Molecular Neurobiology

Oligodendrocytes play an essential role in axonal metabolic support and myelination, and their dysfunction is associated with amyotrophic lateral sclerosis (ALS). LINGO1 is a major inhibitor of oligodendrocyte differentiation and axonal regeneration, identified as a promising therapeutic targeted for inflammatory demyelinating diseases. Here, we investigated the expression pattern of LINGO1 in the well-established SOD1(G93A) transgenic mouse model of ALS. We show that LINGO1 protein expression is significantly elevated in both grey and white matter of the mutant spinal cord during both the symptomatic and end stages of the disease. LINGO1 is also notably co-expressed with astroglial and neuronal markers. However, neither the levels of LINGO1 transcripts, nor levels of microRNAs predicted to regulate its expression were altered in the mutant spinal cord, suggesting the involvement of alternative regulatory mechanisms. Our analysis of microRNAs revealed in the mutant a significant downregulation of miR-138, a microRNA known to promote myelination. The differential expression of LINGO1 and miR-138 in ALS mice thus may drive deleterious processes during disease progression in adulthood. Moreover, our data show that white-matter pathology arises earlier than initially thought, as evidenced by significant alterations of oligodendrogenesis that manifest as early as postnatal day 5. Study of the molecular determinants of this developmental defect shows increased miR-138 expression, which may promote oligodendrocyte progenitor cell maturation and involve compensatory mechanisms. Overall, our work identifies disrupted expression of LINGO1 and/or miR-138 in brain during early oligodendrogenesis and at symptomatic disease stages. These findings open the way to further investigations that may consider miR-138 as a potential presymptomatic biomarker and LINGO1 as a therapeutic target to promote remyelination in the context of ALS.

Background: Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by deficits in social communication and interaction, and restricted interests or repetitive behaviors. ASD is approximately four times more prevalent in males than in females. In this study, we investigated whether sex hormones or sex chromosomes underlie the male bias in ASD susceptibility. Methods: We used the MG4E mouse model, in which microglial eIF4E overexpression produces robust male-biased ASD-like phenotypes. To distinguish the contributions of sex hormones and sex chromosomes, MG4E mice were crossed with four-core-genotype (FCG) mice carrying Sry gene manipulations, generating eight genotypes of experimental mice. Social interaction and repetitive behaviors were assessed using standard behavioral assays. Dendritic spine density was quantified in Thy1-GFP mice. Expression of estrogen receptors (ERs) and androgen receptor (AR) was examined in microglia isolated from control and MG4E mice. Results: Sex hormones, rather than non-Sry genes on sex chromosomes, are responsible for the male-biased deficits in social interaction and the increase in dendritic spine density. At postnatal day 14, ERs were undetectable in microglia, whereas ER expression was readily detected in neurons. Conclusions: These findings demonstrate that sex hormones are a major determinant of the increased susceptibility of males to ASD-like phenotypes in MG4E mice. The absence of ER expression in microglia suggests that sex hormones may act indirectly through hormone-responsive neurons to regulate microglia-neuron interactions during neurodevelopment.

The inability of oligodendrocyte progenitor cells (OPCs) to mature into myelin-making oligodendrocytes (OLs) is a major contributor to disease and disability in multiple sclerosis (MS). Oligodendrocyte maturation is a tightly controlled process with a strong reliance on epigenetic regulation involving DNA methylation and hydroxymethylation. We have previously shown that one carbon metabolism is dysregulated in MS, specifically the methyl donor betaine is depleted in the MS brain. Betaine donates methyl groups to betaine homocysteine methyltransferase (BHMT) in the methionine cycle to increase S-adenosylmethionine (SAM) for epigenetic methylation processes. In the present study we tested the effects of activating the BHMT methylation pathway on preventing MS pathology. We describe a novel mechanism mediated by BHMT and the Ten-eleven translocator enzyme (TET1) that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). We show that this pathway supports oligodendrocyte metabolism to enhance myelin and reduce clinical disability in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. ChIP-seq studies show that BHMT is enriched at genes involved in OPC metabolism and proximal ligation assays (PLAs) demonstrate that BHMT interacts with TET1 on chromatin. This interaction regulates gene expression programs that support a shift in OPC metabolism to glycolysis during neuroinflammatory processes. These data highlight the critical role of methionine metabolism in supporting myelination and have important implications for the development of new therapeutic strategies for MS and other neurodegenerative diseases.

Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by the loss of dopaminergic (DA) neurons and alpha-synuclein aggregation, with ferroptosis playing a critical pathological role. This study investigated the neuroprotective potential of Kocuria rhizophila strain CDMP12, a marine bacterium isolated from the Gulf of Mannar, India, using Caenorhabditis elegans models of PD. Dietary supplementation with K. rhizophila (CDMP12) significantly preserved DA neuron structure, rescued neuro-sensory and motor deficits, and attenuated both alpha-synuclein expression in the C. elegans models. Transcriptomic and qRT-PCR analyses revealed that CDMP12 systematically suppressed ferroptosis by significantly downregulating iron and lipid regulatory genes such as smf-3, ftn-1, and acs-4, while upregulating the protective antioxidant gene gpx-1. Furthermore, BODIPY staining demonstrated that CDMP12 treatment markedly reduced lipid peroxidation, lowering the oxidized-to-non-oxidized lipid ratio in PD worms. Collectively, these findings identify K. rhizophila (CDMP12) as a promising marine-derived neuroprotective candidate that mitigates PD-associated pathology, accompanied by reduced alpha-synuclein burden, preservation of DA neuronal function, and attenuation of ferroptosis-associated molecular and lipid peroxidation signatures.

PurposePerioperative exposure to the volatile anesthetic isoflurane (ISO) has been associated with cognitive and olfactory deficits and may increase the risk of Alzheimers disease (AD). Apolipoprotein E4 (APOE4), the strongest genetic risk factor for AD, contributes to disease pathogenesis through disrupted lipid homeostasis. However, whether and how isoflurane interacts with APOE genotype to influence neurological vulnerability remains unclear. MethodsYoung adult, presymptomatic humanized APOE4 and APOE3 knock-in mice underwent laparotomy under 2 h of isoflurane anesthesia. Microglia and astrocytes were isolated from the olfactory bulb (OB) and hippocampus (HI) by magnetic-activated cell sorting. Lipid composition, transcriptional responses, and functional outcomes were assessed using lipidomic, bulk RNA-seq, and longitudinal behavioral testing. In vivo and ex vivo electrophysiological recordings evaluated neuronal excitability and synaptic transmission in both regions. ResultsBy day 7 post-anesthesia, cell type-specific lipidomic profiling of both OB and HI revealed more pronounced lipid perturbations in microglia and astrocytes from APOE4/ISO mice than from APOE3 mice, characterized by elevated free fatty acids, increased lipid peroxidation, triglyceride depletion, and reduced hippocampal hexosylceramides and cardiolipins. Electrophysiological recordings showed greater olfactory circuit dysfunction in APOE4/ISO mice, accompanied by persistent odor memory deficits, transient olfactory sensitivity loss, early motor coordination impairments, and delayed cognitive deficits. RNA sequencing of the OB identified downregulated lipid metabolism and atherosclerosis-related pathways. ConclusionThese findings establish a mechanistic link between APOE4-dependent glial lipid dysregulation, olfactory circuit dysfunction, and delayed cognitive impairment following isoflurane anesthesia and surgery, highlighting lipid homeostasis as a potential therapeutic target.

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

Spinal muscular atrophy (SMA) is increasingly recognized as a multisystem disorder involving non-neuronal cells, yet the role of Schwann cells (SCs) in late-onset SMA (loSMA) remains unclear. We investigated age-dependent peripheral nerve pathology in a four-copy SMN2 mouse model of loSMA. Sciatic nerves from wild-type and loSMA mice were analyzed at postnatal (P) days 20, 35, 70, and >100 using semi-thin morphometry, immunofluorescence for MBP, Sox10, Sox2, and F4/80, and nerve conduction studies. loSMA nerves showed reduced myelin thickness at all time points and smaller axon diameters at P20 and P35. G- ratios were reduced at P20 but increased from P35 onward, indicating progressively altered axon-myelin relationships. MBP immunofluorescence intensity, compound muscle action potential amplitude, and nerve conduction velocity were reduced in loSMA mice at P>100. The proportion of Sox2+ SCs increased from P35 onward, while Sox10+ cell abundance increased at later stages. F4/80+ macrophages were transiently elevated at P35 and correlated with Sox2+ cell numbers at this stage. These findings demonstrate age-dependent myelin abnormalities, altered SC states, and transient accumulation of macrophages in loSMA peripheral nerves. Whether these changes are SC-autonomous or secondary to chronic axonal dysfunction remains to be determined.

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder caused by loss-of-function mutations in the MECP2 gene and characterized by profound impairments in neuronal maturation and synaptic connectivity. Increasing evidence indicates that astrocyte dysfunction contributes to RTT pathogenesis through non-cell-autonomous mechanisms, although the molecular pathways underlying defective astrocyte-neuron communication are only partially understood. Astrocytes are the primary source of cholesterol in the brain and support neuronal maturation and synaptic function by supplying cholesterol through ApoE-containing lipoproteins. Although alterations in brain cholesterol metabolism have been reported in RTT, the underlying cellular mechanisms and their functional consequences remain poorly investigated. Here, we studied cholesterol homeostasis in Mecp2 knock-out (KO) astrocytes and its impact on neuron-astrocyte communication. Mecp2 KO astrocytes exhibited reduced nuclear localization of the transcriptional regulator Srebp2, together with the downregulation of genes involved in cholesterol biosynthesis and transport. These molecular alterations were associated with intracellular cholesterol and desmosterol accumulation, reduced Abca1 expression and defective ApoE lipidation, despite preserved ApoE expression and cholesterol secretion. Importantly, similar alterations were detected in acutely isolated astrocytes and in the cerebral cortex of Mecp2 deficient mice, demonstrating that impaired cholesterol homeostasis extends beyond in vitro models. Functionally, cholesterol supplementation of astrocyte-conditioned medium rescued the synaptic defects induced in wild-type neurons by Mecp2 KO astrocytes. Moreover, cholesterol treatment restored pre- and post-synaptic density, as well as axon initial segment length in Mecp2 heterozygous (HET) neurons. Together, these findings identify defective astrocyte-to- neuron cholesterol trafficking as a key mechanism contributing to neuronal dysfunction in RTT and suggest that strategies aimed at restoring cholesterol functional availability might represent a promising therapeutic avenue for RTT.

Aim: ROR{gamma}t-transgenic mice provide a model of sustained T helper 17-cell bias, but the associated hippocampal transcriptional profile is incompletely characterized. We investigated gene-level and gene-set expression differences in this model. Methods: Bilateral hippocampal samples from 10-week-old male ROR{gamma}t-transgenic mice and wild-type littermates (n = 3 per genotype) underwent bulk RNA sequencing. Gene-level differential expression was evaluated using the Empirical Analysis of DGE tool in CLC Genomics Workbench. Preranked gene set enrichment analysis examined coordinated expression differences after expression filtering and exclusion of Rorc (13,813 genes). Results: Sixteen genes met a false discovery rate < 0.05 (nine higher and seven lower in transgenic mice), including Rorc, C4b, Xbp1, Nptx2 and Mt2. Of 212 candidates selected using unadjusted P < 0.05 and a twofold-change threshold, 155 (73.1%) had a mean expression below 0.5 reads per kilobase of transcript per million mapped reads. Hallmark interferon--response, interferon-{gamma}-response, and unfolded-protein-response gene sets showed positive enrichment. Thirteen Gene Ontology biological-process sets met a false discovery rate < 0.05, including negatively enriched sets related to excitatory postsynaptic potential, glutamatergic transmission, and calcineurin-mediated signaling. Il17a and Il17f had zero reported counts, and none of the displayed leukocyte or central nervous system cell-marker genes met false discovery rate < 0.05. Conclusion: This exploratory dataset identifies interferon-response, unfolded-protein-response and synaptic transcriptional signatures associated with the ROR{gamma}t-transgenic genotype. The cellular sources, causal mediators and functional consequences remain unresolved.

Experimental bacterial meningitis is often analyzed as microbial burden and inflammation, but the tissue-level organization of host-pathogen interplay remains poorly resolved. Here, we use multimodal imaging to map Escherichia coli meningoencephalitis as a spatially organized process across infected rat brain tissue and cerebrospinal fluid (CSF). A low-dose intracerebral inoculum expands within 24 h into an anatomically structured infection involving ventricular, meningeal, and perivascular interfaces. Scanning electron microscopy reveals biofilm-like multicellular E. coli aggregates embedded in extracellular material, consistent with neutrophil extracellular traps. MALDI mass spectrometry imaging detects aerobactin and salmochelin-derived metabolites, whereas intact enterobactin is not detected despite favorable analytical sensitivity. These bacterial iron-acquisition signals partially overlap with calprotectin proteoforms, defining ventricular and periventricular metal-conflict microenvironments. Spatial peptidomics identifies infection-enriched antimicrobial territories dominated by rat neutrophil peptides RatNP-2, RatNP-3, and RatNP-4. Endogenous proenkephalin-derived neuropeptides are reduced in basal ganglia regions. Finally, cerebrospinal fluid captures both bacterial siderophores and host antimicrobial peptides with lipocalin-2 protein, linking tissue-resolved host-pathogen chemistry to a proximal diagnostic fluid. Together, these data show that experimental E. coli meningoencephalitis is a spatially organized host-pathogen process. Bacterial communities, nutritional immunity, antimicrobial peptides, and neuropeptide networks occupy distinct but connected CNS niches, components of which are recoverable in CSF.

The FoxP family of transcription factors has been linked to neurodevelopmental disorders. FoxP1 and FoxP2 were identified as genes associated with autism spectrum disorders (ASD). FoxP1, FoxP2, and FoxP4 are expressed in many brain regions, including the cerebellum that has been of particular interest in the context of ASD. However, their role in the development of the peripheral nervous system (PNS) has been neglected. In view of the fact that a majority of ASD patients show some signs of sensory perception deficits that could go beyond the central nervous system, we focused on neural circuit formation in the PNS. Our in vivo and in vitro studies revealed a role of all three FoxP transcription factors in neural circuit formation in the hindlimb of the chicken embryo. Silencing of FoxP genes altered motor axon guidance and also impacted neural crest cell migration. This in turn resulted in aberrant formation of dorsal root ganglia and deficits in neural circuits in the PNS. Taken together, our results are in line with the hypothesis that some of the sensory perception deficits in ASD patients, either hyper- or hyposensitivity, are due to aberrant neural circuits in the PNS.

Tackling developmental changes in long-term potentiation (LTP) expression during adolescence is crucial to understand disease susceptibility in neurodevelopmental disorders, epileptogenesis or drug abuse. This paper investigated the alterations in the resting phosphorylation state of synaptic proteins essential for synaptic transmission and hippocampal LTP expression from weaning (3 weeks) to adulthood (12 weeks). Synaptic AMPA GluA1 and GluA2 subunit levels increased, yet to different extents, as the GluA1/GluA2 ratio also increased. Conversely, GluA1 phosphorylation in both Ser831 and Ser845 progressively decreased from weaning to adulthood, suggesting an enhanced availability of these sites for activity-dependent phosphorylation. In accordance, LTP induced by different theta-burst stimulation (TBS) intensities in the CA1 area of rat hippocampal slices enhanced gradually in this developmental period. In contrast, basal phosphorylation of GluN1, GluN2B and CaMKII increased during adolescence. Altogether, these findings suggest that alterations in AMPA receptor subunit composition and basal phosphorylation are crucial for the maturation of hippocampal LTP during adolescence, while a mild enhancement in synaptic CaMKII levels may further provide the necessary structural support to LTP expression and stability. Given the reported involvement of GluA1 phosphorylation changes in epileptogenesis and neurodevelopmental disorders, these findings provide important insights into hippocampal synaptic plasticity in normal and altered brain development and epileptogenesis.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

Cholesterol efflux is an important determinant of cellular lipid homeostasis. However, how microglial excessive cholesterol accumulation affects neuronal synaptic integrity remains poorly understood, particularly in the context of Alzheimer's disease. Here, we utilized a conditional knockout mouse model targeting the cholesterol transporters ABCA1 and ABCG1 in microglia. The microglia-specific ABCA1/ABCG1 deficiency triggered marked cholesterol accumulation, microglial hypertrophy, downregulation of the homeostatic marker P2ry12, and upregulation of the reactivity-associated marker CD11b, indicating shift toward a reactive phenotype. This phenotype was accompanied by increased reactive oxygen species, consistent with enhanced oxidative stress in ABCA1/ABCG1-deficient microglia compared with control. Using organotypic hippocampal slice cultures, we investigated the downstream neuronal outcomes of microglial ABCA1/ABCG1 deficiency. Under basal conditions, microglial ABCA1/ABCG1 knockdown did not significantly alter dendritic spine density in CA1 pyramidal neurons. However, upon exposure to amyloid-beta (A{beta}) stress, microglial ABCA1/ABCG1 deficiency markedly exacerbated dendritic spine loss in CA1 pyramidal neurons. Taken together, our findings highlight an important role for ABCA1/ABCG1-dependent cholesterol efflux in maintaining microglial homeostasis and limiting neuronal synaptic vulnerability to A{beta}-associated stress. These results support further investigation of microglial cholesterol transport as a potential target for preserving synaptic resilience in Alzheimer's disease.

Traumatic brain injury induces profound metabolic reprogramming across neurons, astrocytes, and microglia, yet the spatiotemporal organization of these metabolic responses remains poorly understood. Because pyruvate is uniquely positioned in cerebral metabolism by connecting glycolysis, lactate metabolism, and the tricarboxylic acid cycle, we combined matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging with in vivo administration of isotopically labeled pyruvate and immunohistochemistry to characterize cell-type-wise metabolic remodeling in a controlled cortical impact rat model during the acute and subacute phases of injury. TBI induced distinct spatiotemporal immunometabolic remodeling across neuroglial compartments. Microglia- and macrophage-enriched regions exhibited selective accumulation of citrate, succinate, and itaconate, consistent with inflammatory metabolic rewiring. In contrast, astrocyte enriched regions showed increased glutamine and malate abundance, indicative of altered neuron-astrocyte metabolic coupling such as remodeling of the glutamate-glutamine cycle and enhanced anaplerotic metabolism. These metabolic signatures evolved with distinct regional and temporal distributions, identifying compartmentalized metabolic responses. Notably, labeled isotopologues of selected metabolites, including glutamate and citrate derived from administered pyruvate, changed before the corresponding metabolite pools. Together, these findings describe the spatiotemporal landscape of immunometabolic remodeling following acute TBI, uncover metabolically distinct microglial/macrophage and astrocytic responses during secondary brain injury, and identify candidate metabolic pathways for therapeutic intervention and metabolic imaging.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination, neurodegeneration, and progressive neurological disability. Galanin, a neuropeptide with immunomodulatory properties, signals through G protein-coupled receptors, among which galanin receptor 2 (GALR2) has been implicated with neuroprotective and anti-inflammatory functions. A rare homozygous single nucleotide variant in GALR2 (rs61745847; p.W249L) has been identified in a patient diagnosed with relapsing-remitting MS, however, the biological relevance of this variant in neuroinflammation remains unknown. Here, we investigated the impact of the orthologous GALR2 W248L mutation using a knock-in mouse model and experimental autoimmune encephalomyelitis (EAE). GALR2 W248L knock-in (KI) mice exhibited a more severe clinical course of EAE, accompanied by enhanced inflammatory infiltration, exacerbated demyelination, and increased microglial activation in the spinal cord compared with wild-type (WT) mice. Despite comparable lymphoid and myeloid cell frequencies in the central nervous system, alterations in microglial density and morphology suggested an important contribution of the innate immune system to disease exacerbation in the KI mice. Ex vivo analyses revealed that bone marrow-derived macrophages from KI animals exhibited a pronounced shift toward a pro-inflammatory phenotype, characterized by enhanced M1 polarization, impaired M2-associated responses, and increased NLRP3 inflammasome activation. In parallel, live-cell imaging of primary hippocampal neurons demonstrated reduced galanin binding in mutant cells, consistent with impaired GALR2 functional availability at the plasma membrane. Together, these findings identify GALR2 as a modulator of the neuroinflammatory response and indicate that disruption of galanin-GALR2 signaling promotes sustained innate immune activation, highlighting the relevance of this pathway for MS pathogenesis and its potential as a therapeutic target in neuroinflammatory disorders.

Dysfunction of maternal UBE3A leads to Angelman syndrome (AS), which is characterized by significant intellectual and motor debilities. However, the molecular underpinnings of the behavioral deficits associated with UBE3A dysfunction remain obscure. In this study, we utilized a model mouse of AS and report, for the first time, that the aberrant accumulation of -synuclein may be linked to the development of AS. Firstly, we demonstrated a progressive deterioration of various motor functions in AS mice beginning from the early adolescent phase. Subsequently, we observed an age-dependent increase in the accumulation of both soluble and insoluble -synuclein, including its pathological variant (pSer129), in the striatum and substantia nigra dopaminergic neurons of AS mice. We also found that Ube3a interacts with -synuclein and promotes its proteasome-mediated degradation, as evidenced by decreased levels of K48-linked polyubiquitinated -synuclein in the brain samples of AS mice in comparison to wild-type animals. Finally, using an RT2 Profiler PCR Array that analysed 84 genes specifically related to dopamine and serotonin pathways, we identified altered transcript level of various genes in the striatal tissues of AS mice that are commonly associated with nigrostriatal dysfunctions in Parkinson's disease. These findings highlight -synuclein as a novel target of Ube3a and suggest that -synuclein pathology may contribute to the progressive motor and other behavioral abnormalities witnessed in AS mice.

REV-ERB{beta} is a transcriptional repressor of nuclear receptors that regulates the circadian rhythm and plays an important role in regulation of the proliferation, differentiation, and maturation of neurons. Dysregulation of the circadian rhythm has been associated with neuropsychiatric disorders, and activation of REV-ERBs can induce anxiolytic behavior in mice. Furthermore, hippocampal neurogenesis is important in the effects of antidepressants. However, the role of REV-ERB{beta} in adult hippocampal neurogenesis in vivo at the single-cell level is not known. In this study, protein localization of REV-ERB{beta} in the subgranular zone of the hippocampal dentate gyrus (DG) was mainly shown in NeuN-positive neurons, and the effect of expressing a dominant negative form of REV-ERB{beta} lacking the C-terminal region on newborn neurons in the hippocampal DG of adult mice was examined to investigate the role of REV-ERB{beta} in neurogenesis. A retroviral vector containing the dominant negative REV-ERB{beta} or a control vector was injected into the mouse DG. At 4 weeks after injection, the morphology of dendrites and dendritic spines of newborn neurons labeled by the virus was examined. Expression of the dominant negative form of REV-ERB{beta} inhibited dendrite outgrowth and branching and decreased dendritic spine formation in newborn neurons in the adult mouse hippocampal DG. This study revealed a new role for REV-ERB&{beta} in adult hippocampal neurogenesis at the single cell level, and the results will provide insight into neurogenesis in the adult brain and its relationship with psychiatric disorders.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

Alzheimers disease (AD) is a progressive neurodegenerative disorder with insidious onset. At present, effective early diagnostic biomarkers are scarce, and few studies have explored ultra-early molecular alterations during embryonic development. Urinary proteomics boasts unique strengths including complete non-invasiveness, repeatable sequential sampling and high detection sensitivity, which provides a potential technical strategy for prenatal monitoring of congenital disorders. In this study, we constructed an experimental group by mating wild-type female mice with 3xTg transgenic male mice to obtain pregnant dams carrying heterozygous AD-susceptible fetuses, while wild-type male-female mating was set as the blank control. Urine samples were consecutively collected at 10 time points from gestational day 1 (D1) to D19. Label-free quantitative proteomics was adopted to screen differentially expressed proteins, and Gene Ontology (GO) enrichment analysis was carried out to interpret temporal biological processes. The results showed that stable intergroup differential proteins could be detected as early as the implantation stage (D1), and differential protein profiles existed throughout the whole gestation period. The quantity and expression trend of differential proteins exhibited obvious temporal dynamics, and permutation tests verified that the intergroup differences were not random noise. Paternally inherited AD-causing mutations could trigger systematic molecular responses in maternal mice at the early embryonic stage. This study for the first time characterized the dynamic urinary proteomic landscape of maternal mice that reflects fetal AD susceptibility. It demonstrates that maternal urine can mirror molecular signatures related to fetal AD development, offering fundamental animal experimental data for subsequent screening of prenatal non-invasive monitoring biomarkers and research on the embryonic origin of AD.