The etiology of major depressive disorder (MDD) is multifactorial with both genetic and environmental factors, such as adverse/stressful life events, contributing to risk. There is some evidence suggesting that microRNAs (miRNAs) mediate environmental-genetic interaction leading to the brain dysfunctions that underlie MDD. However, changes in miRNAs expression in human brain regions due to stress and associated with MDD are unclear. To increase the evidence in this regard, miRNA sequencing was performed on tissue samples of subgenual anterior cingulate cortex (sgACC) obtained from depressed patients and control subjects, as well on tissue samples of medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) from mice exposed to chronic social stress (CSS) and control animals. DESeq2 was applied to identify differentially expressed miRNAs (DEMs) and weighted co-expression network preservation analysis to uncover conserved molecular mechanisms between species. Finally, pathways obtained from DESeq2 and preservation analyses were overlapped to robustly identify MDD-related processes across bioinformatic approaches.Eighteen DEMs were identified in the human sgACC, 11 in the mPFC and 9 in the BLA of mice. The human sgACC DEMs were involved mainly in intracellular signaling and immune system-related pathways. The mouse mPFC and BLA DEMs were mainly involved in, respectively, intracellular signaling and nervous system functions. Preservation patterns between humans and mice indicated an over-representation of processes related to cellular signaling. Transcriptional regulation by MECP2 and Protein Kinase A signaling were the two pathways consistently altered across species, brain regions, and bioinformatic approaches. Although further studies are needed, they could represent a novel target for intervention strategies and confirm the dysregulation of intracellular signaling, immune, neuronal and synaptic functions in MDD.
The present manuscript provides a comprehensive overview of neural stem cell (NSC)-derived extracellular vesicles (NSC-EVs( as a cell-free approach to treating central nervous system (CNS) disorders. The study noted that NSCs are regenerative and neuroprotective, but direct transplantation is limited by short survival, immunological rejection, and tumorigenic risk. However, NSC-EVs-nano-sized vesicles loaded with proteins, lipids, and RNAs-can replicate many of their parent cells' benefits without safety or ethical issues. NSC-EVs are the source of numerous biologically active molecular cargoes. Encompassing (BDNF, GDNF, VEGF), signaling lipids, and microRNAs (miR-124, miR-21, miR-146a, miR-219) that are essential in modulating and regulating key processes involved in the induction of neurogenesis, promoting angiogenesis, reducing inflammatory milieu, and improving neuronal survival. In preclinical models of Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), ischemic stroke, spinal cord injury (SCI), and traumatic brain injury (TBI), these vesicles reduce oxidative stress, suppress apoptosis, modulate microglia activation, enhance synaptic plasticity, and promote remyelination. Numerous translational obstacles remain, including heterogeneous EV isolation techniques, limited scalability of clinical-grade manufacturing, and inconsistent elucidation of long-term safety and biodistribution. This review discusses the therapeutic potential of NSC-EVs for neurological and neurodegenerative diseases. Additionally, leveraging the powerful, precise analytical capabilities of Artificial Intelligence (AI) with recent multi-omics data from NSC-EVs will improve the characterization and predictability of therapeutic efficacy. Combining the therapeutic potential of stem cells with the non-invasive, practical, and safe cell-free biologic is expected to transform regenerative neuroscience. A promising aim that requires establishing a multidisciplinary approach among neuroscientists, bioengineers, and clinicians to standardize the isolation process, validate the underlying mechanistic information, and test their therapeutic potential at the clinical level. The present review concludes that NSC-EVs are a promising research topic in regenerative neurotherapy, offering a potential therapeutic strategy for incurable neurological and neurodegenerative diseases.
The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal polarity, yet the developmental roles of its core molecular components-Neurofascin 186 (NF186) and Ankyrin G (AnkG)-remain incompletely defined in cerebellar Purkinje cells. Here, we generated Purkinje cell-specific NF186 and AnkG single- and double-knockout mice to investigate how these adhesion and scaffolding proteins cooperatively regulate AIS formation, ion channel localization, synaptic targeting, and neuronal survival. We found that genetic ablation of either Nfasc NF186 (NFKO) or Ankyrin3 (AnkGKO) disrupted assembly and maintenance of the AIS cytoskeleton, and that this defect was exacerbated by combined loss of both proteins during postnatal development. Other AIS-enriched proteins, including βIV Spectrin (βIVSpec), voltage-gated sodium (Nav), and potassium (Kv1.2) channels, failed to properly localize to the AIS and progressively disintegrated between postnatal days 10 and 30. Notably, Kv1.2 clustering at the pinceau synapse was disrupted, and basket cell axons showed misaligned terminal organization, indicating defective inhibitory synapse innervation. By 2 months of age, degeneration of Purkinje cells was evident, accompanied by cerebellar dysfunction. Notably, AnkG ablation caused a progressive postnatal loss of NF186 at the AIS, whereas NF ablation resulted in much slower loss of AnkG at the AIS in Purkinje cells and closely phenocopied the severe AIS destabilization observed in NF/AnkG double-knockout mice. In addition, our RNA-seq analysis revealed that Purkinje cell-specific loss of NF186 predominantly activated immune-inflammatory pathways; AnkG loss significantly disrupted neuronal developmental and metabolic processes; and the dual loss of NF186/AnkG produced transcriptional changes that were distinct from, and in part intermediate to, those observed in NF186 and AnkG single knockout. Collectively, our results show that NF186 and AnkG have complementary, non-redundant roles in establishing and maintaining the Purkinje cell AIS, and that their loss disrupts synaptic organization at the AIS. These findings advance our understanding of AIS development in cerebellar neurons and have implications for diseases involving AIS dysfunction, including cerebellar ataxia and demyelinating neuropathies.
Myelin sheaths are generated from differentiated plasma membranes of oligodendroglial cells (oligodendrocytes) during development. Despite detailed functional studies, such as aiding nerve conduction velocity, it remains unclear which extracellular signals from neurons interact with oligodendrocytes to control oligodendrocyte development and how these are achieved. Herein, we demonstrate that the Plexin molecule Plexin-B3, generally known as a receptor for Sema family signaling proteins, is specifically expressed in oligodendrocytes and plays a key role in promoting oligodendrocyte maturation and myelination. Oligodendrocyte-specific conditional male knockout mice of Plexin-B3 exhibited decreased myelin thickness and myelin marker protein expression, compared to littermate controls. Unexpectedly, we identified heat shock protein family A member 5 (HSPA5)/Grp78/BiP as the neuron-secreted ligand of Plexin-B3. Indeed, neuron-specific knockdown of HSPA5 in male mice revealed a critical role for myelin thickness not only at the biochemical level but also at the histochemical one. Furthermore, the transcription factor Nkx2.8, which plays a role in oligodendrocytes, was identified as a potential mediator of Plexin-B3 signaling. Our results suggest that the interaction of extracellular HSPA5 with Plexin-B3 on oligodendrocytes, acting possibly through Nkx2.8, regulates oligodendrocyte maturation and myelination. This ligand and receptor interaction and a downstream molecule are newly added to the list of emerging signaling units controlling myelination.Significance Statement Despite the functional importance of myelin sheaths generated by oligodendrocytes, it remains to be established how the combination of extracellular signals, particularly signals from neurons, and receptors on oligodendrocytes, regulates myelination. We have defined that Plexin-B3, a well-known axon guidance receptor member, is uniquely expressed in oligodendrocytes and plays a key role in promoting oligodendrocyte maturation and myelination. Using an affinity-precipitation technique, we have identified HSPA5, a member of the heat shock protein family, as being secreted from neurons and a ligand for Plexin-B3. It has been newly suggested that the Nkx family transcription factor Nkx2.8 can mediate their signaling. These data provide insight into how the relationship between neuronal signals and oligodendrocyte receptors controls axonal myelination by glia.
Endothelial cells (ECs) orchestrate vascular homeostasis and resilience but can undergo reprogramming into a mesenchymal-like phenotype through an endothelial-to-mesenchymal transition (EndMT). Crucially, EndMT is a linchpin underlying several cardiometabolic diseases, but is almost universally studied as an endpoint. The transcription factor ERG (ETS-related gene) is critical to the maintenance of EC identity and function, yet the dynamic transcriptional and functional consequences of ERG loss on EndMT programs, and whether this can be reversed, has not been explored. We modeled both acute and chronic ERG loss in human aortic ECs using siRNA knockdown and CRISPR/Cas9-mediated ERG deletion. We profiled temporal changes in chromatin accessibility (ATAC-seq), transcriptomic responses (RNA-seq), and endothelial phenotypes, including migration and barrier integrity. The temporal kinetics of ERG loss and restoration was assessed by comparing stable ERG knockout to transient ERG knockdown and recovery over time. The implications to human disease were deciphered by examining ERG gene regulatory networks in human atherosclerosis and linkage with genetic variation associated with human cardiovascular disease. Analysis of gene regulatory networks revealed profound and dynamic rewiring of endothelial and mesenchymal transcriptional programs upon loss of ERG. While endothelial identity was rapidly lost by 24 h of ERG knockdown, acquisition of mesenchymal identity, barrier dysfunction, and enhanced cell migration required 72 h to manifest. Loss of ERG was accompanied by a rapid reduction in accessibility of ETS motifs and an extensive gain in open chromatin containing AP1 motifs. Disease-relevant endothelial dysfunction programs were associated with dynamically reorganized transcriptional networks. Importantly, restoration of ERG expression reversed EndMT gene regulatory networks and phenotypes. Overall, this study highlights the ETS factor, ERG, as an essential transcriptional safeguard of endothelial identity and function, and demonstrates that ERG loss initiates a progressive, yet reversible, EndMT program with EC identity loss preceding a gain of mesenchymal gene regulatory networks and phenotypes. This study establishes loss of ERG as an early initiating event in EndMT and suggests that ERG-targeted therapies may hold promise for promoting endothelial resilience.
Tissue repair is a finely organized process that progresses via a series of phases, including hemostasis, inflammation, proliferation, and remodeling, which are coordinated by immune-stromal interactions. Aging profoundly dysregulates these processes through mechanisms such as immunosenescence and inflammaging, cellular senescence, chronic inflammation, and extracellular matrix alterations, ultimately contributing to typical age-related progression. This review discusses the immune mechanisms that govern physiological tissue healing, as well as the age-related perturbations that lead to ulcerative and fibrotic diseases. It also highlights the potential application of extracellular vesicles (EVs), both mammalian and plant-derived, as a stable and low-immunogenicity mediator to modulate and re-establish repair homeostasis. Translational hurdles such as EV standardization, dosing, safety assessment, and manufacturing are critically discussed to promote their use in geroscience, regenerative medicine, and dermatology.
Biological mechanisms underlying heterogeneous antipsychotic response in schizophrenia remain incompletely understood. In this exploratory cross-sectional study, we applied deep data-independent acquisition LC-MS/MS plasma proteomics to 56 adults initially enrolled (14 per group), including healthy controls and patients meeting TRRIP criteria for treatment-sensitive (TSS), treatment-resistant (TRS), and ultra-treatment-resistant schizophrenia (UTRS). Following proteomic quality control and exclusion of outliers, 52 individuals comprised the final analytical cohort. Proteomic data were analyzed using a layered strategy integrating covariate adjustment, variance partitioning, mediation analysis, monotonicity filtering, LASSO stability assessment, and redundancy reduction. More than 1400 plasma proteins were quantified; 450 differed across groups before adjustment, and 310 reviewed proteins remained significant after covariate modeling. A sensitivity-associated profile distinguishing TSS from controls (CRP, CCDC62, FBXW7, GULP1, CALD1, COPS6) was consistent with lower inflammatory tone and relative preservation of proteostatic and cytoskeletal regulation. In contrast, a resistance-associated profile separating TRS/UTRS from TSS (SHROOM1, MYH7, ABR, EZR, SERPINF2) converged on cytoskeletal organization, actin-membrane dynamics, and extracellular regulatory processes. Directionally concordant but quantitatively amplified changes were observed in UTRS relative to TRS, although multivariate separation between resistant subgroups was limited after full covariate adjustment. Several proteins enriched in resistant groups corresponded to intracellular or nuclear factors rarely detected in plasma and require cautious interpretation. Overall, these findings are compatible with a progression-like molecular pattern in which treatment sensitivity and resistance may reflect shifts in cellular adaptability and structural regulation. Replication in larger and longitudinal cohorts is required.
Tau aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. There are disease-causing variants of the tau-encoding gene, MAPT, and the presence of tau aggregates is highly correlated with disease progression. However, the molecular mechanisms linking pathological tau to neuronal dysfunction are not well understood. This is in part due to an incomplete understanding of the normal functions of tau in development and aging, and how the associated molecular and cellular processes change in the context of causal disease variants of tau. To address these questions in an unbiased manner, we conducted multi-omic characterization of iPSC-derived neurons harboring the MAPT V337M mutation or MAPT knockdown. RNA-seq, ATAC-seq, and phosphoproteomics revealed that both the V337M mutation and tau knockdown perturbed levels of transcripts and phosphorylation of proteins related to axonogenesis or axon morphology. When we directly measured axonogenesis, we found that both MAPT V337M and MAPT knockdown caused decreased axon length. Surprisingly, we found that neurons with V337M tau had much lower tau phosphorylation than neurons with WT tau. CRISPR-based screens uncovered regulators of tau phosphorylation in neurons and found that factors involved in axonogenesis modified tau phosphorylation in both MAPT WT and MAPT V337M neurons. Intriguingly, the p38 MAPK pathway specifically modified tau phosphorylation in MAPT V337M neurons. We propose that V337M tau perturbs tau phosphorylation and axon morphology pathways that are relevant to the normal function of tau in development, which could contribute to previously reported cognitive changes in preclinical MAPT variant carriers.
Quantitative colocalization analysis in fluorescence microscopy is widely used to study molecular interactions among proteins, RNAs, and other cellular components. Object-based approaches identify discrete molecular features and quantify distances between neighboring centroids to infer colocalization. A key challenge in this approach is distinguishing true molecular association from incidental overlap arising in crowded or spatially heterogeneous environments. Randomized null models are commonly used to estimate colocalization expected by chance, but they often fail to preserve the heterogeneous spatial density of biomolecules, leading to underestimation of random colocalization and reduced sensitivity to small effect sizes. Here, we introduce Density-aware Spatial Randomization (DenSR), an in silico framework that generates spatially realistic null models by preserving both local clustering and global spatial organization. Applying DenSR to protein and RNA datasets with heterogeneous spatial organization demonstrates that uniform randomization substantially underestimates background proximity, whereas density-aware null models provide more accurate expectations and prevent overestimation of colocalization due to underestimated background proximity. In contrast, analysis of sparse transcript datasets shows that different randomization strategies converge. Together, these results establish DenSR as a general approach for improving estimation of colocalization expected by chance across diverse spatial distributions.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by motor neuron degeneration, oxidative stress, and neuroinflammation. This study evaluated the neuroprotective potential of caffeic acid phenethyl ester (CAPE) against MTME + 5-induced neurotoxicity in an ALS-like pathology model. CAPE (50 and 100 mg/kg., p.o.) demonstrated significant therapeutic efficacy by improving motor and cognitive deficits, restoring oxidative balance, and mitigating neuroinflammatory and apoptotic pathways. Behavioral assessments, including the open field, grip strength, forced swim, and Morris water maze, highlighted CAPE's ability to restore neuromuscular coordination and cognitive function in a dose-dependent manner. Cellular and Molecular analyses revealed that MTME+5 exposure significantly disrupted Klotho/SIRT-1/Nrf2/HO-1 antioxidant signaling, increased pro-inflammatory cytokines (TNF-α, IL-1β), and elevated apoptotic markers (Bax, caspase-3) while depleting anti-inflammatory cytokines (IL-10) and neuroprotective proteins. Furthermore, CAPE treatment restored these parameters, reduced oxidative stress, and enhanced antioxidant defenses (SOD, CAT, r-GSH). Furthermore, CAPE normalized neurotransmitter imbalances, including acetylcholine, dopamine, GABA, serotonin, and glutamate, alleviating excitotoxicity. Histopathological and gross morphological analyses confirmed CAPE50 and CAPE100 ability to preserve neuronal and myelin integrity across key brain regions, including the cerebral cortex, hippocampus, striatum, midbrain, and cerebellum. CAPE also reduced methylmercury accumulation in the brain and cerebrospinal fluid, indicating detoxifying effects. Co-administration of vitamin B1 (VTB1(200)) further amplified CAPE's therapeutic efficacy. Complete blood count (CBC) analysis demonstrated MTME+5-induced hematological abnormalities, including reduced RBCs, hemoglobin, WBCs, and platelets, alongside elevated eosinophils and basophils. CAPE treatment normalized these parameters, indicating systemic recovery. These findings establish CAPE as a promising neuroprotective agent for ALS, capable of targeting neurocomplications.
To study the effect of low-estrogen and the menopause state upon female pituitary function, we used the 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure model to gradually reduce serum estradiol (E2) levels. We utilized single-cell RNA sequencing transcriptomics analysis (scRNA-seq) to determine how reduced E2 levels influence pituitary gonadotrope gene expression and cell state, with a focus on potential contributions from pituitary stem cells to gonadotrope population growth. VCD-treated mice were acyclic and exhibited 8-15-fold increases in serum levels of the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Serum E2 levels were half those of normally cycling proestrous females. Pituitary scRNA-seq identified two gonadotrope populations based upon distinct gene expression signatures (a higher cell number primary cluster, designated here as GON1 and a smaller cell number secondary cluster, designated GON2). The GON1 population increased in number significantly in low-E2, VCD-treated mice, expressing markers indicative of contributions from pituitary stem cell activation. Both gonadotrope populations showed upregulated genes for canonical pathways supporting mRNA translation and hormone secretion, which correlated well with the high serum gonadotropin levels. Consistent with the high serum gonadotropin levels, Fshb and Lhb mRNAs were increased in whole pituitary samples (RT-qPCR). The pituitary Sox2-positive stem cell population exhibited increased expression of more mature transitional progenitors, including Sox9 mRNA, and showed upregulation of pathways involved in stem cell development and mitotic activity. This model provides insight into the cellular and molecular adaptations of the pituitary to the low-E2, menopausal state, with potential relevance for understanding human perimenopausal and menopausal physiology.
Brain organoids represent three-dimensional structures that allow for human-specific studies in brain development, pathology and therapeutics. These self-organizing systems, formed through the differentiation of human pluripotent stem cells, can mimic important cellular and molecular events of brain development and therefore serve as a platform for the investigation of neurodevelopmental and neurodegenerative diseases, brain injuries, and tumorigenesis. Although brain organoids show promising perspectives in the study of human physiology, existing brain organoid platforms are hindered by issues of under vascularization, immaturity and protocol variability. Nevertheless, the rapid development of new bioengineering, microfluidic and multi-omics tools and approaches allows us to overcome existing problems and increase the physiological significance of these organoids. Brain organoid transplantation and functional studies further enhance the applications of brain organoids in drug screening, disease modeling and personalized medicine. Here, we provide an overview of recent developments in the field of brain organoid cultures, functional characteristics and translational applications.
Traumatic brain injury (TBI) frequently leads to chronic neurovascular dysfunction, yet mechanistic insights into human-specific responses have been limited by the absence of long-term, multicellular in vitro models. Here, we report a five-cell-type human neurovascular culture system, comprising endothelial cells, astrocytes, pericytes, microglia, and neurons, engineered within a 3D scaffold to study injury-induced remodeling over multiple weeks. This PENTA-culture platform captures key structural and molecular features of the neurovascular unit and supports compartment-resolved profiling of vascular and neuroimmune responses. Under baseline conditions, PENTA cultures exhibit restricted tracer distribution relative to simpler culture configurations, consistent with the emergence of barrier-like properties within the 3D scaffold. Following mechanical trauma, cultures exhibit a biphasic response characterized by acute endothelial disorganization, mitochondrial structural changes, and neuroimmune alterations, followed by delayed and incomplete structural recovery, accompanied by shifts in angiogenic and immunomodulatory signaling consistent with Tyrosine kinase with immunoglobulin-like epidermal growth factor-like domains 2 (Tie2)- and Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT)-associated signatures. At last, the inclusion of microglia and neurons is associated with improved cytokine resolution and partial recovery of junctional organization, highlighting the influence of neuroimmune complexity on post-injury vascular remodeling. Together, this long-lived, human-derived platform provides a structurally complex and functionally informative system for characterizing neurovascular injury signatures following TBI.
Hereditary optic neuropathies are characterized by bilateral visual loss due to the degeneration of retinal ganglion cells, resulting in optic nerve degeneration and atrophy. Although the genetic origin of the main isolated and syndromic hereditary optic neuropathies have been characterized, the clinical phenotypes exhibit significant and poorly understood variability in both penetrance and expressivity. Additionally, the genetic and environmental factors that influence the onset of these optic neuropathies remain poorly understood, with limited biomarkers to predict disease progression or as readouts for therapeutic trials. Data-driven omics strategies allow deep phenotyping to improve our understanding of pathophysiological mechanisms and to search for new biomarkers and therapeutic targets. We explore whether the omics strategies applied to patients with hereditary optic neuropathies have provided such new insights. MEDLINE, Web of Science and EMBASE databases were screened for studies with terms relating to hereditary optic neuropathies, transcriptomics, epigenomics, proteomics, metabolomics and lipidomics in clinical studies exploring patients' samples. Out of 1244 references identified, 22 articles were included after double-masked data curation. These articles focused only on the 3 main forms of hereditary optic neuropathies, namely, OPA1-related dominant optic atrophy (n = 4), Leber hereditary optic neuropathy (n = 13) and Wolfram syndrome (n = 5). While the methodological designs and results of these studies were highly heterogeneous, they revealed molecular alterations that we have attempted to discuss at the integrated multi-omics level. This data integration highlighted several common pathophysiological mechanisms such as energetic impairment, endoplasmic reticulum stress, proteotoxic and oxidative stresses, lipid remodeling and altered amino acid and purine metabolisms, while suggesting potential new biomarkers and therapeutic targets. These findings underscore the potential of integrated multi-omics approaches to deepen our understanding of the phenotypic complexity of hereditary optic neuropathies and to support the development of innovative diagnostic and therapeutic strategies.
Schizophrenia is a complex neurodevelopmental disorder with cognitive impairment being one of the core features that remains largely unresponsive to current antipsychotic treatments. Histone deacetylase 3 (HDAC3), a negative regulator of memory and synaptic plasticity, has been implicated in neurodegenerative conditions, but its role remains underexplored in psychosis. Here, we hypothesized that aberrant HDAC3 activity contributes to hippocampal dysfunction and learning deficits in schizophrenia. Pregnant SD rats were administered methylazoxymethanol (MAM; 20 mg/kg) and vehicle on GD 17. We characterized the pharmacokinetic profile of selective HDAC3 inhibitor, SP108, to ensure adequate BBB penetration and systemic exposure. Next, adult male offspring were administered SP108 (25 mg/kg, i.p.) and vehicle every day for 3 weeks, followed by behavioral analysis. The MAM-exposed group showed schizophrenia-like behavioral patterns with increased hippocampal HDAC3 expression and activity. HDAC3 inhibitor treatment selectively ameliorated avoidance learning and MK801-induced hyperlocomotion. At the molecular level, HDAC3 inhibition elevated hippocampal H3K9 acetylation and increased the expression of synaptic plasticity markers BDNF and PSD95. To establish a neurodevelopmental link, HDAC3 knockdown was performed in differentiating neurons from mouse embryonic stem cells (mESCs) exposed to MAM at the early differentiating phase in vitro. HDAC3 knockdown in MAM-exposed differentiating neurons enhanced MAP2 intensity and neurite length with improved levels of MAP2, NeuN, TUBB3 (neuronal differentiation and maturation markers), BDNF, and PSD95 (neuroplasticity markers). Collectively, these findings identify HDAC3 as an important regulator of hippocampal dysfunction and cognitive impairment in a schizophrenia-like preclinical model, highlighting its potential to augment the therapeutic outcomes beyond current antipsychotic treatments.
Hyperkinetic movement disorders arise from dysfunction within cortico-basal ganglia-cerebellar loops. They frequently involve psychiatric and cognitive symptoms, reflecting impairment of both motor and non-motor domains within these loops. ADCY5 (MxMD-ADCY5) and SGCE (MYC/DYT-SGCE) related movement disorders are childhood-onset monogenic hyperkinetic conditions, both characterized by myoclonus, dystonia, and frequent psychiatric manifestations. Previous evidence suggests predominant basal ganglia involvement in MxMD-ADCY5 and cerebellar involvement in MYC/DYT-SGCE. The aim was to determine how basal ganglia and cerebellar dysfunction drives cortical dysregulation in hyperkinetic movement disorders. Resting-state functional magnetic resonance imaging (fMRI) was used to examine effective connectivity in motor and non-motor cortico-basal ganglia-cerebellar loops. Findings were validated using leave-one-out cross-validation. Microstructural properties of regions within these loops were assessed with diffusion-weighted imaging, using neurite orientation dispersion and density measures. We enrolled 21 patients with MxMD-ADCY5, 24 with MYC/DYT-SGCE, and matched healthy controls. Both patient groups exhibited elevated rates of psychiatric comorbidities. In MxMD-ADCY5, abnormal basal ganglia connectivity influenced the cerebellum, which in turn modulated cortical activity across motor and non-motor loops. Reduced neurite density was observed in the subthalamic nucleus, a relay between basal ganglia and cerebellum. In MYC/DYT-SGCE, the cerebellum showed predominant influence on cortical activity, with downstream modulation of basal ganglia activity, but no microstructural alterations were detected. Cross-validation largely confirmed the connectivity patterns' reliability. Abnormal cortical modulation in both disorders converges on a shared cerebellar-cortical pathway, with basal ganglia influences in MxMD-ADCY5 transmitted via the cerebellum to the cortex, and cerebellar contributions in MYC/DYT-SGCE directly influencing the cortex. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Recent advancements in neuroproteomics have enabled detailed analysis of protein expression in the human brain, yet resolving synaptic dysfunction-a central feature of many neurological and psychiatric disorders-requires careful methodological consideration. Leveraging the high sensitivity of modern liquid chromatography-tandem mass spectrometry (LC-MS/MS), we evaluated the utility of whole-tissue lysates versus enriched synaptosome preparations for detecting synaptic protein signatures. First, we optimized and standardized a sample preparation protocol for frozen human gray matter (GM) by refining the suspension trapping (sTRAP) digestion method using thin human tissue sections. We accomplished low technical variation by minimizing sample handling and achieved a highly reproducible sample preparation workflow by rigorously applying standardization and randomization across dissection, processing, and LC-MS/MS runs. Second, comparative LC-MS/MS analysis showed that while whole-tissue lysates provide a high-throughput survey of the synaptic proteome, synaptosome isolation is required to investigate synapse-specific proteins to detect alterations at the terminal that are obscured in the soma. Because these methods offer distinct but synergistic levels of information, we recommend a tiered neuroproteomics strategy. This approach utilizes whole-tissue lysates for broad disease-associated screening and consistent quantification in large cohorts, followed by targeted synaptosome proteomics to provide a unique window of insight into synaptic composition and stability. This integrated workflow respects the biological necessity of spatial resolution while maintaining the reproducibility required for robust human brain proteomics. Furthermore, initial tissue-level analysis provides the necessary context to correctly interpret synaptosome data in cases of global synapse loss or gain.
Spiral ganglion neurons (SGNs) relay auditory sensory information from the cochlea to the brain. Their loss results in permanent hearing impairment in humans due to their limited regenerative capacity. Progress in hearing restoration has been constrained by the inaccessibility of human inner ear tissue and challenges in generating functionally mature human SGN-like neurons from stem cells in vitro. To generate human SGN-like neurons from human induced pluripotent stem cells (hiPSCs), we recapitulated key signaling pathways involved in human inner ear development. On day (D) 11 of differentiation, nerve growth factor receptor-positive cells (precursors of pre-placodal ectoderm and neural crest) were isolated using magnetic sorting. From D18 to D25, cultures were treated with sonic hedgehogs to induce otic neural progenitors. Neuronal maturation was subsequently promoted by a cocktail of brain-derived neurotrophic factor, neurotrophin-3, and insulin-like growth factor-1, which supports SGN development. Cellular identity and functionality were assessed using single-cell RNA sequencing, immunocytochemistry, whole-cell patch-clamp electrophysiology, co-culture assays, and calcium ion (Ca²⁺) imaging. hiPSC-derived SGN-like neurons exhibited morphological, molecular, electrophysiological, and functional characteristics of SGNs in vivo. Neurons acquired bipolar morphology and were wrapped by glial cells. Transcriptomic analysis revealed that SGN-like neurons were distinct from other neuronal lineages and showed similarity to type I and type II SGNs based on expression of synaptic and intrinsic excitability-related genes. Electrophysiological recordings revealed progressive hyperpolarization of resting membrane potential and emergence of overshooting action potentials, consistent with neuronal maturation. In co-culture systems, human SGN-like neurons formed functional synaptic connections with mouse cochlear hair cells and cochlear nucleus neurons, evidenced by Ca2+ transients and induction of the immediate early gene c-Fos. This study reports a robust and reproducible protocol for generating human SGN-like neurons from hiPSCs, providing a versatile platform for studying human auditory development, disease modeling, drug screening, and cell-based therapies for hearing restoration.
While single-omics analyses of Parkinson's Disease (PD) have demonstrated their ability in revealing the underlying molecular mechanisms, they often fail to provide a comprehensive view of the complete disease mechanisms. In this study, we leveraged multi-omics data from 64 heterogeneous, well-phenotyped PD patients, generated plasma metabolomics data and Olink proteomics data together with the gut and saliva metagenomics data, and investigated the altered molecular mechanisms and their interactions in association with the severity of motor function disorders in PD patients. Based on our multi-omics approach, we identified a panel of 58 biomarkers comprising one clinical variable, 10 proteins, and 17 metabolites from plasma, 26 gut species, and 4 saliva species for PD severity. These biomarkers exhibited superior predictive performance for assessing PD severity compared to those derived from single-omics datasets. The predictive power of our machine learning models based on these biomarkers was validated using additional multi-omics data from the same group of PD patients after a 3-month follow-up. The contribution of each omics dataset was evaluated by both supervised and unsupervised machine learning approaches, highlighting the importance of plasma metabolomics in disease stratification. Our study unveiled disease-related molecular alterations across multiple omics datasets, offering potential diagnostic and therapeutic insights for PD. Moreover, it underpinned the significance of employing multi-omics analyses when studying complex diseases like PD.
Mitochondrial dysfunction is a critical factor in secondary injury following spinal cord injury (SCI). Mitophagy is an essential mechanism for mitochondrial quality control. Proper and timely activation of mitophagy clears damaged mitochondria, reduces oxidative stress and cell death, and provides neuroprotection. However, excessive activation can cause energy depletion and worsen injury. The effects of mitophagy depend on the specificity of its spatial and temporal activation as well as the cellular microenvironment. This review summarizes novel therapeutic strategies targeting mitophagy, including pharmacological modulators, gene-based interventions, biomaterials, and cell therapies. These approaches precisely regulate mitophagy via distinct molecular pathways. Challenges remain in precise regulation, clarification of cell-specific mechanisms, and real-time monitoring in vivo. Future research should aim to develop precise spatiotemporal regulatory tools, identify relevant biomarkers, and integrate mitophagy-targeted therapies with existing methods, providing new insights into SCI treatment.