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Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy.

Haloperidol is a typical antipsychotic used to treat schizophrenia and induces dopamine D2 receptor antagonism. Long-term use of haloperidol can reduce brain size in animals and humans; however, the underlying mechanism of this effect remains unclear. Notch1 signaling regulates the development and function of the nervous system by balancing stem cell proliferation and differentiation. Therefore, we investigated the effects of long-term exposure to haloperidol on human-derived brain organoids, which served as sophisticated in vitro models of human brain development. Long-term exposure to haloperidol reduced the size of brain organoids and decreased the ventricular zone and Notch1 signaling. When propionate, which protects against haloperidol-induced toxicity, was combined with haloperidol, it rescued both the overall size of brain organoids and Notch1 expression levels. Additionally, treatment with valproic acid, a Notch1 activator, partially restored the size of brain organoids and the thickness of the ventricular layer. Taken together, these data suggest that long-term exposure to haloperidol impairs neurodevelopment via Notch1 signaling in brain organoids. These findings contribute to our understanding of antipsychotic drug safety and provide information for new neurodevelopmental toxicity assessments.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-08855-w.

SummaryCytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in TARDBP (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9, we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease. Graphical abstract Highlights•Mutant MNs maintain viability but are more vulnerable to cellular stress•Mutant MNs do not show TDP-43 pathology•TDP-43 variants lead to a progressive decline in spontaneous neuronal activity•Functional impairments are accompanied by abnormal synaptic marker expression Molecular neuroscience; Cellular neuroscience

SummaryRibosomopathies arise from the disruptions in ribosome biogenesis within the nucleolus, which is organized via liquid-liquid phase separation (LLPS). The roles of LLPS in ribosomopathies remain poorly understood. Here, we generated human induced pluripotent stem cell (hiPSC) models of ribosomopathy caused by mutations in small nucleolar RNA (snoRNA) gene SNORD118. Mutant hiPSC-derived neural progenitor cells (NPCs) or neural crest cells (NCCs) exhibited ribosomopathy hallmark cellular defects resulting in reduced organoid growth, recapitulating developmental delay in patients. SNORD118 mutations in NPCs disrupted nucleolar morphology and LLPS properties coupled with impaired ribosome biogenesis and a translational downregulation of fibrillarin (FBL), the key LLPS effector acting via the intrinsically disordered region (IDR) motif. IDR-depleted FBL failed to rescue NPC defects, whereas a chimeric FBL with swapped IDR motif from an unrelated protein mitigated ribosomopathy and organoid growth defects. Thus, SNORD118 human iPSC models revealed aberrant phase separation and nucleolar functions as potential pathogenic mechanisms in ribosomopathies. Graphical abstract Highlights•SNORD118 mutant hiPSC-derived cells and organoids recapitulate the ribosomopathy defects•Mutations impair ribosome biogenesis and translation of phase separation effector FBL•Phase separation and nucleolar organization are defective in SNORD118 mutant cells•Impaired phase separation causes ribosomopathy and growth defects in hiPSC models Natural sciences; Biological sciences; Cell biology; Stem cell research

Mutations in FUS and TARDBP cause amyotrophic lateral sclerosis (ALS), but the precise mechanisms of selective motor neuron degeneration remain unresolved. To address if pathomechanisms are shared across mutations and related to either gain- or loss-of-function, we performed single-cell RNA sequencing across isogenic induced pluripotent stem cell-derived neuron types, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. Transcriptional changes were far more pronounced in motor neurons than interneurons. About 20% of uniquely dysregulated motor neuron transcripts were shared across FUS mutations, half from gain-of-function. Most indicated mitochondrial impairments, with attenuated pathways shared with mutant TARDBP M337V as well as C9orf72-ALS patient motor neurons. Mitochondrial motility was impaired in ALS motor axons, even with nuclear localized FUS mutants, demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS, uncoupled from protein mislocalization. These early mitochondrial dysfunctions unique to motor neurons may affect survival and represent therapeutic targets in ALS. In this study, the authors performed single-cell RNA-sequencing across various isogenic mutant FUS and TDP43 neurons. Mitochondrial dysfunction emerged as pathway unique to motor neurons demonstrating shared toxic gain of-function mechanisms, uncoupled from protein mislocalization.

AbstractA basic assumption underlying induced pluripotent stem cell (iPSC) models of neurodegeneration is that disease-relevant pathologies present in brain tissue are also represented in donor-matched cells differentiated from iPSCs. However, few studies have tested this hypothesis in matched iPSCs and neuropathologically characterized donated brain tissues. To address this, we assessed iPSC-neuron production of ?-amyloid (A?) A?40, A?42, and A?43 in 24 iPSC lines matched to donor brains with primary neuropathologic diagnoses of sporadic AD (sAD), familial AD (fAD), control, and other neurodegenerative disorders. Our results demonstrate a positive correlation between A?43 production by fAD iPSC-neurons and A?43 accumulation in matched brain tissues but do not reveal a substantial correlation in soluble A? species between control or sAD iPSC-neurons and matched brains. However, we found that the ApoE4 genotype is associated with increased A? production by AD iPSC-neurons. Pathologic tau phosphorylation was found to be increased in AD and fAD iPSC-neurons compared to controls and positively correlated with the relative abundance of longer-length A? species produced by these cells. Taken together, our results demonstrate that sAD-predisposing genetic factors influence iPSC-neuron phenotypes and that these cells are capturing disease-relevant and patient-specific components of the amyloid cascade.