Biology subjects

Kumar, A.

Publications and source records attributed to Kumar, A..

At least 253 records · Page 14Linked to original sources

bioRxiv2024

Lipid Droplet Targeting of ABHD5 and PNPLA3 I148M is required to promote liver steatosis

The storage and release of triacylglycerol (TAG) in lipid droplets (LDs) is regulated by dynamic protein interactions. /{beta} hydrolase domain-containing protein 5 (ABHD5; also known as CGI-58) is a membrane/LD bound protein that functions as a co-activator of Patatin Like Phospholipase Domain Containing 2 (PNPLA2; also known as Adipose triglyceride lipase, ATGL) the rate-limiting enzyme for TAG hydrolysis. The dysregulation of TAG hydrolysis is involved in various metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). We previously demonstrated that ABHD5 interacted with PNPLA3, a closely related family member to PNPLA2. Importantly, a common missense variant in PNPLA3 (I148M) is the greatest genetic risk factor for MASLD. PNPLA3 148M functions to sequester ABHD5 and prevent co-activation of PNPLA2, which has implications for initiating MASLD; however, the exact mechanisms involved are not understood. Here we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. Molecular modeling demonstrates important resides in the C-terminus of PNPLA3 for LD binding and fluorescence cross-correlation spectroscopy demonstrates that PNPLA3 I148M greater associates with ABHD5 than WT PNPLA3. Moreover, the C-terminus of PNPLA3 is sufficient for functional targeting of PNPLAs to LD and the interaction with ABHD5. In addition, ABHD5 is a general binding partner of LD-bound PNPLAs. Finally, PNPLA3 I148M targeting to LD is required to promote steatosis in vitro and in the liver. Overall results suggest that PNPLA3 I148M is a gain of function mutation and that the interaction with ABHD5 on LD is required to promote liver steatosis.

cell biology↗

bioRxiv2024

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y1 and P2Y12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

cell biology↗

bioRxiv2024

Genomic analyses of recently emerging clades of mpox virus reveal gene deletion and single nucleotide polymorphisms that correlate with altered virulence and transmission

Mpox virus (MPXV) has consistently caused human infections since the first reported case in 1970, with the initial outbreaks primarily attributed to sporadic zoonotic transmissions. In recent years, an increase in human-to-human transmission has been observed, particularly during the 2022 outbreak caused by Clade IIb, which was declared a public health emergency by WHO. In 2024, the emergence of Clade Ib from Africa raised global concern once again. While several studies have provided valuable insights into the differences among MPXV clades, research on Clade Ib remains limited. In this study, we have conducted a comprehensive comparative genome sequence analysis and identified unique features across MPXV clade sequences. We report a [~]1141 bp long novel deletion resulting in loss of the complement control protein (CCP) in Clade Ib sequences, a deletion earlier exclusively reported in Clade II sequences. Additionally, B22R, a crucial host receptor-binding protein involved in viral entry and known for its high immunogenicity, shows clade-specific amino acid changes across three MPXV clades. Moreover, multiple extragenic mutations were identified in the 5 UTR of several genes, which may impact gene transcription. Other frequently mutated proteins are linked to immune evasion, translation, and viral entry and exit. The above results and APOBEC signatures associated with sustained human-to-human transmission highlight the viruss potential for rapid adaptation, underscoring the need for vigilance against reverse zoonosis and the risk of spillover to new hosts.

microbiology↗

bioRxiv2024

Widespread brain activity increases in frontal lobe seizures with impaired consciousness

Impaired consciousness is a serious clinical manifestation of epilepsy with negative consequences on quality of life. Little work has investigated impaired consciousness in frontal lobe seizures, a common form of focal epilepsy. In temporal lobe seizures, previous studies showed widespread cortical slow waves associated with depressed subcortical arousal and impaired consciousness. However, in frontal lobe epilepsy, it is not known whether cortical slow waves are present, or whether a very different cortical activity pattern may be related to impaired consciousness. We used intracranial EEG recordings of 65 frontal lobe seizures in 30 patients for quantitative analysis of ictal cortical activity and its relationship to impaired consciousness. Behavioral changes based on blinded review of seizure videos were used to classify focal aware, focal impaired awareness, and focal to bilateral tonic-clonic seizures. Changes in intracranial EEG power from preictal baseline were analyzed in different cortical regions and across frequency ranges in these three categories. We found that frontal lobe focal aware seizures showed approximately 40% increases in intracranial EEG power localized to the frontal lobe of seizure onset across frequency ranges, with relatively smaller changes in other cortical regions. Frontal lobe focal impaired awareness seizures showed approximately 50% increases in intracranial EEG power, not significantly different from focal aware seizures in the frontal lobe of seizure onset (P = 1.038), but significantly greater than focal aware seizures in other broad cortical regions (P < 0.001). Importantly, the widespread cortical increases in EEG power observed in focal impaired awareness versus focal aware seizures were seen not just in the frequency range of slow waves, but were also observed across other frequencies including fast activity. However, the widespread cortical increases in focal impaired awareness seizures differed from focal to bilateral tonic-clonic seizures where intracranial EEG power increased to a much higher level by approximately 600%. The large power increases in focal to bilateral tonic-clonic were significantly greater than in focal impaired awareness seizures both in the frontal lobe of seizure onset and in other cortical regions (P < 0.001). Our findings contrast with focal temporal lobe epilepsy, where impaired consciousness is associated with cortical slow waves. We can speculate that different focal seizure types produce impaired consciousness by impacting widespread cortical regions but through different physiological mechanisms. Insights gained by studying mechanisms of impaired consciousness may be the first step towards developing novel treatments to prevent this important negative consequence of epilepsy.

neuroscience↗

bioRxiv2024

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo, LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

cell biology↗

bioRxiv2024

AyurPhenoClusters define common molecular roots for rare diseases and uncover ciliary dysfunctions in syndromic conditions

The heterogeneity of underlying causes and the variability of symptoms across Multiple organs in rare diseases present significant challenges when approached from an organ-centric perspective. Ayurveda, however, takes a multisystem perspective, assessing diseases through kinetic (Vata:V ), metabolic (Pitta:P ), and structural (Kapha:K ) dimensions, each with distinct phenotypic and molecular correlates. This study explores rare diseases from a systems perspective by integrating Ayurveda and unifying terminologies from both disciplines using Human Phenotype Ontology (HPO). Experts categorized 10,610 HPO terms into Ayurvedic phenotypic groups (V/P/K ) and applied the Expectation Maximization (EM) algorithm to cluster 12,678 diseases. This yielded six distinct clusters, termed AyurPhenoClusters, with 2,814 diseases uniquely classified and enriched for V/P/K phenotypes. Functional annotation of these, highlighted key biological processes: (i) embryogenesis and skeletal morphogenesis, (ii) endocrine and ciliary functions, (iii) DNA damage response and cell cycle regulation, (iv) inflammation and immune response, (v) immune function, hemopoiesis, and telomere aging, and (vi) small molecule metabolism and transport. Notably, the K predominant cluster had the highest ciliary gene enrichment (43%), followed by the V predominant cluster (16%), suggesting potential ciliopathies in V cluster. This systems-based approach enhances the understanding of rare diseases by bridging Ayurveda with modern medicine, for improved diagnostics and therapeutics

genetics↗

bioRxiv2024

Inhibition of glutaminase elicits senolysis in therapy-induced senescent melanoma cells.

The cyclin D1-Cyclin-Dependent Kinases 4 and 6 (CDK4/6) complex is crucial for the development of melanoma. We previously demonstrated that targeting CDK4/6 using small molecule inhibitors (CDK4/6i) suppresses BrafV600E melanoma growth in vitro and in vivo through induction of cellular senescence. Clinical trials investigating CDK4/6i in melanoma have not yielded successful outcomes, underscoring the necessity to enhance the therapeutic efficacy of CDK4/6i. Accumulated research has shown that while senescence initially suppresses cell proliferation, a prolonged state of senescence eventually leads to tumor relapse by altering the tumor microenvironment, suggesting that removal of those senescent cells (in a process referred to as senolysis) is of clinical necessity to facilitate clinical response. We demonstrate that glutaminase 1 (GLS1) expression is specifically upregulated in CDK4/6i-induced senescent BrafV600E melanoma cells. Upregulated GLS1 expression renders BrafV600E melanoma senescent cells vulnerable to GLS1 inhibitor (GLS1i). Furthermore, we demonstrate that this senolytic approach targeting upregulated GLS1 expression is applicable even though those cells developed resistance to the BrafV600E inhibitor vemurafenib, a frequently encountered substantial clinical challenge to treating patients. Thus, this novel senolytic approach may revolutionize current CDK4/6i mediated melanoma treatment if melanoma cells undergo senescence prior to developing resistance to CDK4/6i. Given that we demonstrate that a low dose of vemurafenib induced senescence, which renders BrafV600E melanoma cells susceptible to GLS1i and recent accumulated research shows many cancer cells undergo senescence in response to chemotherapy, radiation, and immunotherapy, this senolytic therapy approach may prove applicable to a wide range of cancer types once senescence and GLS1 expression are induced. Key pointsUpregulated GLS1 expression renders senescent BrafV600E melanoma cells induced by CDK4/6 inhibitor (CDK4/6i) or vemurafenib susceptible to GLS1 inhibitor (GLS1i) even with BrafV600E inhibitor resistance. This senolytic therapy combining CDK4/6i and GLS1i provides insights into potential novel therapeutic strategies for metastatic melanoma and may be applicable to various types of cancers providing alternative therapy options.

cancer biology↗

bioRxiv2024

Functional consequences of fast-spiking interneurons in striatum

The striatum features a distinct network characterized by a high degree of shared feedforward inhibition (FFI) from a mere 1% of fast-spiking interneurons (FSI). We investigate the potential roles of this extensively shared FFI in striatal function beyond inducing synchrony. Our findings reveal that FSIs increase the acrosstrial variability of striatal responses to cortical stimuli and, combined with recurrent inhibition, lead to a correlation attractor of striatal activities, i.e., weakly correlated inputs result in more correlated responses and vice versa. Thus, we uncover a mechanism by which input correlation can be bidirectionally modulated, which is possible only because of high sharing of FSI inputs. We posit that the emergence of a correlation attractor leads to non-zero correlation level and variable rate trajectories of striatal responses across trials, hence beneficial for exploration in learning. However, given their role in across-trial variability, we argue that FSIs should be disengaged from the MSNs during performance where stability across trials is required. Significance StatementStriatum is a network of inhibitory neurons. Fast spiking interneurons constitute about 1% of the striatal neural population and provide feedforward inhibition (FSI). Here, we unravel two novel ways in which FSIs may shape striatal function. Given the recurrent inhibition, it is assumed that striatum can only de-correlate inputs. We show that high sharing of FSI also renders the striatum an ability to correlate inputs. Thus, recurrent and shared FSI create a correlation attractor. Besides, we show that shared FSIs give rise to high across-trial variability. Therefore, we argue that FSIs are more crucial in learning as they provide the neural basis of exploration, but they may impair learned behavior due to high across-trial variability.

neuroscience↗

bioRxiv2024

STRUCTURE-BASED DESIGN OF PROTACS FOR THE DEGRADATION OF SOLUBLE EPOXIDE HYDROLASE

Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker.2 Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimers disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems.

biochemistry↗

bioRxiv2024

LncRNA-536 and RNA Binding Protein RBM25 Interactions in Pulmonary Arterial Hypertension

OBJECTIVEHyperproliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the essential features of the maladaptive inward remodeling of the pulmonary arteries in pulmonary arterial hypertension (PAH). In this study, we define the mechanistic association between long-noncoding RNA: ENST00000495536 (Lnc-536) and anti-proliferative HOXB13 in mediating smooth muscle hyperplasia. METHODSAntisense oligonucleotide-based GapmeRs or plasmid overexpressing lnc-536 were used to evaluate the role of lnc-536 in mediating hyperproliferation of PDGF-treated or idiopathic PAH (IPAH) PASMCs. Further, we pulled down lnc536 to identify the proteins directly interacting with lnc536. The in-vivo role of lnc-536 was determined in Sugen-hypoxia and HIV-transgenic pulmonary hypertensive rats. RESULTSIncreased levels of lnc-536 in PDGF-treated or IPAH PASMCs promote hyperproliferative phenotype by downregulating the HOXB13 expression. Knockdown of lnc-536 in-vivo prevented increased RVSP, Fulton Index, and pulmonary vascular remodeling in Sugen-Hypoxia rats. The lncRNA-536 pull-down assay demonstrated the interactions of RNA binding protein: RBM25 with SFPQ, a transcriptional regulator that has a binding motif on HOXB13 exon Further, The RNA-IP experiment using the SFPQ antibody showed direct interaction of RBM25 with SFPQ and knockdown of RBM25 resulted in increased interactions of SFPQ and HOXB13 mRNA while attenuating PASMC proliferation. Finally, we examined the role of lnc-536 and HOXB13 axis in the PASMCs exposed to the dual hit of HIV and a stimulant: cocaine as well. CONCLUSIONlnc-536 acts as a decoy for RBM25, which in turn sequesters SFPQ, leading to the decrease in HOXB13 expression and hyperproliferation of smooth muscle cells associated with PAH development.

cell biology↗

bioRxiv2024

Reinforcement learning of state representation and value: the power of random feedback and biological constraints

Representation of external and internal states in the brain plays a critical role in enabling suitable behavior. Recent studies suggest that state representation and state value can be simultaneously learnt through Temporal-Difference-Reinforcement-Learning (TDRL) and Backpropagation-Through-Time (BPTT) in recurrent neural networks (RNNs) and their readout. However, neural implementation of such learning remains unclear as BPTT requires offline update using transported downstream weights, which is suggested to be biologically implausible. We demonstrate that simple online training of RNNs using TD reward prediction error and random feedback, without additional memory or eligibility trace, can still learn the structure of tasks with cue-reward delay and timing variability. This is because TD learning itself is a solution for temporal credit assignment, and feedback alignment, a mechanism originally proposed for supervised learning, enables gradient approximation without weight transport. Furthermore, we show that biologically constraining downstream weights and random feedback to be non-negative not only preserves learning but may even enhance it because the non-negative constraint ensures loose alignment - allowing the downstream and feedback weights to roughly align from the beginning. These results provide insights into the neural mechanisms underlying the learning of state representation and value, highlighting the potential of random feedback and biological constraints.

neuroscience↗

bioRxiv2024

Insights from Molecular Docking and Dynamics Simulations of P2RX7-????Syn Complex

Alpha-synucleinopathies, characterized by extracellular alpha-synuclein (Syn) accumulation and aggregation, have been linked to neurological disorders including Parkinsons disease (PD) and multiple system atrophy (MSA). Syn interacts with membrane proteins, phospholipids, and cholesterol, leading to membrane damage and inflammation. This interaction heightens the likelihood of Syn engaging with P2RX7, a non-selective cationic transmembrane receptor that is primarily overexpressed in immune and neural cells. Activation of P2RX7 by Syn is implicated in neuronal degeneration, potentially causing pore dilation and increased inflammation. However, the precise molecular mechanisms and binding sites for this interaction, as well as the conformational dynamics of P2RX7 in response to Syn, remain unclear. We attempted to elucidate the molecular mechanisms and binding sites for P2RX7-Syn interaction, by integrating the data curation, molecular docking, and molecular dynamics (MD) simulations coupled with structural analyses. We elucidated interactions between P2RX7 and the N-terminal domain (NTD) of Syn using cryo-EM structures of P2RX7 in both ATP-bound and unbound states and assessed how Syn influences P2RX7structural and functional dynamics. Initially, the analyses revealed that Syn interactomes are mainly involved in regulating mitochondrial homeostasis, while P2RX7 interactors are linked to receptor internalization and calcium transport. Molecular docking with six tools identified that Syn-NTD fragments preferentially bind to the proximal region of P2RX7 transmembrane domain. Microsecond all atom MD simulations in a POPS lipid bilayer showed significant atomic fluctuations, particularly in the head region, lower body, and large loop of P2RX7 cytoplasmic domain. Secondary structure analysis indicated unfolding in regions related to pore dilation and receptor desensitization. Further by contact-based and solvent accessibility analyses, along with protein structure network (PSN) studies, we identified crucial residues involved in Syn-P2RX7 interactions. This insight deepens our understanding of how Syn and P2RX7 interact, offering a detailed atomic view of the structural and functional changes that occur during these interactions. This understanding could advance our grasp of neurodegenerative diseases and be vital for devising future preventive and therapeutic strategies. Author summaryNeural homeostasis is crucial for neuron survival and function, with protein-protein interactions, particularly between Syn and P2RX7, playing a vital role. Accumulation of Syn can worsen neuronal damage through its interaction with P2RX7. Traditional biological experiments struggle with time-scale limitations, but advances in computational methods--like molecular dynamics (MD) simulations and protein structure network (PSN) analysis--allow detailed atomic-level study of these interactions. This study used these computational tools to explore the dynamics between Syn and P2RX7, aiming to uncover structural and functional changes relevant to protein interactions and neural damage. The findings contribute to understanding the dynamics of P2RX7-Syn interactions.

bioinformatics↗

bioRxiv2024

Low hemoglobin causes hematoma expansion and poor intracerebral hemorrhage outcomes

ObjectivesAlthough lower hemoglobin levels associate with worse intracerebral hemorrhage (ICH) outcomes, causal drivers for this relationship remain unclear. We investigated the hypothesis that lower hemoglobin relates to increased hematoma expansion (HE) risk and poor outcomes using human observational data and assessed causal relationships using a translational murine model of anemia and ICH. MethodsICH patients with baseline hemoglobin measurements and serial CT neuroimaging enrolled between 2010-2016 to a multicenter, prospective observational cohort study were studied. Patients with systemic evidence of coagulopathy were excluded. Separate regression models assessed relationships of baseline hemoglobin with HE ([≥]33% and/or [≥]6mL growth) and poor long-term neurological outcomes (modified Rankin Scale 4-6) after adjusting for relevant covariates. Using a murine collagenase ICH model with serial neuroimaging in anemic vs. non-anemic C57/BL6 mice, intergroup differences in ICH lesion volume, ICH volume changes, and early mortality were assessed. ResultsAmong 1190 ICH patients analyzed, lower baseline hemoglobin levels associated with increased odds of HE (adjusted OR per -1g/dL hemoglobin decrement: 1.10 [1.02-1.19]) and poor 3-month clinical outcomes (adjusted OR per -1g/dL hemoglobin decrement: 1.11 [1.03-1.21]). Similar relationships were seen with poor 6 and 12-month outcomes. In our animal model, anemic mice had significantly greater ICH lesion expansion, final lesion volumes, and greater mortality, as compared to non-anemic mice. ConclusionsThese results, in a human cohort and a mouse model, provide novel evidence suggesting that anemia has causal roles in HE and poor ICH outcomes. Additional studies are required to clarify whether correcting anemia can improve these outcomes.

pathology↗

bioRxiv2024

Faster amylin aggregation on fibrillar collagen hastens diabetic progression through β cell death and loss of function

Amyloid deposition of the neuroendocrine peptide amylin in islet tissues is a hallmark of type 2 diabetes (T2DM), leading to {beta}-cell toxicity through nutrient deprivation, membrane rupture and apoptosis. Though accumulation of toxic amylin aggregates in islet matrices is well documented, the role of the islet extracellular matrix in mediating amylin aggregation and its pathological consequences remains elusive. Here, we address this question by probing amylin interaction with collagen I (Col I)--whose expression in the islet tissue increases during diabetes progression. By combining multiple biophysical techniques, we show that hydrophobic, hydrophilic & cation-{pi} interactions regulate amylin binding to Col I, with fibrillar collagen driving faster amylin aggregation. Amylin-entangled Col I matrices containing high amounts of amylin induce death and loss of function of INS1E {beta}-cells. Together, our results illustrate how amylin incorporation in islet matrices through amylin-Col interactions drives T2DM progression by impacting {beta}-cell viability and insulin secretion.

biophysics↗

bioRxiv2024

Unraveling Structural Disparities in Human and Mycobacterium Tuberculosis Type-I Fatty Acid Synthase

Type-I Fatty acid synthase is an essential enzyme present in diverse species including humans (hFAS-I) and mycobacterium tuberculosis (MtbFAS-I), and is an attractive antibacterial drug target. A structural comparison of the two enzymes is essential in order to identify selective drug targets in MtbFAS-I. In this work, we have analyze equilibrium average structures of hFAS-I and MtbFAS-I obtained from 100 ns long molecular dynamics simulation trajectories. Our structural analysis revealed that two of the seven domains present in both hFAS-I and MtbFAS-I, viz. dehydratase (DH) and enoyl reductase (ER), are significantly dissimilar. We further compared corresponding catalytic pockets in these two domains and analyzed their physicochemical characteristics. In addition to being large in MtbFAS-I, the pockets are significantly different in their physicochemical characteristics and water content.

biophysics↗

bioRxiv2024

Dynamic control of neural manifolds

In the central nervous system, sequences of neural activity form trajectories on low dimensional neural manifolds. The neural computation underlying flexible cognition and behavior relies on dynamic control of these structures. For example different tasks or behaviors are represented on different subspaces, requiring fast timescale subspace rotation to move from one behavior to the next. For flexibility in a particular behavior, the neural trajectory must be dynamically controllable within that behaviorally determined subspace. To understand how dynamic control of neural trajectories and their underlying subspaces may be implemented in neural circuits, we first characterized the relationship between features of neural activity sequences and aspects of the low dimensional projection. Based on this, we propose neural mechanisms that can act within local circuits to modulate activity sequences thereby controlling neural trajectories in low dimensional subspaces. In particular, we show that gain modulation and transient synaptic currents control the speed and path of neural trajectories and clustered inhibition determines manifold orientation. Together, these neural mechanisms may enable a substrate for fast timescale computation on neural manifolds.

neuroscience↗

bioRxiv2024

Heat-killed probiotic Levilactobacillus brevis MKAK9 and its exopolysaccharide promote longevity by modulating aging hallmarks and enhancing immune responses in Caenorhabditis elegans

BackgroundProteostasis is a critical aging hallmark responsible for removing damaged or misfolded proteins and their aggregates by improving proteasomal degradation through the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS). Research on the impact of heat-killed probiotic bacteria and their structural components on aging hallmarks and innate immune responses is scarce, yet enhancing these effects could potentially delay age- related diseases. ResultsThis study introduces a novel heat-killed Levilactobacillus brevis strain MKAK9 (HK MKAK9), along with its exopolysaccharide (EPS), demonstrating their ability to extend longevity by improving proteostasis and immune responses in wild-type Caenorhabditis elegans. We elucidate the underlying mechanisms through a comprehensive approach involving mRNA- and small RNA sequencing, proteomic analysis, lifespan assays on loss-of- function mutants, and quantitative RT-PCR. Mechanistically, HK MKAK9 and its EPS resulted in downregulation of the insulin-like signaling pathway in a DAF-16-dependent manner, enhancing protein ubiquitination and subsequent proteasomal degradation through activation of the ALP pathway, which is partially mediated by microRNA mir-243. Importantly, autophagosomes engulf ubiquitinylated proteins, as evidenced by increased expression of the autophagy receptor sqst-3, and subsequently fuse with lysosomes, facilitated by increased levels of the lysosome-associated membrane protein (LAMP) lmp-1, suggesting the formation of autolysosomes for degradation of the selected cargo. Moreover, HK MKAK9 and its EPS activated the p38 MAPK pathway and its downstream SKN-1 transcription factor, which are known to regulate genes involved in innate immune response (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidation (sod-3 and gst-44), thereby reducing the accumulation of reactive oxygen species (ROS) at both cellular and mitochondrial levels. Notably, SOD-3 emerged as a transcriptional target of both DAF-16 and SKN-1 transcription factors. ConclusionOur research sets a benchmark for future investigations by demonstrating that heat-killed probiotic and its specific cellular component, EPS, can downregulate the insulin- signaling pathway, potentially improving the autophagy-lysosome pathway (ALP) for degrading ubiquitinylated proteins and promoting organismal longevity. Additionally, we discovered that increased expression of microRNA mir-243 regulates insulin-like signaling and its downstream ALP pathway. Our findings also indicate that postbiotic treatment may bolster antioxidative and innate immune responses, offering a promising avenue for interventions in aging-related diseases.

microbiology↗

bioRxiv2024

The Soybean Cyst Nematode Effector Cysteine Protease 1 (CPR1) Targets a Mitochondrial Soybean Branched-Chain Amino Acid Aminotransferase (GmBCAT1) for Degradation

The soybean cyst nematode (SCN; Heterodera glycines) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, presence of a signal peptide, and lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering.

plant biology↗