Biology subjects

Gowrishankar, S.

Publications and source records attributed to Gowrishankar, S..

9 recordsLinked to original sources

bioRxiv2026

Compartment-Specific Lysosomal Heterogeneity in Microglia Is Regulated by Adaptor Protein Complex-4

Microglia rely on lysosomes to clear phagocytosed material and orchestrate immune responses, but the mechanisms governing glial lysosome diversity and function remain largely undefined. Using high-resolution, high-throughput 3D imaging of mouse brain tissue, we find that glial lysosomes are heterogeneous in size and protease content depending on subcellular location. CD68+ and LAMP1+ vesicles were more numerous in glial processes than cell bodies; process LAMP1+ vesicles carried less protease than cell-body vesicles, but CD68+ protease content did not differ between compartments, revealing at least two lysosomal subpopulations within processes. Loss of Adaptor Protein complex 4 (AP-4) selectively remodels this landscape: it increases CD68+ vesicle number and CD68 enrichment, particularly in processes, without changing protease content, and disrupts the normal cell-body-versus-process pattern in protease- and CD68-enrichment. AP-4 loss also lowers protease content in cell-body LAMP1+ vesicles, mirroring a neuronal phenotype. These results provide the first evidence of glial lysosome heterogeneity and implicate AP-4 in shaping microglial lysosome composition and inflammatory function.

cell biology↗

bioRxiv2026

Pathogenic MAPK8IP3 variants drive distinct motor and behavioral phenotypes in humans and mice

Pathogenic variants in MAPK8IP3 (JIP3) cause Neurodevelopmental Disorder with or without variable Brain Abnormalities (NEDBA), characterized by cognitive impairment, global developmental delay, motor dysfunction, abnormal muscle tone, behavioral dysregulation including autism and attention deficit hyperactivity disorder (ADHD), seizures, and structural brain abnormalities. While more than 30 largely de novo MAPK8IP3 variants have been reported, the functional impact of variants across JIP3 protein structural domains is poorly defined. To address this knowledge gap, we compare clinical features of individuals with a truncating (p.E27X) or one of two missense (p.R578C and p.R1146C) variants from distinct JIP3 functional protein domains to corresponding knock-in mouse models. Our findings showed that all individuals, regardless of variant type, exhibited delays in language and gross motor function, but individual variants were associated with distinct motor, cognitive, and psychiatric symptoms. Corresponding homozygous p.E27X and p.R1147C variant mice resulted in embryonic lethality, consistent with essential roles for JIP3 in early neurodevelopment. Behavioral characterization of viable heterozygous mice revealed variant-specific locomotor, motor coordination, and hindlimb clasping defects that closely recapitulate clinical observations. Heterozygous p.R579C mice exhibited reduced locomotion, impaired motor performance, and hypertonia-like clasping, mirroring human motor deficits. In contrast, p.R1147C mice displayed hyperactivity, hindlimb clasping, and decreased brain weight, paralleling human clinical features. Together, our findings demonstrate that while distinct MAPK8IP3 variants lead to some shared phenotypes, they are also associated with distinct phenotypes that could reveal domain-specific aspects of JIP3 function. This work establishes the first domain-resolved in vivo rodent models of NEDBA and provides a validated translational platform for mechanistic investigation and preclinical therapeutic testing.

neuroscience↗

bioRxiv2024

Small molecule modulator of neuronal lysosome positioning and function resolves Alzheimers Disease-linked pathologies in cultured human neurons

Abnormal increase in axonal lysosome abundance is associated with multiple neurodegenerative diseases including Alzheimers disease. However, the underlying mechanisms and disease relevance are not fully understood. We have recently identified RH1115 as a small molecule modulator of the autophagy-lysosomal pathway that regulates lysosome positioning in neurons. This allowed us to manipulate neuronal lysosome distribution including in axons and interrogate its contribution to both optimal neuronal functioning and to disease pathology. We demonstrate that the small molecule not only rescues aberrant buildup of both axonal autophagic and lysosomal intermediates but also reduces secreted A{beta}42 levels in human iPSC-derived neurons lacking the lysosomal adaptor, JIP3. We thus demonstrate that restoring efficient axonal lysosome transport has an anti-amyloidogenic effect in human neurons and is a promising therapeutic strategy for Alzheimers disease. Furthermore, we show that the small molecule enhances neuronal lysosome degradation, requires the lysosomal adaptor JIP4 to rescue the axonal lysosome pathology in the JIP3 KO neurons and increases levels of the JIP4-interacting lysosomal membrane protein, TMEM55B. Lastly, treatment with the small molecule led to a striking rescue of locomotor defects in JIP3 KO zebrafish larvae. Thus, we have identified a small molecule which can be impactful in neurodegenerative diseases that have a lysosomal pathology and have determined its molecular targets in modulating axonal lysosome abundance.

cell biology↗

bioRxiv2024

Axonal organelle buildup from loss of AP-4 complex function causes exacerbation of amyloid plaque pathology and gliosis in Alzheimers disease mouse model

Lysosomes and related precursor organelles robustly build up in swollen axons that surround amyloid plaques and disrupted axonal lysosome transport has been implicated in worsening Alzheimers pathology. Our prior studies have revealed that loss of Adaptor protein-4 (AP-4) complex function, linked primarily to Spastic Paraplegia (HSP), leads to a similar build of lysosomes in structures we term "AP-4 dystrophies". Surprisingly, these AP-4 dystrophies were also characterized by enrichment of components of APP processing machinery, {beta}-site cleaving enzyme 1 (BACE1) and Presenilin 2. Our studies examining whether the abnormal axonal lysosome build up resulting from AP-4 loss could lead to amyloidogenesis revealed that the loss of AP-4 complex function in an Alzheimers disease model resulted in a strong increase in size and abundance of amyloid plaques in the hippocampus and corpus callosum as well as increased microglial association with the plaques. Interestingly, we found a further increase in enrichment of the secretase, BACE1, in the axonal swellings of the plaques of Alzheimer model mice lacking AP-4 complex compared to those having normal AP-4 complex function, suggestive of increased amyloidogenic processing under this condition. Additionally, the exacerbation of plaque pathology was region-specific as it did not increase in the cortex. The burden of the AP-4 linked axonal dystrophies/AP-4 dystrophies was higher in the corpus callosum and hippocampus compared to the cortex, establishing the critical role of AP-4 -dependent axonal lysosome transport and maturation in regulating amyloidogenic amyloid precursor protein processing. Significance StatementA major pathological feature of Alzheimers disease is the accumulation of axonal lysosomes near sites of amyloid plaques. Lysosome accumulation is thought to contribute to amyloid production. In fact, a genetic perturbation that arrests lysosomes in axons exacerbates amyloid plaque pathology. The mechanisms that control axonal lysosome abundance as well the molecular composition of axonal endolysosomes that produce Abeta, however, are not fully understood. Axonal lysosome build-up is emerging as a common pathology in other neurodegenerative disorders such as Hereditary Spastic Paraplegia (HSP), but its relevance to amyloid production is unknown. We find that a model of HSP caused by loss of AP-4 adaptor complex lead to axonal lysosome buildup that differs in some of its content, but still contributes to amyloidogenesis. This demonstrates that different perturbations leading to changes in heterogeneous pool of axonal lysosomes can converge on a common pathology.

cell biology↗

bioRxiv2023

Identification of novel oncogenic transcriptional targets of mutant p53 in Esophageal Squamous Cell Carcinoma

Missense mutations in the DNA binding domain of p53 are observed frequently in Esophageal Squamous Cell Carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common hotspot p53 mutations while rarer mutations are poorly characterized. We had previously identified SMARCD1 as an oncogenic transcriptional target of rare non-hotspot p53 mutants detected from squamous cell carcinoma of the oral tongue (SCCOT). We now report the characterization of non-hotspot p53 mutations from ESCC. In-vitro tumorigenic assays performed following ectopic-expression of non-hotspot mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harbouring mutant p53. Of these, ARF6, C1QBP and TRIM23 were studied further due to their previously reported pro-oncogenic roles. Reverse transcription quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumor samples revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild type and several mutant p53 forms followed by RT-qPCR, Chromatin affinity-purification and Promoter-luciferase assays indicated the exclusive recruitment of p53 mutants - P190T and P278L, to the target genes leading to activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effect of the genes was confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of rare non-hotspot mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor specific p53 mutations.

cancer biology↗

bioRxiv2021

AP-4 regulates neuronal lysosome composition, function and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimers disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.

cell biology↗

bioRxiv2020

JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimers disease.

cell biology↗

bioRxiv2020

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimers disease-related amyloid precursor protein (APP)-derived A{beta}42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology.

cell biology↗

bioRxiv2019

Cytoplasm localized ARID1B promotes oncogenesis in pancreatic cancer by activating RAF-ERK signaling

The ARID1B/BAF250b subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. Immunohistochemistry on a pancreatic cancer tissue microarray revealed significant ARID1B cytoplasmic localization that correlated with advanced tumor stage and lymph node positivity. Identification of the nuclear localization signal (NLS) using in silico prediction and subcellular localization studies facilitated evaluation of a possible cytoplasmic function for ARID1B. A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised to regulate transcription activation and tumor suppression functions, as expected. Surprisingly however, cytoplasm-localized ARID1B could bind c-RAF and PPP1CA causing stimulation of RAS-RAF-ERK signaling and {beta}-catenin transcription activity in pancreatic cancer cells. More importantly, cytoplasmic ARID1B resulted in an induction of cell growth and migration in pancreatic cancer cell lines that was dependent on ERK signaling and caused increased tumorigenesis in nude mice. NLS peptides representing mutations identified from pancreatic cancer samples exhibiting ARID1B cytoplasmic localization or curated from cancer somatic mutation database were significantly compromised to effect nuclear localization of a reporter protein. ARID1B cytoplasmic localization correlated significantly with active forms of ERK and {beta}-catenin in primary pancreatic tumor samples. ARID1B may therefore promote oncogenesis through non-canonical cytoplasm-based gain of function mechanisms in addition to dysregulation in the nucleus.

cell biology↗