C9orf72 hexanucleotide repeat expansion (C9-HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). However, approximately half of the FTD patients are sporadic without a clear genetic background. To compare characteristics of microglia from different FTD subtypes, we generated induced pluripotent stem cell-derived microglia (iMG) from sporadic and C9-HRE-carrying behavioral variant FTD (bvFTD) patients and healthy controls. C9-HRE iMG displayed C9-HRE-associated RNA foci and dipeptide repeat proteins. All bvFTD iMG had fewer LAMP2-A-positive vesicles compared to control iMG. Additionally, C9-HRE iMG showed significantly increased LC3BII/I conversion after bafilomycin A1 treatment and altered phagocytic activity. The gene expression profile of C9-HRE iMG only modestly differed from the control iMG, but was greatly different from the sporadic bvFTD patient iMG. Our data show alterations in phagocytic and autophagosomal/lysosomal pathways and gene expression profiles between C9-HRE and sporadic bvFTD iMG for the first time.

Human-induced pluripotent stem cell-derived midbrain organoids offer a promising platform for modeling Parkinson's disease (PD). Yet, their utility has been limited by the absence of microglia and the development of a necrotic core during maturation. Here, we present an air-liquid interface (ALI) slice culture system for extended cultivation of midbrain organoids (mORGs), enabling efficient microglial integration, improved neuronal viability, and enhanced functional maturation. Compared with conventional mORGs grown in suspension cultures, the ALI method supports more consistent engraftment of microglial progenitors and the development of astrocytes and oligodendrocyte progenitors, as revealed by single-cell RNA sequencing. Functionally, ALI-mORGs exhibited robust and reproducible neural network activity, with N-methyl-D-aspartate (NMDA) stimulation reliably inducing synchronous bursting, as measured by 3D microelectrode array recordings. Importantly, exposure to alpha-synuclein (αSyn) preformed fibrils triggered the progressive accumulation of phosphorylated αSyn inclusions, closely recapitulating key features of PD pathology. The ALI-mORG model addresses major limitations of existing systems and provides a more physiologically relevant platform for investigating cellular mechanisms of PD and for supporting future therapeutic strategies.

Microglia play a major role in the pathophysiology of Parkinson's disease, where they regulate both α-synuclein (αSyn) aggregate clearance and inflammatory responses. Interferon gamma (IFNγ) is a strong immunomodulator, but its role in shaping human microglial phenotypes during αSyn exposure remains incompletely understood. Further, whether the secreted factors from microglia after exposure to αSyn pre-formed fibrils (PFFs) and IFNγ can affect morphology and functionality of dopaminergic neurons has not been studied. We used human stem cell-derived microglia to investigate how αSyn PFFs and IFNγ stimulation influence microglial metabolism, lipid composition, and phenotypic state. IFNγ induced broad metabolic and lipidomic remodeling, affecting glycolytic, tryptophan, and phospholipid pathways. Pre-exposure to IFNγ promoted a resolving-like phenotype upon αSyn PFFs challenge, characterized by increased transglutaminase 2 (TGM2) expression and elevated TGFβ1 secretion. Conditioned media (CM) from exposed microglia were applied to human iPSC-derived dopaminergic neurons, and neuronal morphology, protein expression, mitochondrial function, and electrophysiological activity were evaluated. CM from αSyn PFFs-exposed microglia reduced neuronal branching and neurite length. Furthermore, CM from stimulated microglia decreased TUJ1 and TH expression, altered mitochondrial regulators (cytochrome C, MnSOD, iNOS), and disrupted neuronal activity as measured by changes in c-FOS expression and multielectrode array recordings. IFNγ profoundly modulates human microglial states, enhancing resolving-like features and simultaneously driving secretome-mediated neuronal effects. These findings highlight microglial activation state as a critical determinant of neuronal vulnerability in αSyn-associated pathology. Importantly, IFNγ-induced microglial activation can, at least partially, counteract αSyn-driven adverse effects on dopaminergic neurons.

Ischemic stroke triggers a rapid immune infiltration to the brain, reshaping its cell type composition and possibly confounding the analysis of immune cell enriched molecules. MicroRNAs (miRNAs) act as powerful regulators by fine-tuning messenger RNA (mRNA) expression, thereby modulating cell type specific responses to ischemia. Several bulk tissue analyses suggest elevated levels of anti-inflammatory miRNA, miR-223-3p, in ischemic stroke but pathological relevance of this deregulation remains unclear due to lack of single-cell resolution. We show that miR-223-3p is acutely increased in ischemic stroke patients’ blood and selectively expressed in myeloid cells in mice. Due to myeloid specificity of miR-223-3p, it appears massively elevated in bulk ischemic brain lysates, although the elevation primarily reflects increased myeloid cell abundance rather than per-cell upregulation in the brain. Strikingly, brain infiltrating macrophages exhibited acutely reduced miR-223-3p levels. We hypothesize that these macrophages initially acquire a more proinflammatory phenotype than the brain resident macrophages, and re-analysis of single-cell transcriptomics (GSE234052) further supported a proinflammatory macrophage phenotype after acute cerebral ischemia. Our study exemplifies the inherent limitations of bulk tissue analyses and underscores the need to revisit conclusions drawn from the tissue-level data, especially when immune cell infiltration and enrichment of a molecule is suspected. Overcoming the technical challenges of integrated spatial miRNA and mRNA detection at single-cell resolution will be essential to dissect the regulatory mechanisms underlying ischemic stroke pathology and to inform the development of effective immunomodulatory therapies. Increased brain miR-223-3p in ischemic stroke is caused by changes in cell type composition. In healthy brain, miR-223-3p is expressed by microglia and resident macrophages. Ischemia causes infiltration of miR-223-3p-rich peripheral myeloid cells to the brain, increasing the level of miR-223-3p in tissue lysates. In ischemic macrophages, miR-223-3p was decreased while no deregulation was found in microglia.

Extracellular vesicles (EVs) are increasingly recognized as key mediators of intercellular communication in neurodegenerative disorders, yet their mechanistic contribution to Alzheimer’s disease (AD)-related molecular changes remains poorly understood. Neural progenitor cells (NPCs) are particularly relevant because deficits in their proliferation and differentiation impair neurogenesis and brain repair. To investigate how EVs may influence these processes, we isolated EVs through size exclusion chromatography (SEC) from induced pluripotent stem cells (iPSCs)- derived NPCs carrying the AD-associated PSEN1 ΔE9 mutation and from isogenic controls. EV formulations were characterized by nanoparticle tracking analysis, cryogenic transmission electron microscopy, immunoblotting, and liquid chromatography–tandem mass spectrometry-based proteomics. Their effects on iPSC-derived brain organoids (BOs) were assessed by transcriptomic profiling. AD NPC EVs showed increased 6E10-reactive Aβ-related signal and contained AD-associated protein signatures, including PZP, ALPL, POSTN, APOC3, and transferrin. Application of AD EV preparations to healthy BOs induced transcriptomic changes that partially overlapped with AD BO transcriptomic signatures, including altered developmental and synapse-associated gene programs and downregulation of metabolic pathways. Consistently, PSEN1 ΔE9 NPCs showed reduced oxidative phosphorylation and glycolysis in Seahorse extracellular flux assays, while PSEN1 ΔE9 EV-treated BOs displayed downregulation of TCA cycle and glycolytic pathways at the transcriptomic level. Together, our findings suggest that NPC-derived SEC-enriched EV preparations contain AD-associated molecular signatures that could affect developmental, synapse-associated, and metabolic gene programs in recipient BOs, providing insight into cell-cell communication in AD.

Introduction The proteasome is a critical cellular degradative machinery impaired in late-stage Alzheimer's disease (AD). However, the status and activity of the proteasome in early-stage sporadic AD (sAD) is unknown. Methods A cellular model of human early-stage sAD was generated from sAD patient iPSC-derived cortical neurons by dual-SMAD inhibition. The iPSCs, neuroprogenitors, and cortical neurons were validated by the expressions of key markers. The level of total intraneuronal Aβ was measured by ELISA. Composition and native proteolytic activities of the proteasome in control and sAD cortical neurons were measured using complementary fluorogenic probes. Results Control and sAD patients iPSCs expressed pluripotent markers OCT4, NANOG, and SSEA4 which induced into neuroprogenitors expressing NESTIN and PAX6. The neuroprogenitors terminally differentiated into cortical neurons expressing neuronal markers MAP2 and TUJ1, and cortical layer marker TBR1. The level of intraneuronal Aβ in the sAD cortical neurons was significantly higher compared to control. Control and sAD cortical neurons expressed native 30S, 26S, and 20S proteasome assemblies with the sAD cortical neurons displaying higher 20S assemblies. Increased active 20S assemblies was associated with higher β1, β2, and β5 proteolytic sites activities. Discussion The significant elevation in the proteolytic activities of the β1, β2, and β5 subunits of 20S proteasome in sAD cortical neurons suggests that this may be a possible compensatory response to elevated intraneuronal Aβ.

The ε4 isoform of apolipoprotein E (ApoE) is the most significant genetic risk factor for Alzheimer’s disease. Glial cells are the main source of ApoE in the brain, and in microglia, the ε4 isoform of ApoE has been shown to impair mitochondrial metabolism and the uptake of lipids and Aβ42. However, whether the ε4 isoform alters autophagy or lysosomal activity in microglia in basal and inflammatory conditions is unknown. Altogether, microglia-like cells (iMGs) from eight APOE3/3 and six APOE4/4 human induced pluripotent stem cell (iPSC) lines were used in this study. The responses of iMGs to Aβ42, LPS and IFNγ were studied by metabolomics, proteomics, and functional assays. Here, we demonstrate that iMGs with the APOE4/4 genotype exhibit reduced basal pinocytosis levels compared to APOE3/3 iMGs. Inflammatory stimulation with a combination of LPS and IFNγ or Aβ42 induced PI3K/AKT/mTORC signaling pathway, increased pinocytosis, and blocked autophagic flux, leading to the accumulation of sequestosome 1 (p62) in both APOE4/4 and APOE3/3 iMGs. Exposure to Aβ42 furthermore caused lysosomal membrane permeabilization, which was significantly stronger in APOE4/4 iMGs and positively correlated with the secretion of the proinflammatory chemokine IL-8. Metabolomics analysis indicated a dysregulation in amino acid metabolism, primarily L-glutamine, in APOE4/4 iMGs. Overall, our results suggest that inflammation-induced metabolic reprogramming places lysosomes under substantial stress. Lysosomal stress is more detrimental in APOE4/4 microglia, which exhibit endo-lysosomal defects.

Oligodendrocyte progenitor cells (OPCs) have been implicated in synaptic remodelling in animal models, but the underlying mechanisms and their relevance to human brain development remain unclear. Here, we generate a human multi-lineage forebrain organoid model in which OPCs, together with microglia, form close contacts with synapses and spontaneously internalize synaptic material. Single-nucleus transcriptomic profiling with unbiased cell-cell communication analysis identifies the growth arrest-specific gene 6 (GAS6)-TYRO3, AXL, and MERTK (TAM) receptor axis as a key signalling pathway, with neurons and microglia expressing GAS6 and a subset of OPCs expressing AXL. Further, dose-dependent pharmacological inhibition of TAM receptors demonstrates the importance of AXL, and targeted reduction of AXL expression in OPCs impairs synaptic uptake. These findings reveal a role for GAS6-AXL signalling in driving synaptic internalisation by AXL+ OPCs during early human brain development.

Frontotemporal dementia (FTD) is the second most common cause of dementia in patients under 65 years, characterized by diverse clinical symptoms, neuropathologies, and genetic background. Synaptic dysfunction is suggested to play a major role in FTD pathogenesis. Disturbances in the synaptic function can also be associated with the C9orf72 repeat expansion (C9-HRE), the most common genetic mutation causing FTD. C9-HRE leads to distinct pathological hallmarks, such as C9orf72 haploinsufficiency and development of toxic RNA foci and dipeptide repeat proteins (DPRs). FTD patient brains, including those carrying the C9-HRE, are also characterized by neuropathologies involving accumulation of TDP-43 and p62/SQSTM1 proteins. This study utilized induced pluripotent stem cell (iPSC)-derived cortical neurons from C9-HRE-carrying or sporadic FTD patients and healthy control individuals. We report that the iPSC neurons derived from C9-HRE carriers developed typical C9-HRE-associated hallmarks, including RNA foci and DPR accumulation. All FTD neurons demonstrated increased cytosolic accumulation of TDP-43 and p62/SQSTM1 and changes in nuclear size and morphology. In addition, the FTD neurons displayed reduced number and altered morphologies of dendritic spines and significantly altered synaptic function indicated by a decreased response to stimulation with GABA. These structural and functional synaptic disturbances were accompanied by upregulated gene expression in the FTD neurons related to synaptic function, including synaptic signaling, glutamatergic transmission, and pre- and postsynaptic membrane, as compared to control neurons. Pathways involved in DNA repair were significantly downregulated in FTD neurons. Only one gene, NUPR2, potentially involved in DNA damage response, was differentially expressed between the sporadic and C9-HRE-carrying FTD neurons. Our results show that the iPSC neurons from FTD patients recapitulate pathological changes of the FTD brain and strongly support the hypothesis of synaptic dysfunction as a crucial contributor to disease pathogenesis in FTD.

Multidisciplinary evidence support a neurodevelopmental origin for schizophrenia, yet the mechanistic translation of known risk factors remains poorly understood. In this study, we leverage multi-lineage, forebrain-patterned organoids derived from monozygotic twins discordant for schizophrenia, integrating single-cell transcriptomic and epigenomic profiling to uncover disease-associated gene expression and chromatin accessibility topics. By constructing fate probability maps, we identify an accelerated developmental trajectory emerging from a distinct, schizophrenia-associated radial glia cell state, and reveal unique interactions among schizophrenia risk genes through unbiased multimodal analyses. Further, we confirm that schizophrenia-enriched states persist in differentiated lineages and disrupt synaptic programs, accompanied by molecular and cellular phenotypes mimicking observed disease pathology. Collectively, our findings delineate an early disruption in forebrain development, providing novel mechanistic insights into the origins of schizophrenia risk. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVE:Schizophrenia is a neurodevelopmental disorder characterized by an excessive loss of synapses. Kynurenic acid (KYNA), a neuroactive metabolite of tryptophan along the kynurenine pathway, can induce schizophrenia-related phenotypes in rodents, and clinical studies have revealed elevated KYNA levels in the CNS of individuals with schizophrenia. However, the factors that cause elevated KYNA levels in schizophrenia, and the mechanisms by which KYNA contributes to pathophysiology, remain largely elusive. The authors used patient-derived cellular modeling to test the hypothesis that KYNA can induce microglia-mediated synapse engulfment by reducing neuronal activity. METHODS:Patient-derived induced pluripotent stem cells were used to generate 2D cultures of neurons and microglia-like cells, as well as forebrain organoids with innately developing microglia, to study how KYNA influences synaptic activity and microglial uptake of synaptic structures. To verify the experimental data in a clinical context, large-scale developmental postmortem brain tissue and genetic datasets were used to study coexpression networks for the KYNA-producing kynurenine aminotransferases (KATs) regarding enrichment for common schizophrenia genetic risk variants and functional annotations. RESULTS:In these patient-derived experimental models, KYNA induced uptake of synaptic structures in microglia, and inhibition of the endogenous KYNA production led to a decrease in the internalization of synapses in microglia. The integrated large-scale transcriptomic and genetic datasets showed that KYNA-producing KATs enriched for genes governing synaptic activity and genetic risk variants for schizophrenia. CONCLUSIONS:Together, these results link genetic risk variants for schizophrenia to elevated production of KYNA and excessive and activity-dependent internalization of synaptic material in microglia, while implicating pharmacological inhibition of KATs as a strategy to avoid synapse loss in schizophrenia.

Phospholipase C gamma 2, proline 522 to arginine (PLCγ2-P522R) is a protective variant that reduces the risk of Alzheimer’s disease (AD). Recently, it was shown to mitigate β-amyloid pathology in a 5XFAD mouse model of AD. Here, we investigated the protective functions of the PLCγ2-P522R variant in a less aggressive APP/PS1 mouse model of AD and assessed the underlying cellular mechanisms using mouse and human microglial models. The effects of the protective PLCγ2-P522R variant on microglial activation, AD-associated β-amyloid and neuronal pathologies, and behavioral changes were investigated in PLCγ2-P522R knock-in variant mice crossbred with APP/PS1 mice. Transcriptomic, proteomic, and functional studies were carried out using microglia isolated from mice carrying the PLCγ2-P522R variant. Finally, microglia-like cell models generated from human blood and skin biopsy samples of PLCγ2-P522R variant carriers were employed. The PLCγ2-P522R variant decreased β-amyloid plaque count and coverage in female APP/PS1 mice. Moreover, the PLCγ2-P522R variant promoted anxiety in these mice. The area of the microglia around β-amyloid plaques was also increased in mice carrying the PLCγ2-P522R variant, while β-amyloid plaque-associated neuronal dystrophy and the levels of certain cytokines, including IL-6 and IL-1β, were reduced. These alterations were revealed through [18F]FEPPA PET imaging and behavioral studies, as well as various cytokine immunoassays, transcriptomic and proteomic analyses, and immunohistochemical analyses using mouse brain tissues. In cultured mouse primary microglia, the PLCγ2-P522R variant reduced the size of lipid droplets. Furthermore, transcriptomic and proteomic analyses revealed that the PLCγ2-P522R variant regulated key targets and pathways involved in lipid metabolism, mitochondrial fatty acid oxidation, and inflammatory/interferon signaling in acutely isolated adult mouse microglia and human monocyte-derived microglia-like cells. Finally, the PLCγ2-P522R variant also increased mitochondrial respiration in human iPSC-derived microglia. These findings suggest that the PLCγ2-P522R variant exerts protective effects against β-amyloid and neuronal pathologies by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice. The changes observed in lipid/fatty acid and mitochondrial metabolism revealed by the omics and metabolic assessments of mouse and human microglial models suggest that the protective effects of the PLCγ2-P522R variant are potentially associated with increased metabolic capacity of microglia.

Microglia, brain-resident immune cells, are involved in pathophysiology of several neurodegenerative diseases, including Parkinson’s disease. Given significant species-specific differences in microglia gene expression, particularly in disease-risk genes, as well as the highly reactive nature of these cells, studying human microglia in a whole brain environment is essential. Here, we established a humanized mouse model by transplanting human induced pluripotent stem cell-derived hematopoietic progenitor cells into the striatum of immunodeficient adult mice and injected human alpha-synuclein preformed fibrils to model Parkinson’s disease pathology. Transplanted human cells engraft, mature into microglia and maintain their phenotype for at least three months post-transplantation. These human microglia interact with alpha-synuclein, significantly limiting its propagation from the striatum to the substantia nigra and further reducing local small aggregates; they also mildly protect tyrosine hydroxylase neurons there. Transcriptomic profiling reveals 56 differentially expressed genes in human microglia in response to alpha-synuclein preformed fibrils, while host mouse cells show 202 gene expression changes, including an upregulation of gene Hcrt (fold change = 7.77, p = 0.0015). Immunohistochemistry analysis further confirms the preservation of hypocretin-positive neurons in the hypothalamus of the transplanted mice (p = 0.0079). The findings highlight the neuroprotective role of human microglia and establish a more disease-relevant in vivo model for investigating alpha-synuclein aggregation and therapeutic interventions in Parkinson’s disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to infect areas of the human brain and a subset of neurons in vitro. We have previously demonstrated that the virus enters human induced pluripotent stem cell (hiPSC)-derived neurons via an endosomal-lysosomal pathway. Here, we show that neuronal infection with both SARS-CoV-2 Wuhan and Omicron XBB.1.5 variants is dependent on cathepsins and can be blocked by an inhibitor of cathepsin B. The result was reproducible in non-transgenic hiPSC-derived cortical organoids. We further show that SARS-CoV-2 can replicate in neuron cultures, but the infectivity of the newly produced virions declined at 24 h post-infection despite a further increase in released viral RNA at later time points. The number of infected neurons decreased within five days, suggesting virus-induced neuronal cell death. The infection also caused the accumulation of the hypoxia-inducible stress factor HIF1-α in infected neurons under normoxia. Finally, expanding previous findings, in SARS-CoV-2 infected neurons, the microtubule-associated protein tau was hyperphosphorylated at multiple loci, including S202/T205, and mislocalized to the soma of infected 2D-neuronal cultures, but not in 3D-organotypic models. Hence, the neurodegenerative potential of SARS-CoV-2 infection should be carefully considered in different infection models.

BACKGROUND: Brain endothelial cells (ECs) lining blood vessels are essential for the normal function of the brain. They form the first layer of the blood-brain barrier (BBB) and regulate nutrient exchange, immune responses, and angiogenesis. Numerous studies have reported the disruption of the BBB in neurodegenerative diseases, including Alzheimer’s disease (AD). However, the impact of cell-intrinsic amyloid pathology on EC function remains to be clarified. METHODS: To optimize the method for producing functional ECs from human induced pluripotent stem cells (hiPSCs), we compared two different protocols. The first, a widely used method, relies on spontaneous differentiation after mesoderm specification. The second method involves transient overexpression of ETV2 to guide EC differentiation. To study the impact of beta-amyloid overproduction on EC function, we generated ECs from hiPSC lines carrying the APP Swedish mutation (APPswe), which causes AD. We assessed the functionality of both control and APPswe ECs using in vitro permeability assays, 2D and 3D vessel formation assays, and adhesion assays. RESULTS: ECs generated using transient ETV2 overexpression exhibited higher levels of canonical EC markers, tight junction proteins, transporters, leukocyte adhesion molecules, and angiogenesis-associated receptors than ECs derived by spontaneous differentiation. Additionally, ETV2-ECs responded robustly to inflammatory and angiogenic stimuli, displaying functional and transcriptional changes, whereas spontaneously differentiated ECs did not. Consequently, we chose the ETV2 overexpression protocol to study the impact of APPswe mutation on endothelial function. We found that ETV2-ECs carrying the APPswe mutation displayed a reduced angiogenic potential following exposure to the sprouting mix and elevated expression of leukocyte adhesion molecules following inflammatory stimulation, leading to increased adhesion of monocyte-like cells. CONCLUSIONS: Overall, our study suggests that APPswe mutation in ECs impairs their response to inflammatory and angiogenic stimuli, potentially contributing to AD progression. Additionally, we confirmed that ETV2 overexpression during a critical window effectively guides hiPSCs toward the EC lineage, resulting in a stable and pure population of ECs suitable for disease modeling and drug screening.

Exposure to air pollution is associated with neurological diseases. Traffic is a major source of air pollution, consisting of a complex mixture of ultrafine particles, that can invade the brain and induce a microglia-mediated inflammatory response. However, the exact mechanisms of how traffic-related particles impact human microglia remain poorly understood. This study investigates the effects of diesel exhaust particles (DEPs) on human induced pluripotent stem cell-derived microglia-like cells (iMGL). We exposed iMGLs to three different DEPs and studied the impact on the iMGL transcriptome and functionality, focusing on cytokine secretion, mitochondrial respiration, lysosomal function, and phagocytosis. A20 particles were collected from a heavy-duty engine run with petroleum diesel. For A0, the same engine was run with renewable diesel. E6 was produced with a modern 2019 model diesel passenger car run with renewable diesel. RNAseq revealed activation of the cytokine storm pathway and inhibition of the autophagy pathway in iMGLs after exposure to particles derived from older diesel emission technology (A20, A0). Particles from the modern diesel engine technology (E6) did not alter microglial transcriptome after 24 h exposure. A20 and A0 exposure led to impaired lysosomal functions in iMGLs. In contrast, E6 did not cause major alterations in microglia functions. In addition, we show that response to particles is more pronounced in human iMGLs compared to mouse primary microglia. To conclude, particles from older emission technology impair phago-lysosomal functions of iMGLs, but modern alternatives with filtration do not induce drastic changes in the functionality of iMGLs.

Background The causal factors related to human sexual orientation have remained largely unknown. In several countries, homosexuality has been considered as a degenerative cultural phenomenon and is prohibited under threat of death penalty. Homosexual individuals face discrimination and have substantially higher risk of suicide and mental disorders than heterosexual individuals. Methods We studied the gene and protein expression phenotype in induced pluripotent stem cell (iPSC)- derived cortical neurons, corresponding to maturity at the late first trimester, among Finnish homosexual men (N=6), heterosexual men (N=6), and heterosexual women (N=5). Results Homosexual orientation was strongly linked to down-regulation of HHIP, GLI1, and several histone genes, and up-regulation of ZP3, FIRRE, miR-206, and miR-199a-5p. The findings were associated with DNA methylation and transcriptional repression, sonic hedgehog, and IL-33 signaling pathways. Among the most significant DEPs between homosexual and heterosexual men were downregulated collagen genes (COL8A1, COL6A1, and COL18A1), which were also enriched in the top GO and IPA pathways. Upregulated proteins found in homosexual men were related to neuronal firing (GNAO1), plasticity (CAMK2A), synapse modulation (SULT4A1), and dendrite development (ACTL6B), which could indicate improved neuronal maturation. The most significant DEP was NEDD4 Like E3 Ubiquitin Protein Ligase (NEDD4L) belonging to the ubiquitination machinery, which was upregulated in homosexual men compared to heterosexual men. The miRNA expression pattern and proteomics results indicate enhanced neuronal differentiation in homosexual men. Our findings suggest that sexual orientation is associated with the differential expression of genes coding for histone modification, left-right asymmetry, extracellular matrix modulation and X chromosome inactivation, as well as accelerated neuronal maturation, indicated by proteomic results. Discussion The results suggest that male homosexuality is a neurobiological phenotype of which correlates are present already during pregnancy. Elucidating the causal mechanism of homosexuality may help to reduce stigma and discrimination against sexual minorities.

Schizophrenia is a complex developmental disorder whose molecular mechanisms are not fully understood. The developmental course of schizophrenia can be modeled with human induced pluripotent stem cell (hiPSC) -derived brain cells that carry patient-specific genetic risk factors for the disorder. Although transcriptomic characterization of the patient-derived cells is a standard procedure, microRNA (miRNA) profiling is less frequently performed. To investigate the role of miRNAs in transcriptomic regulation in schizophrenia, we performed miRNA sequencing for hiPSC-derived neurons from five monozygotic twin pairs discordant for schizophrenia and six controls (CTR). We compared the miRNA expression to differentially expressed genes (DEGs) reported for the same cells in our earlier work. We found 21 DEmiRNAs between the affected twins (AT) and CTR with implications for the regulation of neuronal function. In addition, a separate analysis of three AT with treatment-resistant schizophrenia (TRS), their unaffected twins (UT), and CTR revealed an upregulation of four miRNAs in the UT compared to both AT and CTR. The DEmiRNAs found between the UT and CTR were associated with increased cAMP/PKA signaling and synaptogenesis signaling in the UT. We hypothesize that the upregulation of these processes in the UT could be linked to compensatory features against schizophrenia.

Alzheimer's disease (AD) is the most prevalent cause of dementia characterized by a progressive cognitive decline. Addressing neuroinflammation represents a promising therapeutic avenue to treat AD; however, the development of effective antineuroinflammatory compounds is often hindered by their limited blood-brain barrier (BBB) permeability. Consequently, there is an urgent need for accurate, preclinical AD patient-specific BBB models to facilitate the early identification of immunomodulatory drugs capable of efficiently crossing the human AD BBB. This study presents a unique approach to BBB drug permeability screening as it utilizes the familial AD patient-derived induced brain endothelial-like cell (iBEC)-based model, which exhibits increased disease relevance and serves as an improved BBB drug permeability assessment tool when compared to traditionally employed in vitro models. To demonstrate its utility as a small molecule drug candidate screening platform, we investigated the effects of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(atsm)) and a library of metal bis(thiosemicarbazone) complexes─a class of compounds exhibiting antineuroinflammatory therapeutic potential in neurodegenerative disorders. By evaluating the toxicity, cellular accumulation, and permeability of those compounds in the AD patient-derived iBEC, we have identified 3,4-hexanedione bis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(dtsm)) as a candidate with good transport across the AD BBB. Furthermore, we have developed a multiplex approach where AD patient-derived iBEC were combined with immune modulators TNFα and IFNγ to establish an in vitro model representing the characteristic neuroinflammatory phenotype at the patient's BBB. Here, we observed that treatment with CuII(dtsm) not only reduced the expression of proinflammatory cytokine genes but also reversed the detrimental effects of TNFα and IFNγ on the integrity and function of the AD iBEC monolayer. This suggests a novel pathway through which copper bis(thiosemicarbazone) complexes may exert neurotherapeutic effects on AD by mitigating BBB neuroinflammation and related BBB integrity impairment. Together, the presented model provides an effective and easily scalable in vitro BBB platform for screening AD drug candidates. Its improved translational potential makes it a valuable tool for advancing the development of metal-based compounds aimed at modulating neuroinflammation in AD.

Timely relief of edema and clearance of waste products, as well as promotion of anti-inflammatory immune responses, reduce ischemic stroke pathology, and attenuate harmful long-term effects post-stroke. The discovery of an extensive and functional lymphatic vessel system in the outermost meningeal layer, dura mater, has opened up new possibilities to facilitate post-stroke recovery by inducing dural lymphatic vessel (dLV) growth via a single injection of a vector encoding vascular endothelial growth factor C (VEGF-C). In the present study, we aimed to improve post-stroke outcomes by inducing dLV growth in mice. We injected mice with a single intracerebroventricular dose of adeno-associated viral particles encoding VEGF-C before subjecting them to transient middle cerebral artery occlusion (tMCAo). Behavioral testing, Gadolinium (Gd) contrast agent-enhanced magnetic resonance imaging (MRI), and immunohistochemical analysis were performed to define the impact of VEGF-C on the post-stroke outcome. VEGF-C improved stroke-induced behavioral deficits, such as gait disturbances and neurological deficits, ameliorated post-stroke inflammation, and enhanced an alternative glial immune response. Importantly, VEGF-C treatment increased the drainage of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF), as shown by Gd-enhanced MRI. These outcomes were closely associated with an increase in the growth of dLVs around the region where we observed increased vefgc mRNA expression within the brain, including the olfactory bulb, cortex, and cerebellum. Strikingly, VEGF-C-treated ischemic mice exhibited a faster and stronger Gd-signal accumulation in ischemic core area and an enhanced fluid outflow via the cribriform plate. In conclusion, the VEGF-C-induced dLV growth improved the overall outcome post-stroke, indicating that VEGF-C has potential to be included in the treatment strategies of post-ischemic stroke. However, to maximize the therapeutic potential of VEGF-C treatment, further studies on the impact of an enhanced dural lymphatic system at clinically relevant time points are essential.