Biology subjects

Sahin, M.

Publications and source records attributed to Sahin, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Postnatal gene restoration in succinic semialdehyde dehydrogenase deficiency (SSADHD) reveals phenotype reversibility

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder due to loss-of-function ALDH5A1 mutations impairing the catabolism of {gamma}-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain. In SSADHD, pathologic accumulation of GABA and its metabolic by-product {gamma}-hydroxybutyrate (GHB) corresponds to a clinical syndrome dominated by developmental delay and epilepsy in half of patients with risk of sudden death in adolescence and adulthood. Brain-wide ALDH5A1 gene replacement for SSADHD is unavailable, and whether such treatment will reverse the SSADHD phenotype is unknown. We developed an inducible mouse SSADHD model, Aldh5a1lox-STOP, enabling Cre-dependent Aldh5a1 restoration to evaluate gene therapy feasibility. In the absence of SSADH, Aldh5a1lox-STOP mice exhibit hyperactivity and excessive serum GHB levels, culminating in death by [~]postnatal day 22, recapitulating the severe SSADHD condition. Systemic delivery of a blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV) carrying a Cre gene to Aldh5a1lox-STOP mice leads to brain-wide SSADH restoration, serum GHB level reduction, normalization of hyperactivity, and substantial increase in survival. As a step toward clinical translation, we further assessed an AAV encompassing a functional native promoter (FLnP) of ALDH5A1 tethered to its human coding sequence, namely AAV-FLnP-hALDH5A1. Aldh5a1lox-STOP mice were effectively rescued when treated with AAV-FLnP-hALDH5A1 packaged in the blood-brain barrier (BBB)-penetrating capsid PHP.eB. These findings provide preclinical proof that SSADH gene replacement therapy is feasible and potentially effective.

neuroscience↗

bioRxiv2025

An open-source pipeline for calcium imaging and all-optical physiology in human stem cell-derived neurons

High-throughput profiling of neuronal activity at single-cell resolution is essential for advancing our understanding of brain function, enabling large-scale functional screens, and modeling neurological disorders. However, existing approaches are limited by scalability, manual data processing, and variability, thus restricting their ability to detect disease-associated phenotypes. Here, we present a scalable, open-source platform that integrates optogenetic stimulation, calcium imaging, automated data acquisition, and a fully integrated analysis pipeline. By combining spontaneous and evoked activity profiling, the system enables robust quantification of dynamic neuronal responses across hundreds of stem cell-derived human neurons and multiple timepoints, facilitating phenotyping at both cellular and network levels. We validated the platform by recapitulating established activity phenotypes in neurodevelopmental disorders including CDKL5 Deficiency Disorder and SSADH deficiency. In addition, we generated CRISPR-Cas9 knock-in human induced pluripotent stem cell (hiPSC) lines stably expressing the genetically encoded calcium indicator GCaMP6s to model network dysfunction in Tuberous Sclerosis Complex (TSC). Using this system, we further demonstrated functional rescue of the altered neuronal activity observed in the TSC following pharmacological intervention. By linking single-cell dynamics to population-level phenotypes, this framework provides a powerful and broadly applicable tool for disease modeling, mechanistic studies, and therapeutic screening across a range of neurological disorders.

neuroscience↗

bioRxiv2025

Arrayed CRISPR/Cas9 Loss-Of-Function Screen in a Neuronal Model of Adaptor Protein Complex 4 Deficiency Identifies Modulators of ATG9A Trafficking

Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including the autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the druggable genome, in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

cell biology↗

bioRxiv2024

Excitatory Cortical Neurons from CDKL5 Deficiency Disorder Patient-Derived Organoids Show Early Hyperexcitability Not Identified in Neurogenin2 Induced Neurons

CDKL5 deficiency disorder (CDD) is a rare developmental and epileptic encephalopathy resulting from variants in cyclin-dependent kinase-like 5 (CDKL5) that lead to impaired kinase activity or loss of function. CDD is one of the most common genetic etiologies identified in epilepsy cohorts. To study how CDKL5 variants impact human neuronal activity, gene expression and morphology, CDD patient-derived induced pluripotent stem cells and their isogenic controls were differentiated into excitatory neurons using either an NGN2 induction protocol or a guided cortical organoid differentiation. Patient-derived neurons from both differentiation paradigms had decreased phosphorylated EB2, a known molecular target of CDKL5. Induced neurons showed no detectable differences between cases and isogenic controls in network activity using a multielectrode array, or in MAP2+ neurite length, and only two genes were differentially expressed. However, patient-derived neurons from the organoid differentiation showed increased synchrony and weighted mean firing rate on the multielectrode array within the first month of network maturation. CDD patient-derived cortical neurons had lower expression of CDKL5 and HS3ST1, which may change the extracellular matrix around the synapse and contribute to hyperexcitability. Similar to the induced neurons, there were no differences in neurite length across or within patient-control cell lines. Induced neurons have poor cortical specification while the organoid derived neurons expressed cortical markers, suggesting that the changes in neuronal excitability and gene expression are specific to cortical excitatory neurons. Examining molecular mechanisms of early hyperexcitability in cortical neurons is a promising avenue for identification of CDD therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/622878v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9d84b8org.highwire.dtl.DTLVardef@1745073org.highwire.dtl.DTLVardef@14dbd20org.highwire.dtl.DTLVardef@bba058_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Targetable Treatment Resistance in Thyroid Cancer with Clonal Hematopoiesis

Anaplastic thyroid cancer (ATC) is a clinically aggressive malignancy with a dismal prognosis. Combined BRAF/MEK inhibition offers significant therapeutic benefit in patients with BRAFV600E-mutant ATCs. However, relapses are common and overall survival remains poor. Compared with differentiated thyroid cancer, a hallmark of ATCs is significant infiltration with myeloid cells, particularly macrophages. ATCs are most common in the aging population, which also has an increased incidence of TET2-mutant clonal hematopoiesis (CH). CH-mutant macrophages have been shown to accelerate CH-associated pathophysiology including atherosclerosis. However, the clinical and mechanistic contribution of CH-mutant clones to solid tumour biology, prognosis and therapeutic response has not been elucidated. Here we show that TET2-mutant CH is enriched in the tumour microenvironment of patients with solid tumours and associated with adverse prognosis in ATC patients. We find that Tet2-mutant macrophages selectively infiltrate mouse BrafV600E-mutant ATC and that their overexpression of Tgf{beta}-family ligands mediates resistance to BRAF/MEK inhibition. Importantly, inhibition of Tgf{beta} signaling restores sensitivity to MAPK pathway inhibition, opening a path for synergistic strategies to improve outcomes of patients with ATCs and concurrent CH.

cancer biology↗

bioRxiv2024

AKT-mediated phosphorylation of TSC2 controls stimulus and tissue-specific mTORC1 signaling and organ growth

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

cell biology↗

bioRxiv2024

TAD hierarchy restricts poised LTR activation and loss of TAD hierarchy promotes LTR co-option in cancer

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

cancer biology↗

bioRxiv2024

Human TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro, including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

developmental biology↗

bioRxiv2023

Rescue of Impaired Blood-Brain Barrier in Tuberous Sclerosis Complex Patient Derived Neurovascular Unit

Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

neuroscience↗

bioRxiv2023

A Novel Drosophila Model to Investigates Adipose Tissue Macrophage Infiltration (ATM) and Obesity highlights the Therapeutic Potential of Attenuating Eiger/TNF Signaling to Ameliorate Insulin Resistance and ATM.

Obesity is a global health concern associated with various metabolic disorders including insulin resistance and adipose tissue inflammation characterized by adipose tissue macrophage (ATM) infiltration. In this study, we present a novel Drosophila model to investigate the mechanisms underlying ATM infiltration and its association with obesity-related pathologies. Furthermore, we demonstrate the therapeutic potential of attenuating Eiger/TNF signaling to ameliorate insulin resistance and ATM. To study ATM infiltration and its consequences, we established a novel Drosophila model (OBL) that mimics key aspects of human adipose tissue and allows for investigating ATM infiltration and other related metabolic disorders in a controlled experimental system. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic, and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy, and high levels of circulating glucose. Moreover, we observed a significant infiltration of immune cells (hemocytes) in the fat bodies accompanied by insulin resistance and systemic metabolic dysregulation. Furthermore, we found that attenuation of Eiger/TNF signaling and using metformin and anti-oxidant bio-products like anthocyanins led to a reduction in ATM infiltration and improved insulin sensitivity. Our data suggest that the key mechanisms that trigger immune cell infiltration into adipose tissue are evolutionarily conserved and may provide the opportunity to develop Drosophila models to better understand pathways critical for immune cell recruitment into adipose tissue, in relation to the development of insulin resistance in metabolic diseases such as obesity and type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). We believe that our OBL model can also be a valuable tool and provide a platform either to perform genetic screens or to test the efficacy and safety of novel therapeutic interventions for these diseases.

physiology↗

bioRxiv2023

3D Chromatin Dynamics during Innate and Adaptive Immune Memory Acquisition

Immune cells responding to pathogens undergo molecular changes that are intimately linked to genome organization. Recent work has demonstrated that natural killer (NK) and CD8+ T cells experience substantial transcriptomic and epigenetic rewiring during their differentiation from naive to effector to memory cells. Whether these molecular adaptations are accompanied by changes in three-dimensional (3D) chromatin architecture is unknown. In this study, we combine histone profiling, ATAC-seq, RNA-seq and high-throughput chromatin capture (HiC) assay to investigate the dynamics of one-dimensional (1D) and 3D chromatin during the differentiation of innate and adaptive lymphocytes. To this end, we discovered a coordinated 1D and 3D epigenetic remodeling during innate immune memory differentiation, and demonstrate that effector CD8+ T cells adopt an NK-like architectural program that is maintained in memory cells. Altogether, our study reveals the dynamic nature of the 1D and 3D genome during the formation of innate and adaptive immunological memory.

immunology↗

bioRxiv2022

Non-Canonical Functions of a Mutant TSC2 Protein in Mitotic Division

Tuberous Sclerosis Complex (TSC) is a debilitating neurodevelopmental disorder characterized by a variety of clinical manifestations including epilepsy, autism, and intellectual disability. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with mTORC1 inhibition, suggesting that multipolar division is an mTORC1-independent phenotype. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC. HighlightsO_LINovel multipolar division in patient-derived iPSCs with mutant form of tuberin, TSC2 encoded protein C_LIO_LIMutant tuberin may act in a dominant negative, mTORC1-independent manner C_LI eTOCTuberous sclerosis complex (TSC) is a disorder caused by mutations in TSC1 or TSC2 genes leading to mTORC1 hyperactivity. Chalkley and colleagues found that a mutant microdeletion allele of TSC2 causes multipolar division in human induced pluripotent stem cells. Chalkley and colleagues also found that the multipolar division from mutant TSC2 may have a dominant negative mechanism and be mTOR1-independent.

cell biology↗

bioRxiv2022

The Histopathological Assesments of Effecting Fe and Zn Heavy Metals in Cerebrum and Cerebellum Tissues of Rats

Heavy metals left intensively to the environment cause different healthy problems on people by entering the food chain heavy metals. Our aim is to provide determinig effect of in tissues especially in central nervous system as a result of consuming cause foods included heavy metals and water. Five groups were constituded by using 35 adult male Wistar-Albino sexual rats. Hematoxylin-eosin (H&E) staining was applied to determine the histological sides of the damages of heavy metals in cerebrum and cerebellum tissues and effects of given juglone (5-hydroxy-1,4-naphthoquinone) for reducing these damages. Besides, immunohistochemical TUNNEL method was applied to determine DNA damages in cell. Density of damage in cerebrum of Fe and Fe+Juglone groups were higher than in the control group. Apoptotic cells number was decreased in the group consisting of juglone. There was little damage and less apoptotic cells in treated Zn group. In the cerebellum similar cerebrum results were obtained. There were much degeneration Fe and Fe+Juglone groups cerebellum. Apoptotic cells were increased Fe groups. Fe/600 ppm. doses heavy metals have created a toxic effect on the cerebrum and cerebellum tissues.

cell biology↗

bioRxiv2021

Convergent Cerebrospinal Fluid Proteomes and Metabolic Ontologies in Humans and Animal Models of Rett Syndrome

MECP2 loss-of-function mutations cause Rett syndrome, a disorder that results from a disrupted brain transcriptome. How these transcriptional defects are decoded into a disease proteome remains unknown. We studied the proteome in Rett syndrome cerebrospinal fluid (CSF) across vertebrates. We identified a consensus proteome and ontological categories shared across Rett syndrome cerebrospinal fluid (CSF) from three species, including humans. Rett CSF proteomes enriched proteins annotated to HDL lipoproteins, complement, mitochondria, citrate/pyruvate metabolism, as well as synapse compartments. We used these prioritized and shared ontologies to select analytes for orthogonal quantification. These studies independently validated our proteome and ontologies. Ontologically selected CSF hits had genotypic discriminatory capacity as determined by Receiver Operating Characteristic (ROC) analysis and distinguished Rett from a related neurodevelopmental disorder, CDKL5 deficiency disorder. We propose that Mecp2 mutant CSF proteomes and ontologies inform novel putative mechanisms and biomarkers of disease. We suggest that Rett syndrome is a metabolic disorder impacting synapse function.

neuroscience↗

bioRxiv2021

A Tissue-Bioengineering Strategy for Modeling Rare Human Kidney Diseases In Vivo

The lack of animal models for certain human diseases precludes our understanding of disease mechanisms and our ability to test new therapies in vivo. Here we generated kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs to recapitulate a rare kidney tumor called angiomylipoma (AML). Organoids derived from TSC2-/- hiPSCs but not from isogenic TSC2+/- or TSC2+/+ hiPSCs shared a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and developed epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be robustly and consistently recapitulated with transgenic mice. Transplantation of multiple TSC2-/- kidney organoids into the kidneys of immunodeficient rats allowed us to recapitulate AML and cystic kidney disease in vivo, in a scalable fashion and with fidelity, and to test the efficiency of rapamycin-loaded nanoparticles as a novel approach to ablate AMLs by inducing apoptosis triggered by mTOR-inhibition. Collectively, these methods represent a novel tissue-bioengineering strategy for rare disease modeling in vivo.

bioengineering↗

bioRxiv2021

Single-cell dissection of the human cerebrovasculature in health and disease

Despite the importance of the blood-brain barrier in maintaining normal brain physiology and in understanding neurodegeneration and CNS drug delivery, human cerebrovascular cells remain poorly characterized due to their sparsity and dispersion. Here, we perform the first single-cell characterization of the human cerebrovasculature using both ex vivo fresh-tissue experimental enrichment and post mortem in silico sorting of human cortical tissue samples. We capture 31,812 cerebrovascular cells across 17 subtypes, including three distinct subtypes of perivascular fibroblasts as well as vasculature-coupled neurons and glia. We uncover human-specific expression patterns along the arteriovenous axis and determine previously uncharacterized cell type-specific markers. We use our newly discovered human-specific signatures to study changes in 3,945 cerebrovascular cells of Huntingtons disease patients, which reveal an activation of innate immune signaling in vascular and vasculature-coupled cell types and the concomitant reduction to proteins critical for maintenance of BBB integrity. Finally, our study provides a comprehensive resource molecular atlas of the human cerebrovasculature to guide future biological and therapeutic studies.

neuroscience↗

bioRxiv2021

Chromatin interaction aware gene regulatory modeling with graph attention networks

Linking distal enhancers to genes and modeling their impact on target gene expression are longstanding unresolved problems in regulatory genomics and critical for interpreting non-coding genetic variation. Here we present a new deep learning approach called GraphReg that exploits 3D interactions from chromosome conformation capture assays in order to predict gene expression from 1D epigenomic data or genomic DNA sequence. By using graph attention networks to exploit the connectivity of distal elements up to 2Mb away in the genome, GraphReg more faithfully models gene regulation and more accurately predicts gene expression levels than state-of-the-art deep learning methods for this task. Feature attribution used with GraphReg accurately identifies functional enhancers of genes, as validated by CRISPRi-FlowFISH and TAP-seq assays, outperforming both CNNs and the recently proposed Activity-by-Contact model. Sequence-based GraphReg also accurately predicts direct transcription factor (TF) targets as validated by CRISPRi TF knockout experiments via in silico ablation of TF binding motifs. GraphReg therefore represents an important advance in modeling the regulatory impact of epigenomic and sequence elements.

genomics↗

bioRxiv2021

Loss of Tsc1 in cerebellar Purkinje cells induces transcriptional and translation changes in FMRP target transcripts

Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1-deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using Translating Ribosome Affinity Purification (TRAP). Finally, we found that the FMRP target, SHANK2, was reduced in PC synapses, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.

neuroscience↗