Biology subjects

Abdian, N.

Publications and source records attributed to Abdian, N..

12 recordsLinked to original sources

bioRxiv2025

LRRK2 Kinase Mediates Increased GCase Activity in Microglia in Response to Proinflammatory Stimuli

Variants in the LRRK2 and GBA1 genes are among the most common risk factors associated with Parkinsons disease (PD). Both patients carrying PD-associated variants in GBA1, encoding lysosomal enzyme glucocerebrosidase (GCase), and a subset of non-carrier patients have been shown to have reduced GCase enzymatic activity, suggesting that reduced GCase activity may be a feature of both genetic and a subset of sporadic PD. However, the effect of PD-associated variants in LRRK2, encoding a serine/threonine kinase, on GCase activity remains controversial, with conflicting results in various tissues and cell types. Moreover, rare patients carrying both GBA1 and LRRK2 risk alleles seem to have a more benign disease course than carriers of GBA1 variants alone, suggesting a complex interplay between these two genes in PD. Here we evaluate the effect of LRRK2 kinase activity on GCase activity in human induced pluripotent stem cell (iPSC)-derived microglia (iMGs), a PD-relevant brain cell type expressing high levels of LRRK2. Using CRISPR editing, isogenic control iPSC lines were generated to match PD patient-derived iPSC lines harbouring the LRRK2 p.G2019S, p.M1646T, or p.N551K-p.R1398H protective haplotype variants. Whereas iMGs harbouring the p.M1646T variant, and the protective haplotype, respectively increased and decreased phosphorylation of canonical LRRK2 substrate, Rab10, GCase protein levels and activity were not altered in any of the LRRK2 variant lines. Additionally, whereas pharmacological inhibition of LRRK2 kinase activity had no impact on GCase activity in iMGs under basal conditions, it attenuated the increase in GCase activity elicited in response to interferon {gamma} (IFN{gamma}) treatment. Moreover, GCase activity induced by IFN{gamma} was reduced in PD risk LRRK2 p.M1646T iMGs and increased in p.N551K-p.R1398H protective haplotype iMGs compared to their isogenic corrected controls, congruent with their respective effects on LRRK2 kinase activity and PD risk. Thus, our data suggests a role for LRRK2 kinase activity in regulation of GCase activity in response to neuroinflammation.

neuroscience↗

bioRxiv2025

Glucosylceramide induced ectosomes propagate pathogenic α-synuclein in Parkinson's disease

Intercellular transmission of -synuclein contributes to Parkinsons disease pathology. Yet, the mechanisms of -synuclein spread are not fully understood. Here, we used live-cell microscopy to examine the impact of Parkinsons disease associated lipid alterations on -synuclein release. We discovered that increased glucosylceramides induce ectosome shedding from primary neurons, and from dopaminergic neurons derived from Parkinsons disease patient iPSCs harboring mutations in GBA1 (N370S, L444P and W378G) and LRRK2 (G2019S and R1441H) compared to their isogenic control. We show that elevated glucosylceramide similarly increases vesicle release and uptake by other neurons in living mouse brains using 2-photon microscopy. Finally, we show that ectosomes are loaded with pathogenic -synuclein and lead to the transmission of -synuclein pathology to neighbouring neurons. These data reveal ectosomes as the predominant route for -synuclein transmission that can only be appreciated by live-cell imaging technologies.

cell biology↗

bioRxiv2025

Mutations in SOD1 induce ALS-related phenotypes in 3D iPSC-derived motor neuron (MN) spheroids

A significant challenge in ALS research is the heterogeneity of the disease. Even mutations within the same gene can lead to different disease prognosis. For instance, in silico protein modeling predicts distinct properties for distinct SOD1 mutations. With this in mind, in this study, we generated and characterized 3D iPSC-derived MN spheroids carrying homozygous knock-in SOD1 mutations (D90A and G93A), as well as a double mutation (D90A/G93A), to evaluate potential synergistic effects. An isogenic control line with the same genetic background was used for phenotypic comparisons with the knock-in variants. Mutant SOD1 MN spheroids exhibited multiple ALS-related phenotypes including altered SOD1 expression, reduced cell viability, downregulation of neurofilament (NF) subunit expression, hypoactivity, and altered burst activity. Our results highlight the advantages of using 3D MN spheroids as a disease model and stress the importance of considering phenotype variability at the genetic level in ALS.

neuroscience↗

bioRxiv2024

Transcriptome-based screening in TARDBP/TDP-43 knock-in motor neurons identifies the NEDD8-activating enzyme inhibitor MLN4924

A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease, evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues, and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations, we performed whole-transcriptome profiling of motor neurons differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database, we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation, the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity, highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS.

neuroscience↗

bioRxiv2023

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinsons disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

neuroscience↗

bioRxiv2023

Transcriptional dysregulation and impaired neuronal activity in FMR1 knock-out and Fragile X patients iPSC-derived models.

The lack of fragile X mental retardation protein (FMRP) protein, due to a repression of the FMR1 gene, causes Fragile X syndrome (FXS), one of the most prevalent forms of syndromic autisms. The FMR1 gene codes for an RNA binding protein involved in the regulation of gene expression through RNA processing, control of local translation, and protein-protein interactions; processes that are crucial for proper brain development. Taking advantage of induced pluripotent stem cells (iPSCs) and CRISPR-Cas9 genome editing technologies, we generated iPSC-derived cortical neural progenitors and cortical neurons from an FMR1 knock-out and patient cell line with the aim of identifying common phenotypes between the two cellular models. Using RNA sequencing, quantitative PCR and multielectrode array approaches, we assessed how the absence of the functional FMR1 gene affects the transcriptional profiles and the activities of iPSC-derived cortical neuronal progenitor cells (NPCs) and neurons with both models. We observed that FMR1 KO and FXS patient cells have a decrease in their mean firing rate; a cellular activity that can also be blocked by tetrodotoxin (TTX) application in wild-type active neurons. Relative to wild-type neurons, in FMR1 KO neurons, increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and Autism Related Genes found amongst downregulated genes. This implies that an absence of functional FMRP affects transcriptional profiles at the NPC stage, resulting in impaired activity and differentiation of the progenitors into mature neurons over time. These findings from the FMR1 KO lines were also shared with FXS patients iPSC-derived cells that also present with an impairment in activity and neuronal differentiation, illustrating the critical role of FMRP protein in neuronal development.

neuroscience↗

bioRxiv2023

Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to progressive synaptic dysfunction in human iPSC-derived motor neurons

Cytoplasmic 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 the TARDBP gene (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 iPSC lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. 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.

neuroscience↗

bioRxiv2022

Generation of PRKN and PINK1-KO and double KO cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing

Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinsons disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinsons disease.

neuroscience↗

bioRxiv2021

A streamlined CRISPR workflow to introduce mutations and generate isogenic iPSCs for modeling amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) represents a complex neurodegenerative disorder with significant genetic heterogeneity. To date, both the genetic etiology and the underlying molecular mechanisms driving this disease remain poorly understood, although in recent years a number of studies have highlighted a number of genetic mutations causative for ALS. With these mutations pointing to potential pathways that may be affected within individuals with ALS, having the ability to generate human neurons and other disease relevant cells containing these mutations becomes even more critical if new therapies are to emerge. Recent developments with the advent of induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing fields gave us the tools to introduce or correct a specific mutation at any site within the genome of an iPSC, and thus model the specific contribution of risk mutations. In this study we describe a rapid and efficient way to either introduce a mutation into a control line, or to correct a mutation, generating an isogenic control line from patient-derived iPSCs with a given mutation. The mutations introduced were the G93A mutation into SOD1 or H517Q into FUS, and the mutation corrected was a patient iPSC line with I114T in SOD1. A combination of small molecules and growth factors were used to guide a stepwise differentiation of the edited cells into motor neurons in order to demonstrate that disease-relevant cells could be generated for downstream applications. Through a combination of iPSCs and CRISPR editing, the cells generated here will provide fundamental insights into the molecular mechanisms underlying neuron degeneration in ALS.

neuroscience↗

bioRxiv2021

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy

SNCA, the first gene associated with Parkinsons disease, encodes the -synuclein (-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinsons disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of -syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of -syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of -syn and exhibit an age-dependent increase in -syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of -syn. These phosphorylated -syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinsons disease and provide a powerful system to study the pathogenesis of synucleinopathies.

neuroscience↗

bioRxiv2021

Auto-qPCR: A Python based web app for automated and reproducible analysis of qPCR data

Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app (https://auto-q-pcr.com/) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

molecular biology↗

bioRxiv2021

Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic integrity in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based test for the detection of hotspot mutations test. Cell-line identity was authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by qPCR for progenitor, neuronal, cortical, and glial markers. Taken together, we present a multistep quality control workflow to evaluate variability and reproducibility across and between iPSCs.

cell biology↗