The expression and preliminary functional exploration of Olfaxin in the central nervous system of mice

The expression and preliminary functional exploration of Olfaxin in the central nervous system of mice | 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 Research Article The expression and preliminary functional exploration of Olfaxin in the central nervous system of mice Hong Wang, Weihao Zhuang, Xueyuan Niu, Cong Cao, Min Liao, Shi-mo Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6270949/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Prune homolog 2 (Drosophila) (PRUNE2) protein has five subtypes, four of which have been reported to play important roles in the central nervous system (CNS), but the fifth ,Olfaxin still remains unclear. Therefore, this study aims to investigate Olfaxin expression in the CNS of mouse. In vivo and in vitro , experiments indicate Olfaxin is expressed in the olfactory bulb (OB) neurons: glutamatergic neuron marked by Excitatory Amino Acid Carrier 1 (EAAC1), GABA-ergic neuron marked by Glutamic Acid Decarboxylase 67 (GAD67), Cholinergic neuron marked by Choline Acetyltransferase (ChAT) and dopaminergic neuron marked by Tyrosine Hydroxylase (TH). While,in the OB glia, its expression pattern differed between in vivo and in vitro settings. Under low - glucose conditions, Olfaxin and Monocarboxylate Transporter 1 (MCT1) expression increased in microglia, suggesting a role in energy metabolism. In an Alzheimer's disease (AD) mouse model, Olfaxin expression varied in different brain regions. These results indicate that Olfaxin may influence AD progression, possibly through microglial energy metabolism, although the underlying mechanisms remain to be further investigated. Olfaxin PRUNE2 isoform Neurons Glia Alzheimer's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction PRUNE2 is an X-linked methylCpG binding protein 2 (MeCP2) target gene, which may be associated with Rett syndrome(Nectoux et al., 2010). It contains BNIP2 and Cdc42GAP homology (BCH) domains at the C-terminus. The PRUNE2 protein is mainly expressed in the spinal cord and dorsal root ganglion (DRG), and plays an important role in synaptic function, cell transformation, neuronal differentiation, and apoptosis (Pan & Low, 2012).And other research has shown that as a binding protein of 8-oxo-GTP, PRUNE2 may be associated with the maintenance of mature nervous systems (Iwama et al., 2011). There are five subtypes of PRUNE2: PRUNE2, C9orf65, molecules with BCH motifs in carboxyl terminal region 1 (BMCC1), BNIP2 ultra long (BNIPXL), and Olfaxin/BMCC1s.C9orf65 expression is useful for the differentiation between leiomyosarcomas and gastrointestinal stromal tumors(Islam et al., 2018).BMCC1s regulate MAP6 induced microtubule cold stability through direct interaction with MAP6/STOP(Arama et al., 2012), and it can control the transport of renal glutaminase (KGA) to mitochondria(Boulay et al., 2013).BNIPXL is the N-terminal truncated form of BMCC1, which can interact with RhoA and RhoA specific guanine nucleotide exchange factors to promote lymphocyte proliferation crisis, thereby regulating cell transformation(Soh & Low, 2008); Prostate cancer antigen 3 (PCA3) is a prominent cancer biomarker of the prostate, contained in the PRUNE2 gene. PCA3 is transcribed in the opposite chain of PRUNE2, which controls PRUNE2 levels through a unique regulatory mechanism(Salameh et al., 2015). In summary, the first four subtypes have been shown to be is a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively, all of which play crucial roles in the nervous system(Li et al., 2011). In our previous research, we discovered the fifth subtype of PRUNE2,Olfaxin/BMCC1s,and based on the N-terminal 133 amino acid sequence of the protein encoded by the AK038997.1 mRNA in the mouse PRUNE2 gene, we designed an antibody that specifically recognizes 52 kDa Olfaxin. It is predominantly expressed in the olfactory system, shares 56.3% amino acid identity with mouse Caytaxin(Li et al., 2012). But the expression and biological function of Olfaxin in neurons and glia are still unclear. Recent studies have shown that microglia are not only immune cells, but also play a key role in maintaining glucose homeostasis in the CNS(Pan et al., 2022). Both astrocytes and microglia are sensitive to in vitro changes of surrounding glucose levels, which impact their functions and the expression of the glucose transporters (Robb et al., 2020).In addition, Olfaxin deficiency impaired the increasing expression of ACL, which is a key metabolic enzyme that produces the acetyl-CoA for acetylcholine synthesize, in the piriform cortex during development(Islam et al., 2018).Therefore, in this study, we detected the expression of Olfaxin in different types of neurons and glia, and preliminarily explored its biological function in low glucose model of microglia and AD mice model. 2. Materials and Methods 2.1 Materials The following antibodies and chemicals were used in this study: anti-c-fos (Santa Cruz Biotechnology, Cat# sc-166940, RRID:AB_10609634); anti-EAAC1(Sigma-Aldrich, Cat# anti-EAAC1, RRID:AB_3101962); anti-ChAT (Proteintech, Cat# 66816-1-Ig, RRID:AB_2882159); anti-GAD67(Sigma-Aldrich, Cat# G5419, RRID:AB_261978); anti-TH (Cell Signaling Technology, Cat# 2791, RRID:AB_2201522); anti-NeuN antibody(Abcam, Cat# ab104225, RRID:AB_10711153); anti-NG2 antibody (Abcam, Cat# ab275024, RRID:AB_2922401); anti-GFAP (Abcam, Cat#ab7260, RRID:AB_305808); Anti-iba1(Novus, Cat# NB100-1028, RRID:AB_521594); anti-MCT1 (Abcam, Cat#ab93048, RRID:AB_10563650); anti-actin (Abgent, Cat# AC-15, RRID:AB_2223210);anti-ERp72(Proteintech Cat# 14712-1-AP, RRID: AB_2160973);HRP-conjugated goat anti-rat༈Jackson ImmunoResearch Labs, Cat# 112-035-003, RRID:AB_2338128), anti-mouse(Yeasen Biotech, Cat# 33201ES60, RRID:AB_10015289༉, and anti-rabbit(Jackson ImmunoResearch Labs, Cat# 111-035-144, RRID:AB_2307391); Alexa Fluor® 488-AffiniPure Donkey Anti-mouse IgG (H + L)(Jackson ImmunoResearch Labs, Cat# 715-545-150, RRID:AB_2340846); anti-rabbit(Jackson ImmunoResearch Labs, Cat# 711-545-152, RRID:AB_2313584); Alexa Fluor® 594-AffiniPure Donkey Anti-mouse IgG (H + L)(Jackson ImmunoResearch Labs, Cat# 715-585-150, RRID:AB_2340854); Anti-rabbit (Jackson ImmunoResearch Labs, Cat# 711-585-152, RRID:AB_2340621); Chemicals from Gibco, TRANS, Biosharp, Beyotime, and Sigma.Pyruvate(PA) Content Assay Kit (BC2205, Solarbio).To determine the expression of Olfaxin in the neurons and glia, monoclonal and polyclonal antibodies against N-terminal (ter) Olfaxin, respectively, were generated according to the protocol used in a previous study (Li et al., 2012). 2.2 Animals Animal experiments were approved by the Experimental Animal Ethics Committee of Wenzhou Medical University. Mice were purchased from Beijing Weitong Lihua Co., .APP/PS1 mice are used as the AD model with Mo/Hu APP Swedish mutations (K595N/M596L) + Hu PS1 deltaE9 mutation. We housed C57BL/6 and APP/SP1 male mice (9 months old, body weight 27-30g) in standard laboratory cages under a12/12 h light/dark cycle, a temperature of 22 ± 3 ℃`and a temperature of 55%± 5% relative humidity in a controlled environment. 2.3 Primary culture of neurons The OB from P0 C57BL/6 mice was isolated. Meninges were carefully removed from the OB under a dissection microscope. Tissues were trypsinized and dissociated by gentle trituration. Cells were cultured in Neurobasal Medium (Invitrogen, Carlsbad, CA) supplemented with B-27 (Invitrogen), 1 mM L-glutamine (Sigma), and penicillin/streptomycin at 2x10 4 cells on 12-mm cover slips (Matsunami, Osaka, Japan) coated with 20 µg/ml poly-L-lysine (Sigma) in a 24-well culture plate. DIV4-7 cells were treated with AraC (0.25µm) .DIV 14 neurons were fixed for 15 min in 4% PFA/0.1 M phosphate buffer (PB) (pH 7.4), then subjected to ICC and WB. ICC sections were examined under an inverted fluorescent microscope (Olympus) or a confocal microscope (Leica sp8), as previously described (Li et al., 2012). 2.4 Primary culture of glia In the animal facility of the research institute, a male mouse aged 7–9 weeks and a female mouse aged 7–9 weeks were kept. After visually confirming pregnancy, the female mouse was separated from the male mouse and their offspring were observed. On day 0, a litter of newborn mice was collected. OB was obtained after the mouses were terminated and mechanically dissociated. Meninges were carefully removed from the OB under a dissection microscope. Then OB were trypsinized and resuspended in 1× PBS (PBS: phosphate-buffered saline). Filtration using a 70µm strainer was performed. The sample was centrifuged at 1000 g for 5 min at Room Temperature (RT). The supernatant was then removed, which was then resuspended in 10% DMEM/F12 (Gibco) medium. The suspension was again centrifuged at 1000 g for 5 min at RT. The supernatant was removed and resuspended in 10% FBS DMEM/F12 medium. The cells were cultured with 10% DMEM/F12 in T25 flasks coated with 20 g/ml poly-L-lysine (Sigma). Whole medium was changed after 24 h, and the half medium was changed every 3 days. At 10–14 DIV (80–90%), by tapping the T25 flask, the medium with floating microglia was harvested, and it was centrifuged at 1000 g for 5 min at RT; the supernatant was resuspended in 10% DMEM/F12 and cultured in a 6-well culture plat. The cultures were then shaken again at 220 rpm overnight to separate astrocytes and oligodendrocyte progenitor cells (OPCs). Trypsinize the adherent cells in the culture flask, and then it was centrifuged at 1000 g for 5 min at RT; the supernatant was resuspended in 10% DMEM/F12 and cultured in a 6-well culture plat. For further purification of OPCs, the medium obtained after shaking was transferred to uncoated dishes for 1 hour to remove astrocytes and microglia. The non-adherent cells that remained were collected and cultured in a medium containing 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 50 µg/mL apo-transferrin, 0.1% bovine serum albumin (BSA), 5 µg/mL insulin, 30 nmol/L sodium selenite, 10 nmol/L D-biotin,10 ng/mL PDGF-AA and 10 nmol/L hydrocortisone, and 10 ng/mL bFGF. Cells were planted into six- or 24-well plates with 12 mm diameter glass coverslips at a density of 5×104 cells/cm 2 for experiments. 2.5 BV2 cell culture and low-glucose treatment The BV-2 cells employed in this study originated from primary cells cultivated in the laboratory. The cells were cultured with 10% FBS DMEM 4.5g/L glucose medium (Gibco) in a culture dish and maintained in a CO 2 incubator at a temperature of 37℃. The cells were plated in normal growth media (DMEM 4.5g/L glucose + 10% FBS) at a density of 10 5 cells/mL in 6-well plates and allowed to reach 90% confluence, and the medium was then removed, and 10% FBS DMEM 1.0g/L glucose medium was added (Gibco).Cells were cultured for 30-min, 60-min, and 120-min and then harvested. Then they were subjected to western blotting. 2.6 Western blotting First, mouse (C57BL/6 and APP/PS1) tissues or culture cells were collected in each group and added RIPA buffer containing protease inhibitors and phosphatase inhibitor. Then, tissues or cells were totally lysed and centrifuged at 4°C for about 30 min to obtain the supernatant. The protein concentration was determined by the BCA protein assay kit (Beyotime Biotechnology). Total protein was subjected to 10%SDS-PAGE gels and then transferred to the PVDF membrane (Millipore). The membranes were blocked by 5% skim milk for 2h and then were incubated overnight at 4℃ with primary antibody against Olfaxin (polyclonal) (1:1000),MCT1 (1:1000), β-actin (1:3000). After three times washing, the membranes were incubated horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:20000) or HRP-linked anti-mouse IgG (1:20000) at room temperature for 2h. Protein band were visualized by ECL solution (Meilunbio) and detected with an automatic chemiluminescence imaging analysis system (Amersham Imager 680). The intensity of each band was quantified using ImageJ software. 2.7 Immunohistochemical staining After perfusion and fixation, mouse olfactory bulb tissues were embedded in OCT and sectioned into 14µm thick slices. The olfactory bulb tissue of each mouse was used for c-fos and Olfaxin(monoclonal) immunohistochemical staining. The sections underwent antigen retrieval treatment, followed by incubation with a 3% H202-methanol solution for 15 min, and overnight incubation with c-fos and Olfaxin antibodies at 40℃, respectively. A working solution of 1:500 horseradish peroxidase-labeled anti-mouse secondary antibody was added, and the sections were incubated at room temperature for 3 h. Then, the sections were incubated with DAB chromogenic solution, dehydrated using a gradient alcohol series, cleared with xylene, and finally, the slides were sealed. 2.8 Immunofluorescence staining Mice were perfused with PBS followed by 4% paraformaldehyde in 0.1 M PB. Brains were post-fixed for 2h in the same fixative, which was then replaced by 15% sucrose in 0.1 M PB. Sections (14µm) were then rinsed with PBS and permeabilized for 5 minutes with 0.1% Triton-X (Sigma). They were then blocked with 1% Bovine Serum Albumin (BSA) in PBST for 1 hour. Primary antibodies(monoclonal antibodies against N-terminal (ter) Olfaxin,EAAC1,ChAT,GAD67,NG2,iba1,GFAP,ERp72) plus 1% BSA incubated the samples overnight at 4°C. The secondary antibodies (Alexa-488, Alexa-594) were then incubated at room temperature for 1 hour. The nuclei of cells in samples were stained with DAPI at room temperature for 3 min,and 3,30 -diaminobenzidine (DAB; Wako, Osaka, Japan) was employed for visualization(Li et al., 2012). 2.9 Pyruvate content determination Follow the manufacturer’s instructions to process the cell supernatant, then inoculate it into a 96-well plate. Set up 3 replicate wells for each group of samples, blank group, and standard group. Next, add the reagents from the kit according to the instructions, mix thoroughly, and measure the absorbance values of each group at a wavelength of 520nm. Finally, calculate the pyruvate content of each group. 3.0 Statistical analysis GraphPad Prism 8.0 was used for statistical analysis. Data shown is mean ± SD with P < 0.05 considered statistically significant. Two-tailed unpaired t test was applied for comparisons between two groups. The Plot Profile tool of imageJ is used for fluorescence co-localization analysis, and the Colocalization Finder plug-in of imageJ is used to calculate the Pearson co-localization coefficient of the fluorescence map. 3. Results 3.1 Olfaxin expression in neurons The polyclonal N-ter-Olfaxin antibody specifically detected a clear band of approximately 52 kDa (Fig. 1 a). Subsequently, we validated the expression level of Olfaxin in the entire brain. The result showed Olfaxin expression was observed in the OB and CBM but not in the brain stem (BS) (Fig. 1 a).Therefore, we used Cellular Proto-Oncogene Fos(c-fos) antibody was a positive control to validate the availability of our experimental method and antibody, and chose monoclonal N-ter-Olfaxin antibodies to detect the expression of Olfaxin in the OB,CBM and HIP. Olfaxin expression was observed in the OB, CBM, and HIP. In addition, in the OB, Olfaxin was found to be mainly localized in the glomerular layer (GL), external plexiform layer (EPL), and internal plexiform layer (Fig. 1 b)(Li et al., 2012). Our results are consistent with the results of Li et al. in 2012. We observed the GI (Glomerular Input) region (Fig. 2 a and 2 b) of mouse OB tissue and found that Olfaxin was completely co-localized with EAAC1 (Fig. 2 c to 2 f), ChAT (Fig. 2 g to 2 j) positive cells and TH (Fig. 2 o to 2 r), while Olfaxin and GAD67 (Fig. 2 k to 2 n) positive cells were only partially co-localized. Subsequently, in primary cultured OB cells, we observed that Olfaxin was completely co-localized with EAAC1 (Fig. 3 a to 3 d) and ChAT (Fig. 3 e to 3 h) positive cells; Olfaxin and GAD67(Fig. 3 i to 3 l) positive cells only partially co-localized. These are consistent with the results of in vivo experiments. In summary, Olfaxin is expressed in glutamatergic neuron (marked by EAAC1), GABA-ergic neuron (marked by GAD67), cholinergic neuron (marked by ChAT),and dopaminergic neuron (marked by TH). 3.2 Olfaxin expression in glia glia constitute roughly half of the cells of the CNS, and they influence dynamically neuronal birth, migration, axon specification, and growth through circuit assembly and synaptogenesis.(Allen & Lyons, 2018)The FPKM data of RNA-sequencing transcriptome and splicing indicate that PRUNE2 mRNA expression is observed in astrocytes, OPCs, newly formed oligodendrocytes, and microglia(Zhang et al., 2014). Subsequently, we conducted immunofluorescence staining using tissues and primary culture cells to examine the expression of Olfaxin in different types of glial cells. Olfaxin was completely co-localized with NG2 positive cells in the OB (Fig. 4 a to 4 d) and primary culture cells (Fig. 5 a to 5 d).In our study, Olfaxin did not co-locate with iba1 positive cells in the OB (Fig. 4 e to 4 h). But Olfaxin was co-localized with iba1 positive cells in primary culture cells (Fig. 5 e to 5 h). Unexpectedly, Olfaxin was not co-localized with GFAP positive cells in mouse tissue (Fig. 4 i to 4 l). And similar to iba1, Olfaxin was co-localized with GFAP positive cells in primary culture cells (Fig. 5 i to 5 l). The above experimental results indicate that: in vitro , Olfaxin is expressed in all glia: oligodendrocytes (marked by NG2) and microglia (marked by iba1), astrocytes (marked by GFAP). And in vivo Olfaxin is just expressed in the oligodendrocytes (marked by NG2). 3.3 Olfaxin expression and low glucose treatment Olfaxin deficiency impaired the increasing expression of ACL in the piriform cortex during development(Islam et al., 2018). Both astrocytes and microglia are sensitive to in vitro changes of surrounding glucose levels, which impact their functions and the expression of the glucose transporters (Robb et al., 2020). Therefore, we performed low-glucose treatment to the BV2 cell line to examine the function of Olfaxin in microglia. We used MCT1 as a positive marker for low-glucose treatment. The expression patterns of Olfaxin and MCT1 are very similar. After 30-min treatment in a low-glucose medium, Olfaxin protein expression increased remarkably and in a time-dependent manner (Fig. 6 a and 6 b). Futhermore, pyruvic acid content determination results showed that pyruvic acid increased significantly in a time-dependent manner during the first 60 minutes of low glucose treatment, followed by a significant decrease at the 120-min (Fig. 6 c). These results suggest that Olfaxin may have important functions in metabolism. However, we need more experiments to explore the functions of Olfaxin in the glia. 3.4 Olfaxin expression in aged APP/PS1 mice Considering the energy metabolism disorder present in the brain of AD patients, the aforementioned results implied that Olfaxin may significantly affect metabolic processes. We further examined the Olfaxin and MCT1 expression in AD mice model. We performed western blotting using aged APP/PS1 mice. The results indicated that Olfaxin expression is increased in the OB of aged APP/PS1 mice but decreased in CBM and did not change in HIP and PIR (Fig. 6 d and 6 e). Interestingly, similar to Olfaxin, MCT1 expression increased in the OB and decreased in the CBM, but decreased in the HIP (Fig. 6 f and 6 g). In addition, we observed that Olfaxin was co-localized with Endoplasmic Reticulum Protein 72 (ERp72) (endoplasmic reticulum marker) in the OB of WT and APP/PS1 mice, and compared to WT mice, the Pearson co-localization coefficient of ERp72 and Olfaxin in the OB of APP/PS1 mice was higher. (Fig. 6 h and 6 i). These experimental results suggest that Olfaxin may be related to the disease process of AD and functional maintenance and damage of different brain regions, providing a new idea for exploring the biological function of Olfaxin . 4. Discussion The FPKM data of RNA-sequencing transcriptome and splicing in neurons, glia, and vascular cells of the cerebral cortex suggested PRUNE2 mRNA expression was observed in the neurons, astrocytes, OPCs, newly formed oligodendrocytes, and microglia (data: http://www.brainrnaseq.org)(Zhang et al., 2014). In neurons, the studies of Olfaxin revealed that Olfaxin was predominantly expressed in the olfactory bulb (OB) and piriform cortex (PIR) where glutamatergic terminal was localized(Li et al., 2012). The studies of Olfaxin in the Olfaxin-KO mice suggested that Olfaxin deficiency led to impairment of odor-associative learning and odor preference. Moreover ,compared with the control mice at postnatal day14, the level of BDNF and ACL expression were lower in the piriform cortex of KO mice, while Kv4.2 and ChAT expression were higher(Islam et al., 2018).Our studies revealed that Olfaxin is expressed in glutamatergic neurons ,cholinergic neurons GABA-ergic neurons and dopaminergic neurons of the OB (Figs. 2 and 3 ). These results indicate that Olfaxin plays a crucial role in the functional regulation of the OB neural circuit. Its absence can disrupt the function and interaction of these neurons, resulting in impairments in odor association learning and odor preference in mice. This provides significant experimental evidence for further understanding the olfactory-related neural mechanisms and the biological function of Olfaxin. However, we need further research to explore the functions of Olfaxin in these neurons. Glial cells including astrocytes, oligodendrocytes, and microglia support the physiological functions of neurons. In the CNS, these glia cooperate to promote and preserve neuronal health, playing important roles in regulating the activity of neuronal networks during different life stages. Astrocytes and microglia are important for the systemic regulation of energy and glucose homeostasis in the course of normal physiological control and during disease (Robb et al., 2020).Therefore, in this study, we examined the expression of Olfaxin in the glia. Interestingly, in mouse tissues, Olfaxin colocalized with NG2, and not with GFAP and iba1 (Fig. 4 ). But, in primary culture of OB, Olfaxin colocalized with NG2, GFAP, and iba1 (Fig. 5 ). The expression and colocalization of Olfaxin in oligodendrocytes suggest that Olfaxin may play a role in oligodendrocytes. However, the co-localization of Olfaxin with iba1 and GFAP in the mouse olfactory bulb and its primary cultured glial cells exhibits inconsistency, indicating that the expression of Olfaxin in glial cells may depend on the nutritional environment of the cells or cell differentiation status and further exploration is needed. In microglia, the expression of Olfaxin is not entirely unforeseen (Fig. 5 e to 5 h).In previous research on Olfaxin, it was mentioned that Olfaxin may be closely associated with the production of acetyl-CoA(Islam et al., 2018).Glucose serves as the primary energy source for the brain, and maintaining glucose homeostasis is vital for ensuring normal neurological function. MCT1 is a transmembrane protein that facilitates the transport of monocarboxylic compounds (such as lactate and pyruvate) across the cell membrane(Hadjihambi et al., 2023). In low-glucose environments, MCT1 enhances the transport of ketone bodies and lactate, thereby supporting cellular energy supply and metabolic adaptation. Thus, in our study, we used MCT1 as a positive marker for low-glucose treatment. And the results showed at 30-min,60-min and 120-min in a low-glucose medium, the expression of Olfaxin and MCT1 significantly increased in a time-dependent manner, while pyruvic acid significantly decreased at the 120-min (Fig. 6 a to 6 c).This phenomenon may be caused by the significant increase in the expression level of MCT1 in order to meet the energy supply of cells. However, following 30 and 60 minutes of low-glucose treatment, pyruvate levels gradually rose due to the enhanced glycolysis pathway. By 120-min, as metabolic pathways reorganized, microglia shifted towards utilizing alternative energy sources like fatty acids and ketone bodies, leading to a decrease in pyruvate content. These results indicate under low-glucose conditions, microglia enhance the effective transport of pyruvate and alternative energy sources by promoting an upregulation of MCT1 expression levels. and the expression pattern of Ofaxin mirrors that of MCT1, suggesting that Olfaxin may play a significant role in the energy metabolism of microglia. Considering that PRUNE2 has been shown to be a susceptibility gene for Alzheimer's disease (AD)(Li et al., 2011),and metabolic alterations in microglia in AD can contribute to neuroinflammation and disease progression(Tondo et al., 2020).We further investigated the expression of Olfaxin and MCT1 in the brains of AD mice. Compared to the control group of mice, the expression of Olfaxin in the OB of APP/PS1 increased, but in the CBM decreased and remained unchanged in the HIP and PIR (Fig. 6 d and 6 e). The expression of MCT1 raised in the OB, but reduced in the CBM and HIP (Fig. 6 f and 6 g). The OB plays a pivotal role in olfactory information processing, and in early-stage AD patients, the impairment of odor perception appears first (Li et al., 2012). The elevated expression of Olfaxin and MCT1 in the OB may be an attempt by the body to mitigate the impact of pathological changes on olfactory function through energy metabolism regulation. The PIR is closely associated with olfactory processing and olfactory memory functions, but in the APP/PS1 mouse model, the function of this region may not have been significantly impaired yet. Alternatively, the mechanism by which it maintains normal function may not rely on changes in Olfaxin expression. Therefore, compared with age-matched C57BL/6 mice, the expression level of Olfaxin in the piriform cortex of APP/PS1 mice remains relatively stable.The HIP is a critical brain region for learning and memory, and due to pathological changes such as amyloid deposition in Alzheimer's disease, the number and activity of neurons decrease in the region. This reduction may lead to a decreased demand for energy metabolism substrates, subsequently lowering MCT1 expression. However, Olfaxin expression in the HIP remains stable, suggesting that the metabolic regulatory role of Olfaxin may not be the primary response mechanism in the HIP, or later than OB. The CBM is involved in motor control, learning, reflex adaptation, and cognition. Studies have shown that the number of mitochondria at the presynaptic terminals of cerebellar neurons decreases, in Alzheimer's disease patients(Fan et al., 2018) ,which may lead to a decrease in the overall metabolic activity of the cerebellum, thereby inhibiting the expression of MCT1 and Olfaxin. In addition, we observed that Olfaxin was co-localized with ERp72(endoplasmic reticulum marker) in the OB of WT and APP/PS1 mice, and compared to WT mice, the Pearson co-localization coefficient of ERp72 and Olfaxin in the OB of APP/PS1 mice was higher. (Fig. 6 h and 6 i). This phenomenon indicates that Olfaxin may be associated with protein folding disorder in AD. In summary, these findings indeed indicate that Olfaxin may play a critical role in the disease progression of AD. With these results in mind, the role of Olfaxin in AD mice model should be interested. In conclusion, we conducted a general experiment on the in vivo and in vitro expression of Olfaxin in various neurons and glia cells. And we also detected the expression of Olfaxin and MCT1 in a low-glucose model and measured the content of pyruvate. Moreover, the expression levels of Olfaxin in parts of OB, PIR, HIP and CBM, and the expression levels of MCT1 in parts of OB, HIP and CBM were detected in the AD mice model. We also observed the co-localization of ERp72 and Olfaxin in the OB of WT and APP/PS1 mice. These results preliminarily demonstrate that Olfaxin may influence the occurrence and progression of Alzheimer's disease by impacting the energy metabolism of microglia or other aspects within the central nervous system, but the mechanism remains to be studied. Declarations Acknowledgments We thank Dr T. Nakagawa (Gifu University Graduate School of Medicine) for Olfaxin anti-rat antibody samples. This work was supported by Basic Scientific Research project of Wenzhou. We would like to thank Editage (www.editage.cn) for English language editing. Funding statement This work was supported by the Nature Science Foundation of Zhejiang Province, China (Grant No. LY20H090010). Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author Contributions LSM and LM was involved in the project design ,writing – review & editing and writing – original draft.WH,ZWH ,NXY and CC performed the major experimental work and data analysis; WH revised the manuscript. All authors approved the final article before publication. Data A vailability The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. 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Brain Res, 1688, 81-90. https://doi.org/10.1016/j.brainres.2018.03.025 Iwama, E., Tsuchimoto, D., Iyama, T., Sakumi, K., Nakagawara, A., Takayama, K., Nakanishi, Y., & Nakabeppu, Y. (2011). Cancer-related PRUNE2 protein is associated with nucleotides and is highly expressed in mature nerve tissues. J Mol Neurosci, 44(2), 103-114. https://doi.org/10.1007/s12031-010-9490-2 Li, S., Hayakawa-Yano, Y., Itoh, M., Ueda, M., Ohta, K., Suzuki, Y., Mizuno, A., Ohta, E., Hida, Y., Wang, M. X., & Nakagawa, T. (2012). Olfaxin as a novel Prune2 isoform predominantly expressed in olfactory system. Brain Res, 1488, 1-13. https://doi.org/10.1016/j.brainres.2012.10.001 Li, S., Itoh, M., Ohta, K., Ueda, M., Mizuno, A., Ohta, E., Hida, Y., Wang, M. X., Takeuchi, K., & Nakagawa, T. (2011). The expression and localization of Prune2 mRNA in the central nervous system. Neurosci Lett, 503(3), 208-214. https://doi.org/10.1016/j.neulet.2011.08.037 Nectoux, J., Fichou, Y., Rosas-Vargas, H., Cagnard, N., Bahi-Buisson, N., Nusbaum, P., Letourneur, F., Chelly, J., & Bienvenu, T. (2010). Cell cloning-based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes. J Cell Mol Med, 14(7), 1962-1974. https://doi.org/10.1111/j.1582-4934.2010.01107.x Pan, C. Q., & Low, B. C. (2012). Functional plasticity of the BNIP-2 and Cdc42GAP Homology (BCH) domain in cell signaling and cell dynamics. FEBS Lett, 586(17), 2674-2691. https://doi.org/10.1016/j.febslet.2012.04.023 Pan, R. Y., He, L., Zhang, J., Liu, X., Liao, Y., Gao, J., Liao, Y., Yan, Y., Li, Q., Zhou, X., Cheng, J., Xing, Q., Guan, F., Zhang, J., Sun, L., & Yuan, Z. (2022). Positive feedback regulation of microglial glucose metabolism by histone H4 lysine 12 lactylation in Alzheimer's disease. Cell Metab, 34(4), 634-648.e636. https://doi.org/10.1016/j.cmet.2022.02.013 Robb, J. L., Morrissey, N. A., Weightman Potter, P. G., Smithers, H. E., Beall, C., & Ellacott, K. L. J. (2020). Immunometabolic Changes in Glia - A Potential Role in the Pathophysiology of Obesity and Diabetes. Neuroscience, 447, 167-181. https://doi.org/10.1016/j.neuroscience.2019.10.021 Salameh, A., Lee, A. K., Cardó-Vila, M., Nunes, D. N., Efstathiou, E., Staquicini, F. I., Dobroff, A. S., Marchiò, S., Navone, N. M., Hosoya, H., Lauer, R. C., Wen, S., Salmeron, C. C., Hoang, A., Newsham, I., Lima, L. A., Carraro, D. M., Oliviero, S., Kolonin, M. G.,…Arap, W. (2015). PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3. Proc Natl Acad Sci U S A, 112(27), 8403-8408. https://doi.org/10.1073/pnas.1507882112 Soh, U. J., & Low, B. C. (2008). BNIP2 extra long inhibits RhoA and cellular transformation by Lbc RhoGEF via its BCH domain. J Cell Sci, 121(Pt 10), 1739-1749. https://doi.org/10.1242/jcs.021774 Tondo, G., Iaccarino, L., Caminiti, S. P., Presotto, L., Santangelo, R., Iannaccone, S., Magnani, G., & Perani, D. (2020). The combined effects of microglia activation and brain glucose hypometabolism in early-onset Alzheimer's disease. Alzheimers Res Ther, 12(1), 50. https://doi.org/10.1186/s13195-020-00619-0 Zhang, Y., Chen, K., Sloan, S. A., Bennett, M. L., Scholze, A. R., O'Keeffe, S., Phatnani, H. P., Guarnieri, P., Caneda, C., Ruderisch, N., Deng, S., Liddelow, S. A., Zhang, C., Daneman, R., Maniatis, T., Barres, B. A., & Wu, J. Q. (2014). An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci, 34(36), 11929-11947. https://doi.org/10.1523/jneurosci.1860-14.2014 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6270949","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443334903,"identity":"d7a0c44d-90d6-4448-9e5f-adce875085b3","order_by":0,"name":"Hong Wang","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Wang","suffix":""},{"id":443334904,"identity":"fbd820a9-5bb2-4dbc-8939-602ea353755a","order_by":1,"name":"Weihao Zhuang","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weihao","middleName":"","lastName":"Zhuang","suffix":""},{"id":443334906,"identity":"5bef38d1-1d3c-43b4-9ea4-e30c6a4132ee","order_by":2,"name":"Xueyuan Niu","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xueyuan","middleName":"","lastName":"Niu","suffix":""},{"id":443334907,"identity":"a54955c9-5c13-45ae-aada-6be155b5e017","order_by":3,"name":"Cong Cao","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Cao","suffix":""},{"id":443334911,"identity":"1ab1c0dd-75d8-4dfe-a3b8-f0704880eabf","order_by":4,"name":"Min Liao","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liao","suffix":""},{"id":443334913,"identity":"226beccb-47d4-4560-8725-d38d39083a34","order_by":5,"name":"Shi-mo Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIie3PsQrCMBCA4ZPIdQlmTUH6DAEhLn2YBsFJwUk6FBSUdhBx9i06FVcJ6FL3ju3S3U03qbgppm4O+bnxPrgDsNn+sB4BhADAQ0KOZRBGZoIvMmAOjkSZn1qQ5wCo/Y5Kt1qTFsRx6rKa+J1UUxmqJQJLNoHhMDoUKhsToXFeqEMfeH5JTQS5yjQKTbJC5QiCT03EqRtChQY5UzFpQ0A2hLurroSWhDZkLBjBEQ/yEzX+wti5du+Zv4iZPl5vYeSxZPudvEV/W7fZbDbbxx7b5kBACfB1KgAAAABJRU5ErkJggg==","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shi-mo","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-03-20 15:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6270949/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6270949/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81016256,"identity":"69d6373c-43fb-443a-952a-a10ab6c25475","added_by":"auto","created_at":"2025-04-21 08:55:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":953112,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-Olfaxin antibody analysis.\u003cstrong\u003e(a) \u003c/strong\u003eWestern blot analysis to examine anti-Olfaxin antibody. The bands, which are approximately 52 kDa, indicated Olfaxin. Olfaxin expression was observed in the olfactory blub (OB) and cerebellum (CBM) but not in the brain stem (BS); \u003cstrong\u003e(b) \u003c/strong\u003eImmunohistochemistry analysis to examine anti-Olfaxin antibody. Olfaxin expression was observed in the OB, hippocampus (HIP), and CBM. C-fos was used as a positive control to study changes in the morphology of mouse tissues. Scale bars: 1000μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/e816651e4a6ea9067a658101.png"},{"id":81016257,"identity":"8c592226-7c22-4055-a450-800abe457753","added_by":"auto","created_at":"2025-04-21 08:55:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1265622,"visible":true,"origin":"","legend":"\u003cp\u003eOlfaxin expression in various neurons of OB.\u003cstrong\u003e(a) \u003c/strong\u003eImmunohistochemical staining of mouse olfactory bulb;\u003cstrong\u003e(b)\u003c/strong\u003e Immunofluorescence staining of mouse olfactory bulb and selecting the GI region of the olfactory bulb to examine Olfaxin expression in different types neurons of OB.\u003cstrong\u003e(c,d,e)\u003c/strong\u003e Olfaxin colocalized with EAAC1(glutamatergic neuron marker); \u003cstrong\u003e(g,h,i)\u003c/strong\u003e ChAT (cholinergic neurons marker);\u003cstrong\u003e(o,p,q)\u003c/strong\u003eTH (dopaminergic neurons marker); \u003cstrong\u003e(k,l,m) \u003c/strong\u003eOlfaxin partly co-localized with GAD67(GABAergic neurons marker); \u003cstrong\u003e(f,j,n,r)\u003c/strong\u003e Fluorescence co-localization analysis of Olfaxin and EAAC1, ChAT, GAD67,and TH. GI:Glomerular Input. Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/84678842b4e0c103156d79e1.png"},{"id":81016262,"identity":"156b6788-fe9c-4873-af7e-697b1fa8b9b6","added_by":"auto","created_at":"2025-04-21 08:55:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352336,"visible":true,"origin":"","legend":"\u003cp\u003eOlfaxin expression in various of neurons obtained fromprimary culture of OB. \u003cstrong\u003e(a,b,c) \u003c/strong\u003eOlfaxin colocalized with EAAC1 (glutamatergic neuron marker); \u003cstrong\u003e(e,f,g) \u003c/strong\u003eChAT(cholinergic neurons marker); \u003cstrong\u003e(i,j,k) \u003c/strong\u003eOlfaxin partly colocalized with GAD67 (GABAergic neurons marker). \u003cstrong\u003e(d,h,l)\u003c/strong\u003e Fluorescence co-localization analysis of Olfaxin and EAAC1, ChAT, and GAD67.Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/b3b841406fee8add81e987e5.png"},{"id":81016263,"identity":"57df8bff-0562-40fa-ac3f-6f33457aa101","added_by":"auto","created_at":"2025-04-21 08:55:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1378346,"visible":true,"origin":"","legend":"\u003cp\u003eOlfaxin expression in various of glia of OB. \u003cstrong\u003e(a,b,c) \u003c/strong\u003eOlfaxin colocalized with NG2 (NG2-type glia or oligodendrocytes marker) but \u003cstrong\u003e(e,f,g) \u003c/strong\u003edid not colocalize with iba1 (microglia marker) and \u003cstrong\u003e(i,g,k) \u003c/strong\u003eGFAP (astrocyte marker) in mouse OB.\u003cstrong\u003e(d,h,l)\u003c/strong\u003e Fluorescence co-localization analysis of Olfaxin and NG2, iba1,and GFAP. Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/913f253eee0f92e50656acd3.png"},{"id":81016258,"identity":"5f770cd0-5260-4708-8138-ca8bba3bd1c3","added_by":"auto","created_at":"2025-04-21 08:55:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":310024,"visible":true,"origin":"","legend":"\u003cp\u003eOlfaxin expression in various of glia obtained from primary culture of OB. \u003cstrong\u003e(a,b,c)\u003c/strong\u003e Olfaxin colocalized with NG2(NG2-type glia or oligodendrocytes marker); \u003cstrong\u003e(e,f,g) \u003c/strong\u003eiba1(microglia marker) and \u003cstrong\u003e(i,j,k) \u003c/strong\u003eGFAP(astrocyte marker) in mouse OB primary culture. \u003cstrong\u003e(d,h,l)\u003c/strong\u003eFluorescence co-localization analysis of Olfaxin and NG2, iba1,and GFAP. Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/749226d2dfc6874a8cfad414.png"},{"id":81016259,"identity":"7ad97a20-0ccf-46c8-a7cc-78dbb83f6477","added_by":"auto","created_at":"2025-04-21 08:55:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1423695,"visible":true,"origin":"","legend":"\u003cp\u003eOlfaxin expression in the low-glucose treatment of microglia and the mouse model of AD.(a)Western blot results in the low-glucose treatment: Olfaxin expression levels in BV2 cell lines after 0, 30, and 60, 120 minutes of low-glucose treatment. MCT1 serves as a positive control for hypoglycemic treatment. (b) Statistical analysis of western blotting for (a). (c) Statistical analysis of pyruvate content determination in BV2 cell lines after 0, 30, and 60, 120 minutes of low-glucose treatment. (d) Western blotting results showing that the expression of Olfaxin in the OB, HIP, PIR and CBM of the 9 months APP/PS1 mice, the same age C57BL/6J mice as the control (WT). HIP: hippocampus; PIR: piriform cortex; CBM: cerebellum; (e) Statistical analysis of western blotting for (d). (f) Western blotting results showing that the expression of MCT1 in the OB, HIP and CBM of the aged (9 mouths) APP/PS1 mice, the same age C57BL/6J mice as the control (WT) (g) Statistical analysis of western blotting for (f).Error bars are presented as mean ± SD from three repeat lines. Statistical comparisons are performed using unpaired t test. (*p\u0026lt;0.05; **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.001. ns: not significant.)(h) Olfaxin colocalized with ERp72(endoplasmic reticulum marker) in the OB of WT and APP/PS1 mice. (i) Statistical analysis of Pearson coefficient for (h); Scale bars: 20 μm.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/c6b951f551448c181d8bbb01.png"},{"id":93418262,"identity":"15a68592-dd14-4b53-9e83-4a8c99ef64cb","added_by":"auto","created_at":"2025-10-13 15:46:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6303055,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6270949/v1/5f55d1bd-5036-4d8f-a36a-17dc92bd4f95.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The expression and preliminary functional exploration of Olfaxin in the central nervous system of mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePRUNE2 is an X-linked methylCpG binding protein 2 (MeCP2) target gene, which may be associated with Rett syndrome(Nectoux et al., 2010). It contains BNIP2 and Cdc42GAP homology (BCH) domains at the C-terminus. The PRUNE2 protein is mainly expressed in the spinal cord and dorsal root ganglion (DRG), and plays an important role in synaptic function, cell transformation, neuronal differentiation, and apoptosis (Pan \u0026amp; Low, 2012).And other research has shown that as a binding protein of 8-oxo-GTP, PRUNE2 may be associated with the maintenance of mature nervous systems (Iwama et al., 2011).\u003c/p\u003e \u003cp\u003eThere are five subtypes of PRUNE2: PRUNE2, C9orf65, molecules with BCH motifs in carboxyl terminal region 1 (BMCC1), BNIP2 ultra long (BNIPXL), and Olfaxin/BMCC1s.C9orf65 expression is useful for the differentiation between leiomyosarcomas and gastrointestinal stromal tumors(Islam et al., 2018).BMCC1s regulate MAP6 induced microtubule cold stability through direct interaction with MAP6/STOP(Arama et al., 2012), and it can control the transport of renal glutaminase (KGA) to mitochondria(Boulay et al., 2013).BNIPXL is the N-terminal truncated form of BMCC1, which can interact with RhoA and RhoA specific guanine nucleotide exchange factors to promote lymphocyte proliferation crisis, thereby regulating cell transformation(Soh \u0026amp; Low, 2008); Prostate cancer antigen 3 (PCA3) is a prominent cancer biomarker of the prostate, contained in the PRUNE2 gene. PCA3 is transcribed in the opposite chain of PRUNE2, which controls PRUNE2 levels through a unique regulatory mechanism(Salameh et al., 2015). In summary, the first four subtypes have been shown to be is a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively, all of which play crucial roles in the nervous system(Li et al., 2011).\u003c/p\u003e \u003cp\u003eIn our previous research, we discovered the fifth subtype of PRUNE2,Olfaxin/BMCC1s,and based on the N-terminal 133 amino acid sequence of the protein encoded by the AK038997.1 mRNA in the mouse PRUNE2 gene, we designed an antibody that specifically recognizes 52 kDa Olfaxin. It is predominantly expressed in the olfactory system, shares 56.3% amino acid identity with mouse Caytaxin(Li et al., 2012). But the expression and biological function of Olfaxin in neurons and glia are still unclear.\u003c/p\u003e \u003cp\u003eRecent studies have shown that microglia are not only immune cells, but also play a key role in maintaining glucose homeostasis in the CNS(Pan et al., 2022). Both astrocytes and microglia are sensitive to \u003cem\u003ein vitro\u003c/em\u003e changes of surrounding glucose levels, which impact their functions and the expression of the glucose transporters (Robb et al., 2020).In addition, Olfaxin deficiency impaired the increasing expression of ACL, which is a key metabolic enzyme that produces the acetyl-CoA for acetylcholine synthesize, in the piriform cortex during development(Islam et al., 2018).Therefore, in this study, we detected the expression of Olfaxin in different types of neurons and glia, and preliminarily explored its biological function in low glucose model of microglia and AD mice model.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003e The following antibodies and chemicals were used in this study: anti-c-fos (Santa Cruz Biotechnology, Cat# sc-166940, RRID:AB_10609634); anti-EAAC1(Sigma-Aldrich, Cat# anti-EAAC1, RRID:AB_3101962); anti-ChAT (Proteintech, Cat# 66816-1-Ig, RRID:AB_2882159); anti-GAD67(Sigma-Aldrich, Cat# G5419, RRID:AB_261978); anti-TH (Cell Signaling Technology, Cat# 2791, RRID:AB_2201522); anti-NeuN antibody(Abcam, Cat# ab104225, RRID:AB_10711153); anti-NG2 antibody (Abcam, Cat# ab275024, RRID:AB_2922401); anti-GFAP (Abcam, Cat#ab7260, RRID:AB_305808); Anti-iba1(Novus, Cat# NB100-1028, RRID:AB_521594); anti-MCT1 (Abcam, Cat#ab93048, RRID:AB_10563650); anti-actin (Abgent, Cat# AC-15, RRID:AB_2223210);anti-ERp72(Proteintech Cat# 14712-1-AP, RRID: AB_2160973);HRP-conjugated goat anti-rat༈Jackson ImmunoResearch Labs, Cat# 112-035-003, RRID:AB_2338128), anti-mouse(Yeasen Biotech, Cat# 33201ES60, RRID:AB_10015289༉, and anti-rabbit(Jackson ImmunoResearch Labs, Cat# 111-035-144, RRID:AB_2307391); Alexa Fluor\u0026reg; 488-AffiniPure Donkey Anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L)(Jackson ImmunoResearch Labs, Cat# 715-545-150, RRID:AB_2340846); anti-rabbit(Jackson ImmunoResearch Labs, Cat# 711-545-152, RRID:AB_2313584); Alexa Fluor\u0026reg; 594-AffiniPure Donkey Anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L)(Jackson ImmunoResearch Labs, Cat# 715-585-150, RRID:AB_2340854); Anti-rabbit (Jackson ImmunoResearch Labs, Cat# 711-585-152, RRID:AB_2340621); Chemicals from Gibco, TRANS, Biosharp, Beyotime, and Sigma.Pyruvate(PA) Content Assay Kit (BC2205, Solarbio).To determine the expression of Olfaxin in the neurons and glia, monoclonal and polyclonal antibodies against N-terminal (ter) Olfaxin, respectively, were generated according to the protocol used in a previous study (Li et al., 2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals\u003c/h2\u003e \u003cp\u003e Animal experiments were approved by the Experimental Animal Ethics Committee of Wenzhou Medical University. Mice were purchased from Beijing Weitong Lihua Co., .APP/PS1 mice are used as the AD model with Mo/Hu APP Swedish mutations (K595N/M596L)\u0026thinsp;+\u0026thinsp;Hu PS1 deltaE9 mutation. We housed C57BL/6 and APP/SP1 male mice (9 months old, body weight 27-30g) in standard laboratory cages under a12/12 h light/dark cycle, a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;3 ℃`and a temperature of 55%\u0026plusmn; 5% relative humidity in a controlled environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Primary culture of neurons\u003c/h2\u003e \u003cp\u003eThe OB from P0 C57BL/6 mice was isolated. Meninges were carefully removed from the OB under a dissection microscope. Tissues were trypsinized and dissociated by gentle trituration. Cells were cultured in Neurobasal Medium (Invitrogen, Carlsbad, CA) supplemented with B-27 (Invitrogen), 1 mM L-glutamine (Sigma), and penicillin/streptomycin at 2x10\u003csup\u003e4\u003c/sup\u003e cells on 12-mm cover slips (Matsunami, Osaka, Japan) coated with 20 \u0026micro;g/ml poly-L-lysine (Sigma) in a 24-well culture plate. DIV4-7 cells were treated with AraC (0.25\u0026micro;m) .DIV 14 neurons were fixed for 15 min in 4% PFA/0.1 M phosphate buffer (PB) (pH 7.4), then subjected to ICC and WB. ICC sections were examined under an inverted fluorescent microscope (Olympus) or a confocal microscope (Leica sp8), as previously described (Li et al., 2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Primary culture of glia\u003c/h2\u003e \u003cp\u003eIn the animal facility of the research institute, a male mouse aged 7\u0026ndash;9 weeks and a female mouse aged 7\u0026ndash;9 weeks were kept. After visually confirming pregnancy, the female mouse was separated from the male mouse and their offspring were observed. On day 0, a litter of newborn mice was collected. OB was obtained after the mouses were terminated and mechanically dissociated. Meninges were carefully removed from the OB under a dissection microscope. Then OB were trypsinized and resuspended in 1\u0026times; PBS (PBS: phosphate-buffered saline). Filtration using a 70\u0026micro;m strainer was performed. The sample was centrifuged at 1000 g for 5 min at Room Temperature (RT). The supernatant was then removed, which was then resuspended in 10% DMEM/F12 (Gibco) medium. The suspension was again centrifuged at 1000 g for 5 min at RT. The supernatant was removed and resuspended in 10% FBS DMEM/F12 medium. The cells were cultured with 10% DMEM/F12 in T25 flasks coated with 20 g/ml poly-L-lysine (Sigma). Whole medium was changed after 24 h, and the half medium was changed every 3 days. At 10\u0026ndash;14 DIV (80\u0026ndash;90%), by tapping the T25 flask, the medium with floating microglia was harvested, and it was centrifuged at 1000 g for 5 min at RT; the supernatant was resuspended in 10% DMEM/F12 and cultured in a 6-well culture plat. The cultures were then shaken again at 220 rpm overnight to separate astrocytes and oligodendrocyte progenitor cells (OPCs). Trypsinize the adherent cells in the culture flask, and then it was centrifuged at 1000 g for 5 min at RT; the supernatant was resuspended in 10% DMEM/F12 and cultured in a 6-well culture plat. For further purification of OPCs, the medium obtained after shaking was transferred to uncoated dishes for 1 hour to remove astrocytes and microglia. The non-adherent cells that remained were collected and cultured in a medium containing 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 50 \u0026micro;g/mL apo-transferrin, 0.1% bovine serum albumin (BSA), 5 \u0026micro;g/mL insulin, 30 nmol/L sodium selenite, 10 nmol/L D-biotin,10 ng/mL PDGF-AA and 10 nmol/L hydrocortisone, and 10 ng/mL bFGF. Cells were planted into six- or 24-well plates with 12 mm diameter glass coverslips at a density of 5\u0026times;104 cells/cm\u003csup\u003e2\u003c/sup\u003e for experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 BV2 cell culture and low-glucose treatment\u003c/h2\u003e \u003cp\u003eThe BV-2 cells employed in this study originated from primary cells cultivated in the laboratory. The cells were cultured with 10% FBS DMEM 4.5g/L glucose medium (Gibco) in a culture dish and maintained in a CO\u003csub\u003e2\u003c/sub\u003e incubator at a temperature of 37℃. The cells were plated in normal growth media (DMEM 4.5g/L glucose\u0026thinsp;+\u0026thinsp;10% FBS) at a density of 10\u003csup\u003e5\u003c/sup\u003ecells/mL in 6-well plates and allowed to reach 90% confluence, and the medium was then removed, and 10% FBS DMEM 1.0g/L glucose medium was added (Gibco).Cells were cultured for 30-min, 60-min, and 120-min and then harvested. Then they were subjected to western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blotting\u003c/h2\u003e \u003cp\u003eFirst, mouse (C57BL/6 and APP/PS1) tissues or culture cells were collected in each group and added RIPA buffer containing protease inhibitors and phosphatase inhibitor. Then, tissues or cells were totally lysed and centrifuged at 4\u0026deg;C for about 30 min to obtain the supernatant. The protein concentration was determined by the BCA protein assay kit (Beyotime Biotechnology). Total protein was subjected to 10%SDS-PAGE gels and then transferred to the PVDF membrane (Millipore). The membranes were blocked by 5% skim milk for 2h and then were incubated overnight at 4℃ with primary antibody against Olfaxin (polyclonal) (1:1000),MCT1 (1:1000), β-actin (1:3000). After three times washing, the membranes were incubated horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:20000) or HRP-linked anti-mouse IgG (1:20000) at room temperature for 2h. Protein band were visualized by ECL solution (Meilunbio) and detected with an automatic chemiluminescence imaging analysis system (Amersham Imager 680). The intensity of each band was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunohistochemical staining\u003c/h2\u003e \u003cp\u003eAfter perfusion and fixation, mouse olfactory bulb tissues were embedded in OCT and sectioned into 14\u0026micro;m thick slices. The olfactory bulb tissue of each mouse was used for c-fos and Olfaxin(monoclonal) immunohistochemical staining. The sections underwent antigen retrieval treatment, followed by incubation with a 3% H202-methanol solution for 15 min, and overnight incubation with c-fos and Olfaxin antibodies at 40℃, respectively. A working solution of 1:500 horseradish peroxidase-labeled anti-mouse secondary antibody was added, and the sections were incubated at room temperature for 3 h. Then, the sections were incubated with DAB chromogenic solution, dehydrated using a gradient alcohol series, cleared with xylene, and finally, the slides were sealed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eMice were perfused with PBS followed by 4% paraformaldehyde in 0.1 M PB. Brains were post-fixed for 2h in the same fixative, which was then replaced by 15% sucrose in 0.1 M PB. Sections (14\u0026micro;m) were then rinsed with PBS and permeabilized for 5 minutes with 0.1% Triton-X (Sigma). They were then blocked with 1% Bovine Serum Albumin (BSA) in PBST for 1 hour. Primary antibodies(monoclonal antibodies against N-terminal (ter) Olfaxin,EAAC1,ChAT,GAD67,NG2,iba1,GFAP,ERp72) plus 1% BSA incubated the samples overnight at 4\u0026deg;C. The secondary antibodies (Alexa-488, Alexa-594) were then incubated at room temperature for 1 hour. The nuclei of cells in samples were stained with DAPI at room temperature for 3 min,and 3,30 -diaminobenzidine (DAB; Wako, Osaka, Japan) was employed for visualization(Li et al., 2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Pyruvate content determination\u003c/h2\u003e \u003cp\u003eFollow the manufacturer\u0026rsquo;s instructions to process the cell supernatant, then inoculate it into a 96-well plate. Set up 3 replicate wells for each group of samples, blank group, and standard group. Next, add the reagents from the kit according to the instructions, mix thoroughly, and measure the absorbance values of each group at a wavelength of 520nm. Finally, calculate the pyruvate content of each group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3.0 Statistical analysis\u003c/h3\u003e\n\u003cp\u003eGraphPad Prism 8.0 was used for statistical analysis. Data shown is mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Two-tailed unpaired t test was applied for comparisons between two groups. The Plot Profile tool of imageJ is used for fluorescence co-localization analysis, and the Colocalization Finder plug-in of imageJ is used to calculate the Pearson co-localization coefficient of the fluorescence map.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Olfaxin expression in neurons\u003c/h2\u003e \u003cp\u003eThe polyclonal N-ter-Olfaxin antibody specifically detected a clear band of approximately 52 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Subsequently, we validated the expression level of Olfaxin in the entire brain. The result showed Olfaxin expression was observed in the OB and CBM but not in the brain stem (BS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).Therefore, we used Cellular Proto-Oncogene Fos(c-fos) antibody was a positive control to validate the availability of our experimental method and antibody, and chose monoclonal N-ter-Olfaxin antibodies to detect the expression of Olfaxin in the OB,CBM and HIP. Olfaxin expression was observed in the OB, CBM, and HIP. In addition, in the OB, Olfaxin was found to be mainly localized in the glomerular layer (GL), external plexiform layer (EPL), and internal plexiform layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb)(Li et al., 2012). Our results are consistent with the results of Li et al. in 2012.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed the GI (Glomerular Input) region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) of mouse OB tissue and found that Olfaxin was completely co-localized with EAAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), ChAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej) positive cells and TH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003er), while Olfaxin and GAD67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en) positive cells were only partially co-localized.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, in primary cultured OB cells, we observed that Olfaxin was completely co-localized with EAAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea to \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) and ChAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee to \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) positive cells; Olfaxin and GAD67(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei to \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el) positive cells only partially co-localized. These are consistent with the results of \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, Olfaxin is expressed in glutamatergic neuron (marked by EAAC1), GABA-ergic neuron (marked by GAD67), cholinergic neuron (marked by ChAT),and dopaminergic neuron (marked by TH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Olfaxin expression in glia\u003c/h2\u003e \u003cp\u003eglia constitute roughly half of the cells of the CNS, and they influence dynamically neuronal birth, migration, axon specification, and growth through circuit assembly and synaptogenesis.(Allen \u0026amp; Lyons, 2018)The FPKM data of RNA-sequencing transcriptome and splicing indicate that PRUNE2 mRNA expression is observed in astrocytes, OPCs, newly formed oligodendrocytes, and microglia(Zhang et al., 2014). Subsequently, we conducted immunofluorescence staining using tissues and primary culture cells to examine the expression of Olfaxin in different types of glial cells.\u003c/p\u003e \u003cp\u003eOlfaxin was completely co-localized with NG2 positive cells in the OB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea to \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and primary culture cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).In our study, Olfaxin did not co-locate with iba1 positive cells in the OB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee to \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). But Olfaxin was co-localized with iba1 positive cells in primary culture cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Unexpectedly, Olfaxin was not co-localized with GFAP positive cells in mouse tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei to \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). And similar to iba1, Olfaxin was co-localized with GFAP positive cells in primary culture cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe above experimental results indicate that: \u003cem\u003ein vitro\u003c/em\u003e, Olfaxin is expressed in all glia: oligodendrocytes (marked by NG2) and microglia (marked by iba1), astrocytes (marked by GFAP). And \u003cem\u003ein vivo\u003c/em\u003e Olfaxin is just expressed in the oligodendrocytes (marked by NG2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Olfaxin expression and low glucose treatment\u003c/h2\u003e \u003cp\u003eOlfaxin deficiency impaired the increasing expression of ACL in the piriform cortex during development(Islam et al., 2018). Both astrocytes and microglia are sensitive to \u003cem\u003ein vitro\u003c/em\u003e changes of surrounding glucose levels, which impact their functions and the expression of the glucose transporters (Robb et al., 2020). Therefore, we performed low-glucose treatment to the BV2 cell line to examine the function of Olfaxin in microglia.\u003c/p\u003e \u003cp\u003eWe used MCT1 as a positive marker for low-glucose treatment. The expression patterns of Olfaxin and MCT1 are very similar. After 30-min treatment in a low-glucose medium, Olfaxin protein expression increased remarkably and in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Futhermore, pyruvic acid content determination results showed that pyruvic acid increased significantly in a time-dependent manner during the first 60 minutes of low glucose treatment, followed by a significant decrease at the 120-min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). These results suggest that Olfaxin may have important functions in metabolism. However, we need more experiments to explore the functions of Olfaxin in the glia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Olfaxin expression in aged APP/PS1 mice\u003c/h2\u003e \u003cp\u003eConsidering the energy metabolism disorder present in the brain of AD patients, the aforementioned results implied that Olfaxin may significantly affect metabolic processes. We further examined the Olfaxin and MCT1 expression in AD mice model. We performed western blotting using aged APP/PS1 mice. The results indicated that Olfaxin expression is increased in the OB of aged APP/PS1 mice but decreased in CBM and did not change in HIP and PIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Interestingly, similar to Olfaxin, MCT1 expression increased in the OB and decreased in the CBM, but decreased in the HIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). In addition, we observed that Olfaxin was co-localized with Endoplasmic Reticulum Protein 72 (ERp72) (endoplasmic reticulum marker) in the OB of WT and APP/PS1 mice, and compared to WT mice, the Pearson co-localization coefficient of ERp72 and Olfaxin in the OB of APP/PS1 mice was higher. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). These experimental results suggest that Olfaxin may be related to the disease process of AD and functional maintenance and damage of different brain regions, providing a new idea for exploring the biological function of Olfaxin .\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe FPKM data of RNA-sequencing transcriptome and splicing in neurons, glia, and vascular cells of the cerebral cortex suggested PRUNE2 mRNA expression was observed in the neurons, astrocytes, OPCs, newly formed oligodendrocytes, and microglia (data: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.brainrnaseq.org)(Zhang\u003c/span\u003e\u003cspan address=\"http://www.brainrnaseq.org)(Zhang\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e et al., 2014). In neurons, the studies of Olfaxin revealed that Olfaxin was predominantly expressed in the olfactory bulb (OB) and piriform cortex (PIR) where glutamatergic terminal was localized(Li et al., 2012). The studies of Olfaxin in the Olfaxin-KO mice suggested that Olfaxin deficiency led to impairment of odor-associative learning and odor preference. Moreover ,compared with the control mice at postnatal day14, the level of BDNF and ACL expression were lower in the piriform cortex of KO mice, while Kv4.2 and ChAT expression were higher(Islam et al., 2018).Our studies revealed that Olfaxin is expressed in glutamatergic neurons ,cholinergic neurons GABA-ergic neurons and dopaminergic neurons of the OB (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicate that Olfaxin plays a crucial role in the functional regulation of the OB neural circuit. Its absence can disrupt the function and interaction of these neurons, resulting in impairments in odor association learning and odor preference in mice. This provides significant experimental evidence for further understanding the olfactory-related neural mechanisms and the biological function of Olfaxin. However, we need further research to explore the functions of Olfaxin in these neurons.\u003c/p\u003e \u003cp\u003eGlial cells including astrocytes, oligodendrocytes, and microglia support the physiological functions of neurons. In the CNS, these glia cooperate to promote and preserve neuronal health, playing important roles in regulating the activity of neuronal networks during different life stages. Astrocytes and microglia are important for the systemic regulation of energy and glucose homeostasis in the course of normal physiological control and during disease (Robb et al., 2020).Therefore, in this study, we examined the expression of Olfaxin in the glia. Interestingly, in mouse tissues, Olfaxin colocalized with NG2, and not with GFAP and iba1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). But, in primary culture of OB, Olfaxin colocalized with NG2, GFAP, and iba1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The expression and colocalization of Olfaxin in oligodendrocytes suggest that Olfaxin may play a role in oligodendrocytes. However, the co-localization of Olfaxin with iba1 and GFAP in the mouse olfactory bulb and its primary cultured glial cells exhibits inconsistency, indicating that the expression of Olfaxin in glial cells may depend on the nutritional environment of the cells or cell differentiation status and further exploration is needed.\u003c/p\u003e \u003cp\u003eIn microglia, the expression of Olfaxin is not entirely unforeseen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).In previous research on Olfaxin, it was mentioned that Olfaxin may be closely associated with the production of acetyl-CoA(Islam et al., 2018).Glucose serves as the primary energy source for the brain, and maintaining glucose homeostasis is vital for ensuring normal neurological function. MCT1 is a transmembrane protein that facilitates the transport of monocarboxylic compounds (such as lactate and pyruvate) across the cell membrane(Hadjihambi et al., 2023). In low-glucose environments, MCT1 enhances the transport of ketone bodies and lactate, thereby supporting cellular energy supply and metabolic adaptation. Thus, in our study, we used MCT1 as a positive marker for low-glucose treatment. And the results showed at 30-min,60-min and 120-min in a low-glucose medium, the expression of Olfaxin and MCT1 significantly increased in a time-dependent manner, while pyruvic acid significantly decreased at the 120-min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea to \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).This phenomenon may be caused by the significant increase in the expression level of MCT1 in order to meet the energy supply of cells. However, following 30 and 60 minutes of low-glucose treatment, pyruvate levels gradually rose due to the enhanced glycolysis pathway. By 120-min, as metabolic pathways reorganized, microglia shifted towards utilizing alternative energy sources like fatty acids and ketone bodies, leading to a decrease in pyruvate content. These results indicate under low-glucose conditions, microglia enhance the effective transport of pyruvate and alternative energy sources by promoting an upregulation of MCT1 expression levels. and the expression pattern of Ofaxin mirrors that of MCT1, suggesting that Olfaxin may play a significant role in the energy metabolism of microglia.\u003c/p\u003e \u003cp\u003eConsidering that PRUNE2 has been shown to be a susceptibility gene for Alzheimer's disease (AD)(Li et al., 2011),and metabolic alterations in microglia in AD can contribute to neuroinflammation and disease progression(Tondo et al., 2020).We further investigated the expression of Olfaxin and MCT1 in the brains of AD mice. Compared to the control group of mice, the expression of Olfaxin in the OB of APP/PS1 increased, but in the CBM decreased and remained unchanged in the HIP and PIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). The expression of MCT1 raised in the OB, but reduced in the CBM and HIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). The OB plays a pivotal role in olfactory information processing, and in early-stage AD patients, the impairment of odor perception appears first (Li et al., 2012). The elevated expression of Olfaxin and MCT1 in the OB may be an attempt by the body to mitigate the impact of pathological changes on olfactory function through energy metabolism regulation. The PIR is closely associated with olfactory processing and olfactory memory functions, but in the APP/PS1 mouse model, the function of this region may not have been significantly impaired yet. Alternatively, the mechanism by which it maintains normal function may not rely on changes in Olfaxin expression. Therefore, compared with age-matched C57BL/6 mice, the expression level of Olfaxin in the piriform cortex of APP/PS1 mice remains relatively stable.The HIP is a critical brain region for learning and memory, and due to pathological changes such as amyloid deposition in Alzheimer's disease, the number and activity of neurons decrease in the region. This reduction may lead to a decreased demand for energy metabolism substrates, subsequently lowering MCT1 expression. However, Olfaxin expression in the HIP remains stable, suggesting that the metabolic regulatory role of Olfaxin may not be the primary response mechanism in the HIP, or later than OB. The CBM is involved in motor control, learning, reflex adaptation, and cognition. Studies have shown that the number of mitochondria at the presynaptic terminals of cerebellar neurons decreases, in Alzheimer's disease patients(Fan et al., 2018) ,which may lead to a decrease in the overall metabolic activity of the cerebellum, thereby inhibiting the expression of MCT1 and Olfaxin. In addition, we observed that Olfaxin was co-localized with ERp72(endoplasmic reticulum marker) in the OB of WT and APP/PS1 mice, and compared to WT mice, the Pearson co-localization coefficient of ERp72 and Olfaxin in the OB of APP/PS1 mice was higher. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). This phenomenon indicates that Olfaxin may be associated with protein folding disorder in AD. In summary, these findings indeed indicate that Olfaxin may play a critical role in the disease progression of AD. With these results in mind, the role of Olfaxin in AD mice model should be interested.\u003c/p\u003e \u003cp\u003eIn conclusion, we conducted a general experiment on the \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e expression of Olfaxin in various neurons and glia cells. And we also detected the expression of Olfaxin and MCT1 in a low-glucose model and measured the content of pyruvate. Moreover, the expression levels of Olfaxin in parts of OB, PIR, HIP and CBM, and the expression levels of MCT1 in parts of OB, HIP and CBM were detected in the AD mice model. We also observed the co-localization of ERp72 and Olfaxin in the OB of WT and APP/PS1 mice. These results preliminarily demonstrate that Olfaxin may influence the occurrence and progression of Alzheimer's disease by impacting the energy metabolism of microglia or other aspects within the central nervous system, but the mechanism remains to be studied.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr T. Nakagawa (Gifu University Graduate School of Medicine) for Olfaxin anti-rat antibody samples. This work was supported by Basic Scientific Research project of Wenzhou. We would like to thank Editage (www.editage.cn) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the\u0026nbsp;Nature Science Foundation of Zhejiang Province, China (Grant No. LY20H090010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLSM\u0026nbsp;and LM was involved in the project design ,writing – review \u0026amp; editing\u0026nbsp;and writing – original draft.WH,ZWH ,NXY and CC\u0026nbsp;performed the major experimental work and data analysis; WH revised the manuscript. All authors approved the final article before publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003evailability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate all animal procedures were approved by the Animal Research Ethics Committee of China Wenzhou Medical University (ID Number: xmsq2021-0458).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllen, N. J., \u0026amp; Lyons, D. A. (2018). Glia as architects of central nervous system formation and function. 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J Neurosci, 34(36), 11929-11947. https://doi.org/10.1523/jneurosci.1860-14.2014\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Olfaxin, PRUNE2 isoform, Neurons, Glia, Alzheimer's disease","lastPublishedDoi":"10.21203/rs.3.rs-6270949/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6270949/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrune homolog 2 (Drosophila) (PRUNE2) protein has five subtypes, four of which have been reported to play important roles in the central nervous system (CNS), but the fifth ,Olfaxin still remains unclear. Therefore, this study aims to investigate Olfaxin expression in the CNS of mouse. \u003cem\u003eIn vivo and in vitro\u003c/em\u003e, experiments indicate Olfaxin is expressed in the olfactory bulb (OB) neurons: glutamatergic neuron marked by Excitatory Amino Acid Carrier 1 (EAAC1), GABA-ergic neuron marked by Glutamic Acid Decarboxylase 67 (GAD67), Cholinergic neuron marked by Choline Acetyltransferase (ChAT) and dopaminergic neuron marked by Tyrosine Hydroxylase (TH). While,in the OB glia, its expression pattern differed between \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e settings. Under low - glucose conditions, Olfaxin and Monocarboxylate Transporter 1 (MCT1) expression increased in microglia, suggesting a role in energy metabolism. In an Alzheimer's disease (AD) mouse model, Olfaxin expression varied in different brain regions. These results indicate that Olfaxin may influence AD progression, possibly through microglial energy metabolism, although the underlying mechanisms remain to be further investigated.\u003c/p\u003e","manuscriptTitle":"The expression and preliminary functional exploration of Olfaxin in the central nervous system of mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 08:55:27","doi":"10.21203/rs.3.rs-6270949/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd84523b-42a9-467e-8133-5265816f6f93","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T15:38:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 08:55:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6270949","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6270949","identity":"rs-6270949","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}