Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation

Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation | 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 Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation Junghyung Park, Jinyoung Won, Eunyeoung Yang, Jincheol Seo, Jiyeon Cho, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3875281/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Oxidative stress plays an essential role in the progression of Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder. Streptozotocin (STZ)-induced abnormal brain insulin signaling and oxidative stress play crucial roles in the progression of Alzheimer’s disease (AD)-like pathology. Peroxiredoxins (Prxs) are associated with protection from neuronal death induced by oxidative stress. However, the molecular mechanisms underlying Prxs on STZ-induced progression of AD in the hippocampal neurons are not yet fully understood. Here, we investigated the effect of Peroxiredoxin 1 (Prx1) on STZ-induced AD-like pathology. Prx1 expression was increased by STZ treatment in the hippocampus cell line, HT-22 cells. We evaluated whether Prx1 affects STZ-induced HT-22 cells using overexpression. Prx1 successfully protected the forms of STZ-induced AD-like pathology, such as neuronal apoptosis, synaptic loss, and tau phosphorylation. Moreover, Prx1 suppressed STZ-induced increase of mitochondrial dysfunction and fragmentation by down-regulating Drp1 phosphorylation and mitochondrial location. Prx1 plays a role in an upstream signal pathway of Drp1 phosphorylation, cyclin-dependent kinase 5 (Cdk5) by inhibiting the STZ-induced conversion of p35 to p25. We found that STZ-induced of intracellular Ca 2+ accumulation was an important modulator of AD-like pathology progression by regulating Ca 2+ -mediated Calpain activation, and Prx1 down-regulated STZ-induced intracellular Ca 2+ accumulation and Ca 2+ -mediated Calpain activation. Finally, we identified that Prx1 antioxidant capacity affected Ca 2+ /Calpain/Cdk5-mediated AD-like pathology progress. Therefore, these findings demonstrated that Prx1 is a key factor in the STZ-induced hippocampal neuronal death through inhibition of Ca 2+ /Calpain/Cdk5-mediated mitochondrial dysfunction by protecting oxidative stress. Biological sciences/Biochemistry/Neurochemistry Biological sciences/Cell biology/Cell signalling Biological sciences/Cell biology/Organelles Biological sciences/Cell biology/Post translational modifications Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Neuroscience/Molecular neuroscience Peroxiredoxin 1(Prx1) oxidative stress Alzheimer’s disease (AD) streptozotocin calpain mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the continuing memory impairments and cognitive declines, and changes in behavior and personality. Abnormal accumulation of amyloid plaques and hyperphosphorylated tau in the form of neurofibrillary tangles, and oxidative stress are among the most prominent pathological features in AD brains 1 . Insulin signaling in the brain contributes to the maintenance of numerous brain functions, such as nutrient homeostasis, reproduction, cognition, and memory. Therefore, alteration of insulin signaling in the brain closely associated with brain aging, and neurodegeneration 2 . Impaired brain insulin signaling is observed in the brains of AD patients and experimental models 3 . Additionally, anti-diabetic drugs exert protective effects on AD-related pathologies in the AD models of mice and monkey 4 , 5 . Streptozotocin (STZ) is a diabetogenic and a toxic compound for pancreatic β-cell generally used to establish animal models of diabetes owing to its ability to selectively disrupt the insulin signaling pathway 6 . The intracerebroventricular (ICV) injection of STZ into the brain is a well-established non-transgenic AD animal model showing behavioral, pathological, and molecular aspects of AD, including memory impairment, neuronal loss, and oxidative stress 7 , 8 . Oxidative stress is caused by imbalance between production of reactive oxygen species (ROS) and antioxidant defense, and plays a critical role in progression of AD 9 , 10 . The central nervous system (CNS) is particularly susceptible to oxidative stress because of its high demand for and consumption of oxygen, and high concentrations of easily oxidized lipid-rich material 11 . Peroxiredoxins (Prxs) are a family of antioxidant enzymes that eliminate hydrogen peroxide (H 2 O 2 ), and is related with various cellular signal transduction processes by maintaining the redox state balance in various type of neuronal cells 12 , 13 . There are six mammalian Prxs, classified in three subtypes (typical 2-Cys, atypical 2-Cys, and typical 1-Cys) depending on their catalytic mechanism of peroxide reduction. Prx1 belongs to the 2-Cys Prx and is widely distributed within diverse subcellular compartments, including the cytosol and nucleus 14 . Prx1 expression level was elevated in the brains of patients with AD 15 , 16 , whereas it was decreased in other research 17 . Prx1 exerts protective effects in experimental models for AD 18 . However, molecular mechanisms of Prx1 on the protection of AD progression are still lacking. Mitochondria are an essential cellular organelle with key regulatory functions in energy production, oxidative balance, and calcium homeostasis 19 . Several molecular abnormalities involving mitochondria, such as loss of energy metabolism, abnormal morphology, and accumulation of ROS were observed in the brain of patients and animal model with AD 20 – 23 . Mitochondria are highly dynamic subcellular organelles that constantly repeat the process of fusion and fission events. The balance of mitochondrial fission and fusion is crucial for cellular processes in hippocampal neurons 24 , 25 . Cumulative mitochondrial fission induces mitochondrial fragmentation, and is closely associated with pathogenesis of STZ-induced AD models 26 , 27 . Dynamin-1-like protein (Drp1) is a key regulator of mitochondrial fission. It is recruited from the cytosol to mitochondria, then triggers mitochondrial division by increasing their activity 28 . The activity of Drp1 is controlled by post-translational modification, including phosphorylation 29 . In particular, phosphorylation of Drp1 at Ser616 (S616) has been shown to control the activity of Drp1, which plays an important role in various neurodegenerative pathological processes 30 , 31 . However, the molecular relationship between oxidative stress and changes in mitochondrial dynamics induced by STZ in an AD experimental model has not been fully elucidated. Calpains are calcium (Ca 2+ )-dependent cysteine proteinases that are involved in multiple cellular functions, such as proliferation, differentiation, growth, and apoptosis 32 . Abnormal activation of calpains induced by disruption of Ca 2+ homeostasis is observed around amyloid plaque and neurofibrillary tangles of AD patients and experimental models 33 , 34 , and inhibition of calpains improved spatial-working memory and synaptic transmission in AD mice model 35 , 36 . In particular, the elevated expression level of calpain-2 (m-calpain), one of the major calpain isoforms, is known to reflect calpain activity in neuronal cells and is closely associated with progression of AD pathologies. 37 – 39 Cyclin-dependent kinase 5 (Cdk5) belongs to the family of proline-directed serine/threonine kinase is activated by the neuron-specific activator p35 under normal conditions, and can be deregulated by p25, which is the cleaved form of p35 40 . Cdk5 performs an important role in the maintenance of neuronal development, survival, and synaptic plasticity and neurotransmission 41 . However, uncontrolled activity of Cdk5/p25 is involved in the development of AD pathologies, including tau phosphorylation 42 , amyloidogenesis 43 . Therefore, preservation of Cdk5 homeostasis is suggested as a reasonable therapeutic target for ameliorating AD pathological processes 44 , 45 . Interestingly, Cdk5 was also identified as an upstream regulator of mitochondrial fission via phosphorylation of Drp1 at S616 in neurodegenerative conditions 46 , 47 . Calpain is one of the factors involved in the cleavage of p25, which has been linked to AD pathogenesis 48 . In this study, we evaluated whether oxidative stress-induced Ca 2+ accumulation affects calpain-2-mediated progression of AD-like pathologies in STZ-induced hippocampal neuronal HT-22 cells. Therefore, we focused Prx1 as a regulator of STZ-induced oxidative stress. Furthermore, to validate the effect of Prx1 on the molecular signal pathway in the progression of STZ-induced AD-like pathologies (neuronal apoptosis, synaptic function, tau pathology, and mitochondrial fragmentation), we determined the change in calpain, cdk5, and mitochondria morphology using antioxidant molecule and Ca 2+ chelator. These studies suggested that maintenance of oxidative balance via increase in antioxidant defense is a promising target to ameliorate the progress of AD. Materials and Methods Cell culture and treatment HT-22 cells were derived from HT-4 cells, which were immortalized from primary mouse hippocampal neuronal culture 49 . HT-22 cells were maintained at 37°C in DMEM with high glucose (Welgene, Daegu, Korea) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Welgene) in a humidified atmosphere incubator (Thermo Fisher Scientific) with 5% CO 2 . The cells were pretreated with BAPTA-AM (0.5 µM; Thermo Fisher Scientific), NAC (5 mM; Sigma-Aldrich, St. Louis, MO, USA) for 30 min and were incubated with STZ (10 mM; Sigma-Aldrich). Plasmid construction Preparation of stable cell line Prx1 gene including plasmid (pLenti6.3-Prx1-V5; Thermo Fisher Scientific) kindly provided by Dr. Dong-Seok Lee (Kyungpook National University, Daegu, Korea). Prx1 was amplified by performing PCR with LA Taq polymerase (TaKaRa, Shiga, Japan). These genes were cloned into a gateway entry vector pCR8/GW/TOPO (Thermo Fisher Scientific) to generate expression clones by performing LR recombination between the entry vector and gateway destination vectors pLenti6.3/V5-DEST (Thermo Fisher Scientific). pLenti6.3/V5-DEST vector have a C-terminal V5 epitope that aids in detecting recombinant proteins during immunoblotting analysis 50 . The sequences of the constructed vectors were confirmed by performing restriction mapping and DNA sequencing. 1 µg of pLenti6.3-Prx1-V5 plasmid was transfected into the HT-22 cells by using effectene (Qiagen, Hilden, Germany), according to the manufacturer's instructions. After 24 h, the transfected cells were selected using 8 µg/mL blasticidin (Thermo Scientific). Western blot analysis Whole protein lysates were prepared using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Korea), and mitochondrial fractions were performed with a mitochondria isolation kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by electrophoresis on 8–15% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, NJ, USA). The membranes were blocked by incubation in blocking buffer (BD Biosciences) and probed with the following antibodies overnight at 4°C: anti-Prx1 (Ab Frontier, Seoul, Korea), anti-β-actin (Sigma-Aldrich), anti-V5, anti-AT8 (Thermo Fisher Scientific), anti-cleaved caspase-3, anti-PARP, p-Tau(S262), anti-Drp1, anti-p-Drp1(S616), anti-COXIV, anti-Cdk5 (Cell Signaling, MA, USA), anti-NeuN, anti-PSD95, (Abcam, MA, USA), anti-p35, anti-calpain-2 (Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were washed with TBS with 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) for 1 h at room temperature. After washing with TBST, protein bands were visualized using enhanced chemiluminescence reagent in the Chemi DocXRS + imaging system (Bio-Rad, Hercules, CA, USA). Finally, densitometric analysis was performed using Image Lab software, version 3.0 (Bio-Rad). MTT assay Mock- and Prx1-expressed HT-22 cells were cultured on 96-well plates for 24 h. After experiments, each cell sample was incubated for 30 min at 37°C with MTT (0.5 mg/mL; Sigma-Aldrich), and then 100 µL DMSO (Sigma-Aldrich) was added. Absorbance was measured at 550 nm. Measurements of intracellular ATP levels The intracellular ATP levels were determined using an ATP determination kit (Thermo Fisher Scientific) following the manufacturer’s instructions. After experiments, whole protein lysates at a concentration of 1 µg/mL were used for the measurements of intracellular ATP levels. Mitochondrial imaging To observe mitochondrial morphology, DsRed2-mito including plasmid (pLenti6.3-DsRed2-mito) was kindly provided by Dr. Dong-Seok Lee (Kyungpook National University, Daegu, Korea). DsRed2-mito expressed HT-22 cells were seeded on 0.01% poly-D-lysine-coated round coverslips and were incubated for 24 h. After experiments, cells were washed with PBS, and fixed with 4% paraformaldehyde for 1 h. After washing, the coverslips were mounted on slides with mounting medium (VECTOR Laboratories, CA, USA). To observe mitochondrial morphology in Prx1-expressed HT-22 cells, MitoTracker Green (100 µM; Thermo Fisher Scientific) was incubated with the cells. Mitochondrial images were acquired using the LSM-710 confocal microscope (Carl Zeiss, Jena, Germany). Mitochondrial length measurements were performed using image J software as previously described 51 . Determination of intracellular Ca 2+ levels Intracellular Ca 2+ level was measured using Fluo-4 AM (Thermo Fisher Scientific). After experiment, cells were incubated with Fluo-4 AM for 30 min at 37°C. After washing with HBSS, images of Fluo-4 AM were obtained using an ECLIPSE Ti-U microscope (Nikon, Tokyo, Japan), and fluorescence intensity of Fluo-4 AM was measured with image J software. Measurements of intracellular ROS Intracellular ROS generation was assessed using CM-H 2 DCFDA. After experiments, cells were incubated with 5 µM CM-H 2 DCFDA (Thermo Fisher Scientific) for 30 min at 37°C, and then analyzed using an ECLIPSE Ti-U microscope (Nikon) and FACSCalibur flow cytometry (BD Biosciences). Statistical analysis The data represent the mean ± SD from three independent experiments (n = 3). Experimental differences were tested for statistical significance using GraphPad Prism 9 software (San Diego, CA, USA). Multiple group analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for normally distributed datasets. Statistical significance was set at p < 0.05, and is indicated on the graphs using asterisks; P-values < 0.01 and < 0.001 were indicated by two and three asterisks, respectively. Results STZ-mediated Prx1 induction inhibits progression of AD-like pathology We investigated the Prx1 protein expression levels in HT-22 cells following time-dependent STZ treatments. STZ concentration (10 mM) referred to induce AD-like pathology in HT-22 cells 52 . We found that Prx1 protein expression level was gradually increased after 12 h STZ treatments (Figure 1A). To assess the effect of inducible Prx1 expression by STZ on process of AD-like pathology, we generated V5-tagged Prx1 (Prx1-V5) stably expressing HT-22 cells line by using transfection of pLtneti6.3-Prx1-V5. We identified the exogenous expression of Prx1 (Prx1-V5) by western blotting using Prx1 and V5-tag antibodies (Figure 1B). Prx1 expression reversed STZ-induced decreased cell viability and increased apoptotic markers, such as cleaved caspase-3 and cleaved PARP (Figure 1C and D). Furthermore, we determined the effect of Prx1 on STZ-induced neuronal loss and synaptic function using immunoblotting with NeuN (neuronal marker), and PSD95 (post-synapse marker). Our results showed that STZ-induced down-regulation of NeuN and PSD95 were inhibited in Prx1 expression (Figure 1E and F). We also confirmed that Prx1 impacted the phosphorylation of tau epitopes, such as p-Tau(S262) and AT8(S202/T205). Up-regulated p-Tau (S262) and AT8(S202/T205) by STZ treatment was also suppressed by Prx1 expression (Figure 1G). These results suggested that Prx1 induction by STZ regulates STZ-induced process of AD-like pathology, apoptotic neuronal loss, synaptic loss, and tauopathy in hippocampal cell lines. Prx1 prevents STZ-induced mitochondrial fragmentation and dysfunction We previously reported that change in mitochondrial morphology and mitochondrial function relate to STZ-induced progression of AD-like pathology 52 . Therefore, we measured changes in intracellular ATP levels after STZ treatment for 12 h in mock- and Prx1-expressed HT-22 cells. Our results indicated that down-regulated intracellular ATP level by STZ treatment was suppressed by Prx1 expression (Figure 2A). We determined whether Prx1 affects STZ-induced mitochondrial fragmentation. To observe changes in mitochondrial morphology after STZ treatment for 12 h in HT-22 and Prx1-expressed HT-22 cells, we stained with mitotracker green after experiments. Our result indicated that decrease of mitochondrial average length by STZ treatment in HT-22 cells was inhibited in Prx1-expressed HT-22 cells (Figure 2B and C). STZ-induced mitochondrial fragmentation was depended on the Drp1 translocation from the cytoplasm to the mitochondria by phosphorylation of Drp1(S616) via activating Cdk5/p25 signaling pathway 52 . Therefore, we assess the effects of Prx1 on the Cdk5/p25-mediated Drp1 activation by STZ treatment for 12 h. Our results indicated that STZ-induced increase in the level of mitochondrial Drp1 was inhibited by Prx1 expression (Figure 2C). Drp1(S616) phosphorylation was increased by STZ treatment for 12 h, and up-regulated Drp1(S616) phosphorylation was repressed by Prx1 expression (Figure 2D). Moreover, increased level of p25 by STZ treatment for 6 h, which is known to show the highest level of p25 by STZ treatment in HT-22 cells 52 , was inhibited by Prx1 expression (Figure 2E). Therefore, we demonstrate that induction of Prx1 by STZ regulates Cdk5-mediated mitochondrial fragmentation and dysfunction by inhibiting cleavage of p35 to p25. Prx1 regulates STZ-induce Ca 2+ -mediated mitochondrial fragmentation and dysfunction through calpain/Cdk5-mediated Drp1 activation Previous reports indicated that Prx5 inhibits the accumulation of intracellular Ca 2+ , calpain activation, and Cdk5 activation in an amyloid-beta (Aβ) oligomer-mediated AD cellular model 53 . Therefore, we measured intracellular Ca 2+ levels using the intracellular Ca 2+ indicator Fluo-4 AM, in time-dependent STZ-treated HT-22 cells. Intracellular Ca 2+ was measured from 3 h STZ treatment, and maintained to 6 h (Figure 3A). Increased level of STZ-induced intracellular Ca 2+ was down-regulated by Prx1 expression (Figure 3B). The elevated expression level of calpain-2 is known to reflect calpain activation in neuron cells 38,39 . Thus, we then assessed whether Prx1 affected calpain-2 expression. We found that calpain-2 expression level was the highest at 1.5 h STZ treatment time, and was gradually down-regulated to 6 h (Figure 3C). Prx1 inhibited STZ-induced increase of calpain-2 expression (Figure 3D). We then assessed whether Prx1 influenced STZ-induced mitochondrial fragmentation through intracellular Ca 2+ regulation using an intracellular Ca 2+ chelator, BAPTA-AM. We found that inhibition of STZ-induced Ca 2+ accumulation prevented the calpain activation and Cdk5 activation by p35 cleavage to p25 (Figure 4A and B). Furthermore, STZ-induced increase of fragmented mitochondria and mitochondrial dysfunction were restored by intracellular Ca 2+ chelation via controlling Drp1 activation (Figure 4C, D, E, and F). These results suggest that Prx1 regulates calpain/Cdk5-mediated mitochondrial fragmentation by controlling STZ-induced intracellular Ca 2+ accumulation. Effect of intracellular Ca 2+ on the STZ-induced AD-like pathology Past demonstrations indicated that intracellular Ca 2+ or control of mitochondrial morphology were key mediators in AD 53 . As we proved that role of Ca 2+ as a regulator of STZ-induced mitochondrial fragmentation, we determined the effect of STZ-induced Ca 2+ on the progression of AD-like pathology. STZ-induced Ca 2+ inhibition with BAPTA-AM suppressed increased neuronal apoptosis and neuronal loss. The increased level of cleaved caspase-3 and cleaved PARP, reduced level of NeuN by STZ reversed by BAPTA-AM treatment (Figure 5A and B). In addition, the STZ-induced increased synaptic loss and Tau activation were confirmed with PSD95, p-Tau(S262), and AT8(S202/T205). STZ-induced decrease of PSD95 and increase of p-Tau(S262) and AT8(S202/T205) were attenuated by BAPTA-AM treatment (Figure 5C and D). These results indicated that STZ-mediated accumulation of intracellular Ca 2+ can influence the progression of STZ-induced AD-like pathology. Influence of ROS on STZ-induced AD-like pathology via Ca 2+ /calpain/Cdk5-mediated mitochondrial fragmentation Previous reports suggested that oxidative stress relates to the accumulation of intracellular Ca 2+ -mediated calpain activation, and Prx blocks the increase in intracellular Ca 2+ accumulation 54,55 . Therefore, to assess whether Prx1 affected Ca 2+ -mediated calpain activation using Prx1 antioxidant capacity, we inhibited STZ-induced ROS production using antioxidants molecule, N -acetyl-cysteine (NAC). We first determined effect of Prx1 to STZ-induced intracellular ROS level in STZ-induced HT-22 cells and Prx1-expressed HT-22 cells with CM-H 2 DCFDA. Our results indicated that increased levels of intracellular ROS induced by STZ treatment at 12 h were suppressed by Prx1 expression (Figure 6A and B). We investigated the impact of STZ-induced ROS inhibition on calpain activation and p35 cleavage. Up-regulation of calpain-2 and p25 proteins level were restored by NAC treatment (Figure C and D). Increase of mitochondrial fragmentation and dysfunction, a downstream pathway of Ca 2+ /calpain/Cdk5 were rescued by ROS scavenge through inhibition of Drp1(S616) phosphorylation and mitochondrial location (Figure 6E, F, G, and H). We then assessed the effect of STZ-induced ROS inhibition on the STZ-induced progression of AD-like pathology. Our results showed that STZ-induced increase in neuronal apoptosis, neuronal loss, and synaptic loss, and Tau phosphorylation were prevented by NAC treatment (Figure 7). These results suggested that STZ-induced progression of AD-like pathology in HT-22 cells were involved in increased ROS levels through governing Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation. Discussion In our previous research, we developed an STZ-induced effective and clinically relevant AD-like model in non-human primates and rodents through intra-cisternal magna (ICM) route, characterized by cerebral and hippocampal damage, disintegration of neurovascular unit, Aβ deposition, neuroinflammation, and Cdk5 activation 56 – 58 . Recently, we suggested that regulation of Cdk5/Drp1-dependent mitochondrial morphology play roles for potential inhibitor of abnormal metabolic functions associated with the AD-like pathogenesis 52 . Based on these findings, more detailed molecular mechanistic investigation of the STZ-induced AD-like pathologies are warranted. Oxidative stress precedes the onset of significant AD pathology in the brains of patients and animal models with AD 59 , 60 . Antioxidant defense system in the response from oxidative stress, similarly to change in the expression of Prx subtypes seem to be involved in AD pathology, but this remains controversial 61 – 64 . Mounting evidence determined that various type of Prxs, such as Prx5 and Prx6 have been associated with regulation of the progress of AD pathologies 53 , 65 , 66 . Specifically, Prx1 was mainly expressed in oligodendrocytes and astrocytes, and were detected in a few neuronal cells 67 , 68 . However, Prx1 expression was increased in an Aβ-resistant neuronal cell line response to oxidative stress 69 . Therefore, we focused on the role of Prx1 to assess the association with an STZ-mediated antioxidant response in HT-22 hippocampus cell line. We found that Prx1 was upregulated in a time-dependent manner in response to STZ-mediated oxidative stress. Therefore, we produced the Prx1 overexpression cell line in HT-22 cells to assume a situation in which Prx1 was expressed in an early timepoint of STZ treatment. Prx1 overexpression inhibited STZ-mediated neuronal apoptosis, synaptic loss, and tau phosphorylation via preventing cellular ROS accumulation. Furthermore, STZ-mediated mitochondrial fragmentation was suppressed by Prx1 overexpression through prevention of Cdk5-dependent Drp1 phosphorylation. It has been suggested that dysregulation of Cdk5 homeostasis has pathological relevance to AD 44 , 70 – 72 . Oxidative stress is considered to a crucial modulator of Cdk5 activation 73 , 74 . Prx5 involved in Cdk5 activation by modulating oxidative stress 75 , 76 , and Prx1 activation inhibited Aβ-induced impaired axonal transport 77 . However, the precise relationship between Prx1 and Cdk5 activation in STZ-mediated AD-like pathogenesis are still unclear. Our findings reveal that Prx1 is an important suppressor of STZ-induced progression of AD-like pathology via Cdk5 activation and mitochondrial fragmentation. Cdk5 activation by p25 is triggered by activation of calpain, which is related to accumulation of intracellular Ca 2+ 74,78 . Our result also showed that elimination of STZ-induced accumulated intracellular Ca 2+ suppressed STZ-mediated calpain-2 expression, Cdk5 activation, mitochondrial fragmentation, and progression of AD-like pathology. Oxidative stress is associated with dysregulation of Ca 2+ release and signal pathway 79 , 80 , which in turn triggers calpain-2 activation 81 – 83 . Prx1 modulates Aβ-induced increased intracellular Ca 2+ level by inhibiting ROS accumulation 77 . Our results suggested that STZ-induced up-regulated Prx1 caused a decrease in Ca 2+ and calpain-2 level. Apart from antioxidant function of Prx1 that reduce peroxides via a highly reactive catalytic cysteine oxidation to sulfenic acid 84 , Prx1 displays chaperone function by controlling the protein-binding partners 85 . Therefore, we proved that Prx1 decreased the STZ-induced Ca 2+ -mediated calpain-2 expression depending on Prx1 antioxidant capacity. However, it needs to study the direct role of Drp1 phosphorylation in the process of STZ-induced AD-like pathology. Our results demonstrated that the antioxidant capacity of Prx1 is a key factor of the regulation of Ca 2+ level and Ca 2+ -dependent calpain-2 expression, Cdk5-related mitochondria fragmentation. Increased expression level of Prx1 observed in various neurodegenerative conditions 86 , 87 and increased expression of Prx1 contribute to resistance to oxidative stress 69 . Prx1 is known to play a protective role against ROS-mediated brain injury, such as endotoxin‑induced injury 88 , Huntington’s disease 89 , and acute ischemic stroke 90 . Furthermore, microglial Prx1 participated in the protective function against endotoxin-induced pro-inflammatory response by regulating oxidative stress 91 , and Prx1 overexpression reduced neuronal inflammation and apoptosis by affecting microglial and astrocyte mRNA stability 86 . Therefore, these findings along with our results suggested that Prx1 plays a protective role in neurodegenerative environments by affecting neuron, astrocyte, and microglia. Apart from these results, our findings have the limitations that it is not reflected complex environment present in the brains of AD rodents or patients because these findings were investigated from immortalized hippocampal cell line, HT-22 cells. Although several advantages of HT-22 cells, such as AD pathology being well reflected or useful for molecular mechanism study, more research should be conducted in primary hippocampus cells or AD animal models based on the results of this study. Consequently, regulation of Prx1 may be a potential inhibitor of STZ-induced neurodegeneration by preventing Ca 2+ /capain-2/Cdk5 signal pathway, and may consider as a possible strategy for developing therapies to treat the pathogenesis of AD. Declarations Data availability All the data generated and/or analyzed during performing this current study are included in this article [and also in its supplementary dataset files]. However, there is no restriction on the availability of materials and data from the corresponding author on reasonable request. Declaration of Competing Interests The authors declare no competing financial interests. Funding This study was supported by the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (KGM4562431), and by the National Research Council of Science & Technology (NST) grant by the Korea government (MSIT) (No. CPS21101-100), and by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (Project Number: 9991006929, RS-2020-KD000264). Author Contributions JP participated in design of the experiments, performed the experiments, analyzed the data, and drafted the manuscript. JW and EY performed the experiments, and analyzed the data. JS, JC, JBS, H-GY, KK, YGK, MK, C-YJ, and KSY performed and supported the experiments. D-SL, and YL edited manuscript. Acknowledgments We would like to thank Editage (www.editage.co.kr) for English language editing. References Querfurth, H. W. & LaFerla, F. M. Alzheimer's disease. N Engl J Med 362 , 329-344, doi:10.1056/NEJMra0909142 (2010). Spinelli, M., Fusco, S. & Grassi, C. Brain Insulin Resistance and Hippocampal Plasticity: Mechanisms and Biomarkers of Cognitive Decline. Front Neurosci 13 , 788, doi:10.3389/fnins.2019.00788 (2019). Ferreira, L. S. S., Fernandes, C. S., Vieira, M. N. N. & De Felice, F. G. Insulin Resistance in Alzheimer's Disease. Front Neurosci 12 , 830, doi:10.3389/fnins.2018.00830 (2018). Lourenco, M. V. et al. TNF-alpha mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's beta-amyloid oligomers in mice and monkeys. Cell Metab 18 , 831-843, doi:10.1016/j.cmet.2013.11.002 (2013). Batista, A. F. et al. The diabetes drug liraglutide reverses cognitive impairment in mice and attenuates insulin receptor and synaptic pathology in a non-human primate model of Alzheimer's disease. J Pathol 245 , 85-100, doi:10.1002/path.5056 (2018). Lee, W., Wakasugi, H. & Ibayashi, H. Comparison of somatostatin distribution in pancreatic duct ligated rats and streptozotocin diabetic rats. Gastroenterol Jpn 18 , 453-458, doi:10.1007/bf02776585 (1983). Salkovic-Petrisic, M. & Hoyer, S. Central insulin resistance as a trigger for sporadic Alzheimer-like pathology: an experimental approach. J Neural Transm Suppl , 217-233, doi:10.1007/978-3-211-73574-9_28 (2007). Kamat, P. K. Streptozotocin induced Alzheimer's disease like changes and the underlying neural degeneration and regeneration mechanism. Neural Regen Res 10 , 1050-1052, doi:10.4103/1673-5374.160076 (2015). Kim, G. H., Kim, J. E., Rhie, S. J. & Yoon, S. The Role of Oxidative Stress in Neurodegenerative Diseases. Exp Neurobiol 24 , 325-340, doi:10.5607/en.2015.24.4.325 (2015). Wang, X. et al. Oxidative stress and mitochondrial dysfunction in Alzheimer's disease. Biochim Biophys Acta , doi:10.1016/j.bbadis.2013.10.015 (2013). Salim, S. Oxidative Stress and the Central Nervous System. J Pharmacol Exp Ther 360 , 201-205, doi:10.1124/jpet.116.237503 (2017). Zhu, H., Santo, A. & Li, Y. The antioxidant enzyme peroxiredoxin and its protective role in neurological disorders. Exp Biol Med (Maywood) 237 , 143-149, doi:10.1258/ebm.2011.011152 (2012). Szeliga, M. Peroxiredoxins in Neurodegenerative Diseases. Antioxidants (Basel) 9 , doi:10.3390/antiox9121203 (2020). Wood, Z. A., Schroder, E., Robin Harris, J. & Poole, L. B. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28 , 32-40, doi:10.1016/s0968-0004(02)00003-8 (2003). Kim, S. H., Fountoulakis, M., Cairns, N. & Lubec, G. Protein levels of human peroxiredoxin subtypes in brains of patients with Alzheimer's disease and Down syndrome. J Neural Transm Suppl , 223-235, doi:10.1007/978-3-7091-6262-0_18 (2001). Krapfenbauer, K., Engidawork, E., Cairns, N., Fountoulakis, M. & Lubec, G. Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders. Brain Res 967 , 152-160, doi:10.1016/s0006-8993(02)04243-9 (2003). Stepler, K. E. et al. Inclusion of African American/Black adults in a pilot brain proteomics study of Alzheimer's disease. Neurobiol Dis 146 , 105129, doi:10.1016/j.nbd.2020.105129 (2020). Lee, Y. J. et al. Peroxiredoxin I regulates the component expression of gamma-secretase complex causing the Alzheimer's disease. Lab Anim Res 27 , 293-299, doi:10.5625/lar.2011.27.4.293 (2011). Lin, M. Y. & Sheng, Z. H. Regulation of mitochondrial transport in neurons. Exp Cell Res , doi:10.1016/j.yexcr.2015.01.004 (2015). Reddy, P. H. Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease. Exp Neurol 218 , 286-292, doi:10.1016/j.expneurol.2009.03.042 (2009). Dixit, S., Fessel, J. P. & Harrison, F. E. Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer's disease and a novel protective role for ascorbate. Free Radic Biol Med 112 , 515-523, doi:10.1016/j.freeradbiomed.2017.08.021 (2017). Guo, X. D. et al. LX2343 alleviates cognitive impairments in AD model rats by inhibiting oxidative stress-induced neuronal apoptosis and tauopathy. Acta Pharmacol Sin 38 , 1104-1119, doi:10.1038/aps.2016.128 (2017). Latina, V. et al. Tau Cleavage Contributes to Cognitive Dysfunction in Strepto-Zotocin-Induced Sporadic Alzheimer's Disease (sAD) Mouse Model. Int J Mol Sci 22 , doi:10.3390/ijms222212158 (2021). Knott, A. B., Perkins, G., Schwarzenbacher, R. & Bossy-Wetzel, E. Mitochondrial fragmentation in neurodegeneration. Nat Rev Neurosci 9 , 505-518, doi:10.1038/nrn2417 (2008). Jiang, S. et al. Mfn2 ablation causes an oxidative stress response and eventual neuronal death in the hippocampus and cortex. Mol Neurodegener 13 , 5, doi:10.1186/s13024-018-0238-8 (2018). Correia, S. C. et al. Mitochondrial abnormalities in a streptozotocin-induced rat model of sporadic Alzheimer's disease. Curr Alzheimer Res 10 , 406-419, doi:10.2174/1567205011310040006 (2013). Yang, S. et al. Mitochondrial dysfunction driven by the LRRK2-mediated pathway is associated with loss of Purkinje cells and motor coordination deficits in diabetic rat model. Cell Death Dis 5 , e1217, doi:10.1038/cddis.2014.184 (2014). Westermann, B. Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 11 , 872-884, doi:10.1038/nrm3013 (2010). Elgass, K., Pakay, J., Ryan, M. T. & Palmer, C. S. Recent advances into the understanding of mitochondrial fission. Biochim Biophys Acta 1833 , 150-161, doi:10.1016/j.bbamcr.2012.05.002 (2013). Wang, X. et al. Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J Neurosci 29 , 9090-9103, doi:10.1523/JNEUROSCI.1357-09.2009 (2009). Kim, B., Park, J., Chang, K. T. & Lee, D. S. Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation. Free Radic Biol Med 90 , 184-194, doi:10.1016/j.freeradbiomed.2015.11.015 (2016). Ferreira, A. Calpain dysregulation in Alzheimer's disease. ISRN Biochem 2012 , 728571, doi:10.5402/2012/728571 (2012). Tsuji, T., Shimohama, S., Kimura, J. & Shimizu, K. m-Calpain (calcium-activated neutral proteinase) in Alzheimer's disease brains. Neurosci Lett 248 , 109-112, doi:10.1016/s0304-3940(98)00348-6 (1998). Kurbatskaya, K. et al. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain. Acta Neuropathol Commun 4 , 34, doi:10.1186/s40478-016-0299-2 (2016). Trinchese, F. et al. Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. J Clin Invest 118 , 2796-2807, doi:10.1172/JCI34254 (2008). Medeiros, R. et al. Calpain inhibitor A-705253 mitigates Alzheimer's disease-like pathology and cognitive decline in aged 3xTgAD mice. Am J Pathol 181 , 616-625, doi:10.1016/j.ajpath.2012.04.020 (2012). Ahmad, F. et al. Isoform-specific hyperactivation of calpain-2 occurs presymptomatically at the synapse in Alzheimer's disease mice and correlates with memory deficits in human subjects. Sci Rep 8 , 13119, doi:10.1038/s41598-018-31073-6 (2018). Zhang, H. et al. Calpain-2/p35-p25/Cdk5 pathway is involved in the neuronal apoptosis induced by polybrominated diphenyl ether-153. Toxicol Lett 277 , 41-53, doi:10.1016/j.toxlet.2017.05.027 (2017). Haque, A. et al. Calpain mediated expansion of CD4+ cytotoxic T cells in rodent models of Parkinson's disease. Exp Neurol 330 , 113315, doi:10.1016/j.expneurol.2020.113315 (2020). Tsai, L. H., Delalle, I., Caviness, V. S., Jr., Chae, T. & Harlow, E. p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371 , 419-423, doi:10.1038/371419a0 (1994). Cheung, Z. H. & Ip, N. Y. Cdk5: a multifaceted kinase in neurodegenerative diseases. Trends Cell Biol 22 , 169-175, doi:10.1016/j.tcb.2011.11.003 (2012). Kimura, T., Ishiguro, K. & Hisanaga, S. Physiological and pathological phosphorylation of tau by Cdk5. Front Mol Neurosci 7 , 65, doi:10.3389/fnmol.2014.00065 (2014). Wen, Y. et al. Transcriptional regulation of beta-secretase by p25/cdk5 leads to enhanced amyloidogenic processing. Neuron 57 , 680-690, doi:10.1016/j.neuron.2008.02.024 (2008). Piedrahita, D. et al. Silencing of CDK5 reduces neurofibrillary tangles in transgenic alzheimer's mice. J Neurosci 30 , 13966-13976, doi:10.1523/JNEUROSCI.3637-10.2010 (2010). Castro-Alvarez, J. F., Uribe-Arias, S. A., Kosik, K. S. & Cardona-Gomez, G. P. Long- and short-term CDK5 knockdown prevents spatial memory dysfunction and tau pathology of triple transgenic Alzheimer's mice. Front Aging Neurosci 6 , 243, doi:10.3389/fnagi.2014.00243 (2014). Meuer, K. et al. Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis. Cell Death Differ 14 , 651-661, doi:10.1038/sj.cdd.4402087 (2007). Jahani-Asl, A. et al. CDK5 phosphorylates DRP1 and drives mitochondrial defects in NMDA-induced neuronal death. Hum Mol Genet 24 , 4573-4583, doi:10.1093/hmg/ddv188 (2015). Trinchese, F. et al. Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. J Clin Invest 118 , 2796-2807, doi:10.1172/JCI34254 (2008). Davis, J. B. & Maher, P. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res 652 , 169-173 (1994). Southern, J. A., Young, D. F., Heaney, F., Baumgartner, W. K. & Randall, R. E. Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. J Gen Virol 72 ( Pt 7) , 1551-1557 (1991). Park, J. et al. Mitochondrial dynamics modulate the expression of pro-inflammatory mediators in microglial cells. J Neurochem 127 , 221-232, doi:10.1111/jnc.12361 (2013). Park, J. et al. Streptozotocin Induces Alzheimer's Disease-Like Pathology in Hippocampal Neuronal Cells via CDK5/Drp1-Mediated Mitochondrial Fragmentation. Front Cell Neurosci 14 , 235, doi:10.3389/fncel.2020.00235 (2020). Park, J. et al. Peroxiredoxin 5 Decreases Beta-Amyloid-Mediated Cyclin-Dependent Kinase 5 Activation Through Regulation of Ca(2+)-Mediated Calpain Activation. Antioxid Redox Signal 27 , 715-726, doi:10.1089/ars.2016.6810 (2017). De Simoni, S., Linard, D., Hermans, E., Knoops, B. & Goemaere, J. Mitochondrial peroxiredoxin-5 as potential modulator of mitochondria-ER crosstalk in MPP+-induced cell death. Journal of neurochemistry 125 , 473-485, doi:10.1111/jnc.12117 (2013). Sabharwal, S. S., Waypa, G. B., Marks, J. D. & Schumacker, P. T. Peroxiredoxin-5 targeted to the mitochondrial intermembrane space attenuates hypoxia-induced reactive oxygen species signalling. Biochem J 456 , 337-346, doi:10.1042/BJ20130740 (2013). Yeo, H. G. et al. Characterization of Cerebral Damage in a Monkey Model of Alzheimer's Disease Induced by Intracerebroventricular Injection of Streptozotocin. J Alzheimers Dis 46 , 989-1005, doi:10.3233/JAD-143222 (2015). Lee, Y. et al. Insulin/IGF signaling-related gene expression in the brain of a sporadic Alzheimer's disease monkey model induced by intracerebroventricular injection of streptozotocin. J Alzheimers Dis 38 , 251-267, doi:10.3233/JAD-130776 (2014). Ahn, Y. et al. Synaptic loss and amyloid beta alterations in the rodent hippocampus induced by streptozotocin injection into the cisterna magna. Lab Anim Res 36 , 17, doi:10.1186/s42826-020-00049-x (2020). Xie, H. et al. Rapid cell death is preceded by amyloid plaque-mediated oxidative stress. Proc Natl Acad Sci U S A 110 , 7904-7909, doi:10.1073/pnas.1217938110 (2013). Butterfield, D. A., Swomley, A. M. & Sultana, R. Amyloid beta-peptide (1-42)-induced oxidative stress in Alzheimer disease: importance in disease pathogenesis and progression. Antioxid Redox Signal 19 , 823-835, doi:10.1089/ars.2012.5027 (2013). Cumming, R. C., Dargusch, R., Fischer, W. H. & Schubert, D. Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells. J Biol Chem 282 , 30523-30534, doi:10.1074/jbc.M700869200 (2007). Kim, S. H., Fountoulakis, M., Cairns, N. & Lubec, G. Protein levels of human peroxiredoxin subtypes in brains of patients with Alzheimer's disease and Down syndrome. J Neural Transm Suppl , 223-235 (2001). Calkins, M. J., Manczak, M., Mao, P., Shirendeb, U. & Reddy, P. H. Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer's disease. Human molecular genetics 20 , 4515-4529, doi:10.1093/hmg/ddr381 (2011). Krapfenbauer, K., Engidawork, E., Cairns, N., Fountoulakis, M. & Lubec, G. Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders. Brain Res 967 , 152-160 (2003). Kim, I. K., Lee, K. J., Rhee, S., Seo, S. B. & Pak, J. H. Protective effects of peroxiredoxin 6 overexpression on amyloid beta-induced apoptosis in PC12 cells. Free Radic Res 47 , 836-846, doi:10.3109/10715762.2013.833330 (2013). Kim, B., Park, J., Chang, K. T. & Lee, D. S. Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation. Free Radic Biol Med 90 , 184-194, doi:10.1016/j.freeradbiomed.2015.11.015 (2016). Goemaere, J. & Knoops, B. Peroxiredoxin distribution in the mouse brain with emphasis on neuronal populations affected in neurodegenerative disorders. J Comp Neurol 520 , 258-280, doi:10.1002/cne.22689 (2012). Jin, M. H. et al. Characterization of neural cell types expressing peroxiredoxins in mouse brain. Neurosci Lett 381 , 252-257, doi:10.1016/j.neulet.2005.02.048 (2005). Cumming, R. C., Dargusch, R., Fischer, W. H. & Schubert, D. Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells. J Biol Chem 282 , 30523-30534, doi:10.1074/jbc.M700869200 (2007). Shukla, V. et al. A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice. Faseb J 27 , 174-186, doi:10.1096/fj.12-217497 (2013). Sun, K. H., de Pablo, Y., Vincent, F. & Shah, K. Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction. Journal of neurochemistry 107 , 265-278, doi:10.1111/j.1471-4159.2008.05616.x (2008). Lopes, J. P., Oliveira, C. R. & Agostinho, P. Neurodegeneration in an Abeta-induced model of Alzheimer's disease: the role of Cdk5. Aging Cell 9 , 64-77, doi:10.1111/j.1474-9726.2009.00536.x (2010). Sahlgren, C. M. et al. A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death. Embo J 25 , 4808-4819, doi:10.1038/sj.emboj.7601366 (2006). Lee, M. S. et al. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405 , 360-364, doi:10.1038/35012636 (2000). Kim, M. H. et al. Peroxiredoxin 5 Inhibits Glutamate-Induced Neuronal Cell Death through the Regulation of Calcineurin-Dependent Mitochondrial Dynamics in HT22 Cells. Mol Cell Biol 39 , doi:10.1128/MCB.00148-19 (2019). Lee, D. G., Kam, M. K., Lee, S. R., Lee, H. J. & Lee, D. S. Peroxiredoxin 5 deficiency exacerbates iron overload-induced neuronal death via ER-mediated mitochondrial fission in mouse hippocampus. Cell Death Dis 11 , 204, doi:10.1038/s41419-020-2402-7 (2020). Choi, H. et al. Increased acetylation of Peroxiredoxin1 by HDAC6 inhibition leads to recovery of Abeta-induced impaired axonal transport. Mol Neurodegener 12 , 23, doi:10.1186/s13024-017-0164-1 (2017). Hidalgo, C. Cross talk between Ca2+ and redox signalling cascades in muscle and neurons through the combined activation of ryanodine receptors/Ca2+ release channels. Philos Trans R Soc Lond B Biol Sci 360 , 2237-2246, doi:10.1098/rstb.2005.1759 (2005). Sanmartin, C. D., Adasme, T., Hidalgo, C. & Paula-Lima, A. C. The antioxidant N-acetylcysteine prevents the mitochondrial fragmentation induced by soluble amyloid-beta peptide oligomers. Neurodegener Dis 10 , 34-37, doi:10.1159/000334901 (2012). Paula-Lima, A. C. et al. Amyloid beta-peptide oligomers stimulate RyR-mediated Ca2+ release inducing mitochondrial fragmentation in hippocampal neurons and prevent RyR-mediated dendritic spine remodeling produced by BDNF. Antioxid Redox Signal 14 , 1209-1223, doi:10.1089/ars.2010.3287 (2011). Adamec, E., Mohan, P., Vonsattel, J. P. & Nixon, R. A. Calpain activation in neurodegenerative diseases: confocal immunofluorescence study with antibodies specifically recognizing the active form of calpain 2. Acta Neuropathol 104 , 92-104, doi:10.1007/s00401-002-0528-6 (2002). Austin, S. A. & Katusic, Z. S. Loss of Endothelial Nitric Oxide Synthase Promotes p25 Generation and Tau Phosphorylation in a Murine Model of Alzheimer's Disease. Circ Res 119 , 1128-1134, doi:10.1161/CIRCRESAHA.116.309686 (2016). Yin, Y. et al. Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor alpha4 via activating calpain-2. Sci Rep 6 , 27283, doi:10.1038/srep27283 (2016). Jarvis, R. M., Hughes, S. M. & Ledgerwood, E. C. Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells. Free Radic Biol Med 53 , 1522-1530, doi:10.1016/j.freeradbiomed.2012.08.001 (2012). Hopkins, B. L. et al. A Peroxidase Peroxiredoxin 1-Specific Redox Regulation of the Novel FOXO3 microRNA Target let-7. Antioxid Redox Signal 28 , 62-77, doi:10.1089/ars.2016.6871 (2018). Yang, G. Q. et al. Prdx1 Reduces Intracerebral Hemorrhage-Induced Brain Injury via Targeting Inflammation- and Apoptosis-Related mRNA Stability. Front Neurosci 14 , 181, doi:10.3389/fnins.2020.00181 (2020). Huang, S. et al. Expression of Peroxiredoxin 1 After Traumatic Spinal Cord Injury in Rats. Cell Mol Neurobiol 35 , 1217-1226, doi:10.1007/s10571-015-0214-6 (2015). Sun, H. N. et al. Peroxiredoxin I deficiency increases LPSinduced lethal shock in mice. Mol Med Rep 18 , 2427-2432, doi:10.3892/mmr.2018.9170 (2018). Pitts, A. et al. Dithiol-based compounds maintain expression of antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin. J Biol Chem 287 , 22717-22729, doi:10.1074/jbc.M111.334565 (2012). Kim, S. et al. The antioxidant enzyme Peroxiredoxin-1 controls stroke-associated microglia against acute ischemic stroke. Redox Biol 54 , 102347, doi:10.1016/j.redox.2022.102347 (2022). Kim, S. U. et al. Peroxiredoxin I is an indicator of microglia activation and protects against hydrogen peroxide-mediated microglial death. Biol Pharm Bull 31 , 820-825, doi:10.1248/bpb.31.820 (2008). Additional Declarations No competing interests reported. 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09:00:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3875281/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3875281/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50293770,"identity":"f45889f7-8fc7-4464-8308-23518af9c896","added_by":"auto","created_at":"2024-01-29 09:20:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":311775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Prx1 on STZ-induced AD-like pathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The protein expression level of Prx1 was determined by western blotting in STZ (10 mM)-treated HT-22 cells following indicated time points (6–24 h). (B) Prx1-expressed HT-22 cells were verified using western blotting with Prx1 and V5-tag antibodies. (C) Cell viability was determined using the MTT assay in mock- and Prx1-expressed HT-22 cells according to STZ treatment for 24 h. (D) Cleaved Caspase-3, cleaved PARP, (E) NeuN, (F) PSD95, and (G) p-Tau (S262), AT8 (S202/T205) protein expression level were confirmed by western blotting in STZ (24 h)-treated mock- and Prx1-expressed HT-22 cells. The data are presented as mean values ± SD (n = 3). * denotes p \u0026lt; 0.05, ** denotes p \u0026lt; 0.01, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/301f8038437ab095e3cfaeac.png"},{"id":50293318,"identity":"e8f41b96-a251-4f5f-8ca7-403e1a51360c","added_by":"auto","created_at":"2024-01-29 09:12:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":396212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Prx1 on STZ-induced mitochondrial dysfunction and change in mitochondrial morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Intracellular ATP levels were measured using the ATP determination kit in STZ (12 h)-treated mock- and Prx1-expressed HT-22 cells. (B) Mitochondrial morphology was observed with mitotracker (100 μM) staining using confocal microscopy in STZ (12 h)-treated HT-22 and Prx1-expressed cells. The bottom panels showed the magnified images of regions indicated by white squares in the top panels; scale bar, 5 μm. The graph showed distribution of all mitochondrial particles and average mitochondrial length. (C) Mitochondrial location of Drp1 was analyzed by western blotting from mitochondrial isolated protein in mock- and Prx1-expressed HT-22 cells treated with STZ for 12 h. COXⅣ was used as the loading control for the mitochondria. (D) The protein expression levels of p-Drp1(S616) and Drp1 were determined using western blot analysis in STZ (12 h)-treated mock- and Prx1-expressed HT-22 cells. Drp1 was used as the loading control for p-Drp1(S616). (E) Protein levels of p25/35 and Cdk5 in STZ (6 h)-treated mock- and Prx1-expressed HT-22 cells were confirmed using western blotting. The data are presented as mean values ± SD (n = 3). ** denotes p \u0026lt; 0.01, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/96a0403fde3afd73af5a362d.png"},{"id":50293311,"identity":"4f9f4a62-58e5-4f49-bd61-aff63d12b7f1","added_by":"auto","created_at":"2024-01-29 09:12:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":507633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Prx1 on the STZ-induced Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/Calpain signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e level in STZ-treated HT-22 cells following indicated time points (1–6 h) was observed with Fluo-4 AM (5 μM) staining using fluorescent microscopy; scale bar, 200 μm. Graph showing Fluo-4 AM fluorescent spectrophotometer results. (B) Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels in STZ (3 h)-treated HT-22 and Prx1-expressed HT-22 cells with Fluo-4 AM observed using fluorescent microscopy; scale bar, 200 μm. Fluorescent spectrophotometer results of Fluo-4 AM represented as graphs. (C) Calpain-2 protein expression was confirmed by western blotting in HT-22 cells following indicated time point (1–6 h) treated STZ. (D) Calpain-2 expression was confirmed using western blotting in STZ (1.5h)-treated HT-22 and Prx1-expressed HT-22 cells. The data are presented as mean values ± SD (n = 3). * denotes p \u0026lt; 0.05, ** denotes p \u0026lt; 0.01, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/ac9e3d889305703c7a667076.png"},{"id":50294166,"identity":"2b2b8a80-b996-4561-88fe-f787dc6e1e58","added_by":"auto","created_at":"2024-01-29 09:28:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":377088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e on STZ-induced calpain/Cdk5/Drp1 activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Calpain-2 protein expression level in STZ (1.5 h)-treated HT-22 cells pretreated with or without BAPTA-AM (0.5 μM) were confirmed with western blotting. (B) The proteins expression level of p25/35 and Cdk5 in STZ (6 h)-treated HT-22 cells pretreated with or without BAPTA-AM were confirmed with western blotting. (C) p-Drp1(S616), and (D) Mitochondrial Drp1 proteins expression level in STZ (12 h)-treated HT-22 cells pretreated with or without BAPTA-AM were confirmed with western blotting. COXⅣ was used as the loading control for the mitochondria. (E) Change in mitochondrial morphology was observed using confocal microscopy in STZ (12 h)-treated DsRed2-mito expressed HT-22 cells pretreated with or without BAPTA-AM. The bottom panels showed the magnified images of regions indicated by white squares in the top panels; scale bar, 5 μm. The graph showed distribution of all mitochondrial particles and average mitochondrial length. (F) Intracellular ATP levels were measured in STZ (24 h)-treated HT-22 cells pretreated with or without BAPTA-AM. The data are presented as mean values ± SD (n = 3). ** denotes p \u0026lt; 0.01, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/b3b0ad35a9c0dd8a74e4abdc.png"},{"id":50293315,"identity":"705ed6b5-4925-41dc-9493-a08c0bc2b0c5","added_by":"auto","created_at":"2024-01-29 09:12:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":190050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e on STZ-induced AD-like pathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cleaved Caspase-3 and cleaved PARP, (B) NeuN, (C) PSD95, (D) p-Tau(S262) and AT8(S202/T205) protein expression level were confirmed by western blotting analysis in STZ (24 h)-treated HT-22 cells pretreated with or without BAPTA. The data are presented as mean values ± SD (n = 3). * denotes p \u0026lt; 0.05, ** denotes p \u0026lt; 0.01, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/13daab211ec57f7ee278684d.png"},{"id":50293316,"identity":"9dab06c6-6a26-4611-954e-238a8b0435d9","added_by":"auto","created_at":"2024-01-29 09:12:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":504718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Prx1 antioxidant capacity on STZ-induced calpain/Cdk5/Drp1 activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Intracellular ROS was observed in STZ (24 h)-treated HT-22 and Prx1-expressed cells with CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA (5 μM) staining using fluorescent microscopy; scale bar, 200 μm. (B) Intracellular ROS level was analyzed in STZ (24 h)-treated HT-22 and Prx1-expressed HT-22 cells using flow cytometry with CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA staining. (C) Calpain-2 protein expression level in STZ (1.5h)-treated HT-22 cells pretreated with or without NAC (5 mM) were confirmed with western blotting analysis. (D) p25/35 and Cdk5 proteins expression level in STZ (6 h)-treated HT-22 cells pretreated with or without NAC were confirmed with western blotting analysis. (E) p-Drp1 (S616), (F) Mitochondrial Drp1 levels in STZ (12 h)-treated HT-22 cells pretreated with or without NAC were confirmed with western blotting. COXⅣ was used as the loading control for the mitochondria. (G) Change in mitochondrial morphology was observed using confocal microscopy in STZ (12 h)-treated DsRed2-mito expressed HT-22 cells pretreated with or without NAC. The bottom panels showed the magnified images of regions indicated by white squares in the top panels; scale bar, 5 μm. The graph showed distribution of all mitochondrial particles and average mitochondrial length. (H) Intracellular ATP levels were measured in STZ (12 h)-treated HT-22 cells pretreated with or without NAC. The data are presented as mean values ± SD (n = 3). * denotes p \u0026lt; 0.05, ** denotes p \u0026lt; 0.01, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/edfadad4dea2b3114963fc03.png"},{"id":50293317,"identity":"df61c68b-b540-408a-9225-1ff419cd1476","added_by":"auto","created_at":"2024-01-29 09:12:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":187592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect Prx1 antioxidant capacity on STZ-induced AD-like pathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cleaved Caspase-3 and cleaved PARP, (B) NeuN, (C) PSD95, (D) p-Tau(S262) and AT8(S202/T205) protein expression level were confirmed by western blotting analysis in STZ-treated HT-22 cells pretreated with or without NAC. The data are presented as mean values ± SD (n = 3). * denotes p \u0026lt; 0.05, and *** denotes p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/1552aae2b7d04e537d62f1c5.png"},{"id":50294675,"identity":"0875ad7a-a072-4845-8ff5-1abe1fc7a966","added_by":"auto","created_at":"2024-01-29 09:36:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2080628,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/d6c094d2-14df-42b4-9a26-d694a98f7eea.pdf"},{"id":50293314,"identity":"e61a67d5-96bb-478b-997c-f7e6993a8711","added_by":"auto","created_at":"2024-01-29 09:12:02","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":179230,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractFigure.png","url":"https://assets-eu.researchsquare.com/files/rs-3875281/v1/6caa9146ebc67753325ca8c5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is a progressive neurodegenerative disease characterized by the continuing memory impairments and cognitive declines, and changes in behavior and personality. Abnormal accumulation of amyloid plaques and hyperphosphorylated tau in the form of neurofibrillary tangles, and oxidative stress are among the most prominent pathological features in AD brains \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Insulin signaling in the brain contributes to the maintenance of numerous brain functions, such as nutrient homeostasis, reproduction, cognition, and memory. Therefore, alteration of insulin signaling in the brain closely associated with brain aging, and neurodegeneration \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Impaired brain insulin signaling is observed in the brains of AD patients and experimental models \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Additionally, anti-diabetic drugs exert protective effects on AD-related pathologies in the AD models of mice and monkey \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Streptozotocin (STZ) is a diabetogenic and a toxic compound for pancreatic β-cell generally used to establish animal models of diabetes owing to its ability to selectively disrupt the insulin signaling pathway \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The intracerebroventricular (ICV) injection of STZ into the brain is a well-established non-transgenic AD animal model showing behavioral, pathological, and molecular aspects of AD, including memory impairment, neuronal loss, and oxidative stress \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOxidative stress is caused by imbalance between production of reactive oxygen species (ROS) and antioxidant defense, and plays a critical role in progression of AD \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The central nervous system (CNS) is particularly susceptible to oxidative stress because of its high demand for and consumption of oxygen, and high concentrations of easily oxidized lipid-rich material \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Peroxiredoxins (Prxs) are a family of antioxidant enzymes that eliminate hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and is related with various cellular signal transduction processes by maintaining the redox state balance in various type of neuronal cells \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. There are six mammalian Prxs, classified in three subtypes (typical 2-Cys, atypical 2-Cys, and typical 1-Cys) depending on their catalytic mechanism of peroxide reduction. Prx1 belongs to the 2-Cys Prx and is widely distributed within diverse subcellular compartments, including the cytosol and nucleus \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Prx1 expression level was elevated in the brains of patients with AD \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, whereas it was decreased in other research \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Prx1 exerts protective effects in experimental models for AD \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, molecular mechanisms of Prx1 on the protection of AD progression are still lacking.\u003c/p\u003e \u003cp\u003eMitochondria are an essential cellular organelle with key regulatory functions in energy production, oxidative balance, and calcium homeostasis \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Several molecular abnormalities involving mitochondria, such as loss of energy metabolism, abnormal morphology, and accumulation of ROS were observed in the brain of patients and animal model with AD \u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Mitochondria are highly dynamic subcellular organelles that constantly repeat the process of fusion and fission events. The balance of mitochondrial fission and fusion is crucial for cellular processes in hippocampal neurons \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Cumulative mitochondrial fission induces mitochondrial fragmentation, and is closely associated with pathogenesis of STZ-induced AD models \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Dynamin-1-like protein (Drp1) is a key regulator of mitochondrial fission. It is recruited from the cytosol to mitochondria, then triggers mitochondrial division by increasing their activity \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The activity of Drp1 is controlled by post-translational modification, including phosphorylation \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In particular, phosphorylation of Drp1 at Ser616 (S616) has been shown to control the activity of Drp1, which plays an important role in various neurodegenerative pathological processes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, the molecular relationship between oxidative stress and changes in mitochondrial dynamics induced by STZ in an AD experimental model has not been fully elucidated.\u003c/p\u003e \u003cp\u003eCalpains are calcium (Ca\u003csup\u003e2+\u003c/sup\u003e)-dependent cysteine proteinases that are involved in multiple cellular functions, such as proliferation, differentiation, growth, and apoptosis \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Abnormal activation of calpains induced by disruption of Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis is observed around amyloid plaque and neurofibrillary tangles of AD patients and experimental models \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and inhibition of calpains improved spatial-working memory and synaptic transmission in AD mice model \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In particular, the elevated expression level of calpain-2 (m-calpain), one of the major calpain isoforms, is known to reflect calpain activity in neuronal cells and is closely associated with progression of AD pathologies. \u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCyclin-dependent kinase 5 (Cdk5) belongs to the family of proline-directed serine/threonine kinase is activated by the neuron-specific activator p35 under normal conditions, and can be deregulated by p25, which is the cleaved form of p35 \u003csup\u003e40\u003c/sup\u003e. Cdk5 performs an important role in the maintenance of neuronal development, survival, and synaptic plasticity and neurotransmission \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, uncontrolled activity of Cdk5/p25 is involved in the development of AD pathologies, including tau phosphorylation \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, amyloidogenesis \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, preservation of Cdk5 homeostasis is suggested as a reasonable therapeutic target for ameliorating AD pathological processes \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Interestingly, Cdk5 was also identified as an upstream regulator of mitochondrial fission via phosphorylation of Drp1 at S616 in neurodegenerative conditions \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Calpain is one of the factors involved in the cleavage of p25, which has been linked to AD pathogenesis \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated whether oxidative stress-induced Ca\u003csup\u003e2+\u003c/sup\u003e accumulation affects calpain-2-mediated progression of AD-like pathologies in STZ-induced hippocampal neuronal HT-22 cells. Therefore, we focused Prx1 as a regulator of STZ-induced oxidative stress. Furthermore, to validate the effect of Prx1 on the molecular signal pathway in the progression of STZ-induced AD-like pathologies (neuronal apoptosis, synaptic function, tau pathology, and mitochondrial fragmentation), we determined the change in calpain, cdk5, and mitochondria morphology using antioxidant molecule and Ca\u003csup\u003e2+\u003c/sup\u003e chelator. These studies suggested that maintenance of oxidative balance via increase in antioxidant defense is a promising target to ameliorate the progress of AD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eHT-22 cells were derived from HT-4 cells, which were immortalized from primary mouse hippocampal neuronal culture \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. HT-22 cells were maintained at 37\u0026deg;C in DMEM with high glucose (Welgene, Daegu, Korea) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/ml penicillin, and 100 \u0026micro;g/ml streptomycin (Welgene) in a humidified atmosphere incubator (Thermo Fisher Scientific) with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were pretreated with BAPTA-AM (0.5 \u0026micro;M; Thermo Fisher Scientific), NAC (5 mM; Sigma-Aldrich, St. Louis, MO, USA) for 30 min and were incubated with STZ (10 mM; Sigma-Aldrich).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction Preparation of stable cell line\u003c/h2\u003e \u003cp\u003ePrx1 gene including plasmid (pLenti6.3-Prx1-V5; Thermo Fisher Scientific) kindly provided by Dr. Dong-Seok Lee (Kyungpook National University, Daegu, Korea). Prx1 was amplified by performing PCR with LA Taq polymerase (TaKaRa, Shiga, Japan). These genes were cloned into a gateway entry vector pCR8/GW/TOPO (Thermo Fisher Scientific) to generate expression clones by performing LR recombination between the entry vector and gateway destination vectors pLenti6.3/V5-DEST (Thermo Fisher Scientific). pLenti6.3/V5-DEST vector have a C-terminal V5 epitope that aids in detecting recombinant proteins during immunoblotting analysis \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The sequences of the constructed vectors were confirmed by performing restriction mapping and DNA sequencing. 1 \u0026micro;g of pLenti6.3-Prx1-V5 plasmid was transfected into the HT-22 cells by using effectene (Qiagen, Hilden, Germany), according to the manufacturer's instructions. After 24 h, the transfected cells were selected using 8 \u0026micro;g/mL blasticidin (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eWhole protein lysates were prepared using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Korea), and mitochondrial fractions were performed with a mitochondria isolation kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by electrophoresis on 8\u0026ndash;15% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, NJ, USA). The membranes were blocked by incubation in blocking buffer (BD Biosciences) and probed with the following antibodies overnight at 4\u0026deg;C: anti-Prx1 (Ab Frontier, Seoul, Korea), anti-β-actin (Sigma-Aldrich), anti-V5, anti-AT8 (Thermo Fisher Scientific), anti-cleaved caspase-3, anti-PARP, p-Tau(S262), anti-Drp1, anti-p-Drp1(S616), anti-COXIV, anti-Cdk5 (Cell Signaling, MA, USA), anti-NeuN, anti-PSD95, (Abcam, MA, USA), anti-p35, anti-calpain-2 (Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were washed with TBS with 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) for 1 h at room temperature. After washing with TBST, protein bands were visualized using enhanced chemiluminescence reagent in the Chemi DocXRS\u0026thinsp;+\u0026thinsp;imaging system (Bio-Rad, Hercules, CA, USA). Finally, densitometric analysis was performed using Image Lab software, version 3.0 (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eMock- and Prx1-expressed HT-22 cells were cultured on 96-well plates for 24 h. After experiments, each cell sample was incubated for 30 min at 37\u0026deg;C with MTT (0.5 mg/mL; Sigma-Aldrich), and then 100 \u0026micro;L DMSO (Sigma-Aldrich) was added. Absorbance was measured at 550 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of intracellular ATP levels\u003c/h2\u003e \u003cp\u003eThe intracellular ATP levels were determined using an ATP determination kit (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions. After experiments, whole protein lysates at a concentration of 1 \u0026micro;g/mL were used for the measurements of intracellular ATP levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial imaging\u003c/h2\u003e \u003cp\u003eTo observe mitochondrial morphology, DsRed2-mito including plasmid (pLenti6.3-DsRed2-mito) was kindly provided by Dr. Dong-Seok Lee (Kyungpook National University, Daegu, Korea). DsRed2-mito expressed HT-22 cells were seeded on 0.01% poly-D-lysine-coated round coverslips and were incubated for 24 h. After experiments, cells were washed with PBS, and fixed with 4% paraformaldehyde for 1 h. After washing, the coverslips were mounted on slides with mounting medium (VECTOR Laboratories, CA, USA). To observe mitochondrial morphology in Prx1-expressed HT-22 cells, MitoTracker Green (100 \u0026micro;M; Thermo Fisher Scientific) was incubated with the cells. Mitochondrial images were acquired using the LSM-710 confocal microscope (Carl Zeiss, Jena, Germany). Mitochondrial length measurements were performed using image J software as previously described \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels\u003c/h2\u003e \u003cp\u003eIntracellular Ca\u003csup\u003e2+\u003c/sup\u003e level was measured using Fluo-4 AM (Thermo Fisher Scientific). After experiment, cells were incubated with Fluo-4 AM for 30 min at 37\u0026deg;C. After washing with HBSS, images of Fluo-4 AM were obtained using an ECLIPSE Ti-U microscope (Nikon, Tokyo, Japan), and fluorescence intensity of Fluo-4 AM was measured with image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of intracellular ROS\u003c/h2\u003e \u003cp\u003eIntracellular ROS generation was assessed using CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA. After experiments, cells were incubated with 5 \u0026micro;M CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA (Thermo Fisher Scientific) for 30 min at 37\u0026deg;C, and then analyzed using an ECLIPSE Ti-U microscope (Nikon) and FACSCalibur flow cytometry (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments (n\u0026thinsp;=\u0026thinsp;3). Experimental differences were tested for statistical significance using GraphPad Prism 9 software (San Diego, CA, USA). Multiple group analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for normally distributed datasets. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and is indicated on the graphs using asterisks; P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u0026lt;\u0026thinsp;0.001 were indicated by two and three asterisks, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003eSTZ-mediated Prx1 induction inhibits progression of AD-like pathology\u003c/h2\u003e\n\u003cp\u003eWe investigated the Prx1 protein expression levels in HT-22 cells following time-dependent STZ treatments. STZ concentration (10 mM) referred to induce AD-like pathology in HT-22 cells\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e.\u0026nbsp;We found that Prx1 protein expression level was gradually increased after 12 h STZ treatments (Figure 1A). To assess the effect of\u0026nbsp;inducible\u0026nbsp;Prx1\u0026nbsp;expression\u0026nbsp;by STZ on process of AD-like pathology, we generated\u0026nbsp;V5-tagged\u0026nbsp;Prx1\u0026nbsp;(Prx1-V5)\u0026nbsp;stably\u0026nbsp;expressing\u0026nbsp;HT-22 cells\u0026nbsp;line\u0026nbsp;by\u0026nbsp;using\u0026nbsp;transfection of\u0026nbsp;\u0026nbsp;pLtneti6.3-Prx1-V5. We identified the exogenous expression of Prx1 (Prx1-V5)\u0026nbsp;by\u0026nbsp;western blotting using Prx1\u0026nbsp;and V5-tag antibodies (Figure 1B). Prx1 expression reversed STZ-induced decreased cell viability and increased apoptotic markers, such as cleaved caspase-3 and cleaved PARP (Figure 1C and D).\u0026nbsp;Furthermore, we determined the effect of Prx1 on STZ-induced neuronal loss and synaptic function using immunoblotting with NeuN (neuronal marker), and PSD95 (post-synapse marker). Our results showed that STZ-induced down-regulation of NeuN and PSD95 were inhibited in Prx1 expression (Figure 1E and F). We also confirmed that Prx1 impacted the phosphorylation of tau epitopes, such as p-Tau(S262) and AT8(S202/T205). Up-regulated p-Tau (S262) and AT8(S202/T205) by STZ treatment was also suppressed by Prx1 expression (Figure 1G). These results suggested that Prx1 induction by STZ regulates STZ-induced process of AD-like pathology, apoptotic neuronal loss, synaptic loss, and tauopathy in hippocampal cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrx1 prevents STZ-induced mitochondrial fragmentation and dysfunction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously reported that change in mitochondrial morphology and mitochondrial function relate to STZ-induced progression of AD-like pathology\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e. Therefore, we\u0026nbsp;measured changes in intracellular ATP levels after STZ treatment for 12 h in mock- and Prx1-expressed HT-22 cells. Our results indicated that down-regulated intracellular ATP level by STZ treatment was suppressed by Prx1 expression (Figure 2A).\u0026nbsp;We\u0026nbsp;determined whether Prx1 affects STZ-induced mitochondrial fragmentation. To observe changes in mitochondrial morphology after STZ treatment for 12 h in HT-22 and Prx1-expressed HT-22 cells, we stained with mitotracker green after experiments. Our result indicated that decrease of mitochondrial average length by STZ treatment in HT-22 cells was inhibited in Prx1-expressed HT-22 cells (Figure 2B and C). STZ-induced mitochondrial fragmentation was depended on the Drp1 translocation from the cytoplasm to the mitochondria by phosphorylation of Drp1(S616) via activating Cdk5/p25 signaling pathway\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e. Therefore, we assess the effects of Prx1 on the Cdk5/p25-mediated Drp1 activation by STZ treatment for 12 h. Our results indicated that STZ-induced increase in the level of mitochondrial Drp1 was inhibited by Prx1 expression (Figure 2C). Drp1(S616) phosphorylation was increased by STZ treatment for 12 h, and up-regulated Drp1(S616) phosphorylation was repressed by Prx1 expression (Figure 2D). Moreover, increased level of p25 by STZ treatment for 6 h, which is known to show the highest level of p25 by STZ treatment in HT-22 cells\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e, was inhibited by Prx1 expression (Figure 2E).\u0026nbsp;Therefore, we demonstrate that induction of Prx1 by STZ regulates Cdk5-mediated mitochondrial fragmentation and dysfunction by inhibiting cleavage of p35 to p25.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrx1 regulates STZ-induce Ca\u003csup\u003e2+\u003c/sup\u003e-mediated mitochondrial fragmentation and dysfunction through calpain/Cdk5-mediated Drp1 activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious reports indicated that Prx5 inhibits the accumulation of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e, calpain activation, and Cdk5 activation in an amyloid-beta (A\u0026beta;) oligomer-mediated AD cellular model\u0026nbsp;\u003csup\u003e53\u003c/sup\u003e.\u0026nbsp;Therefore, we measured intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels using the intracellular Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eindicator Fluo-4 AM, in time-dependent STZ-treated HT-22 cells. Intracellular\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e was measured from 3 h STZ treatment, and maintained to 6 h (Figure 3A). Increased level of STZ-induced\u0026nbsp;intracellular Ca\u003csup\u003e2+\u003c/sup\u003e was down-regulated by Prx1 expression (Figure 3B).\u0026nbsp;The elevated expression level of calpain-2 is known to reflect calpain activation in neuron cells\u0026nbsp;\u003csup\u003e38,39\u003c/sup\u003e. Thus, we then assessed whether Prx1 affected calpain-2 expression. We found that calpain-2 expression level was the highest at 1.5 h STZ treatment time, and was gradually down-regulated to 6 h (Figure 3C). Prx1 inhibited STZ-induced increase of calpain-2 expression (Figure 3D). We then assessed whether Prx1 influenced STZ-induced mitochondrial fragmentation through intracellular Ca\u003csup\u003e2+\u003c/sup\u003e regulation using an intracellular Ca\u003csup\u003e2+\u003c/sup\u003e chelator, BAPTA-AM. We found that inhibition of STZ-induced Ca\u003csup\u003e2+\u003c/sup\u003e accumulation prevented the calpain activation and Cdk5 activation by p35 cleavage to p25 (Figure 4A and B). Furthermore, STZ-induced increase of fragmented mitochondria and mitochondrial dysfunction were restored by intracellular Ca\u003csup\u003e2+\u003c/sup\u003e chelation via controlling Drp1 activation (Figure 4C, D, E, and F). These results suggest that Prx1 regulates calpain/Cdk5-mediated mitochondrial fragmentation by controlling STZ-induced intracellular Ca\u003csup\u003e2+\u003c/sup\u003e accumulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e on the STZ-induced AD-like pathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePast demonstrations indicated that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e or control of mitochondrial morphology were key mediators in AD\u0026nbsp;\u003csup\u003e53\u003c/sup\u003e.\u0026nbsp;As we proved that role of Ca\u003csup\u003e2+\u003c/sup\u003e as a regulator of STZ-induced mitochondrial fragmentation, we determined the effect of STZ-induced Ca\u003csup\u003e2+\u003c/sup\u003e on the progression of AD-like pathology. STZ-induced Ca\u003csup\u003e2+\u003c/sup\u003e inhibition with BAPTA-AM\u0026nbsp;suppressed increased neuronal apoptosis and neuronal loss. The increased level of cleaved caspase-3 and cleaved PARP, reduced level of NeuN by STZ reversed by BAPTA-AM treatment (Figure 5A and B). In addition, the STZ-induced increased synaptic loss and Tau activation were confirmed with PSD95, p-Tau(S262), and AT8(S202/T205). STZ-induced decrease of PSD95 and increase of p-Tau(S262) and AT8(S202/T205) were attenuated by BAPTA-AM treatment (Figure 5C and D). These results indicated that STZ-mediated accumulation of intracellular\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e can influence the progression of STZ-induced AD-like pathology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of ROS on STZ-induced AD-like pathology via Ca\u003csup\u003e2+\u003c/sup\u003e/calpain/Cdk5-mediated mitochondrial fragmentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious reports suggested that oxidative stress relates to the accumulation of intracellular\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e-mediated calpain activation, and\u0026nbsp;Prx blocks the increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e accumulation\u0026nbsp;\u003csup\u003e54,55\u003c/sup\u003e. Therefore, to assess whether Prx1 affected Ca\u003csup\u003e2+\u003c/sup\u003e-mediated calpain activation using Prx1 antioxidant capacity, we inhibited STZ-induced ROS production using antioxidants molecule, \u003cem\u003eN\u003c/em\u003e-acetyl-cysteine (NAC). We first determined effect of Prx1 to STZ-induced intracellular ROS level in STZ-induced HT-22 cells and Prx1-expressed HT-22 cells with CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA. Our results indicated that increased levels of intracellular ROS induced by STZ treatment at 12 h were suppressed by Prx1 expression (Figure 6A and B). We investigated the impact of STZ-induced ROS inhibition on calpain activation and p35 cleavage. Up-regulation of calpain-2 and p25 proteins level were restored by NAC treatment (Figure C and D). Increase of mitochondrial fragmentation and dysfunction, a downstream pathway of Ca\u003csup\u003e2+\u003c/sup\u003e/calpain/Cdk5 were rescued by ROS scavenge through inhibition of Drp1(S616) phosphorylation and mitochondrial location (Figure 6E, F, G, and H). We then assessed the effect of STZ-induced ROS inhibition on the STZ-induced progression of AD-like pathology. Our results showed that STZ-induced increase in neuronal apoptosis, neuronal loss, and synaptic loss, and Tau phosphorylation were prevented by NAC treatment (Figure 7). These results suggested that STZ-induced progression of AD-like pathology in HT-22 cells were involved in increased ROS levels through governing Ca\u003csup\u003e2+\u003c/sup\u003e/Calpain/Cdk5-mediated mitochondrial fragmentation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our previous research, we developed an STZ-induced effective and clinically relevant AD-like model in non-human primates and rodents through intra-cisternal magna (ICM) route, characterized by cerebral and hippocampal damage, disintegration of neurovascular unit, Aβ deposition, neuroinflammation, and Cdk5 activation \u003csup\u003e\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Recently, we suggested that regulation of Cdk5/Drp1-dependent mitochondrial morphology play roles for potential inhibitor of abnormal metabolic functions associated with the AD-like pathogenesis \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Based on these findings, more detailed molecular mechanistic investigation of the STZ-induced AD-like pathologies are warranted.\u003c/p\u003e \u003cp\u003eOxidative stress precedes the onset of significant AD pathology in the brains of patients and animal models with AD \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Antioxidant defense system in the response from oxidative stress, similarly to change in the expression of Prx subtypes seem to be involved in AD pathology, but this remains controversial \u003csup\u003e\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Mounting evidence determined that various type of Prxs, such as Prx5 and Prx6 have been associated with regulation of the progress of AD pathologies \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Specifically, Prx1 was mainly expressed in oligodendrocytes and astrocytes, and were detected in a few neuronal cells \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. However, Prx1 expression was increased in an Aβ-resistant neuronal cell line response to oxidative stress \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Therefore, we focused on the role of Prx1 to assess the association with an STZ-mediated antioxidant response in HT-22 hippocampus cell line. We found that Prx1 was upregulated in a time-dependent manner in response to STZ-mediated oxidative stress. Therefore, we produced the Prx1 overexpression cell line in HT-22 cells to assume a situation in which Prx1 was expressed in an early timepoint of STZ treatment. Prx1 overexpression inhibited STZ-mediated neuronal apoptosis, synaptic loss, and tau phosphorylation via preventing cellular ROS accumulation. Furthermore, STZ-mediated mitochondrial fragmentation was suppressed by Prx1 overexpression through prevention of Cdk5-dependent Drp1 phosphorylation. It has been suggested that dysregulation of Cdk5 homeostasis has pathological relevance to AD \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Oxidative stress is considered to a crucial modulator of Cdk5 activation \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Prx5 involved in Cdk5 activation by modulating oxidative stress \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, and Prx1 activation inhibited Aβ-induced impaired axonal transport \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. However, the precise relationship between Prx1 and Cdk5 activation in STZ-mediated AD-like pathogenesis are still unclear. Our findings reveal that Prx1 is an important suppressor of STZ-induced progression of AD-like pathology via Cdk5 activation and mitochondrial fragmentation.\u003c/p\u003e \u003cp\u003eCdk5 activation by p25 is triggered by activation of calpain, which is related to accumulation of intracellular Ca\u003csup\u003e2+ 74,78\u003c/sup\u003e. Our result also showed that elimination of STZ-induced accumulated intracellular Ca\u003csup\u003e2+\u003c/sup\u003e suppressed STZ-mediated calpain-2 expression, Cdk5 activation, mitochondrial fragmentation, and progression of AD-like pathology. Oxidative stress is associated with dysregulation of Ca\u003csup\u003e2+\u003c/sup\u003e release and signal pathway \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, which in turn triggers calpain-2 activation \u003csup\u003e\u003cspan additionalcitationids=\"CR82\" citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Prx1 modulates Aβ-induced increased intracellular Ca\u003csup\u003e2+\u003c/sup\u003e level by inhibiting ROS accumulation \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Our results suggested that STZ-induced up-regulated Prx1 caused a decrease in Ca\u003csup\u003e2+\u003c/sup\u003e and calpain-2 level. Apart from antioxidant function of Prx1 that reduce peroxides via a highly reactive catalytic cysteine oxidation to sulfenic acid \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e, Prx1 displays chaperone function by controlling the protein-binding partners \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Therefore, we proved that Prx1 decreased the STZ-induced Ca\u003csup\u003e2+\u003c/sup\u003e-mediated calpain-2 expression depending on Prx1 antioxidant capacity. However, it needs to study the direct role of Drp1 phosphorylation in the process of STZ-induced AD-like pathology. Our results demonstrated that the antioxidant capacity of Prx1 is a key factor of the regulation of Ca\u003csup\u003e2+\u003c/sup\u003e level and Ca\u003csup\u003e2+\u003c/sup\u003e-dependent calpain-2 expression, Cdk5-related mitochondria fragmentation.\u003c/p\u003e \u003cp\u003eIncreased expression level of Prx1 observed in various neurodegenerative conditions \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e and increased expression of Prx1 contribute to resistance to oxidative stress \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Prx1 is known to play a protective role against ROS-mediated brain injury, such as endotoxin‑induced injury \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e, Huntington\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e, and acute ischemic stroke \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. Furthermore, microglial Prx1 participated in the protective function against endotoxin-induced pro-inflammatory response by regulating oxidative stress \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e, and Prx1 overexpression reduced neuronal inflammation and apoptosis by affecting microglial and astrocyte mRNA stability \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Therefore, these findings along with our results suggested that Prx1 plays a protective role in neurodegenerative environments by affecting neuron, astrocyte, and microglia.\u003c/p\u003e \u003cp\u003eApart from these results, our findings have the limitations that it is not reflected complex environment present in the brains of AD rodents or patients because these findings were investigated from immortalized hippocampal cell line, HT-22 cells. Although several advantages of HT-22 cells, such as AD pathology being well reflected or useful for molecular mechanism study, more research should be conducted in primary hippocampus cells or AD animal models based on the results of this study. Consequently, regulation of Prx1 may be a potential inhibitor of STZ-induced neurodegeneration by preventing Ca\u003csup\u003e2+\u003c/sup\u003e/capain-2/Cdk5 signal pathway, and may consider as a possible strategy for developing therapies to treat the pathogenesis of AD.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated and/or analyzed during performing this current study are included in this article [and also in its supplementary dataset files]. However, there is no restriction on the availability of materials and data from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (KGM4562431), and by the National Research Council of Science \u0026amp; Technology (NST) grant by the Korea government (MSIT) (No. CPS21101-100), and by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health \u0026amp; Welfare, the Ministry of Food and Drug Safety) (Project Number: 9991006929, RS-2020-KD000264).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJP participated in design of the experiments, performed the experiments, analyzed the data, and drafted the manuscript. JW and EY performed the experiments, and analyzed the data. JS, JC, JBS, H-GY, KK, YGK, MK, C-YJ, and KSY performed and supported the experiments. D-SL, and YL edited manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.co.kr) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQuerfurth, H. W. \u0026amp; LaFerla, F. M. Alzheimer\u0026apos;s disease. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e362\u003c/strong\u003e, 329-344, doi:10.1056/NEJMra0909142 (2010).\u003c/li\u003e\n\u003cli\u003eSpinelli, M., Fusco, S. \u0026amp; Grassi, C. Brain Insulin Resistance and Hippocampal Plasticity: Mechanisms and Biomarkers of Cognitive Decline. \u003cem\u003eFront Neurosci\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 788, doi:10.3389/fnins.2019.00788 (2019).\u003c/li\u003e\n\u003cli\u003eFerreira, L. S. S., Fernandes, C. S., Vieira, M. N. N. \u0026amp; De Felice, F. G. Insulin Resistance in Alzheimer\u0026apos;s Disease. \u003cem\u003eFront Neurosci\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 830, doi:10.3389/fnins.2018.00830 (2018).\u003c/li\u003e\n\u003cli\u003eLourenco, M. V.\u003cem\u003e et al.\u003c/em\u003e TNF-alpha mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer\u0026apos;s beta-amyloid oligomers in mice and monkeys. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 831-843, doi:10.1016/j.cmet.2013.11.002 (2013).\u003c/li\u003e\n\u003cli\u003eBatista, A. F.\u003cem\u003e et al.\u003c/em\u003e The diabetes drug liraglutide reverses cognitive impairment in mice and attenuates insulin receptor and synaptic pathology in a non-human primate model of Alzheimer\u0026apos;s disease. \u003cem\u003eJ Pathol\u003c/em\u003e \u003cstrong\u003e245\u003c/strong\u003e, 85-100, doi:10.1002/path.5056 (2018).\u003c/li\u003e\n\u003cli\u003eLee, W., Wakasugi, H. \u0026amp; Ibayashi, H. Comparison of somatostatin distribution in pancreatic duct ligated rats and streptozotocin diabetic rats. \u003cem\u003eGastroenterol Jpn\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 453-458, doi:10.1007/bf02776585 (1983).\u003c/li\u003e\n\u003cli\u003eSalkovic-Petrisic, M. \u0026amp; Hoyer, S. Central insulin resistance as a trigger for sporadic Alzheimer-like pathology: an experimental approach. \u003cem\u003eJ Neural Transm Suppl\u003c/em\u003e, 217-233, doi:10.1007/978-3-211-73574-9_28 (2007).\u003c/li\u003e\n\u003cli\u003eKamat, P. K. Streptozotocin induced Alzheimer\u0026apos;s disease like changes and the underlying neural degeneration and regeneration mechanism. \u003cem\u003eNeural Regen Res\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1050-1052, doi:10.4103/1673-5374.160076 (2015).\u003c/li\u003e\n\u003cli\u003eKim, G. H., Kim, J. E., Rhie, S. J. \u0026amp; Yoon, S. The Role of Oxidative Stress in Neurodegenerative Diseases. \u003cem\u003eExp Neurobiol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 325-340, doi:10.5607/en.2015.24.4.325 (2015).\u003c/li\u003e\n\u003cli\u003eWang, X.\u003cem\u003e et al.\u003c/em\u003e Oxidative stress and mitochondrial dysfunction in Alzheimer\u0026apos;s disease. \u003cem\u003eBiochim Biophys Acta\u003c/em\u003e, doi:10.1016/j.bbadis.2013.10.015 (2013).\u003c/li\u003e\n\u003cli\u003eSalim, S. Oxidative Stress and the Central Nervous System. \u003cem\u003eJ Pharmacol Exp Ther\u003c/em\u003e \u003cstrong\u003e360\u003c/strong\u003e, 201-205, doi:10.1124/jpet.116.237503 (2017).\u003c/li\u003e\n\u003cli\u003eZhu, H., Santo, A. \u0026amp; Li, Y. The antioxidant enzyme peroxiredoxin and its protective role in neurological disorders. \u003cem\u003eExp Biol Med (Maywood)\u003c/em\u003e \u003cstrong\u003e237\u003c/strong\u003e, 143-149, doi:10.1258/ebm.2011.011152 (2012).\u003c/li\u003e\n\u003cli\u003eSzeliga, M. Peroxiredoxins in Neurodegenerative Diseases. \u003cem\u003eAntioxidants (Basel)\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, doi:10.3390/antiox9121203 (2020).\u003c/li\u003e\n\u003cli\u003eWood, Z. A., Schroder, E., Robin Harris, J. \u0026amp; Poole, L. B. Structure, mechanism and regulation of peroxiredoxins. \u003cem\u003eTrends Biochem Sci\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 32-40, doi:10.1016/s0968-0004(02)00003-8 (2003).\u003c/li\u003e\n\u003cli\u003eKim, S. H., Fountoulakis, M., Cairns, N. \u0026amp; Lubec, G. Protein levels of human peroxiredoxin subtypes in brains of patients with Alzheimer\u0026apos;s disease and Down syndrome. \u003cem\u003eJ Neural Transm Suppl\u003c/em\u003e, 223-235, doi:10.1007/978-3-7091-6262-0_18 (2001).\u003c/li\u003e\n\u003cli\u003eKrapfenbauer, K., Engidawork, E., Cairns, N., Fountoulakis, M. \u0026amp; Lubec, G. Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders. \u003cem\u003eBrain Res\u003c/em\u003e \u003cstrong\u003e967\u003c/strong\u003e, 152-160, doi:10.1016/s0006-8993(02)04243-9 (2003).\u003c/li\u003e\n\u003cli\u003eStepler, K. E.\u003cem\u003e et al.\u003c/em\u003e Inclusion of African American/Black adults in a pilot brain proteomics study of Alzheimer\u0026apos;s disease. \u003cem\u003eNeurobiol Dis\u003c/em\u003e \u003cstrong\u003e146\u003c/strong\u003e, 105129, doi:10.1016/j.nbd.2020.105129 (2020).\u003c/li\u003e\n\u003cli\u003eLee, Y. J.\u003cem\u003e et al.\u003c/em\u003e Peroxiredoxin I regulates the component expression of gamma-secretase complex causing the Alzheimer\u0026apos;s disease. \u003cem\u003eLab Anim Res\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 293-299, doi:10.5625/lar.2011.27.4.293 (2011).\u003c/li\u003e\n\u003cli\u003eLin, M. Y. \u0026amp; Sheng, Z. H. Regulation of mitochondrial transport in neurons. \u003cem\u003eExp Cell Res\u003c/em\u003e, doi:10.1016/j.yexcr.2015.01.004 (2015).\u003c/li\u003e\n\u003cli\u003eReddy, P. H. Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer\u0026apos;s disease. \u003cem\u003eExp Neurol\u003c/em\u003e \u003cstrong\u003e218\u003c/strong\u003e, 286-292, doi:10.1016/j.expneurol.2009.03.042 (2009).\u003c/li\u003e\n\u003cli\u003eDixit, S., Fessel, J. P. \u0026amp; Harrison, F. E. Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer\u0026apos;s disease and a novel protective role for ascorbate. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 515-523, doi:10.1016/j.freeradbiomed.2017.08.021 (2017).\u003c/li\u003e\n\u003cli\u003eGuo, X. D.\u003cem\u003e et al.\u003c/em\u003e LX2343 alleviates cognitive impairments in AD model rats by inhibiting oxidative stress-induced neuronal apoptosis and tauopathy. \u003cem\u003eActa Pharmacol Sin\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 1104-1119, doi:10.1038/aps.2016.128 (2017).\u003c/li\u003e\n\u003cli\u003eLatina, V.\u003cem\u003e et al.\u003c/em\u003e Tau Cleavage Contributes to Cognitive Dysfunction in Strepto-Zotocin-Induced Sporadic Alzheimer\u0026apos;s Disease (sAD) Mouse Model. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, doi:10.3390/ijms222212158 (2021).\u003c/li\u003e\n\u003cli\u003eKnott, A. B., Perkins, G., Schwarzenbacher, R. \u0026amp; Bossy-Wetzel, E. Mitochondrial fragmentation in neurodegeneration. \u003cem\u003eNat Rev Neurosci\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 505-518, doi:10.1038/nrn2417 (2008).\u003c/li\u003e\n\u003cli\u003eJiang, S.\u003cem\u003e et al.\u003c/em\u003e Mfn2 ablation causes an oxidative stress response and eventual neuronal death in the hippocampus and cortex. \u003cem\u003eMol Neurodegener\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 5, doi:10.1186/s13024-018-0238-8 (2018).\u003c/li\u003e\n\u003cli\u003eCorreia, S. C.\u003cem\u003e et al.\u003c/em\u003e Mitochondrial abnormalities in a streptozotocin-induced rat model of sporadic Alzheimer\u0026apos;s disease. \u003cem\u003eCurr Alzheimer Res\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 406-419, doi:10.2174/1567205011310040006 (2013).\u003c/li\u003e\n\u003cli\u003eYang, S.\u003cem\u003e et al.\u003c/em\u003e Mitochondrial dysfunction driven by the LRRK2-mediated pathway is associated with loss of Purkinje cells and motor coordination deficits in diabetic rat model. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e1217, doi:10.1038/cddis.2014.184 (2014).\u003c/li\u003e\n\u003cli\u003eWestermann, B. Mitochondrial fusion and fission in cell life and death. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 872-884, doi:10.1038/nrm3013 (2010).\u003c/li\u003e\n\u003cli\u003eElgass, K., Pakay, J., Ryan, M. T. \u0026amp; Palmer, C. S. Recent advances into the understanding of mitochondrial fission. \u003cem\u003eBiochim Biophys Acta\u003c/em\u003e \u003cstrong\u003e1833\u003c/strong\u003e, 150-161, doi:10.1016/j.bbamcr.2012.05.002 (2013).\u003c/li\u003e\n\u003cli\u003eWang, X.\u003cem\u003e et al.\u003c/em\u003e Impaired balance of mitochondrial fission and fusion in Alzheimer\u0026apos;s disease. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 9090-9103, doi:10.1523/JNEUROSCI.1357-09.2009 (2009).\u003c/li\u003e\n\u003cli\u003eKim, B., Park, J., Chang, K. T. \u0026amp; Lee, D. S. Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 184-194, doi:10.1016/j.freeradbiomed.2015.11.015 (2016).\u003c/li\u003e\n\u003cli\u003eFerreira, A. Calpain dysregulation in Alzheimer\u0026apos;s disease. \u003cem\u003eISRN Biochem\u003c/em\u003e \u003cstrong\u003e2012\u003c/strong\u003e, 728571, doi:10.5402/2012/728571 (2012).\u003c/li\u003e\n\u003cli\u003eTsuji, T., Shimohama, S., Kimura, J. \u0026amp; Shimizu, K. m-Calpain (calcium-activated neutral proteinase) in Alzheimer\u0026apos;s disease brains. \u003cem\u003eNeurosci Lett\u003c/em\u003e \u003cstrong\u003e248\u003c/strong\u003e, 109-112, doi:10.1016/s0304-3940(98)00348-6 (1998).\u003c/li\u003e\n\u003cli\u003eKurbatskaya, K.\u003cem\u003e et al.\u003c/em\u003e Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer\u0026apos;s disease brain. \u003cem\u003eActa Neuropathol Commun\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 34, doi:10.1186/s40478-016-0299-2 (2016).\u003c/li\u003e\n\u003cli\u003eTrinchese, F.\u003cem\u003e et al.\u003c/em\u003e Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 2796-2807, doi:10.1172/JCI34254 (2008).\u003c/li\u003e\n\u003cli\u003eMedeiros, R.\u003cem\u003e et al.\u003c/em\u003e Calpain inhibitor A-705253 mitigates Alzheimer\u0026apos;s disease-like pathology and cognitive decline in aged 3xTgAD mice. \u003cem\u003eAm J Pathol\u003c/em\u003e \u003cstrong\u003e181\u003c/strong\u003e, 616-625, doi:10.1016/j.ajpath.2012.04.020 (2012).\u003c/li\u003e\n\u003cli\u003eAhmad, F.\u003cem\u003e et al.\u003c/em\u003e Isoform-specific hyperactivation of calpain-2 occurs presymptomatically at the synapse in Alzheimer\u0026apos;s disease mice and correlates with memory deficits in human subjects. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 13119, doi:10.1038/s41598-018-31073-6 (2018).\u003c/li\u003e\n\u003cli\u003eZhang, H.\u003cem\u003e et al.\u003c/em\u003e Calpain-2/p35-p25/Cdk5 pathway is involved in the neuronal apoptosis induced by polybrominated diphenyl ether-153. \u003cem\u003eToxicol Lett\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 41-53, doi:10.1016/j.toxlet.2017.05.027 (2017).\u003c/li\u003e\n\u003cli\u003eHaque, A.\u003cem\u003e et al.\u003c/em\u003e Calpain mediated expansion of CD4+ cytotoxic T cells in rodent models of Parkinson\u0026apos;s disease. \u003cem\u003eExp Neurol\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 113315, doi:10.1016/j.expneurol.2020.113315 (2020).\u003c/li\u003e\n\u003cli\u003eTsai, L. H., Delalle, I., Caviness, V. S., Jr., Chae, T. \u0026amp; Harlow, E. p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e371\u003c/strong\u003e, 419-423, doi:10.1038/371419a0 (1994).\u003c/li\u003e\n\u003cli\u003eCheung, Z. H. \u0026amp; Ip, N. Y. Cdk5: a multifaceted kinase in neurodegenerative diseases. \u003cem\u003eTrends Cell Biol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 169-175, doi:10.1016/j.tcb.2011.11.003 (2012).\u003c/li\u003e\n\u003cli\u003eKimura, T., Ishiguro, K. \u0026amp; Hisanaga, S. Physiological and pathological phosphorylation of tau by Cdk5. \u003cem\u003eFront Mol Neurosci\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 65, doi:10.3389/fnmol.2014.00065 (2014).\u003c/li\u003e\n\u003cli\u003eWen, Y.\u003cem\u003e et al.\u003c/em\u003e Transcriptional regulation of beta-secretase by p25/cdk5 leads to enhanced amyloidogenic processing. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 680-690, doi:10.1016/j.neuron.2008.02.024 (2008).\u003c/li\u003e\n\u003cli\u003ePiedrahita, D.\u003cem\u003e et al.\u003c/em\u003e Silencing of CDK5 reduces neurofibrillary tangles in transgenic alzheimer\u0026apos;s mice. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 13966-13976, doi:10.1523/JNEUROSCI.3637-10.2010 (2010).\u003c/li\u003e\n\u003cli\u003eCastro-Alvarez, J. F., Uribe-Arias, S. A., Kosik, K. S. \u0026amp; Cardona-Gomez, G. P. Long- and short-term CDK5 knockdown prevents spatial memory dysfunction and tau pathology of triple transgenic Alzheimer\u0026apos;s mice. \u003cem\u003eFront Aging Neurosci\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 243, doi:10.3389/fnagi.2014.00243 (2014).\u003c/li\u003e\n\u003cli\u003eMeuer, K.\u003cem\u003e et al.\u003c/em\u003e Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis. \u003cem\u003eCell Death Differ\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 651-661, doi:10.1038/sj.cdd.4402087 (2007).\u003c/li\u003e\n\u003cli\u003eJahani-Asl, A.\u003cem\u003e et al.\u003c/em\u003e CDK5 phosphorylates DRP1 and drives mitochondrial defects in NMDA-induced neuronal death. \u003cem\u003eHum Mol Genet\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 4573-4583, doi:10.1093/hmg/ddv188 (2015).\u003c/li\u003e\n\u003cli\u003eTrinchese, F.\u003cem\u003e et al.\u003c/em\u003e Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 2796-2807, doi:10.1172/JCI34254 (2008).\u003c/li\u003e\n\u003cli\u003eDavis, J. B. \u0026amp; Maher, P. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. \u003cem\u003eBrain Res\u003c/em\u003e \u003cstrong\u003e652\u003c/strong\u003e, 169-173 (1994).\u003c/li\u003e\n\u003cli\u003eSouthern, J. A., Young, D. F., Heaney, F., Baumgartner, W. K. \u0026amp; Randall, R. E. Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. \u003cem\u003eJ Gen Virol\u003c/em\u003e \u003cstrong\u003e72 ( Pt 7)\u003c/strong\u003e, 1551-1557 (1991).\u003c/li\u003e\n\u003cli\u003ePark, J.\u003cem\u003e et al.\u003c/em\u003e Mitochondrial dynamics modulate the expression of pro-inflammatory mediators in microglial cells. \u003cem\u003eJ Neurochem\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 221-232, doi:10.1111/jnc.12361 (2013).\u003c/li\u003e\n\u003cli\u003ePark, J.\u003cem\u003e et al.\u003c/em\u003e Streptozotocin Induces Alzheimer\u0026apos;s Disease-Like Pathology in Hippocampal Neuronal Cells via CDK5/Drp1-Mediated Mitochondrial Fragmentation. \u003cem\u003eFront Cell Neurosci\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 235, doi:10.3389/fncel.2020.00235 (2020).\u003c/li\u003e\n\u003cli\u003ePark, J.\u003cem\u003e et al.\u003c/em\u003e Peroxiredoxin 5 Decreases Beta-Amyloid-Mediated Cyclin-Dependent Kinase 5 Activation Through Regulation of Ca(2+)-Mediated Calpain Activation. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 715-726, doi:10.1089/ars.2016.6810 (2017).\u003c/li\u003e\n\u003cli\u003eDe Simoni, S., Linard, D., Hermans, E., Knoops, B. \u0026amp; Goemaere, J. Mitochondrial peroxiredoxin-5 as potential modulator of mitochondria-ER crosstalk in MPP+-induced cell death. \u003cem\u003eJournal of neurochemistry\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 473-485, doi:10.1111/jnc.12117 (2013).\u003c/li\u003e\n\u003cli\u003eSabharwal, S. S., Waypa, G. B., Marks, J. D. \u0026amp; Schumacker, P. T. Peroxiredoxin-5 targeted to the mitochondrial intermembrane space attenuates hypoxia-induced reactive oxygen species signalling. \u003cem\u003eBiochem J\u003c/em\u003e \u003cstrong\u003e456\u003c/strong\u003e, 337-346, doi:10.1042/BJ20130740 (2013).\u003c/li\u003e\n\u003cli\u003eYeo, H. G.\u003cem\u003e et al.\u003c/em\u003e Characterization of Cerebral Damage in a Monkey Model of Alzheimer\u0026apos;s Disease Induced by Intracerebroventricular Injection of Streptozotocin. \u003cem\u003eJ Alzheimers Dis\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 989-1005, doi:10.3233/JAD-143222 (2015).\u003c/li\u003e\n\u003cli\u003eLee, Y.\u003cem\u003e et al.\u003c/em\u003e Insulin/IGF signaling-related gene expression in the brain of a sporadic Alzheimer\u0026apos;s disease monkey model induced by intracerebroventricular injection of streptozotocin. \u003cem\u003eJ Alzheimers Dis\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 251-267, doi:10.3233/JAD-130776 (2014).\u003c/li\u003e\n\u003cli\u003eAhn, Y.\u003cem\u003e et al.\u003c/em\u003e Synaptic loss and amyloid beta alterations in the rodent hippocampus induced by streptozotocin injection into the cisterna magna. \u003cem\u003eLab Anim Res\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 17, doi:10.1186/s42826-020-00049-x (2020).\u003c/li\u003e\n\u003cli\u003eXie, H.\u003cem\u003e et al.\u003c/em\u003e Rapid cell death is preceded by amyloid plaque-mediated oxidative stress. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 7904-7909, doi:10.1073/pnas.1217938110 (2013).\u003c/li\u003e\n\u003cli\u003eButterfield, D. A., Swomley, A. M. \u0026amp; Sultana, R. Amyloid beta-peptide (1-42)-induced oxidative stress in Alzheimer disease: importance in disease pathogenesis and progression. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 823-835, doi:10.1089/ars.2012.5027 (2013).\u003c/li\u003e\n\u003cli\u003eCumming, R. C., Dargusch, R., Fischer, W. H. \u0026amp; Schubert, D. Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 30523-30534, doi:10.1074/jbc.M700869200 (2007).\u003c/li\u003e\n\u003cli\u003eKim, S. H., Fountoulakis, M., Cairns, N. \u0026amp; Lubec, G. Protein levels of human peroxiredoxin subtypes in brains of patients with Alzheimer\u0026apos;s disease and Down syndrome. \u003cem\u003eJ Neural Transm Suppl\u003c/em\u003e, 223-235 (2001).\u003c/li\u003e\n\u003cli\u003eCalkins, M. J., Manczak, M., Mao, P., Shirendeb, U. \u0026amp; Reddy, P. H. Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer\u0026apos;s disease. \u003cem\u003eHuman molecular genetics\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 4515-4529, doi:10.1093/hmg/ddr381 (2011).\u003c/li\u003e\n\u003cli\u003eKrapfenbauer, K., Engidawork, E., Cairns, N., Fountoulakis, M. \u0026amp; Lubec, G. Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders. \u003cem\u003eBrain Res\u003c/em\u003e \u003cstrong\u003e967\u003c/strong\u003e, 152-160 (2003).\u003c/li\u003e\n\u003cli\u003eKim, I. K., Lee, K. J., Rhee, S., Seo, S. B. \u0026amp; Pak, J. H. Protective effects of peroxiredoxin 6 overexpression on amyloid beta-induced apoptosis in PC12 cells. \u003cem\u003eFree Radic Res\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 836-846, doi:10.3109/10715762.2013.833330 (2013).\u003c/li\u003e\n\u003cli\u003eKim, B., Park, J., Chang, K. T. \u0026amp; Lee, D. S. Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 184-194, doi:10.1016/j.freeradbiomed.2015.11.015 (2016).\u003c/li\u003e\n\u003cli\u003eGoemaere, J. \u0026amp; Knoops, B. Peroxiredoxin distribution in the mouse brain with emphasis on neuronal populations affected in neurodegenerative disorders. \u003cem\u003eJ Comp Neurol\u003c/em\u003e \u003cstrong\u003e520\u003c/strong\u003e, 258-280, doi:10.1002/cne.22689 (2012).\u003c/li\u003e\n\u003cli\u003eJin, M. H.\u003cem\u003e et al.\u003c/em\u003e Characterization of neural cell types expressing peroxiredoxins in mouse brain. \u003cem\u003eNeurosci Lett\u003c/em\u003e \u003cstrong\u003e381\u003c/strong\u003e, 252-257, doi:10.1016/j.neulet.2005.02.048 (2005).\u003c/li\u003e\n\u003cli\u003eCumming, R. C., Dargusch, R., Fischer, W. H. \u0026amp; Schubert, D. Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 30523-30534, doi:10.1074/jbc.M700869200 (2007).\u003c/li\u003e\n\u003cli\u003eShukla, V.\u003cem\u003e et al.\u003c/em\u003e A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer\u0026apos;s disease phenotypes in model mice. \u003cem\u003eFaseb J\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 174-186, doi:10.1096/fj.12-217497 (2013).\u003c/li\u003e\n\u003cli\u003eSun, K. H., de Pablo, Y., Vincent, F. \u0026amp; Shah, K. Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction. \u003cem\u003eJournal of neurochemistry\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 265-278, doi:10.1111/j.1471-4159.2008.05616.x (2008).\u003c/li\u003e\n\u003cli\u003eLopes, J. P., Oliveira, C. R. \u0026amp; Agostinho, P. Neurodegeneration in an Abeta-induced model of Alzheimer\u0026apos;s disease: the role of Cdk5. \u003cem\u003eAging Cell\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 64-77, doi:10.1111/j.1474-9726.2009.00536.x (2010).\u003c/li\u003e\n\u003cli\u003eSahlgren, C. M.\u003cem\u003e et al.\u003c/em\u003e A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death. \u003cem\u003eEmbo J\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 4808-4819, doi:10.1038/sj.emboj.7601366 (2006).\u003c/li\u003e\n\u003cli\u003eLee, M. S.\u003cem\u003e et al.\u003c/em\u003e Neurotoxicity induces cleavage of p35 to p25 by calpain. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e405\u003c/strong\u003e, 360-364, doi:10.1038/35012636 (2000).\u003c/li\u003e\n\u003cli\u003eKim, M. H.\u003cem\u003e et al.\u003c/em\u003e Peroxiredoxin 5 Inhibits Glutamate-Induced Neuronal Cell Death through the Regulation of Calcineurin-Dependent Mitochondrial Dynamics in HT22 Cells. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, doi:10.1128/MCB.00148-19 (2019).\u003c/li\u003e\n\u003cli\u003eLee, D. G., Kam, M. K., Lee, S. R., Lee, H. J. \u0026amp; Lee, D. S. Peroxiredoxin 5 deficiency exacerbates iron overload-induced neuronal death via ER-mediated mitochondrial fission in mouse hippocampus. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 204, doi:10.1038/s41419-020-2402-7 (2020).\u003c/li\u003e\n\u003cli\u003eChoi, H.\u003cem\u003e et al.\u003c/em\u003e Increased acetylation of Peroxiredoxin1 by HDAC6 inhibition leads to recovery of Abeta-induced impaired axonal transport. \u003cem\u003eMol Neurodegener\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 23, doi:10.1186/s13024-017-0164-1 (2017).\u003c/li\u003e\n\u003cli\u003eHidalgo, C. Cross talk between Ca2+ and redox signalling cascades in muscle and neurons through the combined activation of ryanodine receptors/Ca2+ release channels. \u003cem\u003ePhilos Trans R Soc Lond B Biol Sci\u003c/em\u003e \u003cstrong\u003e360\u003c/strong\u003e, 2237-2246, doi:10.1098/rstb.2005.1759 (2005).\u003c/li\u003e\n\u003cli\u003eSanmartin, C. D., Adasme, T., Hidalgo, C. \u0026amp; Paula-Lima, A. C. The antioxidant N-acetylcysteine prevents the mitochondrial fragmentation induced by soluble amyloid-beta peptide oligomers. \u003cem\u003eNeurodegener Dis\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 34-37, doi:10.1159/000334901 (2012).\u003c/li\u003e\n\u003cli\u003ePaula-Lima, A. C.\u003cem\u003e et al.\u003c/em\u003e Amyloid beta-peptide oligomers stimulate RyR-mediated Ca2+ release inducing mitochondrial fragmentation in hippocampal neurons and prevent RyR-mediated dendritic spine remodeling produced by BDNF. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1209-1223, doi:10.1089/ars.2010.3287 (2011).\u003c/li\u003e\n\u003cli\u003eAdamec, E., Mohan, P., Vonsattel, J. P. \u0026amp; Nixon, R. A. Calpain activation in neurodegenerative diseases: confocal immunofluorescence study with antibodies specifically recognizing the active form of calpain 2. \u003cem\u003eActa Neuropathol\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 92-104, doi:10.1007/s00401-002-0528-6 (2002).\u003c/li\u003e\n\u003cli\u003eAustin, S. A. \u0026amp; Katusic, Z. S. Loss of Endothelial Nitric Oxide Synthase Promotes p25 Generation and Tau Phosphorylation in a Murine Model of Alzheimer\u0026apos;s Disease. \u003cem\u003eCirc Res\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 1128-1134, doi:10.1161/CIRCRESAHA.116.309686 (2016).\u003c/li\u003e\n\u003cli\u003eYin, Y.\u003cem\u003e et al.\u003c/em\u003e Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor alpha4 via activating calpain-2. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 27283, doi:10.1038/srep27283 (2016).\u003c/li\u003e\n\u003cli\u003eJarvis, R. M., Hughes, S. M. \u0026amp; Ledgerwood, E. C. Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1522-1530, doi:10.1016/j.freeradbiomed.2012.08.001 (2012).\u003c/li\u003e\n\u003cli\u003eHopkins, B. L.\u003cem\u003e et al.\u003c/em\u003e A Peroxidase Peroxiredoxin 1-Specific Redox Regulation of the Novel FOXO3 microRNA Target let-7. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 62-77, doi:10.1089/ars.2016.6871 (2018).\u003c/li\u003e\n\u003cli\u003eYang, G. Q.\u003cem\u003e et al.\u003c/em\u003e Prdx1 Reduces Intracerebral Hemorrhage-Induced Brain Injury via Targeting Inflammation- and Apoptosis-Related mRNA Stability. \u003cem\u003eFront Neurosci\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 181, doi:10.3389/fnins.2020.00181 (2020).\u003c/li\u003e\n\u003cli\u003eHuang, S.\u003cem\u003e et al.\u003c/em\u003e Expression of Peroxiredoxin 1 After Traumatic Spinal Cord Injury in Rats. \u003cem\u003eCell Mol Neurobiol\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 1217-1226, doi:10.1007/s10571-015-0214-6 (2015).\u003c/li\u003e\n\u003cli\u003eSun, H. N.\u003cem\u003e et al.\u003c/em\u003e Peroxiredoxin I deficiency increases LPSinduced lethal shock in mice. \u003cem\u003eMol Med Rep\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 2427-2432, doi:10.3892/mmr.2018.9170 (2018).\u003c/li\u003e\n\u003cli\u003ePitts, A.\u003cem\u003e et al.\u003c/em\u003e Dithiol-based compounds maintain expression of antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e287\u003c/strong\u003e, 22717-22729, doi:10.1074/jbc.M111.334565 (2012).\u003c/li\u003e\n\u003cli\u003eKim, S.\u003cem\u003e et al.\u003c/em\u003e The antioxidant enzyme Peroxiredoxin-1 controls stroke-associated microglia against acute ischemic stroke. \u003cem\u003eRedox Biol\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 102347, doi:10.1016/j.redox.2022.102347 (2022).\u003c/li\u003e\n\u003cli\u003eKim, S. U.\u003cem\u003e et al.\u003c/em\u003e Peroxiredoxin I is an indicator of microglia activation and protects against hydrogen peroxide-mediated microglial death. \u003cem\u003eBiol Pharm Bull\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 820-825, doi:10.1248/bpb.31.820 (2008).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Peroxiredoxin 1(Prx1), oxidative stress, Alzheimer’s disease (AD), streptozotocin, calpain, mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-3875281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3875281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidative stress plays an essential role in the progression of Alzheimer\u0026rsquo;s disease (AD), the most common age-related neurodegenerative disorder. Streptozotocin (STZ)-induced abnormal brain insulin signaling and oxidative stress play crucial roles in the progression of Alzheimer\u0026rsquo;s disease (AD)-like pathology. Peroxiredoxins (Prxs) are associated with protection from neuronal death induced by oxidative stress. However, the molecular mechanisms underlying Prxs on STZ-induced progression of AD in the hippocampal neurons are not yet fully understood. Here, we investigated the effect of Peroxiredoxin 1 (Prx1) on STZ-induced AD-like pathology. Prx1 expression was increased by STZ treatment in the hippocampus cell line, HT-22 cells. We evaluated whether Prx1 affects STZ-induced HT-22 cells using overexpression. Prx1 successfully protected the forms of STZ-induced AD-like pathology, such as neuronal apoptosis, synaptic loss, and tau phosphorylation. Moreover, Prx1 suppressed STZ-induced increase of mitochondrial dysfunction and fragmentation by down-regulating Drp1 phosphorylation and mitochondrial location. Prx1 plays a role in an upstream signal pathway of Drp1 phosphorylation, cyclin-dependent kinase 5 (Cdk5) by inhibiting the STZ-induced conversion of p35 to p25. We found that STZ-induced of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e accumulation was an important modulator of AD-like pathology progression by regulating Ca\u003csup\u003e2+\u003c/sup\u003e-mediated Calpain activation, and Prx1 down-regulated STZ-induced intracellular Ca\u003csup\u003e2+\u003c/sup\u003e accumulation and Ca\u003csup\u003e2+\u003c/sup\u003e-mediated Calpain activation. Finally, we identified that Prx1 antioxidant capacity affected Ca\u003csup\u003e2+\u003c/sup\u003e/Calpain/Cdk5-mediated AD-like pathology progress. Therefore, these findings demonstrated that Prx1 is a key factor in the STZ-induced hippocampal neuronal death through inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e/Calpain/Cdk5-mediated mitochondrial dysfunction by protecting oxidative stress.\u003c/p\u003e","manuscriptTitle":"Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 09:11:57","doi":"10.21203/rs.3.rs-3875281/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-29T09:59:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-25T16:12:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-20T00:43:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-15T03:36:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b3493454-ca05-447e-9fab-f4fa1e6ef59f","date":"2024-02-05T18:29:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24ea210c-8cab-4b14-8c91-504965415239","date":"2024-02-05T01:27:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7b516faf-00f8-444b-9c9a-279e516df41f","date":"2024-02-03T16:53:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-03T16:51:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-28T16:24:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-24T19:45:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-24T19:41:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-18T08:46:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"672a0683-a50a-4d03-8df5-8f9060f4dc2d","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28412822,"name":"Biological sciences/Biochemistry/Neurochemistry"},{"id":28412823,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":28412824,"name":"Biological sciences/Cell biology/Organelles"},{"id":28412825,"name":"Biological sciences/Cell biology/Post translational modifications"},{"id":28412826,"name":"Biological sciences/Neuroscience/Cellular neuroscience"},{"id":28412827,"name":"Biological sciences/Neuroscience/Molecular neuroscience"}],"tags":[],"updatedAt":"2024-07-01T05:57:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-29 09:11:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3875281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3875281","identity":"rs-3875281","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}