Molecular Neurobiology

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

Background/ObjectivesNeuroinflammation-driven iron dysregulation and neurotoxic astrocyte polarization are increasingly recognized as interconnected pathological mechanisms in neurodegenerative diseases. Systemic inflammation triggered by strenuous exercise or infection can engage the central nervous system and astrocytic inflammatory responses and perturb iron homeostasis; however, targeted nutritional strategies to counteract these processes remain limited. Inflamate(R) is a multi-component botanical supplement comprising boswellic acids, astilbin, xanthohumol, and cinnamaldehyde, each with documented anti-inflammatory properties. However, whether this combined formulation can modulate the inflammatory-iron metabolic axis and astrocyte phenotypic polarization remains unexplored. This study aimed to investigate the effects of Inflamate(R) on LPS-induced pro-inflammatory gene expression, iron metabolism-related gene regulation, and A1/A2 astrocyte phenotypic polarization in mouse astrocytes. MethodsMouse astrocytes (AWT) were pre-treated with Inflamate(R) (0.0375 g/mL) or DMSO vehicle for 24 h, followed by lipopolysaccharide (LPS; 1 g/mL) stimulation for an additional 24 h. The non-cytotoxic working concentration was determined by morphological assessment, CCK-8 cell viability, and LDH cytotoxicity assays. Expression of 14 target genes spanning pro-inflammatory mediators (NOS2, IL6, C3, COX2, PLA2g15, SOCS3), iron metabolism regulators (FTH1, Hepcidin, TFRC, SLC40A1, RGMa, RGMb), and astrocyte polarization markers (S100A10, GFAP) was quantified by qRT-PCR. ResultsUnder normal culture conditions, Inflamate(R) did not significantly alter the expression of any target gene except S100A10, confirming the absence of baseline cytotoxicity or transcriptional homeostatic perturbation. Upon LPS stimulation, Inflamate(R) selectively suppressed NOS2 (approximately 64% reduction, p < 0.0001), IL6 (approximately 37% reduction, p < 0.0001), and C3 (approximately 47% reduction, p < 0.0001), while COX2, PLA2g15, and SOCS3 remained unaffected. Concurrently, Inflamate(R) significantly reduced LPS-induced Hepcidin expression to approximately 17% of the control level (p < 0.05) and attenuated FTH1 upregulation (p < 0.01), without altering the expression of iron transporters (TFRC, SLC40A1) or BMP-SMAD pathway components (RGMa, RGMb). Furthermore, Inflamate(R) upregulated the neuroprotective A2 marker S100A10 under both basal (p < 0.05) and LPS-stimulated conditions (p < 0.01), while the general reactivity marker GFAP remained unchanged. ConclusionsInflamate(R) exerts a selective, multi-target modulatory effect at the transcriptional level in LPS-stimulated astrocytes, encompassing suppression of the iNOS-NO and IL-6 signaling axes, attenuation of inflammation-driven hepcidin-ferritin iron dysregulation via the IL-6-STAT3 pathway, and promotion of a phenotypic shift from neurotoxic A1 toward neuroprotective A2 astrocyte polarization. Given that the IL-6-JAK-STAT3-hepcidin axis is also activated during exercise-induced systemic inflammation, these findings suggest that Inflamate(R) may represent a targeted nutritional strategy for preserving CNS iron homeostasis and supporting neuroprotective astrocyte function in both neurodegenerative and exercise-related neuroinflammatory contexts. Further validation in in vivo neurodegenerative and exercise models, including protein-level analyses, is warranted to confirm these transcriptional findings.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

Cav1.2 is an L-type voltage-gated Ca2+ channel (VGCC) that supports Ca2+ influx in response to membrane depolarization. Ca2+ entering via Cav1.2 alters gene expression, activates Ca2+-dependent enzymes and has been implicated in synaptic plasticity. ORL-1 is a Gi/o-coupled G protein-coupled receptor (GPCR) that is expressed in the peripheral and central nervous systems. Both Cav1.2 and ORL-1 are expressed in the hippocampus, where they have been implicated in learning and memory. It is well-documented that ORL-1 interacts with another VGCC, Cav2.2. However, less is known about potential interactions between Cav1.2 and ORL-1. Here, we examine the interplay between Cav1.2 (Cav1c, Cav2{delta}-1, Cav{beta}1) and ORL-1 co-expressed in tsA-201 cells by using biochemical, electrophysiological and confocal imaging analysis. Co-immunoprecipitations revealed that ORL-1 independently interacts with Cav1c and Cav2{delta}-1 subunits of the Cav1.2 channel complex. Electrophysiological recordings revealed that co-expression with ORL-1 reduced Cav1.2 peak current density without altering its biophysical properties. Acute perfusion with the ORL-1 receptor agonist nociceptin (1 M) did not alter Cav1.2 current density. Confocal imaging experiments revealed that ORL-1 significantly decreases Cav1.2 plasma membrane expression by disrupting forward trafficking. Interestingly, ORL-1 did not affect Cav1.2 endocytosis. Overall, our results demonstrate a previously unrecognized interaction between ORL-1 and Cav1.2 that alters Cav1.2 membrane expression without affecting biophysical properties.

BackgroundBeyond disrupted neurodevelopment, autism spectrum disorder (ASD) encompasses progressive neurodegenerative alterations that remain refractory to standard of care behavioral therapies, highlighting the need for biologically targeted adjunctive interventions. Although, Mesenchymal stem cell (MSC) derived small extracellular vesicles (sEVs) have emerged as potent neuroprotective agents; however, the optimal MSC tissue source and strategies enhancing their efficacy for clinical translation remain undefined. Hypoxic preconditioning potentiates their therapeutic effects, however its specific impact on ASD-related impairments is yet to be established. This study conducts a head to head evaluation of sEVs from bone marrow (BM) and Whartons jelly (WJ) MSCs under normoxic and hypoxic conditions to delineate their comparative neuroregenerative efficacy in ASD. MethodsWe investigated the neuroprotective efficacy of sEVs derived from human tissue-specific mesenchymal stem cells (BM/WJ) cultured under normoxic (21% O2) and hypoxic (1% O2) conditions. In vitro assays evaluated sEV uptake, cell proliferation, neurodifferentiation, mitochondrial health, and immunomodulatory effects using neural stem cell (C17.2) and microglial (N9) cell lines. For in vivo studies, ASD-like phenotypes were induced in C57BL/6 mice via prenatal exposure to valproic acid (600 mg/kg), followed by the biodistribution of fluorescently labeled hypoxia-primed WJ MSCs sEVs (WJ-H-sEVs) following intranasal administration was assessed using IVIS imaging. Behavioural outcomes after intranasal treatment were evaluated. Additionally, post-mortem brain tissues were analyzed for neuroinflammation, oxidative stress, synaptic integrity, and downstream signalling pathways using immunofluorescence, Western blotting, and qRT-PCR, while systemic cytokine pro-inflammatory and anti-inflammatory levels were quantified by ELISA. Mechanistic involvement of Nrf2 and NF-{kappa}B signaling pathways was examined using pharmacological inhibitors. ResultsComparative analyses revealed that hypoxic priming significantly enhanced sEV yield and enriched neuroprotective miRNAs, with WJ-H-sEVs exhibiting elevated levels of miR-125b-5p, miR-21a-5p, and miR-145a-5p. Consistent with this enrichment, WJ-H-sEVs demonstrated superior cellular uptake as observed in neural stem cells and microglia, enhanced neurodifferentiation, robust antioxidant activity, effective immunomodulation, and improved mitochondrial health, thereby outperforming their normoxic and BM-derived counterparts. Intranasally administered WJ-H-sEVs efficiently localized to the brain within 12 h and significantly improved anxiety-like behavior, recognition memory, and spatial learning in ASD mice. These effects were accompanied by reduced neuroinflammation and oxidative damage, enhanced neuronal survival, decreased serum IL-6 levels, and increased IL-10 and TGF-{beta}. Mechanistically, therapeutic benefits were mediated through activation of the Nrf2 antioxidant pathway and suppression of NF-{kappa}B and JAK-STAT signaling. ConclusionIn this study, hypoxia emerges as a clinically exploitable priming strategy to enhance the neuroregenerative performance of tissue-specific MSC-derived sEVs, with potential applicability across MSC sources and other neurological disorders. Moreover, intranasal delivery of WJ-H-sEVs demonstrates translational feasibility, reinforcing their potential as an adjunctive, cell-free, pediatric-friendly neurotherapeutic modality for ASD management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/721255v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@dd0f7forg.highwire.dtl.DTLVardef@1247d24org.highwire.dtl.DTLVardef@93e6deorg.highwire.dtl.DTLVardef@1a349d_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDysregulated peptidyl deiminase 2 (PAD2) and aberrant protein citrullination (PC), a posttranslational modification (PTM), are involved in various inflammatory and neurodegenerative diseases. We previously showed in transgenic mice and postmortem human tissues that PC and PAD2 are altered in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neurons loss, paralysis, and death. Herein, we investigated the role of PAD2 in ALS by PAD2 knockout in a SOD1-ALS mouse model. MethodsTo investigate the role of PAD2-induced citrullination in ALS pathogenesis, we generated PAD2 knockout (PAD2KO) in SOD1G93A ALS mouse model and investigated the consequent modulation on the neuropathology and clinical symptoms, using molecular biology techniques such as qPCR, Western blotting, confocal microscopy, and electron microscopy. Additionally, we identified C3 as being citrullinated in human ALS using ionFinder. ResultsOur results show that PAD2KO blocked the increased PC and reduced myelin basic protein (MBP) aggregates in the ALS model. PAD2KO also improved motor neuron survival and the integrity of myelin, axons, and neuromuscular junctions, and reduced microgliosis in the white matter and C3 protein levels in astrocytes. Clinically, data from monitoring the body weight changes suggests that PAD2KO modulates the course of the disease in the ALS mouse model, accelerating the onset while slowing the progression after the onset, and modestly extending the survival of male mice. ConclusionThese results show that PAD2 is responsible for the increased PC in ALS and PC contributes to neuroinflammation and degeneration of motor neurons and myelinated axons. The modest modulation of the disease phenotype suggests that the role of PC in ALS is complex, involving altered PC in numerous proteins and in multiple cell types. Future studies are needed to investigate how PC modulates individual protein functions in various cell types to understand the contribution of PC to ALS pathogenesis.

BACKGROUNGBisphenol A (BPA) has been linked to hypertension and disturbances in lipid metabolism; however, limited evidence is available regarding its association with hypertensive intracerebral hemorrhage (ICH). METHODSA multicenter, retrospective case-control study was conducted involving 129 participants, including individuals from an ICH group and healthy controls. Standard assays were employed to assess serum thyroid function, lipid profiles, serum fatty acid-binding [x]protein 4 (FABP4), oxidative stress markers, gap junction proteins, Wnt/{beta}-catenin signaling pathway activity, and expression changes of S100A8-mediated inflammatory cytokines involved in gut-brain interactions. Correlation analyses using Pearson and Spearman methods revealed that both BPA exposure and low T3 levels were significantly associated with elevated diastolic blood pressure, altered lipid metabolism, gut microbiota composition, and microglial activation. RESULTSGender-based disparities in lipid metabolism were identified. Changes in {beta}3-adrenergic receptor and neuromodulin-1 expression appear to influence fat regulation and attenuate oxidative stress responses. Subsequently, increased expression of gap junction proteins and activation of the Wnt/{beta}-catenin signaling pathway contribute to metabolic reprogramming and alterations in biochemical kinetics. Gut microbiota analysis demonstrated that, compared to controls, the ICH group exhibited significant dysbiosis and reduced alpha diversity. Further correlation analyses indicated that BPA levels were positively associated with FABP4 and oxidative stress markers, while S100A8 showed a strong dependence on microglial expression. CONCLUSIONThe interplay between lipid metabolism dysfunction and pro-inflammatory cytokines enhances vascular vulnerability. Collectively, BPA exposure, oxidative stress, and microglia-mediated neuroinflammation are significantly associated with an elevated risk of hypertensive ICH. China Clinical Trial Registry registration noticeFrom: China Clinical Trials Registry <chictr@vip.qq.com>+To:guopingwang60a<guopingwang60a@163.com> yunyanshuangfei <yunyanshuangfei@126.com> FUNDINGThis work was supported by the Natural Science Foundation of Shanxi Province (grant no. 201701D121177) Key informationGender-specific differences were observed in lipid metabolism and oxidative stress parameters; BPA exposure was shown to induce lipid metabolic disturbances, promote excessive production of oxidative stress byproducts, and consequently elevate oxidative stress responses; BPA was associated with stress-induced alterations in thyroid hormone function, further exacerbating dysregulation of lipid metabolism and oxidative stress; Fatty acid binding protein 4 (FABP4), a key adipokine implicated in metabolic disorders and adipose tissue inflammation, exhibited a significant positive correlation with serum BPA levels, whereas low levels of triiodothyronine (T3) were negatively correlated with FABP4. These findings suggest that serum FABP4 may serve as a biochemical marker for chronic low-grade adipose tissue inflammation and metabolic dysfunction; Gap junction proteins and the Wnt/{beta}-catenin signaling pathway may contribute to microglial activation and mediate neuroinflammatory responses, nerve injury, and secondary pathological processes in obesity-related cerebral hemorrhage.

PurposeTo investigate whether rapamycin can regulate microglial activation and polarization via mTOR and its downstream signals via autophagy both in vivo and in vitro. MethodsThe in vivo study used wild type C57BL/6 mice that were intraperitoneally injected with rapamycin (2 mg/kg) plus ONC. The BV2 cell line was used in the in vitro study and the cells were incubated with rapamycin (50 nM) or transfected with a specific mTOR-targeting small interfering RNA (si-mTOR). Immunohistochemical staining was used to observe the changes in the morphology and cell surface area of microglia and Weste blotting analysis was used for detection of the changes in the proteins related autophagy, microglia polarization and mTOR pathway after the retinal tissue or the cell samples were collected. ResultsThese results indicate that rapamycin increases autophagy and M2 polarization by inhibiting p-mTOR in wild-type C57BL/6 mice in vivo. In the BV2 cell line, rapamycin and si-mTOR can enhance autophagy and promote M2 polarization by inhibiting the p-mTOR/p-Unc-51-like kinase 1 (p-ULK1) pathway. ConclusionsIn conclusion, this work contributes to the understanding of the complex interplay among rapamycin, autophagy and microglial activation/polarization, highlights the downstream signaling pathway of mTOR, and highlights the potential therapeutic effects of autophagy-modulating drugs in retinal neuroinflammation and neurodegeneration after TON.

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

Social isolation is commonly used to model social stress and is a known risk factor for depression, with impacts on hippocampal function and postnatal neurogenesis. However, most studies focus on social isolation in juvenile mice isolation during adolescence, leaving the effects of prolonged adult isolation less understood. Post-transcriptional regulation of gene expression by microRNAs (miRNAs) plays a role in hippocampal function, and altered miRNA, as well as gene expression, has been reported in the hippocampus of mice exposed to social isolation. A single-nucleotide polymorphism in miR-30e in humans is associated with increased expression of the mature miRNA, impaired cognition, electroencephalogram waveform latency, depression, and schizophrenia. We investigated whether adult isolation in mice alters gene regulation via microRNAs, particularly miR-30e-5p, and affects hippocampal function. In adult BALB/c male mice, 10 weeks of isolation increased miR-30e-5p expression in the ventral hippocampus, reduced its target gene Neurod1, and impaired hippocampal-dependent cognition (object pattern separation), without clear anxiety- or depression-like behaviours. Isolated mice also showed a blunted response to acute stress. These findings suggest that adult social isolation affects hippocampal function through post-transcriptional gene regulation, highlighting a role for miR-30e-5p in neurogenesis and cognition in response to psychological stress.

Background: Norepinephrine (NE) released from locus coeruleus (LC) projections regulates astrocyte structure and function through adrenergic receptor signaling. We previously showed that increasing noradrenergic tone with the NE reuptake inhibitor desipramine increases astrocyte ramification in the molecular layer of the dentate gyrus. However, whether tonic LC-derived noradrenergic tone is required to maintain astrocyte morphological complexity in vivo, and whether {beta}-adrenergic receptor activation is the effector pathway, remained unclear. Methods: Adult male C57BL/6J mice received DSP-4 (50 mg/kg X 3 days i.p.), a selective LC neurotoxin, with or without concurrent isoproterenol that continued for 21 additional days post cessation of DSP-4 treatment (ISO; 2 mg/kg/day X 24 days), or saline (n = 4 mice per group). Animals were sacrificed 22 days after the final DSP-4 injection. Noradrenergic denervation was confirmed by dopamine {beta}-hydroxylase (DBH) immunostaining. GFAP-immunostained astrocytes in the molecular layer of the dentate gyrus were morphologically characterized using Sholl analysis. Astrocyte density was quantified by SOX9 immunostaining. Results: DSP-4 produced >83% reduction in DBH fiber coverage in the molecular layer. Sholl analysis revealed significant reductions in astrocyte branching complexity in both treatment groups, with the reductions concentrated at distances of 5-15 m from the soma. The maximum number of intersections was also significantly reduced in both groups. Unexpectedly, ISO did not rescue morphological complexity. While DSP-4 alone did not alter astrocyte density, as measured by the number of SOX9-expressing astrocytes, DSP-4+ISO increased SOX9-positive cell density, dissociating the effects of adrenergic signaling on morphology from those on cell numbers. Conclusions: LC-derived noradrenergic tone is required for the maintenance of astrocyte arbour complexity in the dentate gyrus molecular layer. {beta}-adrenergic receptor activation alone is insufficient to restore structural integrity following noradrenergic denervation, yet promotes astrocyte density independently of structural remodeling. These findings have implications for understanding how LC neurodegeneration in Alzheimer's disease and depression may compromise hippocampal astrocyte structure and function.

ObjectiveCisplatin, a life-saving chemotherapeutic drug, causes ototoxicity. Although sodium thiosulfate is used to prevent ototoxicity in pediatric patients, no other intervention has been approved for clinical use against cisplatin-induced hearing loss. Hence, there is an urgent need to identify drugs that prevent cisplatin ototoxicity. MethodsCBA/J mice were treated with cisplatin (3 mg/kg, i.p., daily for 5 days), and MnTBAP (10 mg/kg, i.p., daily for 8 days) was used to inhibit cisplatin-induced ototoxicity. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were recorded before and after treatment to assess hearing loss, while immunohistochemistry was performed to examine hair cells and spiral ganglion neuron (SGN) loss. ResultsCisplatin treatment elevated the nitrotyrosine levels in hair cells and SGNs and increased the loss of these cells in the middle and basal cochlear regions. A negative correlation was observed between cisplatin-induced changes in the hair cell count or SGN density and nitrotyrosine levels. Cisplatin elevated the hearing thresholds and lowered the DPOAE amplitudes. However, MnTBAP cotreatment prevented the cisplatin-induced changes in the hearing sensitivity and reversed the morphological changes. ConclusionThe otoprotection observed with MnTBAP cotreatment indicates its potential as a therapeutic drug against cisplatin-induced ototoxicity.

BackgroundGut dysbiosis has been increasingly implicated in post-stroke cognitive impairment (PSCI), yet the causal contribution and therapeutic potential of gut microbiota-derived metabolites remain unclear. This study aimed to identify key microbiota-derived metabolites involved in PSCI and to elucidate their underlying mechanisms. ResultsWe found that both PSCI patients and middle cerebral artery occlusion (MCAO) mice exhibited distinct gut microbial alterations, characterized by a marked reduction in tryptophan-metabolizing bacteria and indole-3-propionic acid (IPA), a gut microbiota-derived tryptophan metabolite. Exogenous IPA administration alleviated PSCI-like phenotypes in MCAO mice. Mechanistically, IPA preserved tyrosine hydroxylase-positive (Th) fibers and catecholamine levels in the dorsal hippocampus. Further analyses showed that IPA binds to the adaptor protein Ywhab, promotes ERK activation, and enhances neuronal survival, thereby counteracting neuronal apoptosis-associated inflammation and subsequent Th fiber degeneration. ConclusionThese findings identify IPA as a gut microbiota-derived neuromodulator that mitigates PSCI by preserving dorsal hippocampal catecholaminergic transmission. IPA may therefore serve as a promising predictive biomarker and therapeutic candidate for PSCI.

The ultimate cause of blindness in glaucoma is the death of retinal ganglion cells, and understanding the mechanism behind retinal ganglion cell loss during glaucoma could lead to the development of novel treatments for glaucoma. Endothelin-1 has been shown to mediate retinal ganglion cell death during glaucoma through impairment of mitochondrial function. Retinal ganglion cells are highly metabolically active, and susceptible to oxidative damage and decreased respiratory capacity. Mitophagy is the process whereby damaged mitochondria are degraded to prevent further propagation of oxidative damage. The current study evaluates the effect of endothelin-1 on mitophagy in retinal ganglion cells. Electron microscopy revealed endothelin-1 administration lead to a decrease in healthy mitochondria in the optic nerve. The MitoQC mouse was used to evalute mitophagy in response to endothelin-1, along with immunohistochemical analysis of mitophagy proteins. Mitophagy follows different trends in the optic nerve and retinal ganglion cell bodies following endothelin-1 administration, mitophagy was increased in the optic nerve but decreased in the retina following endothelin administration. With elevation of intraocular pressure, mitophagy was increased in the retina but decreased in the optic nerve. In retinal ganglion cells, parkin expression and activation was unchanged 24 hours after endothelin-1 administration, but was decreased 72 hours following endothelin-1 administration. Taken together, these results suggest that endothelin-1 impacts mitophagy through parkin-independent mechanisms in retinal ganglion cell bodies, and the ganglion cell bodies and optic nerve appear to have different responses to endothelin-1.

Heterozygous de novo missense mutations in the EEF1A2 gene encoding translation elongation factor eEF1A2 result in neurodevelopmental disorders, typically characterised by early onset epilepsy and intellectual disability (ID). The E122K mutation is the most commonly reported missense mutation and is amongst the more severe in terms of epilepsy and ID. Here we made use of a recently developed mouse model which recapitulates the E122K mutation to examine how mutations in EEF1A2 might disrupt neuronal gene expression. Primary neurons from mutant mice and transfected HEK293T cells were used to examine effects on global protein synthesis. In contrast to previous reports, we were unable to detect a change in global protein synthesis using either of two different assay systems. TRAP-seq and mass spectrometry were then employed to study the effects of the mutation on the translatome and proteome respectively. These analyses revealed perturbation of expression of a subset of genes, with a slight skew towards downregulation, particularly for longer transcripts. Further analysis indicated a down regulation of proteins involved in synaptic function in both the translatomic and proteomic datasets.

Purinergic 2X7 receptor (P2X7R) is considered to play a critical role in neurological diseases, including epilepsy, and has also been proposed as a potential marker for neuroinflammation. This study aimed to validate the binding properties of the novel P2X7R radiotracer, [3H]JNJ-64413739, in rat brain using in vitro autoradiography, and additionally to explore spatial and temporal changes in P2X7R binding levels in a rat model of temporal lobe epilepsy using intrahippocampal administration of kainic acid (KA). Saturation of [3H]JNJ-64413739 to brain sections yielded a KD of approximately 3 nM, with full saturation around 10 nM. The radiotracer was displaced with a structurally different P2X7R ligand, JNJ-47965567, indicating high affinity and specificity to rat P2X7R. In post epileptic rats, region-specific [3H]JNJ-64413739 binding revealed a bilateral increase in the hippocampal formation and its subregions few days after status epilepticus, peaking at day 30, and remained stable at this high level until day 90. Similar temporal profiles were identified in subcortical regions such as the thalamus. Interestingly, no change in binding was observed in the temporal and piriform cortices until day 30 where a dramatic increase occurred. Also, in the corpus callosum, significant increase was detected 30 days after the seizure. These results show that P2X7R binding, likely reflecting inflammation, is increased at delayed time points and exhibit region-specific patterns that is different from acute effects. Our findings suggest that P2X7R may contribute to sustained neuroinflammation and may be involved in those changes leading to epileptogenesis and the development of chronic epilepsy. Highlights[3H]JNJ-64413739 binds specifically to the purinergic P2X7 receptor (P2X7R) and saturates in the rat brain. P2X7R binding increases in a region- and time-dependent manner following status epilepticus. P2X7R binding remains elevated during chronic epilepsy in all examined brain regions. P2X7R is considered a link between early seizures and sustained neuroinflammation and epileptogenesis.

Neurodegeneration is a major determinant of disability progression in multiple sclerosis (MS), yet the pathophysiological mechanisms associating inflammation to neuronal insult remain poorly understood. We recently identified Dendritic Syntaphilin Intrusion (DSI), a novel excitoxicity pathway in which the axonal mitochondrial anchor syntaphilin (SNPH) aberrantly translocates into dendrites, causing neurodegeneration in a non-inflammatory model of MS. However, whether this protein intrudes abruptly into dendrites in inflammatory MS pathology is still not clear. Here, we investigated the role of synaptic and extrasynaptic NMDA receptors (NMDAR) in regulating the intrusion of Syntaphilin into dendrites. Using primary hippocampal neuronal cultures, we examined how the balance between synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs influences DSI under inflammatory conditions. Pharmacological and viral-mediated approaches were employed to manipulate NMDAR subtype activity and evaluate their impact on DSI. Inflammatory cytokines discernibly sensitized neurons to DSI. Our results revealed that blockade of synaptic NMDARs significantly increased DSI, whereas inhibition of extrasynaptic NMDARs reduced DSI. These findings demonstrate opposing roles of NMDAR subtypes, with GluN2A-containing synaptic receptors inhibiting DSI and fostering neuronal survival, while GluN2B-containing extrasynaptic receptors enhancing DSI and neurodegenerative signaling. Manipulation of the GluN2A/GluN2B balance showed opposite effect on DSI, suggesting a relationship between NMDAR subtype signaling and SNPH mislocalization. Overall, our findings extend the relevance of DSI from non-inflammatory MS to inflammatory MS and identify DSI as a downstream convergence point linking inflammatory cytokines and excitotoxic NMDAR signaling to neuronal insult. These results reveal DSI as a potential mechanistic link between inflammatory signaling and excitotoxic neuronal injury and indicate that modulation of GluN2B-dependent pathways warrants further investigation in inflammatory neurodegenerative disorders.

Familial Amyotrophic Lateral Sclerosis (fALS) is a fatal neurodegenerative disease, mainly caused by mutations in the superoxide dismutase 1 (SOD1) protein. Mitochondrial dysfunction is a primary hallmark of ALS pathogenesis. However, the molecular mechanism by which SOD1 mutants impair organellar health remains enigmatic. This study demonstrates that mutant SOD1 associates with the TIM23 complex in Saccharomyces cerevisiae via its intermembrane space (IMS) domain. In ALS-associated SOD1 mutants, both binding and expression of TIM23 complex proteins were downregulated, leading to altered translocation of the substrate protein Sdh3, a component of the electron transport chain (ETC) complex II. Disrupted Sdh3 translocation leads to mitochondrial dysfunction, evidenced by decreased ETC complex II activity, reduced functional mass, and compromised organelle integrity. Overexpression of Tim23 partially rescued mitochondrial integrity by increasing ETC complex activity and functional mass and restoring reticular morphology. Strikingly, the improved mitochondrial homeostasis in Tim23-overexpressing cells partially rescued the growth defects caused by mutant SOD1. Collectively, these findings reveal a previously unrecognized regulatory axis between mutant SOD1 and the mitochondrial pre-sequence translocase machinery, highlighting this pathway as a promising target for future ALS therapies and opening new avenues for mechanistic and translational research. Author SummaryFamilial Amyotrophic Lateral Sclerosis (fALS) is a progressive, fatal neuromuscular disorder marked by motor neuron degeneration. The exact cause of ALS remains unclear. Previous research links familial ALS to mutations in the superoxide dismutase 1 (SOD1) gene. SOD1 mutants in ALS disrupt mitochondrial protein translocation, a key mitochondrial process. The mechanism by which SOD1 mutants affect mitochondrial function and integrity by modulating presequence translocase (TIM23 complex) import is not yet understood. The current study addresses a critical gap in ALS research by demonstrating a novel, direct interaction between SOD1 and Tim23 that regulates mitochondrial function in yeast. We found that SOD1 binds Tim23 via Tim23 IMS domain, stabilizes the Tim23CORE complex, enabling Sdh3 import. Loss of SOD1, Tim23, or Tim50 destabilizes the TIM23CORE complex, leading to impaired Sdh3 import and decreased ETC complex-II activity. These changes disrupt mitochondrial structure, causing fragmentation and a loss of functional mitochondrial mass in {Delta}sod1. ALS-linked SOD1 mutants show similar effects: they diminish Sdh3 import by weakening SOD1-Tim23 interaction and lowering TIM23 complex stability, resulting in punctate mitochondria and reduced mitochondrial mass. Collectively, our study identifies the SOD1-TIM23 interaction as a key regulator of mitochondrial health through Sdh3 import via the TIM23CORE complex and indicates this pathway as a potential early intervention target for ALS therapy.

We showed before that the endogenous peptide Nociceptin blocks the premature differentiation of oligodendrocytes (OLGs), preventing untimely precocious myelination in the developing brain. Consistent with this early function, Nociceptin brain expression is developmentally regulated, sharply decreasing with the initiation and progression of myelination. However, we now found that at difference with controls and relapsing-remitting multiple sclerosis (RRMS), Nociceptin levels are highly elevated in cerebrospinal fluid from patients with the most severe progressive MS (PMS) forms. This questioned whether Nociceptin early developmental effects could be latter recapitulated, interfering with remyelination in PMS. This possibility was tested by inducing experimental autoimmune encephalomyelitis in older mice, at an age equivalent to that with increased risk of RRMS transition into PMS. Older animals develop persistently highly debilitating clinical symptoms, and display both brain and spinal cord demyelination. Importantly, these mice exhibit elevated brain Nociceptin levels, and their treatment with an antagonist of the Nociceptin receptor (NOR) elicits a regression of clinical scoring that is accompanied by higher ratios of OLGs/OLG progenitor cells, increased myelination, and reduction of reactive astrocytes. These findings suggest that Nociceptin may be a crucial player in the age-related progression of MS; interfering with OLG maturation and remyelination, and perhaps further exacerbating neurological dysfunction by targeting astrocyte populations. The upregulation of Nociceptin secretion by human astrocytes in response to proinflammatory cytokines, also points to this peptide as a mediator of microglia-astrocyte interactions supporting MS progression with aging. NOR may offer a novel pharmacological target for ameliorating the devastating effects of MS progression.

Endosomal sorting complexes required for transport (ESCRT) regulate membrane protein trafficking through coordinated cargo selection and endosomal processing, yet their contribution to neurotransmitter receptor sorting remains to be defined. Here, we examined how modulation of distinct complex components influences the surface expression of excitatory and inhibitory neurotransmitter receptors. Using surface biotinylation and imaging approaches in heterologous cells and primary neurons, we altered tumour susceptibility gene 101 (TSG101), a core complex I component, and vacuolar protein sorting-associated protein 4A (VPS4a), an ATPase required for complex III disassembly. Reduction of tumour susceptibility gene 101 increased receptor association with early endosomes and enhanced receptor surface localisation, whereas disruption of VPS4A promoted receptor accumulation within late endosomal compartments and impaired degradative progression. Inhibitory receptor subtypes displayed variable sensitivity. Together, these findings demonstrate that endosomal sorting complex components regulate receptor surface expression through stage-specific trafficking mechanisms associated with altered receptor recycling and degradative processing. Graphical abstractDistinct ESCRT components regulate neurotransmitter receptor trafficking through stage-specific control of the endosomal pathway. ESCRT-I disruption promotes early endosomal retention and recycling, whereas ESCRT-III impairment causes late endosomal accumulation and reduced degradation, together increasing receptor surface expression (created using Biorender). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732891v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1fe66b9org.highwire.dtl.DTLVardef@10a29d7org.highwire.dtl.DTLVardef@4109c4org.highwire.dtl.DTLVardef@1e84f19_HPS_FORMAT_FIGEXP M_FIG C_FIG