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AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV, mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation, neuronal dysfunction, and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia, we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1, ISG15, ISG20, IFI27, IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART, consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However, S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells, mature and immature ChP cells, neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons, accompanied by increased expression of genes promoting cellular senescence and cell death. Together, these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons.

PurposeOxaliplatin-induced peripheral neuropathy (OIPN) is a chronic, debilitating late effect following oxaliplatin treatment. Neurofilament light chain (NfL) is a structural protein found in nerve axons that was investigated upon oxaliplatin exposure in vitro and in vivo correlated to symptoms of OIPN in colorectal cancer patients receiving oxaliplatin.MethodsHuman sensory neurons, derived from induced pluripotent stem cells, were exposed to clinically relevant concentrations of oxaliplatin in vitro, with NfL concentrations measured in the cell medium. The prospective clinical study included patients with colorectal cancer undergoing chemotherapy therapy with or without oxaliplatin. Possible OIPN was defined as bilateral presence of numbness and/or presence of pricking sensations in the feet documented in an interview at the time of blood sampling prior to, 3, and 6 months after initiating treatment.ResultsOxaliplatin exposure led to a dose-dependent NfL increase in vitro. In the clinical cohort of 30 patients (18 in the oxaliplatin group), NfL levels rose at 3 and 6 months compared to controls. NfL level changes correlated to OIPN symptoms at the 6-month timepoint (rho 0.81, p?<?0.001). However, the interindividual variation was substantial, and most patients showed only a minor increase in NfL.ConclusionBoth in vitro and clinical data indicate that oxaliplatin exposure results in elevated NfL levels. Further prospective studies are needed to evaluate NfL as an early biomarker for OIPN, specifically focusing on the timing of blood sampling during chemotherapy treatment to enable the timely reduction of oxaliplatin.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00280-025-04773-w.

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, which were depleted upon increased energy demands. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health.

Cell signaling plays a critical role in neurodevelopment, regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify cytoplasmic and nuclear proteins, including those with post-translational modifications, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines, induced pluripotent stem cells, and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories, shed light on gene regulatory relationships, and help explore the causes and effects of diverse cell signaling in neurodevelopment. Here, the authors demonstrate Phospho-seq, a single-cell multiomics method capable of quantifying chromatin accessibility alongside intracellular proteins, including post-translationally modified proteins. Then, they apply Phospho-seq to organoid models of neurodevelopment.

Citron Kinase (CITK) is a protein encoded by the CIT gene, whose pathogenic variants underlie microcephalic phenotypes that characterize MCPH17 syndrome. In neural progenitors, CITK loss leads to microtubule instability, resulting in mitotic spindle positioning defects, cytokinesis failure, and accumulation of DNA double strand breaks (DSBs), ultimately resulting in TP53-dependent senescence and apoptosis. Although DNA damage accumulation has been associated with impaired homologous recombination (HR), the role of CITK in this process and whether microtubule dynamics are involved is still unknown. In this report we show that CITK is required for proper BRCA1 localization at sites of DNA DSBs. We found that CITK’s scaffolding, rather than its catalytic activity, is necessary for maintaining BRCA1 interphase levels in progenitor cells during neurodevelopment. CITK regulates the nuclear levels of HDAC6, a modulator of both microtubule stability and DNA damage repair. Targeting HDAC6 in CITK-deficient cells increases microtubule stability and recovers BRCA1 localization defects and DNA damage levels to that detected in controls. In addition, the CIT-HDAC6 axis is functionally relevant in a MCPH17 zebrafish model, as HDAC6 targeting recovers the head size phenotype produced by interfering with the CIT orthologue gene. These data provide novel insights into the functional interplay between HR and microtubule dynamics and into the pathogenesis of CITK based MCPH17, which may be relevant for development of therapeutic strategies.