Biology subjects

Muddashetty, R.

Publications and source records attributed to Muddashetty, R..

6 recordsLinked to original sources

bioRxiv2023

Astroglia proliferate upon biogenesis of tunneling nanotubes and clearance of α-synuclein toxicities

Astrocytic cells are a subtype of glial cells that engulf pathogenic aggregates derived from degenerative neurons to facilitate its degradation. Here, we show that exposure to -SYN protofibrils caused a transient increase in biogenesis of tunneling nanotubes (TNTs) in primary astrocytes and astrocyte-origin cancer cell-lines (U-87 MG, U251). Biogenesis of nascent TNTs corresponds to -SYN protofibril-induced organelle toxicities, increased reactive oxygen species (ROS), and oxidative stress-induced premature cellular senescence. These TNTs mediate cell-to-cell transfer of -SYN protofibrils, toxic lysosomes and mitochondria. Biogenesis of TNTs precedes clearance of -SYN-induced organelle toxicities, cellular ROS levels and reversal of cellular senescence. Consequences of cellular clearance results in enhanced cell proliferation. Further, we have shown -SYN-induced senescence promotes transient localization of focal adhesion kinase (FAK) in the nucleus. FAK mediated regulation of Rho-associated kinases may have a role in the biogenesis of TNTs, successively proliferation. Our study emphasizes that TNT biogenesis may have a potential role in the clearance of -SYN toxicities and reversal of stress-induced cellular senescence, consequences of which cause enhanced proliferation in the post-recovered astroglia cells. HighlightsO_LI-SYN protofibrils treated astroglia cells proliferate upon transient biogenesis of TNTs. C_LIO_LITransient TNT biogenesis precedes clearance of -SYN toxicities and reversal of senescence. C_LIO_LIStress-induced senescence results in nuclear localization of FAK and ROCK mediated TNT biogenesis. C_LIO_LIThe rescued cells enhance proliferation through ROCK mediated ERK1/2 and NF{kappa}B signalling cascades. C_LI Synopsis O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/554645v1_figa1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@9dc5bcorg.highwire.dtl.DTLVardef@20e924org.highwire.dtl.DTLVardef@aa7920org.highwire.dtl.DTLVardef@1f6274b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO -SYN protofibrils-induced biogenesis of tunneling nanotubes (TNTs) aids to enhance cellular clearance of toxic burdens as a cellular survival strategy. -SYN protofibrils treated toxic senescence cells regulate FAK mediated modulation of ROCK signalling cascades to promote TNT biogenesis and rescue the cellular toxicities. The rescued cells eventually enhance cell proliferation. C_FIG

cell biology↗

bioRxiv2023

Proteomic analysis reveals APOE isoform-specific regulation of ribosomes in neural precursor cells

ApoE4 isoform contributes to increased risk for Alzheimers Disease (AD) over the life course of individuals. Much remains unknown about the biological pathways that connect APOE4 genotype with the development of pathology that eventually leads to AD, nor do we know how early in life these cellular alterations begin. To answer these questions, we derived neural precursor cells (NPCs) from induced pluripotent stem cells (IPSCs) that were CRISPR-edited at the APOE locus. We intended to characterize the protein expression landscape in the NPCs subsequent to targeted deletion of E4 from a parent IPSC line of APOE3/4 genotype. Differentially expressed proteins (DEPs) following mass spectrometric analysis were determined from the protein abundance fold change values obtained for each protein. Proteins which showed >1.5-fold difference with FDR adjusted P-value < 0.05 were considered differentially expressed. DEPs were mapped to the STRING database (v11.5) for retrieval of interacting proteins and functional enrichment. CRISPR-editing of E4 from the parent line revealed 98 differential expressed proteins. Of these, 54 were upregulated, and 44 were downregulated. Further analysis of the DEPs via STRING database showed that these changes primarily affect pathways linked to RNA processing, plasma membrane repair, and cytoskeleton organization. Indeed, we find the effects of E4 extend beyond proteins considered central to AD pathology. Knowing more about the protein interactions regulated by ApoE, in an isoform-specific manner, can reveal new mechanistic insights into development of AD.

neuroscience↗

bioRxiv2022

Golgi Fragmentation - One of the Earliest Organelle Phenotypes in Alzheimer's Disease Neurons

Alzheimers disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied Amyloid beta (A{beta}) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD disease phenotypes, such as increased A{beta} secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/519571v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@98a8eeorg.highwire.dtl.DTLVardef@7aa7b7org.highwire.dtl.DTLVardef@991e33org.highwire.dtl.DTLVardef@8dabb4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

NMDAR mediated dynamic changes in m6A inversely correlates with neuronal translation

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (m6A) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of m6A modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of m6A modification in response to NMDAR stimulation. We have consistently observed that upon 5-minute NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the m6A signal at 5 minutes time point, correlating with the global translation inhibition. The NMDAR induced increase in the m6A signal is accompanied by the redistribution of the m6A marked RNAs from translating to the non-translating pool of ribosomes. The increased m6A levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in m6A levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.

neuroscience↗

bioRxiv2021

The Fragile X Mental Retardation Protein protects the lung from xenobiotic stress by facilitating the Integrated Stress Response

Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the Fragile X Mental Retardation Protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established Naphthalene (Nap) injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. We find that FMRP-deficient cells fail to actuate the Integrated Stress Response Pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9, 10-Phenanthrenequinone air pollutant model, to find that FMRP-deficient BEAS-2B also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR.

cell biology↗

bioRxiv2019

Distinct Regulaton of Bioenergetics and Translation by Gp I mGluRs and NMDARs

Neuronal activity is responsible for large energy consumption within the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize the mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in the neuronal ATP level, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely to the kinetics of protein synthesis for both the receptors. We observe a persistent elevation in protein synthesis within 5 minutes of mGluR activation and robust inhibition of the same within 2 minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15 minutes of NMDAR activation. We identify a central role for AMPK in this correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation while it was rapidly phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts the receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Therefore, our observations suggest that the glutamate receptors required modulating the AMPK-eEF2 signaling axis to alter neuronal protein synthesis and bioenergetics. Short SummaryStimulation of glutamate receptors induces robust protein synthesis within cortical neurons and consumes a significantly large fraction of cellular ATP. Glutamate receptors viz. mGlulR and NMDAR modulate AMPK-eEF2 signaling uniquely leading to the dynamic regulation of protein synthesis and bioenergetics. Key HighlightsO_LIProtein synthesis following glutamate receptor activation is responsible for the bulk of the activity-induced ATP consumption in cortical neurons. C_LIO_LImGluR and NMDAR regulate protein synthesis with distinct kinetics and dictate the subsequent impacts over neuronal ATP level. C_LIO_LIDynamic modulation of AMPK and eEF2 phosphorylation is key to create unique temporal features of receptor-specific protein synthesis and bioenergetics. C_LI

neuroscience↗