Neuronal Cultures Research Articles

Gene therapy for epilepsy targeting neuropeptide Y and its Y2 receptor to dentate gyrus granule cells

Gene therapy is emerging as an alternative option for individuals with drug-resistant focal epilepsy. Here, we explore the potential of a novel gene therapy based on Neuropeptide Y (NPY), a well-known endogenous anticonvulsant. We develop a lentiviral vector co-expressing NPY with its inhibitory receptor Y2 in which, for the first time, both transgenes are placed under the control of the minimal CamKIIa(0.4) promoter, biasing expression toward excitatory neurons and allowing autoregulation of neuronal excitability by Y2 receptor-mediated inhibition. Vector-induced NPY and Y2 expression and safety are first assessed in cultures of hippocampal neurons. In vivo experiments demonstrate efficient and nearly selective overexpression of both genes in granule cell mossy fiber terminals following vector administration in the dentate gyrus. Telemetry video-EEG monitoring reveals a reduction in the frequency and duration of seizures in the synapsin triple KO model. This study shows that targeting a small subset of neurons (hippocampal granule cells) with a combined overexpression of NPY and Y2 receptor is sufficient to reduce the occurrence of spontaneous seizures.

Dynamic Organization of Neuronal Extracellular Matrix Revealed by HaloTag-HAPLN1.

The brain's extracellular matrix (ECM) regulates neuronal plasticity and animal behavior. ECM staining shows a net-like structure around a subset of neurons, a ring-like structure at the nodes of Ranvier, and diffuse staining in the interstitial matrix. However, understanding the structural features of ECM deposition across various neuronal types and subcellular compartments remains limited. To visualize the organization pattern and assembly process of the hyaluronan-scaffolded ECM in the brain, we fused a HaloTag to hyaluronan proteoglycan link protein 1, which links hyaluronan and proteoglycans. Expression or application of the probe in primary rat neuronal cultures enables us to identify spatial and temporal regulation of ECM deposition and heterogeneity in ECM aggregation among neuronal populations. Dual-color birthdating shows the ECM assembly process in culture and in vivo. Sparse expression in mouse brains of either sex reveals detailed ECM architectures around excitatory neurons and developmentally regulated dendritic ECM. Our study uncovers extensive structural features of the brain's ECM, suggesting diverse roles in regulating neuronal plasticity.

Peering into the mind: unraveling schizophrenia's secrets using models.

Schizophrenia (SCZ) is a complex mental disorder characterized by a range of symptoms, including positive and negative symptoms, as well as cognitive impairments. Despite theextensive research, the underlying neurobiology of SCZ remain elusive. To overcome this challenge, the use of diverse laboratory modeling techniques, encompassing cellular and animal models, and innovative approaches like induced pluripotent stem cell (iPSC)-derived neuronal cultures or brain organoids and genetically engineered animal models, has been crucial. Immortalized cellular models provide controlled environments for investigating the molecular and neurochemical pathways involved in neuronal function, while iPSCs and brain organoids, derived from patient-specific sources, offer significant advantage in translational research by facilitating direct comparisons of cellular phenotypes between patient-derived neurons and healthy-control neurons. Animal models can recapitulate the different psychopathological aspects that should be modeled, offering valuable insights into the neurobiology of SCZ. In addition, invertebrates' models are genetically tractable and offer a powerful approach to dissect the core genetic underpinnings of SCZ, while vertebrate models, especially mammals, with their more complex nervous systems and behavioral repertoire, provide a closer approximation of the human condition to study SCZ-related traits. This narrative review provides a comprehensive overview of the diverse modeling approaches, critically evaluating their strengths and limitations. By synthesizing knowledge from these models, this review offers a valuable source for researchers, clinicians, and stakeholders alike. Integrating findings across these different modelsmay allow us to build a more holistic picture of SCZ pathophysiology, facilitating the exploration of new research avenues and informed decision-making for interventions.

Mitigation of synaptic and memory impairments via F-actin stabilization in Alzheimer’s disease

BackgroundSynaptic dysfunction, characterized by synapse loss and structural alterations, emerges as a prominent correlate of cognitive decline in Alzheimer’s disease (AD). Actin cytoskeleton, which serves as the structural backbone of synaptic architecture, is observed to be lost from synapses in AD. Actin cytoskeleton loss compromises synaptic integrity, affecting glutamatergic receptor levels, neurotransmission, and synaptic strength. Understanding these molecular changes is crucial for developing interventions targeting synaptic dysfunction, potentially mitigating cognitive decline in AD.MethodsIn this study, we investigated the synaptic actin interactome using mass spectrometry in a mouse model of AD, APP/PS1. Our objective was to explore how alterations in synaptic actin dynamics, particularly the interaction between PSD-95 and actin, contribute to synaptic and cognitive impairment in AD. To assess the impact of restoring F-actin levels on synaptic and cognitive functions in APP/PS1 mice, we administered F-actin stabilizing agent, jasplakinolide. Behavioral deficits in the mice were evaluated using the contextual fear conditioning paradigm. We utilized primary neuronal cultures to study the synaptic levels of AMPA and NMDA receptors and the dynamics of PSD-95 actin association. Furthermore, we analyzed postmortem brain tissue samples from subjects with no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer’s dementia (AD) to determine the association between PSD-95 and actin.ResultsWe found a significant reduction in PSD-95-actin association in synaptosomes from middle-aged APP/PS1 mice compared to wild-type (WT) mice. Treatment with jasplakinolide, an actin stabilizer, reversed deficits in memory recall, restored PSD-95-actin association, and increased synaptic F-actin levels in APP/PS1 mice. Additionally, actin stabilization led to elevated synaptic levels of AMPA and NMDA receptors, enhanced dendritic spine density, suggesting improved neurotransmission and synaptic strength in primary cortical neurons from APP/PS1 mice. Furthermore, analysis of postmortem human tissue with NCI, MCI and AD subjects revealed disrupted PSD-95-actin interactions, underscoring the clinical relevance of our preclinical studies.ConclusionOur study elucidates disrupted PSD-95 actin interactions across different models, highlighting potential therapeutic targets for AD. Stabilizing F-actin restores synaptic integrity and ameliorates cognitive deficits in APP/PS1 mice, suggesting that targeting synaptic actin regulation could be a promising therapeutic strategy to mitigate cognitive decline in AD.

The Extract of Camellia japonica L. Protects Against Mice Cerebral Ischemia/Reperfusion Injury via Promoting the H2S-BKCa Pathway

Background and Purpose The effect and mechanism of the extract of Camellia japonica L. (ECJ) on brain injury following cerebral ischemia/reperfusion (I/R) were demonstrated in the present study. Methods We detected mice’s brain damage after cerebral I/R and tested neuronal injury following oxygen-glucose deprivation/re-oxygenation (OGD/R) to evaluate the neuroprotection of ECJ. Besides, we tested the expressions of hydrogen sulfide (H2S) synthase cystathionine-β-synthase (CBS) and α subunit of large-conductance Ca2+-activated K+ channels (BKα) both in brain tissues and culture neurons. Importantly, the roles of iberiotoxin (IbTX), BKCa channel inhibitor, and CBS inhibitor aminooxyacetic acid (AOAA) on ECJ-mediated neuroprotection were assessed to explore the neuroprotective mechanism of ECJ. Results ECJ treatment could alleviate the mice’s brain injury following cerebral I/R and reduce the neuronal damage caused by OGD/R in vitro, which was inhibited by IbTX and AOAA. In addition, ECJ could increase the expression of CBS and the α subunit of BKCa channel (BKα) in mice brain tissues and the culture neurons, as well as improve the H2S production. Furthermore, exogenous H2S donor NaHS also improved the BKα expression in OGD/R neurons. Importantly, NaHS alleviated the neuronal injury in vitro, which was inhibited by IbTX as well. Conclusion ECJ can protect against mouse cerebral I/R injury, the mechanism of which is correlated with promoting the CBS/H2S-BKCa pathway.

Inflammatory Intracellular Signaling in Neurons Is Influenced by Glial Soluble Factors in iPSC-Based Cell Model of PARK2-Associated Parkinson's Disease.

Neuroinflammation is considered to be one of the driving factors in Parkinson's disease (PD). This study was conducted using neuronal and glial cell cultures differentiated from induced pluripotent stem cells (iPSC) of healthy donors (HD) and PD patients with different PARK2 mutations (PD). Based on the results of RNA sequencing, qPCR and ELISA, we revealed transcriptional and post-transcriptional changes in HD and PD neurons cultivated in HD and PD glial-conditioned medium. We demonstrated that if one or both of the components of the system, neurons or glia, is Parkin-deficient, the interaction resulted in the down-regulation of a number of key genes related to inflammatory intracellular pathways and negative regulation of apoptosis in neurons, which might be neuroprotective. In PD neurons, the stress-induced up-regulation of APLNR was significantly stronger compared to HD neurons and was diminished by glial soluble factors, both HD and PD. PD neurons in PD glial conditioned medium increased APLN expression and also up-regulated apelin synthesis and release into intracellular fluid, which represented another compensatory action. Overall, the reported results indicate that neuronal self-defense mechanisms contribute to cell survival, which might be characteristic of PD patients with Parkin-deficiency.

Tyrosine phosphorylation and palmitoylation of TRPV2 ion channel tune microglial beta-amyloid peptide phagocytosis

Alzheimer’s disease (AD) is the leading form of dementia, characterized by the accumulation and aggregation of amyloid in brain. Transient receptor potential vanilloid 2 (TRPV2) is an ion channel involved in diverse physiopathological processes, including microglial phagocytosis. Previous studies suggested that cannabidiol (CBD), an activator of TRPV2, improves microglial amyloid-β (Aβ) phagocytosis by TRPV2 modulation. However, the molecular mechanism of TRPV2 in microglial Aβ phagocytosis remains unknown. In this study, we aimed to investigate the involvement of TRPV2 channel in microglial Aβ phagocytosis and the underlying mechanisms. Utilizing human datasets, mouse primary neuron and microglia cultures, and AD model mice, to evaluate TRPV2 expression and microglial Aβ phagocytosis in both in vivo and in vitro. TRPV2 was expressed in cortex, hippocampus, and microglia.Cannabidiol (CBD) could activate and sensitize TRPV2 channel. Short-term CBD (1 week) injection intraperitoneally (i.p.) reduced the expression of neuroinflammation and microglial phagocytic receptors, but long-term CBD (3 week) administration (i.p.) induced neuroinflammation and suppressed the expression of microglial phagocytic receptors in APP/PS1 mice. Furthermore, the hyper-sensitivity of TRPV2 channel was mediated by tyrosine phosphorylation at the molecular sites Tyr(338), Tyr(466), and Tyr(520) by protein tyrosine kinase JAK1, and these sites mutation reduced the microglial Aβ phagocytosis partially dependence on its localization. While TRPV2 was palmitoylated at Cys 277 site and blocking TRPV2 palmitoylation improved microglial Aβ phagocytosis. Moreover, it was demonstrated that TRPV2 palmitoylation was dynamically regulated by ZDHHC21. Overall, our findings elucidated the intricate interplay between TRPV2 channel regulated by tyrosine phosphorylation/dephosphorylation and cysteine palmitoylation/depalmitoylation, which had divergent effects on microglial Aβ phagocytosis. These findings provide valuable insights into the underlying mechanisms linking microglial phagocytosis and TRPV2 sensitivity, and offer potential therapeutic strategies for managing AD.

Alteration of mature neuronal marker of β-III tubulin expression in differentiated SH-SY5Y cells by refinement of foetal bovine serum concentration

BackgroundThe process of differentiating neuroblastoma cells (SH-SY5Y) is crucial for obtaining mature neuronal markers. However, there is variability in the concentrations of foetal bovine serum (FBS) used in the differentiation media, ranging from 1 to 10%. This inconsistency in FBS concentrations may contribute to the inconsistent differentiation of cells. To improve the utility of the SH-SY5Y cell line as a model for neuronal cell culture, we investigated the impact of FBS concentrations in the differentiation media using Dulbecco's modified eagle medium and 10 μM all-trans-retinoic acid (ATRA). The aim of this study was to optimise the concentrations of FBS in the differentiation media of SH-SY5Y cells. Our study focused on assessing the length of dendrites in neuronal cells and the expression of β-III tubulin, a marker indicative of mature neurons. SH-SY5Y cells were differentiated with 10 µM of ATRA for 7 days. Four treatment groups with different FBS concentrations (1%, 3%, 5%, and 10%) were examined to assess the alteration of cellular morphology and dendritic length. The expression of the mature neuron marker β-III tubulin was evaluated using immunocytochemistry technique.ResultsSH-SY5Y cells' dendrite length was significantly longer (p < 0.05) when there was a higher concentration of FBS in the differentiation medium. The result was confirmed with the significant increase of β-III tubulin expression (p < 0.001) of the differentiated SH-SY5Y cells that have been incubated with higher concentrations of FBS in the differentiation medium.ConclusionsWe concluded that optimised concentrations of FBS in the differentiation media display longer length of the dendrites and express higher production of β-III tubulin in the differentiated SH-SY5Y cells. The consistency of serum concentration used in the differentiation media is important to produce a sustainable in vitro neuronal model of SH-SY5Y cells for neurodegenerative studies.

Elastic Properties of the Cell Surface and Metabolic Profile of an Embryonic Primary Mixed Culture of Hippocampal Neurons under Conditions of P2X3 Receptor Blockade

Elastic Properties of the Cell Surface and Metabolic Profile of an Embryonic Primary Mixed Culture of Hippocampal Neurons under Conditions of P2X3 Receptor Blockade

Abstract P430: Angiotensin-(1-7) Induces A Decrease In Proinflammatory Genes Expression Through A Mas Receptor-Mediated Translocation To Brainstem Nuclei Of Spontaneously Hypertensive Rats

Mas receptor (MasR) displays differential trafficking in brainstem neurons from spontaneously hypertensive rats (SHRs): the fraction of dynamin-mediated internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in Wistar neurons. Surprisingly, in SHR neurons but not in Wistar neurons, Ang-(1-7) induced MasR translocation to the nucleus. Our aim was to investigate the biological response elicited by MasR in the nucleus and whether MasR translocation was due to interaction with other receptors. We evaluated: 1) nitric oxide (NO) generation by DAF (diaminofluorescein) fluorescence; 2) interleukin (IL) 6, IL-1β, cyclooxygenase 2 (COX2) and NFkB expression by real time PCR; and 3) MasR interaction with AT1R and the dopamine type 2 receptor (D2R) by proximity ligation assay in brainstem neuronal primary cultures from Wistar and SHR rats incubated in the presence or absence of Ang-(1-7) or Ang-(1-7) + dynasore (DYN) (dynamin inhibitor) or Ang-(1-7) + A779 (MasR antagonist) during 30 min. Ang-(1-7) (100 nM) induced a decrease in the pro-inflammatory factors IL-6, IL-1β, COX2 and NFkB expression in brainstem neurons from Wistar and SHR rats which was blocked by A779, suggesting a MasR-mediated response. The effect of Ang-(1-7) was prevented when MasR internalization was prevented by DYN only in SHRs neurons but not in Wistar neurons, suggesting that the effect of Ang-(1-7) in SHR neurons is mediated by MasR translocation to the nucleus. Treatment of neurons with Ang-(1-7) induced an increase in NO in the nucleus of SHR but not of Wistar neurons and this effect was prevented by DYN and A779, suggesting that the Ang-(1-7)-induced NO production in the nucleus was due to MasR translocation to this compartment. MasR stimulation with Ang-(1-7) induced an increase in MasR-D2R heteromerization in SHR but not in Wistar neurons, and no change in MasR-AT1R heteromerization. Ou study showed that MasR translocation to the nucleus resulted in a reduction in pro-inflammatory genes expression and an increase in NO generation in SHR neurons. A fraction of MasR was translocated through the interaction with D2Rs, suggesting that this heteomer may be involved in the anti-inflammatory responses elicited by Mas translocation to the nucleus.

3D model for human glia conversion into subtype-specific neurons, including dopamine neurons

Two-dimensional neuronal cultures have a limited ability to recapitulate the invivo environment of the brain. Here, we introduce a three-dimensional invitro model for human glia-to-neuron conversion, surpassing the spatial and temporal constrains of two-dimensional cultures. Focused on direct conversion to induced dopamine neurons (iDANs) relevant to Parkinson disease, the model generates functionally mature iDANs in 2weeks and allows long-term survival. As proof of concept, we use single-nucleus RNA sequencing and molecular lineage tracing during iDAN generation and find that all glial subtypes generate neurons and that conversion relies on the coordinated expression of three neural conversion factors. We also show the formation of mature and functional iDANs over time. The model facilitates molecular investigations of the conversion process to enhance understanding of conversion outcomes and offers a system for invitro reprogramming studies aimed at advancing alternative therapeutic strategies in the diseased brain.

The Effect of ACTH/MSH N-Terminal Fragment Analogs on the Anxiety Level, Pain Sensitivity and Levels of Neurotrophic Factors BDNF and VEGF in Primary Neuronal Cultures of Rats

The Effect of ACTH/MSH N-Terminal Fragment Analogs on the Anxiety Level, Pain Sensitivity and Levels of Neurotrophic Factors BDNF and VEGF in Primary Neuronal Cultures of Rats

Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, β tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

Interindividual Variation in Gut Nitrergic Neuron Density Is Regulated By GDNF Levels and ETV1

The size and function of the enteric nervous system (ENS) can vary substantially between individuals. Because ENS function is involved in the etiology of a growing number of common human diseases, understanding mechanisms that regulate ENS variation is important. We analyzed RNAseq data from 41 normal adult human colon biopsies and single-cell RNA-seq data from human and mouse developing gut. To establish cause-consequence relationship we used alleles in mice that allow levels change of the candidate effector molecule in the comparable range to human samples. We used siRNA and primary neuronal cultures to define downstream molecular events and characterized gut functional changes in mice where molecular phenotypes paralleled findings in humans. We found that glial cell line-derived neurotrophic factor (GDNF) levels in the human colon vary about 5-fold and correlate strongly with nitrergic marker expression. In mice, we defined that GDNF levels are regulated via its 3' untranslated region (3' UTR) in the gastrointestinal tract and observed similar correlation between GDNF levels and nitrergic lineage development. We identified miR-9 and miR-133 as evolutionarily conserved candidates for negative regulation of GDNF expression in the gastrointestinal tract. Functionally, an increase in inhibitory nitrergic innervation results in an increase in gastrointestinal tract transit time, stool size, and water content accompanied with modestly reduced epithelial barrier function. Mechanistically, we found that GDNF levels regulate nitrergic lineage development via induction of transcription factor ETV1, corroborated by single-cell gene expression data in human and mouse developing enteric neurons. Our results reveal how normal variation in GDNF levels influence ENS size, composition, and gut function, suggesting a mechanism for well-known interindividual variation among those parameters.

Neuroregeneration Improved by Sodium-D,L-Beta-Hydroxybutyrate in Primary Neuronal Cultures.

Ketone bodies are considered alternative fuels for the brain when glucose availability is limited. To determine the neuroregenerative potential of D,L-sodium-beta-hydroxybutyrate (D/L-BHB), Sprague Dawley rat primary cortical neurons were exposed to simulated central nervous system injury using a scratch assay. The neuronal cell migration, cell density and degree of regeneration in the damaged areas (gaps) in the absence (control) and presence of BHB (2 mM) were documented with automated live-cell imaging by the CytoSMART system over 24 h, which was followed by immunocytochemistry, labeling synapsin-I and β3-tubulin. The cell density was significantly higher in the gaps with BHB treatment after 24 h compared to the control. In the control, only 1.5% of the measured gap areas became narrower over 24 h, while in the BHB-treated samples 49.23% of the measured gap areas became narrower over 24 h. In the control, the gap expanded by 63.81% post-injury, while the gap size decreased by 10.83% in response to BHB treatment, compared to the baseline. The cell density increased by 97.27% and the gap size was reduced by 74.64% in response to BHB, compared to the control. The distance travelled and velocity of migrating cells were significantly higher with BHB treatment, while more synapsin-I and β3-tubulin were found in the BHB-treated samples after 24 h, compared to the control. The results demonstrate that D/L-BHB enhanced neuronal migration and molecular processes associated with neural regeneration and axonogenesis. These results may have clinical therapeutic applications in the future for nervous system injuries, such as for stroke, concussion and TBI patients.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Pathological characteristics of axons and alterations of proteomic and lipidomic profiles in midbrain dopaminergic neurodegeneration induced by WDR45-deficiency

BackgroundAlthough WD repeat domain 45 (WDR45) mutations have been linked to β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document}-propeller protein-associated neurodegeneration (BPAN), the precise molecular and cellular mechanisms behind this disease remain elusive. This study aims to shed light on the impacts of WDR45-deficiency on neurodegeneration, specifically axonal degeneration, within the midbrain dopaminergic (DAergic) system. We hope to better understand the disease process by examining pathological and molecular alterations, especially within the DAergic system.MethodsTo investigate the impacts of WDR45 dysfunction on mouse behaviors and DAergic neurons, we developed a mouse model in which WDR45 was conditionally knocked out in the midbrain DAergic neurons (WDR45cKO). Through a longitudinal study, we assessed alterations in the mouse behaviors using open field, rotarod, Y-maze, and 3-chamber social approach tests. We utilized a combination of immunofluorescence staining and transmission electron microscopy to examine the pathological changes in DAergic neuron soma and axons. Additionally, we performed proteomic and lipidomic analyses of the striatum from young and aged mice to identify the molecules and processes potentially involved in the striatal pathology during aging. Further more, primary midbrain neuronal culture was employed to explore the molecular mechanisms leading to axonal degeneration.ResultsOur study of WDR45cKO mice revealed a range of deficits, including impaired motor function, emotional instability, and memory loss, coinciding with the profound reduction of midbrain DAergic neurons. The neuronal loss, we observed massive axonal enlargements in the dorsal and ventral striatum. These enlargements were characterized by the accumulation of extensively fragmented tubular endoplasmic reticulum (ER), a hallmark of axonal degeneration. Proteomic analysis of the striatum showed that the differentially expressed proteins were enriched in metabolic processes. The carbohydrate metabolic and protein catabolic processes appeared earlier, and amino acid, lipid, and tricarboxylic acid metabolisms were increased during aging. Of note, we observed a tremendous increase in the expression of lysophosphatidylcholine acyltransferase 1 (Lpcat1) that regulates phospholipid metabolism, specifically in the conversion of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC) in the presence of acyl-CoA. The lipidomic results consistently suggested that differential lipids were concentrated on PC and LPC. Axonal degeneration was effectively ameliorated by interfering Lpcat1 expression in primary cultured WDR45-deficient DAergic neurons, proving that Lpcat1 and its regulated lipid metabolism, especially PC and LPC metabolism, participate in controlling the axonal degeneration induced by WDR45 deficits.ConclusionsIn this study, we uncovered the molecular mechanisms underlying the contribution of WDR45 deficiency to axonal degeneration, which involves complex relationships between phospholipid metabolism, autophagy, and tubular ER. These findings greatly advance our understanding of the fundamental molecular mechanisms driving axonal degeneration and may provide a foundation for developing novel mechanistically based therapeutic interventions for BPAN and other neurodegenerative diseases.