTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eThe sagittal plane of cerebellum from PC\u003csup\u003eTdTomato\u003c/sup\u003e, CBND\u003csup\u003eTdTomato\u003c/sup\u003e and CBND\u003csup\u003eTdTomato\u003c/sup\u003e +Mito mice at 3 weeks after mitochondria therapy. Bar (top) = 500 mm and Bar (bottom) = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b) \u003c/strong\u003eCerebellum area was counted from \u003cstrong\u003eA\u003c/strong\u003e, showing the recovery of PCs after mitochondria transfer. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 6 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e The key steps of CUBIC tissue clearing for PC\u003csup\u003eTdTomato\u003c/sup\u003e, CBND\u003csup\u003eTdTomato\u003c/sup\u003e and CBND\u003csup\u003eTdTomato\u003c/sup\u003e +Mito mice at 3 weeks after mitochondria therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e The 3D images performed by tissue clearing. Bar = 100 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e The magnification of Purkinje cells processed by Imaris.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e Mitochondria transfer rescues the size of PCs of CBND\u003csup\u003eTdTomato\u003c/sup\u003e mice. One-way ANOVA and dunnett’s \u003cem\u003et\u003c/em\u003e test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; The data was shown by mean ± SD.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/d11222f702853b5a82cb4722.png"},{"id":59576528,"identity":"832f1705-f644-491d-9fcf-d85694af0924","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1939020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnter and residence of exogenous mitochondria into Purkinje cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The strategy for constructing the ObRb-Mito-GFP mice whose mitochondira in ObRb positive cells express green fluoresence protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e The liver slice obtained from the ObRb-Mito-GFP mouse was stained with Albumin where the green mitochondria widely distributes. Bars = 50 mm or 25 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e The timeline showing that CBND\u003csup\u003egeneral\u003c/sup\u003e mice were transferred with the mitochondria from ObRb-Mito-GFP mouse and sacrificed after 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e the result from C presenting the exogenous green mitochondria is able to enter Purkinje cells and the section images prove the green mitochondria are colocalized to Purkinje cells. Bar = 20 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e The timeline showing the mito-AAV labeled Pcp2-Cre mice were transferred with the mitochondria from ObRb-Mito-GFP mouse and sacrificed after 7 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e The results from E showing the endogenous mCherry-labeled mitochondria colocalize with exogenous green mitochondria. Bar = 20 mm.\u003c/p\u003e\n\u003cp\u003eThe data was shown by mean ± SD.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/0786f81cf6860e6854a1142d.png"},{"id":59577038,"identity":"5fcc75b7-e256-4988-9196-47a0407291eb","added_by":"auto","created_at":"2024-07-03 11:33:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2882749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransferred mitochondria support degenerated Purkinje cells by inhibiting caspase 3 - related cell apoptosis and parkin - related mitophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Q-PCR results of \u003cem\u003eDrp1\u003c/em\u003e, \u003cem\u003eSCA1\u003c/em\u003e, \u003cem\u003eSCA6\u003c/em\u003e, \u003cem\u003eSCA7\u003c/em\u003e and \u003cem\u003eSCA17\u003c/em\u003e mRNA from CBDN\u003csup\u003egeneral\u003c/sup\u003e mice tested at 1-month-old. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Western blotting results of DRP1, DRP1\u003csup\u003eS616\u003c/sup\u003e, DRP1\u003csup\u003eS637\u003c/sup\u003e, FIS1, MFF and SOD from CBDN\u003csup\u003egeneral\u003c/sup\u003e mice tested at 1-month-old. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 5 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Confocal images of Caspase3, Cox4 and Parkin stained on CBDN\u003csup\u003eTdToamto\u003c/sup\u003e mice at 1, 2, 3 and 4-month-old (left) or on mito-treated CBDN\u003csup\u003eTdToamto\u003c/sup\u003e mice (right) after 3 weeks. Bar =5 mm. n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d, f and h)\u003c/strong\u003e Quantification of Caspase3 (\u003cstrong\u003eD\u003c/strong\u003e), Cox4 (\u003cstrong\u003eF\u003c/strong\u003e) and Parkin (\u003cstrong\u003eH\u003c/strong\u003e) expressed on Purkinje cells of CBDN\u003csup\u003eTdToamto\u003c/sup\u003e mice at 1, 2, 3 and 4-month-old. One-way ANOVA and dunnett’s \u003cem\u003et\u003c/em\u003e test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e, g and i) \u003c/strong\u003eQuantification of Caspase3 (\u003cstrong\u003eE\u003c/strong\u003e), Cox4 (\u003cstrong\u003eG\u003c/strong\u003e) and Parkin (\u003cstrong\u003eI\u003c/strong\u003e) expressed on Purkinje cells of mito-treated CBDN\u003csup\u003eTdToamto\u003c/sup\u003e mice (right) after 3 weeks. Two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, n = 6 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(j)\u003c/strong\u003e Calbindin, GFAP and Iba1 tri-stained on cerebellum of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice at 1, 2, 3 and 4-month-old. Bar = 20 mm. n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(k, l and m)\u003c/strong\u003e Quantification of Calbindin (\u003cstrong\u003eK\u003c/strong\u003e), GFAP (\u003cstrong\u003eL\u003c/strong\u003e) and Iba1 (\u003cstrong\u003eM\u003c/strong\u003e) expressed on cerebellum of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice at 1, 2, 3 and 4-month-old. One-way ANOVA and dunnett’s \u003cem\u003et\u003c/em\u003e test, n = 3 animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(n)\u003c/strong\u003e Transferred mitochondria support degenerated Purkinje cells by inhibiting caspase 3 and parkin at the early stage.\u003c/p\u003e\n\u003cp\u003eThe data was shown by mean ± SD.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/346be174afbea00a4b7845fe.png"},{"id":79034133,"identity":"7aae9131-4b08-435a-b7a0-b21ed1f7c658","added_by":"auto","created_at":"2025-03-23 07:05:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14796879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/5ad2aec5-930e-4d95-afa5-4cea93b9cecd.pdf"},{"id":59576524,"identity":"908bfbb5-1b66-4b8a-9f93-f8083751969f","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16834499,"visible":true,"origin":"","legend":"Supplementary video 1","description":"","filename":"VideoS1balancebeam.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/944669a669c713a9461f9ae6.mp4"},{"id":59577041,"identity":"1f82a76f-c93f-4375-8066-a4726b170843","added_by":"auto","created_at":"2024-07-03 11:33:37","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":73206164,"visible":true,"origin":"","legend":"Supplementary video 2","description":"","filename":"VideoS2.PC4XCB.1Movie.avi","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/c4e605069ce35d35684db5c9.avi"},{"id":59576536,"identity":"e360f42d-16dc-42ba-a068-337ca68ccca4","added_by":"auto","created_at":"2024-07-03 11:25:39","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":206862116,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary video 3\u003c/p\u003e","description":"","filename":"VideoS3.KO4XCBMovie.avi","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/e698651ae89d949ae82398a4.avi"},{"id":59577039,"identity":"23a3cb6f-31db-4c30-9770-d422138bcc35","added_by":"auto","created_at":"2024-07-03 11:33:37","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":39276066,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary video 4\u003c/p\u003e","description":"","filename":"VideoS4.KOMITOCB.aivia.tifMovie.avi","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/c67c7210c9431b7882083b37.avi"},{"id":59576529,"identity":"5fe5b944-ac97-46f8-bbec-f25a4a1f714f","added_by":"auto","created_at":"2024-07-03 11:25:36","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":26811228,"visible":true,"origin":"","legend":"Supplementary video 5","description":"","filename":"PC.1.avi","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/33485a8d457dae63f8315517.avi"},{"id":59576532,"identity":"2e1722f2-9bfd-47f5-a4e9-1eaf636bd432","added_by":"auto","created_at":"2024-07-03 11:25:37","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":42687388,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary video 6\u003c/p\u003e","description":"","filename":"KO.avi","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/f82a500e2d03c9abaaa1e04d.avi"},{"id":59576534,"identity":"3f8dbc66-7562-452e-9e38-ff8aa251225c","added_by":"auto","created_at":"2024-07-03 11:25:37","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":26196294,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary video 7\u003c/p\u003e","description":"","filename":"KOMITO.avi","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/549deb0cc480f2b84304b601.avi"},{"id":59576533,"identity":"9756fbeb-4826-404c-ab77-7fa775fbdcd5","added_by":"auto","created_at":"2024-07-03 11:25:37","extension":"doc","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15311947,"visible":true,"origin":"","legend":"","description":"","filename":"2024.6.20SupplementarymaterialsNC.doc","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/8f6da21fec264533c2591c82.doc"},{"id":59576530,"identity":"643e864d-0888-4bc5-9b99-830c56ee2ca6","added_by":"auto","created_at":"2024-07-03 11:25:37","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":15404493,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-4612386/v1/b71d245640f4639a013703ef.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mitochondria transfer transiently rescues cerebellar neurodegeneration at early stage by alleviating mitophagy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNeurodegenerative diseases (ND) are characterized by a progressive loss of neuronal function. Cerebellar neurodegenerative diseases are characterized by cerebellar ataxia, including ataxia telangiectasia\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, spinocerebellar ataxia\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, etc. The main mechanism of cerebellar neurodegenerative diseases is still unknown, thus preventing the development of its drugs. There are no existing treatments to totally correct the underlying neurodegenerative disease process. Current treatments can only relieve symptoms. As the underlying disease progresses, the effectiveness of drug treatment decreases despite increased drug doses. As a result, the side-effect to benefit ratio increases\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Purkinje cells, as the only efferent neurons in the cerebellar cortex, play an important role in cerebellar neurodegenerative diseases. Purkinje cell degeneration is an inherited or sporadic disease characterized by progressive gait and coordination disorders\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Although immunotherapy\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, electrical stimulation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, gene therapy\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, stem cell transplantation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and other means have been studied to treat animal models of neurodegenerative diseases, the curative effect is not ideal.\u003c/p\u003e \u003cp\u003eChronic mitochondrial dysfunction accompanied by biological energy depletion is a pathological marker of major neurodegenerative diseases. Brain injury triggers acute mitochondrial damage and a local energy crisis that accelerates neuronal death. Defective mitochondrial maintenance and insufficient axon energy have become central problems in neurodegenerative diseases and brain injuries\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Alterations in mitochondrial DNA have been described in diseases, including deletions, point mutations, depletion, and maintenance alterations. In Alzheimer\u0026rsquo;s disease or Parkinson\u0026rsquo;s disease, mutations in genes directly related to mitochondrial function, such as Parkin and PINK1, are responsible for rare inherited forms of the disease\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. And PINK1 regulates PARKIN translocation in damaged mitochondria and is involved in neurodegenerative diseases by driving its removal through selective autophagy, a process called mitochondrial autophagy\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Gasdermin protein, a hangman pore-forming molecule that mediates cell death, plays a role in mitochondrial damage and axon loss, and GSDME knockout prevents neurite loss in iPSC-derived motor neurons in ALS patients\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Current studies have shown that the main mechanism is closely related to mitochondrial dysfunction, and the mutation of multiple mitochondria-related genes will lead to the dysfunction and death of Purkinje cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, targeting Purkinje cell mitochondria for the treatment of degenerative changes may be a reliable treatment\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitochondrial transplantation is a therapeutic approach developed by McCully and colleagues that entails the injection of normal mitochondria harvested from unaffected tissue into an ischemic organ of the same subject\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It has recently been applied to human pediatric patients with myocardial ischemia, receiving widespread media attention\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In the central nervous system, an increasing number of reports in animal models or cultured cell lines have suggested the effectiveness of the mitochondrial transplant. Direct injection through veins\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e or arteries\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, or other channels\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, or local region\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, or indirect injections of modified and packaged mitochondrial complexes\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, for various neurological diseases, including Parkinson's disease\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, brain trauma\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, schizophrenia\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, spinal cord injury\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, nerve injury\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and stroke\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We have noticed the same disadvantage in these studies: mitochondrial dysfunction is not the only factor contributing to neurological defects. Thus, the subsequent recovery of neurological function due to mitochondrial transplantation is helpful but raises questions since it is difficult to assess the effectiveness of the treatment among complex factors.\u003c/p\u003e \u003cp\u003eOur study reveals that Purkinje cell degeneration caused by Drp1 knockout results in ataxia through increased mitochondrial autophagy and apoptosis, as well as induced activation of astrocytes and microglia around Purkinje cells. However, upregulation of Drp1 in cerebellar Purkinje cells by the transgenic technique did not alleviate progressive Purkinje cell loss and motor dysfunction. Interestingly, we showed that in-situ exogenous healthy mitochondria injection into the cerebellum at an early stage of cerebellar neurodegeneration in mice significantly inhibited progressive Purkinje cell loss and behavioral impairment. We identified Purkinje cell and mitochondrial function status as key indicators for ameliorating cerebellar neurodegeneration, and mitochondrial transfer as an effective treatment for early-stage cerebellar neurodegeneration.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Generation of cerebellar neurodegeneration model (CBND) by conditional knock-out of Drp1 in Purkinje cells (PCKO)\u003c/h2\u003e \u003cp\u003eBased on previous studies\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, we generated Drp1\u003csup\u003eflox/flox\u003c/sup\u003e::Pcp2-Cre mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e whose Drp1 gene was specifically knocked out in cerebellar Purkinje cells (PCs) (PCKO), also named CBDN\u003csup\u003egeneral\u003c/sup\u003e mice. Their wild-type (WT) littermates were used as controls. PCKO and control mice were viable, fertile, and normal in size and weight \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. The cerebellum of 3-month-old (3M) PCKO mice showed atrophy \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Sagittal plane of mouse cerebellum (1M-4M) were subjected to immunofluorescence with Calbindin, and the PC number quantified \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. The number of PCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e was decreased at 2M to 22.55%, 3M to 6.19%, and 4M to 3.55%. And the thickness of molecular layer (ML) also decreased sharply since their age of 2 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Next, immunofluorescent staining and RNAscope experiment showed that DRP1 protein and \u003cem\u003eDrp1\u003c/em\u003e mRNA both reduced obviously in PC of PCKO mice at 3M \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-k\u003cb\u003e)\u003c/b\u003e. Furthermore, we performed behavioral testes to detect the motor function of PCKO mice, including gait analysis, rotarod and balance beam. Compared to WT-3M mice, the PCKO mice appeared swaying gait at the age of 3-month \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el\u003cb\u003e)\u003c/b\u003e. The results of rotarod and balance beam tests indicated that PCKO mice developed impaired coordination and inbalanced motion since their age of 4 weeks and the symptoms worsened over time \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003em and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003en\u003cb\u003e)\u003c/b\u003e. These results demonstrated that PCKO mice presented cerebellum atrophy, Purkinje cells neurodegeneration and progressive ataxia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Generation of TdTomato-expressing CBND (CBND\u003csup\u003eTdTomato\u003c/sup\u003e) by TdTomato knock-out mice (PC\u003csup\u003eTdTomato\u003c/sup\u003eKO)\u003c/h2\u003e \u003cp\u003eIn order to ensure the stability of the experiment and exclude the non-characteristic interference of immunofluorescence staining, we constructed PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice whose cerebellar Purkinje cells genetically express TdTomato protein \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, also named TdTomato-expressing CBND (CBND\u003csup\u003eTdTomato\u003c/sup\u003e) mice. The middle sagittal planes were detected and the PCs also degenerated at 2M \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. similarly, the cerebellum area on middle sagittal plane had atrophied at 2M \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Based on the behavioral results of PCKO mice, we performed rotarod and balance beam tests on PC\u003csup\u003eTdTomato\u003c/sup\u003eKO mice at the age of 1 month, who similarly exhibited ataxia \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Pearson correlation Analysis was used to explore the relationship between PCs and motor behavior, showing that the number of PCs and the number of hindfoot slips were negatively correlated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these results, it can be inferred that motor dysfunction can be reversibly alleviated before PC significantly dies. In other words, therapeutic interventions at an early stage of mouse development, such as 1 month of age, may be able to delay the progression of cerebellar neurodegeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Targeted overexpression of DRP1 in PCs failed to rescue ataxia in adolescent Drp1-PCKO mice\u003c/h2\u003e \u003cp\u003eWe designed gain-of function experiments \u003cb\u003e(Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea)\u003c/b\u003e to explore whether Drp1 supplement to PCs could ameliorate ataxia. The PCKO mice at 1-month-old were injected with Drp1-DIO virus at the cerebellum. After 4 weeks, the virus were widely expressed on the cerebellum \u003cb\u003e(Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb)\u003c/b\u003e, and Drp1 mRNA obviously upregulated successfully in the cerebellum of PCKO mice \u003cb\u003e(Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec and S1d)\u003c/b\u003e. The rotarod test showed that the coordination activity of PCKO mice had no improvement after Drp1-targeted OE treatment \u003cb\u003e(Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee)\u003c/b\u003e. Unfortunately, there also was no improvement of balance ability of PCKO mice following Drp1 upregulation compared with Drp1 NC treatment \u003cb\u003e(Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 Strategy of liver-originated free mitochondria transfer into degenerated cerebellum of CBND mice (Mito-transfer) and mitochondrial quality control\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn consideration of the mitochondrial deficit of PCKO mice and the favorable therapeutic effect of mitochondria transfer on neuronal disease, we explored the potential of mitochondria transfer on cerebellar neurodegeneration. We injected hepatic-derived mitochondria from WT mouse into the cerebellum of PCKO mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Healthy mitochondria isolated from 100 mg of liver tissues were stained with Mito-tracker to elucidate their bioactive functions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d\u003cb\u003e)\u003c/b\u003e. The VDAC protein results further testified the mitochondria compound \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Considering the vulnerability of mitochondria, we controlled the process of mitochondria transfer within 1 hour. And we proved that the mitochondrial function will not change within 1 hour after mitochondria extracting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Thus, the mitochondria were transferred at a concentration of 50 mg/ml by direct injection into 4 sites of cerebellar cortex coordinates, as described in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 Mito-transfer, at early stage, significantly alleviates neuron loss and ataxia disorder of both CBND\u003c/b\u003e \u003csup\u003e \u003cb\u003egeneral\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice and CBND\u003c/b\u003e\u003csup\u003e\u003cb\u003eTdTomato\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt 1, 2, 3 and 4-week after mitochondira transfer, the PCKO mice were assessed by behavioral tests \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Fortunately, the mice receiving mitochondria transfer for 3 weeks at the age of 1M (CBDN\u003csup\u003egeneral\u003c/sup\u003e+Mito) could walk quickly and well crossing the beam, while the hindfoot of PCKO mice showed sustained continuous slipping down on the balance beam \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb \u003cb\u003eand Video S1)\u003c/b\u003e. The balance ability tested by balance beam of CBDN\u003csup\u003egeneral\u003c/sup\u003e+Mito mice improved significantly at first week after mitochondria transfer. And this effectiveness maintained 3 weeks and faded away in the 4th week \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. We further identified GFAP-positive astroglia and Iba1-positive microglia in cerebellum \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Compared to the PCKO group, the CBDN\u003csup\u003egeneral\u003c/sup\u003e+Mito mice showed less GFAP- and Iba1-positive cells as well as luxuriant Purkinje cells after 3 weeks of mito-transfer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-h\u003cb\u003e)\u003c/b\u003e. Moreover, we injected sparse virus into cerebellum and found that CBDN\u003csup\u003egeneral\u003c/sup\u003e+Mito can delay the progression of PCs degeneration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBasides, to explore the effects of mito-transfer on whole cerebllum, the CBND\u003csup\u003eTdTomato\u003c/sup\u003e mice were also treated with mitochondria and performed CUBIC brain clearing experiment. The number and morphology of PCs showed obviously improvement at 3 weeks after mito-transfer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. For the CUBIC experiment, after fixation, delipidation and refractive index matching, the brain from CBND\u003csup\u003eTdTomato\u003c/sup\u003e mice become transparent totally \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. The whole brain was imaged using light-sheet microscope and we found the number of PCs significantly increased in CBND\u003csup\u003eTdTomato\u003c/sup\u003e+Mito group compared with CBND\u003csup\u003eTdTomato\u003c/sup\u003e mice \u003cb\u003e(Fig S2 and S3 and Video S2-4)\u003c/b\u003e. The cerebellum were magnified locally, of which the results showed the number of PCs of CBND\u003csup\u003eTdTomato\u003c/sup\u003e mice increased significantly and the dendritic tree were still flourish after mito-transfer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed \u003cb\u003eand Video S5-7)\u003c/b\u003e. Though the imaris-processed analysis, the size of PCs also recovered \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.6 Mito-transfer, at middle and late stages, can not improve neuron loss and ataxia disorder in CBND\u003c/b\u003e \u003csup\u003e \u003cb\u003egeneral\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether mitochondria transfer contributes to CBDN\u003csup\u003egeneral\u003c/sup\u003e mice at middle and late stages of neurodegeneration. We performed mitochondria transfer to CBDN\u003csup\u003egeneral\u003c/sup\u003e mice at 2 or 3-month-old and detected behavioral test at 3 weeks after that \u003cb\u003e(Fig S4a and S4f )\u003c/b\u003e. Calbindin staining showed that Purkinje cells of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice lost severely and the results of cerebellum atrophy had no difference after mitochondria transfer \u003cb\u003e(Fig S4b, S4c, S4g and S4h )\u003c/b\u003e. The astroglial and microglial activation of cerebellum from CBDN\u003csup\u003egeneral\u003c/sup\u003e mice did not present any remission after mitochondria therapy \u003cb\u003e(Fig S4b and S4g )\u003c/b\u003e. Similarly, there was no significant difference in coordination or balance ability between CBDN\u003csup\u003egeneral\u003c/sup\u003e mice and mito-treated CBDN\u003csup\u003egeneral\u003c/sup\u003e mice at 2 \u003cb\u003e(Fig S4d and S4e )\u003c/b\u003e or 3-month-old \u003cb\u003e(Fig S4i and S4j )\u003c/b\u003e. These results indicated that the necessary condition for the effectiveness of mitochondria transfer to delay the progression of Purkinje cells degeneration is that the number of Purkinje cells is enough.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Enter and residence of exogenous mitochondria into Purkinje cells\u003c/h2\u003e \u003cp\u003eTo make sure that exogenous mitochondria were capable of entering Purkinje cells, we generated GFP-Mito tagfloxed mice and crossed them with ObRb-Cre mice to generate ObRb-Mito-GFP mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, whose ObRb\u0026thinsp;+\u0026thinsp;cells in liver exhibited bright mito-GFP fluorescence localized specifically to the mitochondrial compartment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. We transplanted the isolated green mitochondria from the liver of ObRb-Mito-GFP mice to the cerebellum of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice according to the same protocol, and the results confirmed that the green exogenous mitochondria entered the soma of PCs after 24 hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. To identify whether the exogenous mitochondria communicated with the endogenous mitochondria of Purkinje cells, we injected the cerebellum of Pcp2-Cre mice with mitochondria-driving virus. And 3 weeks later, the mitochondria were transferred to that mouse \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. The results manifested that the exogenous green mitochondria were able to enter and interacted with Purkinje cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.8 Transferred mitochondria support degenerated Purkinje cells by inhibiting caspase 3 - related cell apoptosis and parkin - related mitophagy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next investigated whether the well effectiveness of mitochondria transfer on CBDN\u003csup\u003egeneral\u003c/sup\u003e mice was attributed to DRP1 recovery in the Purkinje cells. Calbindin and DRP1 co-staining showed that mitochondria transfer could not up-regulate DRP1 of Purkinje cells \u003cb\u003e(Fig S5)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo explore the possible mechanism of mitochondria transfer, we detected the molecule-related experiment in Purkinje cells of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice. Firstly, the Drp1 mRNA decreased in the cerebellum of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice at the age of 1 month when the ataxia-related molecule, like SCA1 (ATXN1), SCA6 (CACNA1A), SCA7 (ATXN7) and SCA17 (TBP), also reduced in the cerebellum \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Next, we found that DRP1, FIS1 and MFF protein in the cerebellum of CBDN\u003csup\u003egeneral\u003c/sup\u003e mice showed prominent reduction, while there was no difference in phosphated DRP1, like DRP1\u003csup\u003eS616\u003c/sup\u003e and DRP1\u003csup\u003eS637\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. And SOD level reduced in CBDN\u003csup\u003egeneral\u003c/sup\u003e mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, which showed the increased level of oxidative stress in mitochondria. These results indicated that the mitochondrial dysfunction led to Purkinje cells degeneration. We further used CBDN\u003csup\u003eTdTomato\u003c/sup\u003e mice to investigate the mitochondria function in Purkinje cells. We found that the level of caspase3 on Purkinje cells increased markedly in CBDN\u003csup\u003eTdTomato\u003c/sup\u003e mice since their age of 1 month \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. And mitochondria transfer were capable of slowing the progression of apoptosis of Purkinje cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. COX4, a marker presenting the mitochondrial function, decreased significantly in Purkinje cells of CBDN\u003csup\u003eTdTomato\u003c/sup\u003e mice from 2-month-old \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e, which can be reversed by mitochondria transfer at the age of 1 month \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Similarly, the key molecule of mitophagy, PARKIN, showed remarkable increment in Purkinje cells of CBDN\u003csup\u003eTdTomato\u003c/sup\u003e mice at their 2-month-old \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e and noteworthy decrease after mitochondria therapy \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e. These results demonstrated that exogenous mitochondria are able to slow the progression of neurodegeneration by reducing apoptosis and increasing the mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBesides, Calbindin, GFAP and Iba1 tri-staining were used to estimate the intensity of astrocytes and microglia in cerebellum of PCKO mice. In the cerebellum of WT mice, little GFAP and Iba1 positive cells was found \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej\u003cb\u003e)\u003c/b\u003e. Accompanying the progression of PC loss \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ek\u003cb\u003e)\u003c/b\u003e, GFAP intensity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el\u003cb\u003e)\u003c/b\u003e and Iba1 intensity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003em\u003cb\u003e)\u003c/b\u003e gradually and significantly increased since the age of 1 month. These results stated that motor dysfunction and glial activation tend to occur earlier than PCs loss.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eMitochondrial damage plays a pivotal role in maintaining and promoting neurodegeneration, such as the impairment of respiratory chain, mitochondrial DNA and mitophagy\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, the therapy targeted to mitochondria may play a key role in neurodegenerative process. In this study, we described that mitochondrial transfer technology can reduce the level of mitophagy of Purkinje cells by improving mitochondrial function, thereby controlling apoptosis of Purkinje cells and delaying the development of cerebellar neurodegenerative diseases. In 2012, Yusuke Kageyama et al.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e constructed the mice with DRP1-specific knockout in cerebellar Purkinje cells, in which the loss of Purkinje cells occurred over time. We have selected this model as the neurodegeneration of the cerebellum. And we demonstrated a behavioral correlation between the degeneration of cerebellar Purkinje cells and ataxia. In addition, with the progressive loss of Purkinje cells, a large number of astrocytes and microglia in the cerebellum were activated. The molecules related to apoptosis or mitophagy in Purkinje cells were significantly increased and mitochondrial function was significantly impaired. Although we up-regulated DRP1 in Purkinje cells of PCKO mice, this operation did not improve the degeneration of Purkinje cells and the ataxia of mice. Fortunately, when liver-derived mitochondria of healthy mice were injected into the cerebellum, the apoptosis process and mitochondrial dysfunction in Purkinje cells were alleviated. Due to the recovery of Purkinje cells, the motor impairment of the PCKO mouse was significantly alleviated. However, our current study found that the effects of mitochondrial transfer on neurodegeneration only lasted for three weeks and the apoptosis process of Purkinje cells was restarted in the forth week after operation.\u003c/p\u003e \u003cp\u003eConsistent with the results of previous studies, Mitochondria can enter neuron in free form and improve the state of mitochondria in the brain\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our results demonstrated that exogenous healthy mitochondria entered into Purkinje cells, but whether they can improve the physiological function of Purkinje cells and alleviate the motor dysfunction depends on the state of Purkinje cells and their mitochondria. We used Cox4 and Parkin to reflect the the level of mitochondrial respiratory chain and mitophagy, respectively\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The state of Purkinje cells was reflected by Calbindin and caspase3. Even though cerebellar glial cells proliferated and the caspase3 of Purkinje cells increased, the mitochondrial function was compensatory and Purkinje cells were still there. Therefore, the premise that mitochondria transfer can play a role was that the number of Purkinje cells and the function of mitochondria have not been seriously damaged. This was the most critical stage. Exogenous mitochondria can be acquired by host neurons, and then played a function to support the development and growth of host neurons. Therefore, using the Cox4 and Parkin to reflect the mitochondrial state, as well as Calbindin and caspase3 to reflect the neuron state, may gain a valuable time window and win an opportunity to treat neuronal degenerative diseases with natural loss of mitochondrial genes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003en\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMitophagy can selectively wrap and degrade damaged mitochondria through autophagy mechanisms, thereby maintaining mitochondrial and cellular homeostasis. So dysfunction of mitophagy can lead to accumulation of damaged mitochondria and cellular dysfunction, leading to aging and age-prone neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Our study found increased mitophagy in the Purkinje cells of PCKO mice compared to normal mice, and decreased mitophagy in Purkinje cells was found after mitochondrial transplantation. We think this is the result of mitochondrial transplantation improving mitochondrial function. As exogenous healthy mitochondria entered into dysfunctional Purkinje cells, the proportion of damaged mitochondria in Purkinje cells was reduced contributing to the decline of overall mitohagy level. Therefore, the reduction of mitophagy indirectly proved that mitochondrial transplantation can improve the mitochondrial function for recipient cells.\u003c/p\u003e \u003cp\u003eExisting studies have shown that the survival cycle of mitochondria is about 28 days, which is consistent with our findings. After 3 weeks, the metabolism capability of exogenous mitochondria was exhausted leading to a rebound of ataxia. Whether exogenous mitochondria were able to communicate with endogenous mitochondria during mitochondrial transplantation was unclear. The results so far supported two possibilities. One is to integrate, but not replace. The other is not to blend in, do their own thing. However, the existing results showed that mitochondrial transplantation could not make up for the loss of Drp1 in the original nuclear gene. Finally, these mitochondria could not effectively divide\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and the damaged mitochondria in the cell could not be effectively removed\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In the end, neither exogenous healthy mitochondria nor endogenous defective mitochondria could survive for one month. However, the future of mitochondrial transplantation is still exciting, after all, for neurodegenerative diseases, the precious window of time still let people see the light.\u003c/p\u003e \u003cp\u003eTherefore, correcting the lack of Drp1 has become our biggest challenge. Previous studies have shown that gene therapy is a potential protocol for the treatment of neurodegenerative diseases, such as Parkinson\u0026rsquo;s disease and Alzheimer\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In 2014, Michael S Rafii et al. conducted stereotactic gene delivery of AAV2-NGF on a patient with early and middle stage of Alzheimer\u0026rsquo;s disease. The results showed that AAV2-NGF was safe and well-tolerated for 2 years\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In 2018, Michael S Rafii et al. performed AAV2-NGF or sham surgery on 49 patients with early and middle stage of Alzheimer\u0026rsquo;s disease. There was no significant difference in efficacy between the gene therapy group and the placebo group\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In addition, for congenital genetic defects, the time condition of gene therapy are very demanding as patients grow older\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. These studies showed that the efficacy of gene therapy has varied greatly between individuals. Our laboratory also attempted to deliver Drp1 overexpressing AAV to the cerebellum. Although the delivery was satisfactory, the overexpression of Drp1 failed to correct the fate of neuronal degeneration. We hypothesized that this may attribute to late genetic intervention or low doses.\u003c/p\u003e \u003cp\u003eGlial cells also play an important role in neurodegenerative diseases. Consistent with previous research, this study also verified that the activation of surrounding astrocytes and microglia was manifested before mitochondrial damage and neuronal function damage\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. It showed that the cells around neurons and neurons themselves formed a community-like overall structure, which is highly related to each other. However, it is important to note that the activation of these glial cells is the result, not the cause, of the denatalized neurons. When the mitochondria transfer corrected the neuronal state, these activated glial cells disappeared and returned to their original resting state. Therefore, we believe that rather than overestimating the potential of support cells in the community, it may be more reliable to use their status as a measure of treatment effectiveness.\u003c/p\u003e \u003cp\u003eThere are some limitations in this study. Ataxia symptoms appeared in PCKO mice at the age of one month. Although the number of Purkinje cells did not decrease significantly at this time, we speculated that the function of Purkinje cells had been damaged at this time. However, we did not conduct relevant experiments on the function of Purkinje cells in our study. Since the purity of mitochondrial extract was not verified in our study, it cannot be ruled out that other substances also play a role like mitochondria. The validity period of mitochondrial transplantation in this study is only 3 weeks, and we will try to extend the validity period of mitochondria in the next experiment, hoping that mitochondria will become a long-term effective stage for the treatment of cerebellar neurodegeneration.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1 Animals\u003c/h2\u003e\n \u003cp\u003eWe purchase \u003cem\u003ePcp2\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e mice from the Jackson Laboratory (America, Stock No: 004146). Drp1 Cre-triggered conditional knockout mice were designed by inserting a loxP site after the sequence of Drp1, and obtained from Cyagen (serial number: CKOAIS191230RT5, China). ObRb-Cre mice were purchased from Cyagen (CKOCMP-74011-Slc25a27-B6N-VA, China). Rosa26-CAG-LSL-GFP-Mito tag mice (Mito-GFP mice) were constructed by Gempharmatech (cas9-ki(Rosa26), China), of which outer mitochondria membrane carries green fluorescent protein. B6/JGpt-H11\u003csup\u003eem1Cin(CAG\u0026minus;LoxP\u0026minus;ZsGreen\u0026minus;Stop\u0026minus;LoxP\u0026minus;tdTomato)\u003c/sup\u003e/Gpt (B6-G/R) mice were purchased from GemPharmatech (T006163, China). And C\u003cem\u003e57BL/6J\u003c/em\u003e Gpt mice were also gotten from GemPharmatech. \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice were produced by crossing \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/+\u003c/em\u003e\u003c/sup\u003e mice with \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice to generate homozygous control. \u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice were crossed with \u003cem\u003ePcp2\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e mice for two generations to generate \u003cem\u003ePcp2\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eDrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice (PCKO mice) as knockout mice. PC\u003csup\u003etdTomato\u003c/sup\u003e KO mice were constructed by PCKO mice and B6-G/R mice. ObRb-Mito-GFP mice were generated by hybridizing ObRb-Cre mice and Mito-GFP mice. All mice were housed (maximum 5 mice in a cage) with food and water freely available under a 12-h day/night cycle (8 am to 8 pm, day). Roughly equal numbers of male and female mice were used for the experiments. All of the animal protocols complied with the Animal Care and Use Committee of the Air Force Military Medical University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (permit number IACUC-20190107).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2 Genotyping and confirmation\u003c/h2\u003e\n \u003cp\u003eThe mouse genotype was identified by mouse tail PCR with genomic DNA. The primers used to identify the Pcp2 Cre sequence with 576 bp were shown as: F1: 5\u0026rsquo;-ATTCTCGTGGAACTGGATGG-3\u0026rsquo; and R1: 5\u0026rsquo;-GGACAGGTAATGGTTGTCTGG-3\u0026rsquo;. The primers used to identify the Drp1 loxP sequence with 292 bp for mutant and 231 bp for wild type were shown as: F2: 5\u0026rsquo;-CCACTTTAGGTACTCATAACACA-3\u0026rsquo; and R2: 5\u0026rsquo;-ATTGTCATACATTCAGATAGGG-3\u0026rsquo;. The offspring with concurrence of 576 bp and 292 bp was regarded as the Drp1 conditional knockout mouse. The primers used to identify the B6-G/R sequence with 1465 bp were shown as: F3: 5\u0026rsquo;-ATGCCCACCAAAGTCATCAGTGTAG-3\u0026rsquo; and R3: 5\u0026rsquo;-AGGCGGGCCATTTACCGTAAGTTA-3\u0026rsquo;. The primers used to identify the ObRb Cre sequence were shown as: F4: 5\u0026rsquo;-GCTGGAAGATGGCGATTAGC-3\u0026rsquo; and R4: 5\u0026rsquo;-TCTTCTTTCCAGAGTTCAGATGT-3\u0026rsquo;. The primers used to identify the Mito-GFP sequence were shown as: P1: 5\u0026rsquo;-CCCAAAGTCGCTCTGAGTTGTTA-3\u0026rsquo;, P2: 5\u0026rsquo;-TGGCGTTACTATGGGAACATACGTC-3\u0026rsquo; and P3: 5\u0026rsquo;-TCGGGTGAGCATGTCTTTAATCT-3\u0026rsquo;. ObRb-Mito-GFP mice were generated by hybridizing ObRb-Cre mice and Mito-GFP mice. And their offspring, ObRb-Mito-GFP mice, show PCR result of one band of 436 bp and 232 bp. The cycling condition of PCR reaction was as follows: 94\u0026deg;C for 3 min, 35 cycles of 94\u0026deg;C for 30 s for denaturation, 60\u0026deg;C for 35 s for annealing, and 72\u0026deg;C for 35 s for extension, and after the 35 cycles with 72\u0026deg;C for additional extension for 5 min. The primers and PCR conditions were designed by Tsingke Biotechnology Co., Ltd.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3 Tissue preparation\u003c/h2\u003e\n \u003cp\u003eFirstly, mice were anesthetized by isoflurane. And then the mice were perfused transcardially with 0.01 M phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS for fixation. The brain was removed carefully and submerged in 4% PFA overnight at 4\u0026deg;C for postfixation. Subsequently, we immersed the brain in gradient dehydration with sucrose solution (10%, 20%, and 30% in order) for more than 48 hours. The brain was sectioned at 20-\u0026micro;m thicknesses for Immunofluorescent staining, 10-\u0026micro;m for RNAscope in situ hybridization on a cryostat (Leica CM1850, Germany). Finally, we mounted these sections onto the glass slides directly and returned them into \u0026minus;\u0026thinsp;20\u0026deg;C cryostat chamber immediately.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e4.4 Immunofluorescent staining\u003c/h2\u003e\n \u003cp\u003eImmunofluorescent staining was complied with the previous instruction. Firstly, the tissues were washed in the PBS, blocked with 3% fetal bovine serum and permeabilized with 0.3% TritonX-100 at room temperature for 30 minutes. Then, we incubated these tissues with the primary antibodies at 4\u0026deg;C for up to 18 hours (anti-PARKIN was incubated at 4\u0026deg;C for up to 48 hours). And the working concentrations of primary antibodies were as follows: anti-Drp1 (Abcam, AB184247, Rabbit, 1: 50), Anti-Calbindin (Alexa Fluor\u0026reg; 594 Conjugate) (Cell Signaling Technology, #88831, Rabbit, 1: 50); Anti-GFAP (Alexa Fluor\u0026reg; 488 Conjugate) (Cell Signaling Technology, 3655S, Mouse, 1:50); anti-IBA1 (Woka, 011-27991, Goat, 1:400), anti-caspase3 (Absin, abs13825, Rabbit, 1:200), anti-PSD95 (Cell Signaling Technology, #34050S, Rabbit, 1:200), anti-COX4 (Cell Signaling Technology, #4850, Rabbit, 1: 500), anti-VDAC (Abcam, ab15895, Rabbit, 1:500), anti-PARKIN (Invitrogen, PA5-13399, Rabbit, 1:50), anti-LAMP1 (Abcam, AB208943, Rabbit, 1: 100), anti-IP3R (Invitrogen, PA1-901, Rabbit, 1: 250) and anti-Albumin (Abcam, AB207327, Rabbit, 1:500). Alexa Fluor\u0026reg; 647 AffiniPure Donkey Anti-Goat IgG (H\u0026thinsp;+\u0026thinsp;L) (Jackson Immunoresearch, 705-605-147) and Dylight 649, Goat Anti-Rabbit IgG (A23620, Abbkine, USA) were used as the secondary antibodies, which were diluted in PBS and incubated for 2 hours at room temperature. Staining 49,69-diamidino-2-phenylindole dihydrochloride (DAPI) (C1005, Beyotime, China) for 15 minutes was the last procedure of immunofluorescence to stain the nucleus. High-resolution images of section from PCKO mice were performed using confocal microscopy (FV3000, Olympus, Japen). And Leica Stellaris 5 (German) were used to imaging for PC\u003csup\u003etdTomato\u003c/sup\u003eKO mice. The results were quantified by ImageJ. For colocalization, we used JACop plugin to get Manders\u0026rsquo; Colocalization Coefficients (MCC). The total fluorescence intensity of protein in Purkinje cells was presented as PC\u003csub\u003ei,colocal\u003c/sub\u003e. And PC\u003csub\u003ei\u003c/sub\u003e replaced the total fluorescence intensity of Purkinje cells. So the quantification of each protein in Purkinje cells could be regarded as MCC\u003csub\u003ePC\u003c/sub\u003e. The formula of MCC\u003csub\u003ePC\u003c/sub\u003e is \u003cimg src=\"data:image/png;base64,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\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e4.5 RNAscope in situ hybridization\u003c/h2\u003e\n \u003cp\u003eThe staining protocol of RNAScope was referred to previous methods. We baked the tissues at 37\u0026deg;C for 6 h and then washed them with 0.01 M PBS for 5 min. Each tissue was covered by hydrogen peroxide (322281, ACD, USA) at room temperature for 10 min following washing twice in distilled water. The tissues were immersed in the boiled Target Retrieval reagents (322000, ACD, USA) at 97\u0026deg;C for 10 min. Immediately, the tissues were transferred to the room-temperature distilled water and then to the absolute ethanol for an additional 3 min. Next, when the tissues were air-dried, we outline them with a hydrophobic pen (CIRISC PAP pen, I.S. CIRCLE WRITER, Japan). After hydrophobic boundaries completely dried, protease III reagent (322281, ACD, USA) was added to slides to cover the whole tissue. Subsequently, the slides were incubated in a preheated HybEZ oven (ACD, USA) at 40\u0026deg;C for 30 min, followed by washing twice in distilled water. These tissues were used for hybridization. A mixture of three probes was then added to each slide until the tissue was fully covered. The present RNAscope in situ hybridizations were divided into three types of combinations of probes. In the first combination, channel 1 probe was defined as Drp1 (434671, ACD, USA), channel 2 as Pcp2 (509991-C2, ACD, USA), and channel 3 as Nclx (1066901-C3, ACD, USA). In the second combination, channel 1 probe was defined as Mfn1 (578731, ACD, USA), channel 2 as Pcp2, and channel 3 as Mfn2 (581891-C3, ACD, USA). In the third combination, channel 1 probe was defined as Ucp2 (443781, ACD, USA), channel 2 as Pcp2, and channel 3 as Ucp4 (1073441-C3, ACD, USA).\u003c/p\u003e\n \u003cp\u003eAfter hybridizing in a HybEZ oven at 40\u0026deg;C for 2 h, the slides were washed twice in 1\u0026times; RNAscope\u0026reg; washing buffer (310091, ACD, USA) for 2 min each time. The slides were returned to the oven for 30 min following submersion in AMP-1 reagent. This step was repeated with AMP-2 and AMP-3 reagents for 30 min and 15 min, respectively. The HRP-C1, HRP-C2, and HRP-C3 signals were processed in order. Opal 520 (ASOP520, ASbio, USA) was applied to mark the channel 1 probe, Opal 570 (ASOP570, ASbio, USA) was applied to mark the channel 2 probe, and Opal 690 (ASOP690, ASbio, USA) was applied to mark the channel 3 probe. Finally, the tissue was submerged with Prolong Gold Antifade Mountant with DAPI. High-resolution imaging was performed using confocal microscopy (Zeiss, Germany). The results were quantified by ImageJ.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e4.6 Behavior\u003c/h2\u003e\n \u003cp\u003eMice were used to conduct all of the below behavioral tests. Each mouse was grouped and numbered by ground of genotype, which was completed by a third person. The experimenters were ignorant of the mouse genotype. All the behavioral experiments were conducted between 6 p.m. and 10 p.m.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e4.7 Rotarod test.\u003c/h2\u003e\n \u003cp\u003eTo study balance and coordination affected on mice by Drp1 of Purkinje cells, we placed the transgenic mice on an accelerated rotating rod (BZY007, Jiliang, China) with a diameter of 3 cm, which started rolling at 4 rpm and increased to 40 rpm within 180 seconds. Each trial lasted 10 minutes, with at least a 15-minutes interval. We trained the mice for 3 consecutive days, 3 times a day, and collected data on the fourth day. The latency to fall from the rod were recorded for analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e4.8 Balance beam\u003c/h2\u003e\n \u003cp\u003eTo further measure the balance influenced by Drp1, the mice were placed on a horizontal beam (1-meter length and 1.2-centimeter width). We performed the experiment following the protocol of previous research. We trained the mice for 3 consecutive days, 3 times per day, and collected data on the fourth day. The latency to cross the beam and the number of hind foot slips were recorded for analysis. The hindfoot slips were counted if either the left or right hind paw slipped off the beam\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e4.9 Foot print\u003c/h2\u003e\n \u003cp\u003eThe feet of the mice were stained with blue ink. Each mouse was placed on a white sheet and made to walk from the beginning to the end of the sheet. If the mouse leaves the paper from the median position without reaching the end, the round is removed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e4.10 Mitochondria isolation\u003c/h2\u003e\n \u003cp\u003eThe procedure of mitochondria isolation was performed according to the instruction (SM0020, Solarbio, China). The tissue was rinsed with saline and the mitochondrial lysis buffer was added for grinding and homogenizing. The homogenization was centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was transferred to a new tube and centrifuged at the same condition. The secondary supernatant was then centrifuged at 12,000 g for 10 min at 4 ℃. The precipitate was resuspended with wash buffer and centrifuged at 1000 g for 5 min at 4 ℃. The supernatant was centrifuged at 12,000 g for another 10 min at 4 ℃. Finally, mitochondria were resuspended with Store Buffer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e4.11 Mitochondria membrane potential detection\u003c/h2\u003e\n \u003cp\u003eThe functional mitochondria were evaluated by JC-1 detection using isolated mitochondria from cerebellum, which strictly comply with the manufacturer\u0026rsquo;s instruction (C2006, Beyotime, China). In simple, 900 \u0026micro;l JC-1 working solution was added to 100 \u0026micro;l isolated mitochondria suspension and the concentration of total protein ranged from 10 \u0026micro;g to 100 \u0026micro;g. Then added 100 \u0026micro;l of them in a 96-well plate for test. The fluorescence measured in a Microplate reader (Spark, Tecan, Switzerland) was set as below: excitation wavelength, 485 nm; emission wavelength, 590 nm. The fluorescence (F) produced per sample was recorded, and total fluorescence per microgram of proteins (mg) was calculated and analyzed. The F/mg is an indication of the relative amount of J-aggregate formation from isolated mitochondria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, which reflects the potential of active mitochondria. In one control mouse, this was set to 1. The amount of relative fluorescence production of each sample was indicated as a percentage of the F/mg in control mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e4.12 ROS level assay\u003c/h2\u003e\n \u003cp\u003eReactive Oxygen Species Assay Kit (S0033S, Beyotime, China) was used to detect the ROS level. Mitochondria were extracted according to the above method. DCFH-DA was diluted in serum-free medium at 10 \u0026micro;M. After collection, the mitochondria were suspended in diluted DCFH-DA at a concentration of 10 to 100 \u0026micro;g and incubated in a cell incubator at 37\u0026ordm;C for 20 minutes. Inverted and mixed every 3\u0026ndash;5 minutes and then washed three times with serum-free cell culture solution. The fluorescence measured in a Microplate reader (Spark, Tecan, Switzerland) was set as below: excitation wavelength, 488 nm; emission wavelength, 525 nm. The fluorescence (F) produced per sample was recorded, and total fluorescence per microgram of proteins (mg) was calculated and analyzed. The F/mg is an indication of the relative amount of ROS produced from isolated mitochondria. In one control mouse, this was set to 1. The amount of relative fluorescence production of each sample was indicated as a percentage of the F/mg in control mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e4.13 Viral injection\u003c/h2\u003e\n \u003cp\u003eThe mice were anesthetized with pentobarbital and fixed on a stereoscopic brain locator (RWD 69100, Shenzhen, China). Cut the skin on the top of the head of the mouse and expose the skull. As previously described, the RAAV-ef1\u0026alpha;-DIO-Mito-mCherry (BC-1577, Braincase, China) was injected into the cerebellum of mice to label mitochondria of Purkinje cells. The concentrations of AAVs used in our study were all diluted to 1*10\u003csup\u003e12\u003c/sup\u003e v.g./mL. The injection site was determined from brain mapping and shown as below: (\u0026plusmn;0.50, -6.25, -2.25) and (\u0026plusmn;0.50, -7.20, -3.00). AAV was injected using a microsyringe (RWD 79013, Shenzhen, China) with a target injection volume of 1 \u0026micro;L and an injection speed of 150 nL/min. The needle remains in the cerebellum for 8 minutes before removal. The skin was sutured intermittently, and iodine disinfectant was applied to the closed wound. And 4 weeks later, the mice were used to perform behavior test, including rotarod test and balance beam test.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e4.14 Mitochondria transfer\u003c/h2\u003e\n \u003cp\u003eC57BL/6J mice were sacrificed by neck dissection, of which about 100 mg liver was removed immediately to obtain isolated mitochondria. Nanodrop (701-058112, Thermo Scientific, USA) was used to detect the protein concentration in extracted mitochondria, which were diluted to 50 mg/ml. The Drp1 knockout mice were anesthetized with pentobarbital and fixed on a stereoscopic brain locator (RWD 69100, Shenzhen, China). Cut the skin on the top of the head of the mouse and expose the skull. The injection site was determined from brain mapping and shown as below: (\u0026plusmn;0.50, -6.25, -2.25) and (\u0026plusmn;0.50, -7.20, -3.00). Mitochondria were injected using a microsyringe (RWD 79013, Shenzhen, China) with a target injection volume of 200 nL and an injection speed of 100 nl/min. The needle remains in the cerebellum for 8 minutes before removal. And the control mice were treated with the store buffer in the same volume. The skin was sutured intermittently, and iodine disinfectant was applied to the closed wound. The entire transfusion experiment was finished within 1 hour. To testing the bioactivity of mitochondria, we stained it with MitoTacker Red (1:1000, M22426, Invitrogen, USA) under 37 ℃ for 30 minutes. Then, the mitochondrial solution was dropped onto the slide and observed under confocal microscope (FV3000, Olympus, Japen).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e4.15 Sparse virus injection\u003c/h2\u003e\n \u003cp\u003eAAV virus were injected into cerebellum of mice at the age of 1-month-old. In the mean time, PCKO mice were transferred with mitochondria, and the control were transferred with store buffer. The injection sites were shown as: (\u0026plusmn;0.50, -6.25, -2.25; 1 \u0026micro;L)) and (\u0026plusmn;0.50, -7.20, -3.00; 1 \u0026micro;L). The concentrations of AAVs used in our study were all diluted to 1*10\u003csup\u003e12\u003c/sup\u003e v.g./mL. The detailed steps were similar as viral injection. The mice brain was removed after 3 weeks later and sectioned at 100-\u0026micro;m thicknesses and then floated them in PBS. DAPI was stained for 15 minutes. Lastly, confocal microscopy (FV3000, Olympus, Japen) was used to view images.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e4.16 CUBIC tissue clearing\u003c/h2\u003e\n \u003cp\u003eThe thorough steps of tissue clearing were complied with the hydrophilic tissue clearing reagent kit (#210701, Nuohai Life Science, China). After sacrificing, the mice were perfused with PBS, fixed with 4% paraformaldehyde, and post-fixed with 4% paraformaldehyde again at 4 ℃ for 24 hours. Next, washed with PBS for 2 hours and repeated 2 times. Then, immersed the brain in the delipidated solution (solution A : solution B\u0026thinsp;=\u0026thinsp;1:1) and shook it at 37 ℃ for 6 days at a shaking speed of 60 rpm. Consequently, the refractive index was matched with solution C and the brain was shaken at the speed of 60 rpm at 25 ℃ for 2 days. Finally, the brain was immobilized with AGAR gel and photographed with Nuohai LS 18 Tiling Light Sheet Microscope (Nuohai Life Science, China). The corresponding videos were produced with Aivia (version 12.1.0, Leica, Germany).\u003c/p\u003e\n \u003cp\u003eThe raw data for 3D image analysis was captured by LiTScan (Light Innovation Technology Limited) and subsequently converted into an am format readable by Amira software (Thermo Fisher Scientific, USA). Subsampling of the 3D image samples was conducted to adjust resolution, utilizing the Resample module to set appropriate voxel sizes. In the preprocessing stage, the background detection and correction module of filters was employed to eliminate background noise. Structural enhancement filters, particularly 3D filters, were then applied to enhance structural features, followed by segmentation of regions of interest using the Interactive Thresholding module based on specified intensity ranges. Subsequently, manual labeling of segmented regions was performed, followed by removal of small spots to eliminate defects. It is noteworthy that the PC cell and cerebellar regions are treated independently, with segmentation of the cerebellar region accomplished through manual labeling based on atlases. Measurement and analysis modules, especially the label analysis function, were employed for data quantification, with the selection of spreadsheet and spatial map options for result export. Finally, selected data was imported into Imaris software (Oxford Instruments, UK) for visualization representation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e4.17 Real time-quantitative polymerase chain reaction (RT\u0026ndash;qPCR)\u003c/h2\u003e\n \u003cp\u003eMice were sacrificed by neck dissection, whose cerebellar cortex was removed to tube and rinsed with saline immediately. All following procedure were complied with the industrial instruction. The cerebellar cortex were dissected and homogenized. Trizol reagent (DP419, TIANGEN, China) was used to extracted the total RNA. The total RNA was then reversely transcripted to cDNA according to cDNA synthesis kit (TSK314S, TSINGKE, China). Finally, the cDNA was used to perform PCR test (TSE201, TSINGKE, China), including the gene of \u003cem\u003eDrp1\u003c/em\u003e, ATXN1, ATXN6, ATXN7 and ATXN17. GAPDH was used as the reference gene. All these primer sequences were exhibited in the \u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e. We followed the 3-steps reaction procedure, shown as follow: the phase of pre-denaturation was performed under 95 ℃ for 1 min, the reaction phase firstly denaturated at 95℃ for 10 s, then annealed at 60 ℃ for 10 s, and finally extended at 72 ℃ for 10 s, and the reaction phase was repeated for 40 cycles. After the phases of amplification, the DNA products were detected using lysis curves. The quantitative was statistically calculated using the 2\u003csup\u003e\u0026minus;△△\u003c/sup\u003e Ct.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e4.18 Western Blotting\u003c/h2\u003e\n \u003cp\u003eThe cerebellums were removed instantly after neck dissection. The protein concentration was detected using BCA assay (E-BC-K318-M, Elabscience, China). Then the samples were electrophoreted on 7.5% SDS-polyacrylamide gel (PG111, Epizyme, China) or 12.5% SDS-polyacrylamide gel (PG113, Epizyme, China) and transferred to PVDF membrane (IPVH00010, Millipore, USA). Primary antibodies were diluted in 1:1000 and incubated at 4℃ over 16 hours. The information of primary antibodies were shown as: anti-DRP1 (8570, Cell Signaling Technology, USA), anti-DRP1\u003csup\u003eS616\u003c/sup\u003e (ab314755, Abcam, USA), anti-DRP1\u003csup\u003eS637\u003c/sup\u003e (ab193216, Abcam, USA), anti-FIS1 (ab156865, Abcam, USA), anti-MFF (84580s, Cell Signaling Technology, USA), anti-SOD (A12537, Abclonal, China) and anti-\u0026beta;-Actin (AT0001, engibody, USA). The secondary antibody was incubated at room temperature for 2 hours. Their details were presented as: ECL kit (SQ201, EpiZyme, China) and FUSION FX.EDGE (Vilber, French) were used for observing the bands. The results were quantified by Image J software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e4.19 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical comparison of each experiment was presented in the figure legend. The data was shown by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. All data was firstly analyzed for the Normality test and Homogeneity of variance test. For three samples comparison, one-way ANOVA was used to assess differences between groups and the Dunnett\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test was used for multiple comparisons between groups. And for two samples comparison, Unpaired two-tailed student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for analysis. Kruskal-Wallis test or Mann-Whitney U test were used to analyze the data which did not conform to the Normalit test or the Homogeneity of variance test. Statistical analyses were performed using GraphPad Prism 8.0.2. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a statistically significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviation","content":"\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eAnterior\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eAAV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eAdeno-Associated Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eATXN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eAtaxin 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eATXN7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eAtaxin 7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eCACNA1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003esubunit-\u0026alpha; of the Cav2.1 voltage-gated calcium channel\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eCBND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eCerebellar neurodegeneration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eCb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eCerebellar lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eCox4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eCytochrome c oxidase 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eDrp1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eDynamin-related protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eFIS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eMitochondrial fission 1 protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eGFAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eGlial fibrillary acidic protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eInferior\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eIba1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eIonized calcium binding adapter molecule 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eiPSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eInduced pluripotent stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eMFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eMitochondrial fission factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eMito\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eMitochondria\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eMolecular layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eNeurodegenerative diseases\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eNGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eNerve growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eKO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eKnock out\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eObRb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eLeptin receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eOE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eOver expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eposterior\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003ePCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003ePurkinje cells layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003ePCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003ePurkinje cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003ePcp2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003ePurkinje cell protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003ePINK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003ePTEN-induced putative kinase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eSuperior\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eSCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eSpinocerebellar ataxia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eTBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eTATA-box-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eVDAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eVoltage-dependent anion-selective channel\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.922242314647377%\" valign=\"top\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.07775768535262%\" valign=\"top\"\u003e\n \u003cp\u003eWild type\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor imformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally: Shujiao Li,\u0026nbsp;Qian-wen Zheng,\u0026nbsp;Jie Zheng, and Jinbao Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYayun Wang, Yanling Yang, and Xin Sun conceptualized the project. Yayun Wang and Shujiao Li designed experiments and wrote the manuscript. Shujiao Li carried out behavior tests, mitochondrial transfer\u0026nbsp;experiments and virus regulation experiment.\u0026nbsp;Qianwen Zheng and\u0026nbsp;Jie Zheng conducted behavior tests\u0026nbsp;and data analysis, mice genotype test and draw the cartoon images; Hui Liu performed the RNAscope experiment. Jinbao Zhang analyzed the data. Kunlong Zhang\u0026nbsp;and Feifei Wu conducted\u0026nbsp;virus regulation experiment.\u0026nbsp;Xiaodong Li\u0026nbsp;and\u0026nbsp;Shuai Zhang\u0026nbsp;performed images analysis. All of the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Yayun Wang, Yanling Yang and Xin Sun.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all members of\u0026nbsp;Ya-Yun Wang’s\u0026nbsp;lab for their insightful and helpful discussions during the course of the study. This project was supported by the Military Medicine Upgrade Program of Air Force Military Medical University (2020SWAQ04) from Ya-Yun Wang, the Shaanxi Innovation Capability Support Plan (2023-CX-PT-33) from Ya-Yun Wang, the Shaanxi Basic Research Program of Natural Sciences (2024JC-ZDXM-60) from Yan-Ling Yang, the Xijing Hospital Clinical New technology (2023XJSY27) from Yan-Ling Yang, the Shaanxi Basic Research Program of Natural Science (2022JQ-820) from Fei-fei Wu, and the National Natural Science Foundation of China (82201627) from Fei-fei Wu.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they do not have any competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTakahashi T\u003cem\u003e, et al.\u003c/em\u003e LINE-1 activation in the cerebellum drives ataxia. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 3278-3287.e3278 (2022).\u003c/li\u003e\n\u003cli\u003eSwitonski PM\u003cem\u003e, et al.\u003c/em\u003e Altered H3 histone acetylation impairs high-fidelity DNA repair to promote cerebellar degeneration in spinocerebellar ataxia type 7. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 110062 (2021).\u003c/li\u003e\n\u003cli\u003eSudhakar V, Richardson RM. 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And treatments targeting the cerebellum need further research. We constructed a model of cerebellar PCs degeneration, characterized by ataxia, through conditional knockout of Drp1 in PCs (PCKO mice). And we further explored the pathogenesis and possible effective treatment of cerebellar degenerative diseases. Drp1 knockout results in pervasive and progressive apoptosis of PCs, accompanied by severe glial cell activation surrounding them. Mitochondrial dysfunction, as a cause of mitophagy, is a key pathogenic factor of PCs morphological damage and dysfunction. Transfer of liver-derived mitochondria into the cerebellum of PCKO mice at 1 month improved mitochondrial function and reduced mitophagy, resulting in a delay of PCs apoptosis and cerebellar ataxia for 3 weeks. This study demonstrates that mitochondria transfer may be an potential treatment for cerebellar degenerative diseases.","manuscriptTitle":"Mitochondria transfer transiently rescues cerebellar neurodegeneration at early stage by alleviating mitophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 11:25:31","doi":"10.21203/rs.3.rs-4612386/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"43472571-3931-4cdf-a35a-270803914191","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34031618,"name":"Health sciences/Neurology/Neurological disorders/Neurodegeneration"},{"id":34031619,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration"}],"tags":[],"updatedAt":"2025-03-23T07:05:33+00:00","versionOfRecord":{"articleIdentity":"rs-4612386","link":"https://doi.org/10.1038/s41467-025-58189-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-22 04:00:00","publishedOnDateReadable":"March 22nd, 2025"},"versionCreatedAt":"2024-07-03 11:25:31","video":"","vorDoi":"10.1038/s41467-025-58189-4","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58189-4","workflowStages":[]},"version":"v1","identity":"rs-4612386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4612386","identity":"rs-4612386","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}