Biology subjects

Richhariya, S.

Publications and source records attributed to Richhariya, S..

7 recordsLinked to original sources

bioRxiv2026

Glycolytic compensation rather than NAD+/NADH balance sustains neuronal function during mitochondrial stress

Neurons engage compensatory pathways that promote survival when confronted with mitochondrial dysfunction. Indeed, we recently showed that Drosophila neurons upregulate Ldh transcription to help survive the loss of the key mitochondrial fusion gene Opa1. Here, we further characterize this metabolic flexibility and show that it reflects a more general increase in glycolytic activity. A distinct mitochondrial perturbation, TFAM overexpression, similarly induces glycolytic gene expression including Ldh and also elevates lactate levels. LDH is also required to maintain neuronal function under TFAM overexpression. Notably, raising NAD+/NADH ratio by expressing the bacterial NADH oxidase LbNOX does not substitute for LDH function. On the contrary, it further compromises neuronal function in Opa1-deficient and TFAM-overexpressing neurons. Moreover, mitochondria-targeted LbNOX expression alone induces mitochondrial dysfunction and the compensatory glycolytic response. Together, these findings indicate that LDH-mediated rescue does not reflect an increase in NAD+/NADH ratio but is part of a broader neuroprotective metabolic reprogramming which enables neurons to withstand diverse forms of mitochondrial impairment.

neuroscience↗

bioRxiv2024

Chronic mitochondrial fragmentation elicits a neuroprotective Warburg-like effect in Drosophila neurons

Mitochondrial fission and fusion are dynamic and important cellular processes, but the roles of these two very different mitochondrial forms - predominantly spherical and tubular - are not well-characterized in neurons of animals and especially in aging neurons. This is important because neurons are long-lived and mitochondrial dynamics is associated with neurodegenerative diseases. We used here an efficient cell type-specific CRISPR approach to knockout key fission-fusion genes and disrupt mitochondrial dynamics within the inessential clock neurons of Drosophila. Surprisingly, fusion is much more important than fission for maintaining long-term neuronal function. Neurons survive chronic mitochondrial fragmentation due to loss of fusion by triggering a cancer-like transcriptomic response. This Warburg effect includes ATF4-mediated upregulation of the aerobic glycolysis gene Lactate dehydrogenase (Ldh), and LDH is essential to prevent neurodegeneration of neurons deficient in the fusion gene Opa1. These results and others provide insights into the intersection of neuronal metabolism, aging and neurodegeneration.

neuroscience↗

bioRxiv2024

The Circadian Neuropeptide PDF has Sexually Dimorphic Effects on Activity Rhythms

The circadian system regulates the timing of multiple molecular, physiological, metabolic, and behavioral phenomena. In Drosophila, as in other species, most of the research on how the timekeeping system in the brain controls the timing of behavioral outputs has been conducted in males, or sex has not been included as a biological variable. A critical set of circadian pacemaker neurons in Drosophila release the neuropeptide pigment-dispersing factor (PDF), which functions as a key output factor in the network with complex effects on other clock neurons. Lack of Pdf or its receptor, PdfR, results in most flies displaying arrhythmicity in activity-rest cycles under constant conditions. However, our results show that female circadian rhythms are less affected by mutations in both Pdf and PdfR. Crispr-Cas9-mediated mutagenesis of Pdf, specifically in ventral lateral neurons (LNvs), also has a greater effect on male rhythms. We tested the influence of M-cells on the circadian network and showed that speeding up the molecular clock specifically in M-cells led to sexually dimorphic phenotypes, with a more pronounced effect on male rhythmic behavior. Our results suggest that the female circadian system is more resilient to manipulations of M-cells and the PDF pathway, suggesting that circadian timekeeping is more distributed across the clock neuron network in females.

neuroscience↗

bioRxiv2023

Orai mediated Calcium entry sets the excitability threshold of central dopaminergic neurons by regulation of gene expression

Maturation and fine-tuning of neural circuits frequently requires neuromodulatory signals that set the excitability threshold, neuronal connectivity and synaptic strength. Here we present a mechanistic study of how neuromodulator stimulated intracellular Ca2+ signals, through the store - operated Ca2+ channel Orai, regulate intrinsic neuronal properties by control of developmental gene expression in flight promoting central dopaminergic neurons (fpDANs). The fpDANs receive cholinergic inputs for release of dopamine at a central brain tripartite synapse that sustains flight (Sharma and Hasan, 2020). Cholinergic inputs act on the muscarinic acetylcholine receptor to stimulate intracellular Ca2+ release through the endoplasmic reticulum (ER) localised inositol 1,4,5-trisphosphate receptor followed by ER-store depletion and Orai mediated store-operated Ca2+ entry (SOCE). Analysis of gene expression in fpDANs followed by genetic, cellular and molecular studies identified Orai-mediated Ca2+ entry as a key regulator of excitability in fpDANs during circuit maturation. SOCE activates the transcription factor Trithorax-like (Trl) which in turn drives expression of a set of genes including Set2, that encodes a histone 3 Lysine 36 methyltransferase (H3K36me3). Set2 function establishes a positive feedback loop, essential for receiving neuromodulatory cholinergic inputs and sustaining SOCE. Chromatin modifying activity of Set2 changes the epigenetic status of fpDANs and drives expression of key ion channel and signaling genes that determine fpDAN activity. Loss of activity reduces the axonal arborisation of fpDANS within the MB lobe, and prevents dopamine release required for maintenance of long flight. HighlightsO_LIStore-operated Ca2+ entry (SOCE) through Orai is required in a set of flight-promoting central dopaminergic neurons (fpDANs) during late pupae and early adults to establish their gene expression profile. C_LIO_LISOCE activates a homeobox transcription factor, Trithorax-like and thus regulates expression of histone modifiers Set2 and E(z) to generate a balance between opposing epigenetic signatures of H3K36me3 and H3K27me3 on downstream genes. C_LIO_LISOCE drives a transcriptional feedback loop to ensure expression of key genes required for neuronal function including the muscarinic acetylcholine receptor (mAChR) and the inositol 1,4,5-trisphosphate receptor (itpr). C_LIO_LIThe transcriptional program downstream of SOCE is key to functional maturation of the dopaminergic neurons, enabling their neuronal excitability, axonal arborization and synaptic transmission required for adult flight. C_LI

neuroscience↗

bioRxiv2023

Dissecting cell-specific functions of circadian genes using modified cell-specific CRISPR approaches

Circadian behavioral rhythms in Drosophila melanogaster are regulated by about 75 pairs of brain neurons. They all express the core clock genes but have distinct functions and gene expression profiles. To understand the importance of these distinct molecular programs, neuron-specific gene manipulations are essential. Although RNAi based methods are standard to manipulate gene expression in a cell-specific manner, they are often ineffective, especially in assays involving smaller numbers of neurons or weaker Gal4 drivers. We and others recently exploited a neuron-specific CRISPR-based method to mutagenize genes within circadian neurons. Here we further explore this approach to mutagenize three well-studied clock genes: the transcription factor gene vrille, the photoreceptor gene Cryptochrome (cry) and the neuropeptide gene Pdf. The CRISPR-based strategy not only reproduced their known phenotypes but also assigned cry function for different light mediated phenotypes to discrete, different subsets of clock neurons. We further tested two recently published methods for temporal regulation in adult neurons, inducible Cas9 and auxin-inducible gene expression system (AGES). The results were not identical, but both approaches successfully showed that the adult-specific knockout of the neuropeptide Pdf reproduces the canonical loss-of-function mutant phenotypes. In summary, a CRISPR-based strategy is a highly effective, reliable, and general method to temporally manipulate gene function in specific adult neurons. Significance statementMost animals have specific brain neurons that regulate sleep-wake cycles and other aspects of circadian behavior. Drosophila has only about 150 of these clock neurons. Despite their small numbers, they have remarkably diverse anatomy and gene expression profiles. To address the different functions of these neurons, we used highly specific and efficient CRISPR-based methods to create cell type-specific disruptions of three traditional circadian genes. We were able to assign the function of the photoreceptor cryptochrome to two tiny subsets of clock neurons. In addition, two independent methods assigned the neuropeptide PDF to the adult stage. In summary, we find that the CRISPR-based methods are very efficient at studying adult specific functions of genes in small, discrete sets of neurons.

genetics↗

bioRxiv2021

Dopamine and GPCR-mediated modulation of DN1 clock neurons gates the circadian timing of sleep

The metronome-like circadian regulation of sleep timing must still adapt to an uncertain environment. Recent studies in Drosophila indicate that neuromodulation not only plays a key role in clock neuron synchronization but also affects interactions between the clock network and brain sleep centers. We show here that the targets of neuromodulators, G-Protein Coupled Receptors (GPCRs), are highly enriched in the fly brain circadian clock network. Single cell sequencing indicates that they are not only differentially expressed but also define clock neuron identity. We generated a comprehensive guide library to mutagenize individual GPCRs in specific neurons and verified the strategy with a targeted sequencing approach. Combined with a behavioral screen, the mutagenesis strategy revealed a novel role of dopamine in sleep regulation by identifying two dopamine receptors and a clock neuron subpopulation that gate the timing of sleep.

neuroscience↗

bioRxiv2020

IP3/Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase dSET2

Persistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae, that have crossed "critical weight" and can pupariate to form viable adults, such a metabolic shut-down would needlessly lead to death. IP3/Ca2+ signals in certain interneurons (vGlutVGN6341) allow Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. Here we show that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of dSET2, a Drosophila Histone 3 Lysine 36 methyltransferase. Further, dSET2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven dSET2 expression upregulates key Ca2+ signaling genes through a novel positive feedback loop. Transcriptomic studies coupled with analysis of existing ChIP-seq datasets identified genes from larval and pupal stages, that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either an intracellular Ca2+ release channel the IP3R or dSET2. IP3/Ca2+ signals thus regulate gene expression through dSET2 mediated H3K36 marks on select neuronal genes for the larval to pupal transition.

developmental biology↗