Biology subjects

Thakur, P.

Publications and source records attributed to Thakur, P..

11 recordsLinked to original sources

bioRxiv2026

Chlorogenic acid fails to confer neuroprotection in a chronic mouse model of Parkinsons disease

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterized by -Synuclein (-Syn) aggregation, dopaminergic neuronal loss, and chronic neuroinflammation. Chlorogenic acid (CA), a dietary polyphenol abundant in coffee, exhibits antioxidant and anti-inflammatory properties and has shown neuroprotective effects in acute toxin-based PD models. However, its efficacy in chronic, -Syn-driven PD models remains unclear. Here, we evaluated the therapeutic potential of CA using an -Syn-based in vitro system and a chronic -Syn overexpression mouse model that recapitulates key pathological features of human PD. In vitro, CA significantly improved cell viability, reduced -Syn aggregation, and attenuated H2O2-induced apoptosis in U118 and N2a cells. In contrast, chronic oral administration of CA (100 mg/kg for 16 weeks) in C57BL/6J mice (male and female) failed to improve motor behavior, attenuate -Syn pathology, preserve nigrostriatal dopaminergic neurons, or reduce oxidative stress-associated DNA double-strand breaks in vivo. Notably, CA elicited a modest reduction in microglial and astrocytic activation in female mice, highlighting a sex-dependent immunomodulatory response. Collectively, these findings reveal a clear dissociation between robust in vitro neuroprotection and limited in vivo efficacy in a chronic -Syn-driven PD mouse model, emphasizing the importance of incorporating progressive disease paradigms and sex as a biological variable in preclinical therapeutic evaluation.

neuroscience↗

bioRxiv2025

Integrative lipidomics of brain and plasma uncovers sex-specific metabolic signatures in Parkinson's disease

Lipid dysregulation is increasingly recognized as a key feature of Parkinsons disease (PD). A central unresolved question, however, is whether lipidomic signatures identified in accessible peripheral biofluids faithfully recapitulate the pathogenic alterations within the central nervous system (CNS). This gap impedes the development of reliable biomarkers and constrains a comprehensive understanding of PD pathophysiology. To address this challenge, we employed a cross-species lipidomic approach. We modeled PD in both male and female mice by injecting human -synuclein preformed fibrils into the substantia nigra. Three months post-injection, lipidomic profiles of the midbrain and plasma were generated and compared. These findings were further validated in plasma from male and female PD patients and age-matched controls, enabling the identification of conserved alterations. We identified shared dysregulation of sphingolipids, glycerophospholipids, and fatty acids in the brains and plasma of diseased mice as well as in plasma from PD patients. Notably, lipids associated with lipid droplet biogenesis, including triacylglycerols and monoacylglycerols, were elevated in diseased mouse brains and patient plasma. These alterations coincided with a marked accumulation of lipid droplets in the mouse midbrain, and were further corroborated by increased lipid droplet abundance in macrophages derived from PD patients. Interestingly, lipid droplet accumulation exhibited sex-specific patterns: male mice displayed greater microglial accumulation, whereas female mice showed enhanced neuronal deposition. Together, these findings demonstrate that peripheral lipidomic signatures reflect CNS pathology in PD, highlighting new opportunities for biomarker discovery and therapeutic intervention. Furthermore, sex-specific lipid droplet accumulation in innate immune cells and neurons implicates these pathways as mechanistic contributors to PD and underscores the necessity of sex-stratified strategies in biomarker discovery and disease modeling.

neuroscience↗

bioRxiv2025

Metabolic regulation of copper homeostasis governs the Sec61-dependent protein translocation process in Saccharomyces cerevisiae

Concentration of cellular labile pool of copper must be strictly regulated because disruption in copper homeostasis results in diseases. In Saccharomyces cerevisiae, elevated levels of labile copper impair cell viability by inhibiting Sec61-mediated protein translocation into the endoplasmic reticulum. We investigated how metabolic pathways, specifically mitochondrial respiration and autophagy, contribute to copper homeostasis and the translocation of secretory proteins. We show that copper selectively inhibits protein translocation in yeast cells grown in minimal medium but not in rich medium, highlighting a critical role of nutrients in modulating copper toxicity. Supplementation of specific amino acids suppresses the copper-induced defects in protein translocation and cell death, identifying amino acids suppressors of the copper toxicity. Using a panel of gene deletion mutants affecting mitochondrial functions, autophagy, peroxisomes, and lipid droplets, we demonstrate that metabolic pathways regulate sub-cellular distribution of copper and translocation of secretory proteins. Further, disruption of redox and pH homeostasis, and pharmacological inhibition of respiration, reveals that correct subcellular trafficking of copper is essential to prevent inhibitory effects on protein translocation process. Together, our findings provide mechanistic insights into how metabolic status influences cellular copper homeostasis and the secretory pathway of proteins, with broader implications for understanding diseases of copper metabolism.

cell biology↗

bioRxiv2025

Glycated alpha-synuclein assemblies cause distinct Parkinsons disease pathogenesis in mice

Alpha-synuclein (-Syn) misfolding and aggregation are key drivers of Parkinsons disease (PD) pathology. Mutations and certain post-translational modifications impact its aggregation propensity and pathogenicity. Glycation, a non-enzymatic modification enhanced during hyperglycemia and aging, both known risk factors for PD, has been implicated in -Syn pathology. Although preformed -Syn-fibrils induce PD-like phenotypes in mice, the impact of glycation on their pathogenicity is unclear. In the current study, we glycated -Syn using methylglyoxal (MGO), a potent glycating agent, resulting in altered biophysical characteristics in comparison to non-glycated -Syn. Glycation inhibited the formation of typical beta sheet structures under aggregating conditions. Despite that, glycated -Syn assemblies induced dopaminergic neurodegeneration and neuroinflammation to a similar extent as the non-glycated -Syn fibrils upon their injection in the mouse substantia nigra (SN). However, these glycated assemblies triggered higher neuroinflammation and increased accumulation of receptors for advanced glycation end products (RAGE) compared to non-glycated fibrils. Consequently, an earlier onset of neuromuscular deficits and anxiety was observed in these mice. Thus, glycation of -Syn causes distinct PD-associated pathology compared to non-glycated -Syn, causing an earlier onset of motor symptoms. These findings provide insight into how glycation of -Syn due to hyperglycemia may contribute to an increased risk of PD in diabetic populations.

neuroscience↗

bioRxiv2025

A Novel Mouse Model of Parkinson's Disease for Investigating Progressive Pathology and Neuroprotection

Developing animal models that successfully recapitulate the features of progressive Parkinsons disease (PD) is crucial for understanding disease progression mechanisms and creating effective therapeutic interventions. In this study, we created a mouse model of PD by overexpressing -synuclein through a combined injection of AAV6--synuclein and preformed fibrils (PFFs) into the medial and lateral substantia nigra (SN). We also demonstrated that chronic administration of the c-Abl inhibitor PD180970 provides neuroprotection in this model. Mice injected with the AAV6--synuclein and PFF combination showed a progressive loss of dopaminergic (DA) neurons in the SN and their projections in the striatum over 24 weeks. This neuronal loss coincided with a time-dependent accumulation of phosphorylated -synuclein (p-syn) in the SN. The p-syn aggregates spread to synaptically connected DARPP-32-positive neurons in the striatum and further extended to the cortex. We also observed a contralateral spread of p-syn aggregates. Additionally, -synuclein overexpression led to a significant increase in activated microglia and astrocytes at all timepoints, with the strongest activation occurring early and gradually diminishing over time. Daily administration of PD180970 significantly reduced the loss of DA neurons caused by -synuclein injection and decreased the accumulation of p-syn in the SN. PD180970 treatment also reduced the neuroinflammation significantly. Overall, the combined injection of AAV6--synuclein and preformed fibrils into the mouse brain establishes a robust PD model, enabling detailed mechanistic studies of the disease. We further demonstrate the models utility for chronic neuroprotection studies using the potential drug PD180970, highlighting its broad applicability. Significance StatementThis study establishes a robust mouse model of Parkinsons disease (PD) by combining AAV6-mediated -synuclein overexpression and preformed fibrils (PFFs) to replicate key features of PD, such as progressive dopaminergic neuron loss, phosphorylated -synuclein accumulation, and neuroinflammation. The model captures the spread of pathological aggregates to synaptically connected brain regions, closely mimicking the human disease. By testing the c-Abl inhibitor PD180970, we demonstrate its neuroprotective effects, including reduced neuronal loss, decreased -synuclein accumulation, and neuroinflammation highlighting its therapeutic potential. This model offers a valuable platform for investigating PD mechanisms and evaluating novel interventions, bridging the gap between preclinical and clinical applications.

neuroscience↗

bioRxiv2022

Mapping phenotypic heterogeneity in melanoma onto the epithelial-hybrid-mesenchymal axis

Epithelial to mesenchymal transition (EMT) is a well-studied hallmark of epithelial-like cancers that is characterized by loss of epithelial markers and gain of mesenchymal markers. Interestingly, melanoma, which is derived from melanocytes of the skin, also undergo phenotypic plasticity toward mesenchymal-like phenotypes under the influence of various micro-environmental cues. Our study connects EMT to the phenomenon of de-differentiation (i.e., transition from proliferative to more invasive phenotypes) observed in melanoma cells during drug treatment. By analyzing 78 publicly available transcriptomic melanoma datasets, we found that de-differentiation in melanoma is accompanied by upregulation of mesenchymal genes, but not necessarily a concomitant loss of an epithelial program, suggesting a more "one-dimensional" EMT that leads to a hybrid epithelial/ mesenchymal phenotype. Samples lying in the hybrid epithelial/mesenchymal phenotype also correspond to the intermediate phenotypes in melanoma along the proliferative-invasive axis - neural crest and transitory ones. Interestingly, as melanoma cells progress along the invasive axis, the mesenchymal signature does not increase monotonically. Instead, we observe a peak in mesenchymal scores followed by a decline, as cells further de-differentiate. This biphasic response recapitulates the dynamics of melanocyte development, suggesting close interactions among genes controlling differentiation and mesenchymal programs in melanocytes. Similar trends were noted for metabolic changes often associated with EMT in carcinomas in which progression along mesenchymal axis correlates with the downregulation of oxidative phosphorylation, while largely maintaining glycolytic capacity. Overall, these results provide an explanation for how EMT and dedifferentiation axes overlap with respect to their transcriptional and metabolic programs in melanoma.

cancer biology↗

bioRxiv2022

EMC10 reduction in human neurons and adult mouse brain rescues cellular and behavioral deficits linked to 22q11.2 deletion

Adults and children with the 22q11.2 Deletion Syndrome demonstrate cognitive, social and emotional impairments and high risk for schizophrenia. Work in mouse model of the 22q11.2 deletion provided compelling evidence for abnormal expression and processing of microRNAs. A major transcriptional effect of the microRNA dysregulation is up-regulation of Emc10, a component of the ER membrane complex, which promotes membrane insertion of a subset of polytopic and tail-anchored membrane proteins. We previously uncovered a key contribution of EMC10 in mediating the behavioral phenotypes observed in 22q11.2 deletion mouse models. Here we show that expression and processing of miRNAs is abnormal and EMC10 expression is elevated in neurons derived from 22q11.2 deletion carriers. Reduction of EMC10 levels restores defects in neurite outgrowth and calcium signaling in patient neurons. Furthermore, antisense oligonucleotide administration and normalization of Emc10 in the adult mouse brain not only alleviates cognitive deficits in social and spatial memory but remarkably sustains these improvements for over two months post injection, indicating its therapeutic potential. Broadly, our study integrates findings from both animal models and human neurons to elucidate the translational potential of modulating EMC10 levels and downstream targets as a specific venue to ameliorate disease progression in 22q11.2 Deletion Syndrome.

neuroscience↗

bioRxiv2021

Transcriptomic-based quantification of the epithelial-hybrid-mesenchymal spectrum across biological contexts

Epithelial-mesenchymal plasticity (EMP) underlies embryonic development, wound healing, and cancer metastasis and fibrosis. Cancer cells exhibiting EMP often have more aggressive behavior, characterized by drug resistance, and tumor-initiating and immuno-evasive traits. Thus, the EMP status of cancer cells can be a critical indicator of patient prognosis. Here, we compare three distinct transcriptomic-based metrics - each derived using a different gene list and algorithm - that quantify the EMP spectrum. Our results for 96 cancer-related RNA-seq datasets reveal a high degree of concordance among these metrics in quantifying the extent of EMP. Moreover, each metric, despite being trained on cancer expression profiles, recapitulates the expected changes in EMP scores for non-cancer contexts such as lung fibrosis and cellular reprogramming into induced pluripotent stem cells. Thus, we offer a scoring platform to quantify the extent of EMP in vitro and in vivo for diverse biological applications including cancer.

bioinformatics↗

bioRxiv2021

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells

Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as trouble with cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. Apart from genome editing efficiency, hiPSC survival following single-cell cloning has proved to be challenging and has thus restricted the capability to easily isolate homogeneous clones from edited hiPSCs. To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Our reliable single-cell cloning and expansion workflow did not affect the biology of the hiPSCs as the cells retained their growth and morphology, expression of various pluripotency markers and normal karyotype. This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

cell biology↗

bioRxiv2019

Selective K-ATP channel-dependent loss of pacemaking in vulnerable nigrostriatal dopamine neurons by α-synuclein aggregates

Parkinson disease (PD), one of the most common neurodegenerative disorder, is believed to be driven by toxic -synuclein aggregates eventually resulting in selective loss of vulnerable neuron populations, prominent among them, nigrostriatal dopamine (DA) neurons in the lateral substantia nigra (l-SN). How -synuclein aggregates initiate a pathophysiological cascade selectively in vulnerable neurons is still unclear. Here, we show that the exposure to low nanomolar concentrations of -synuclein aggregates (i.e. fibrils) but not its monomeric forms acutely and selectively disrupted the electrical pacemaker function of the DA subpopulation most vulnerable in PD. This implies that only dorsolateral striatum projecting l-SN DA neurons were electrically silenced by -synuclein aggregates, while the activity of neither neighboring DA neurons in medial SN projecting to dorsomedial striatum nor mesolimbic DA neurons in the ventral tegmental area (VTA) were affected. Moreover, we demonstrate functional K-ATP channels comprised of Kir6.2 subunit in DA neurons to be necessary to mediate this acute pacemaker disruption by -synuclein aggregates. Our study thus identifies a molecularly defined target that quickly translates the presence of -synuclein aggregates into an immediate impairment of essential neuronal function. This constitutes a novel candidate process how a protein-aggregation-driven sequence in PD is initiated that might eventually lead to selective neurodegeneration.

neuroscience↗

bioRxiv2019

Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a-deficient mice

SETD1A, a histone methyltransferase, is a key schizophrenia susceptibility gene. Mutant mice carrying a heterozygous loss-of-function mutation of the orthologous gene exhibit alterations in axonal branching and cortical synaptic dynamics, accompanied by specific deficits in working memory that recapitulates SCZ-related alterations. We show that Setd1a targets mostly enhancers and reveal a striking overlap between Setd1a and Mef2 chromatin targets. Setd1a targets are highly expressed in pyramidal neurons and enriched for genes with postnatally-biased expression involved in synaptic structure and function. Notably, evolutionary conserved Setd1a binding sites and target genes are strongly associated with neuropsychiatric genetic risk burden. Reinstating Setd1a expression in adulthood rescues working memory deficits. We identify LSD1 as a major demethylase counteracting the effects of Setd1a methyl transferase activity and show that LSD1 antagonism in adult Setd1a-deficient mice results in a full rescue of the behavioral abnormalities and axonal branching deficits. Our findings advance our understanding of how SETD1A mutations predispose to SCZ and point to therapeutic interventions.

neuroscience↗