Biology subjects

Pradeep, A.

Publications and source records attributed to Pradeep, A..

4 recordsLinked to original sources

bioRxiv2023

Enhanced spine stability and survival lead to increases in dendritic spine density as an early response to local alpha-synuclein overexpression in mouse prefrontal cortex

Lewy Body Dementias (LBD), including Parkinsons disease dementia and Dementia with Lewy Bodies, are characterized by widespread accumulation of intracellular alpha-Synuclein protein deposits in regions beyond the brainstem, including in the cortex. Patients with LBDs develop cognitive changes, including abnormalities in executive function, attention, hallucinations, slowed processing, and cognitive fluctuations. The causes of these non-motor symptoms remain unclear; however, accumulation of alpha-Synuclein aggregates in the cortex and subsequent interference of synaptic and cellular function could contribute to psychiatric and cognitive symptoms. It is unknown how the cortex responds to local pathology in the absence of significant secondary effects of alpha-Synuclein pathology in the brainstem. To investigate this, we employed viral overexpression of human alpha-Synuclein protein targeting the mouse prefrontal cortex (PFC). We then used in vivo 2-photon microscopy to image awake head-fixed mice via an implanted chronic cranial window to assess the early consequences of alpha-Synuclein overexpression in the weeks following overexpression. We imaged apical tufts of Layer V pyramidal neurons in the PFC of Thy1-YFP transgenic mice at 1-week intervals from 1-2 weeks before and 9 weeks following viral overexpression, allowing analysis of dynamic changes in dendritic spines. We found an increase in the relative dendritic spine density following local overexpression of alpha-Synuclein, beginning at 5 weeks post-injection, and persisting for the remainder of the study. We found that alpha-Synuclein overexpression led to an increased percentage and longevity of newly-persistent spines, without significant changes in the total density of newly formed or eliminated spines. A follow up study utilizing confocal microscopy revealed that the increased spine density is found in cortical cells within the alpha-Synuclein injection site, but negative for alpha-Synuclein phosphorylation at Serine-129, highlighting the potential for effects of dose and local circuits on spine survival. These findings have important implications for the physiological role and early pathological stages of alpha-Synuclein in the cortex.

neuroscience↗

bioRxiv2023

SKIN REINNERVATION BY COLLATERAL SPROUTING FOLLOWING SPARED NERVE INJURY IN MICE

Following peripheral nerve injury, denervated tissues can be reinnervated via regeneration of injured neurons or via collateral sprouting of neighboring uninjured afferents into the denervated territory. While there has been substantial focus on mechanisms underlying regeneration, collateral sprouting has received relatively less attention. In this study, we used immunohistochemistry and genetic neuronal labeling to define the subtype specificity of sprouting-mediated reinnervation of plantar hind paw skin in the mouse spared nerve injury (SNI) model, in which productive regeneration cannot occur. Following an initial loss of cutaneous afferents in the tibial nerve territory, we observed progressive centripetal reinnervation by multiple subtypes of neighboring uninjured fibers into denervated glabrous and hairy plantar skin. In addition to dermal reinnervation, CGRP-expressing peptidergic fibers slowly but continuously repopulated the denervated epidermis, Interestingly, GFR2-expressing nonpeptidergic fibers exhibited a transient burst of epidermal reinnervation, followed by trend towards regression. Presumptive sympathetic nerve fibers also sprouted into the denervated territory, as did a population of myelinated TrkC lineage fibers, though the latter did so less efficiently. Conversely, rapidly adapting A{beta} fiber and C fiber low threshold mechanoreceptor (LTMR) subtypes failed to exhibit convincing collateral sprouting up to 8 weeks after nerve injury. Optogenetics and behavioral assays further demonstrated the functionality of collaterally sprouted fibers in hairy plantar skin with restoration of punctate mechanosensation without hypersensitivity. Our findings advance understanding of differential collateral sprouting among sensory neuron subpopulations and may guide strategies to promote the progression of sensory recovery or limit maladaptive sensory phenomena after peripheral nerve injury. Significance StatementFollowing nerve injury, whereas one mechanism for tissue reinnervation is regeneration of injured neurons, another, less well studied mechanism is collateral sprouting of nearby uninjured neurons. In this study, we examined collateral sprouting in denervated mouse skin and showed that it involves some, but not all neuronal subtypes. Despite such heterogeneity, a significant degree of restoration of punctate mechanical sensitivity is achieved. These findings highlight the diversity of collateral sprouting among peripheral neuron subtypes and reveal important differences between pre- and post-denervation skin that might be appealing targets for therapeutic correction to enhance functional recovery from denervation and prevent unwanted sensory phenomena such as pain or numbness.

neuroscience↗

bioRxiv2023

Acetyl transferase EP300 deficiency leads to chronic replication stress mediated by defective fork protection at stalled replication forks

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a model to study EP300 deficiency and an p300-selective PROTAC degrader, degrader as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in EP300-deficient cells. We demonstrate that the DNA replication dynamics of EP300-mutated cells ATLL cells recapitulate features of BRCA-deficient cancers. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress and defective replication fork restart results in persistent genomic instability that underlie aggressive chemo-resistant tumorigenesis in humans.

cancer biology↗

bioRxiv2022

Myeloid-biased HSC require Semaphorin 4A from the bone marrow niche for self-renewal under stress and life-long persistence

Tissue stem cells are hierarchically organized. Those that are most primitive serve as key drivers of regenerative response but the signals that selectively preserve their functional integrity are largely unknown. Here, we identify a secreted factor, Semaphorin 4A (Sema4A), as a specific regulator of myeloid-biased hematopoietic stem cells (myHSC), which are positioned at the top of the HSC hierarchy. Lack of Sema4A leads to exaggerated myHSC (but not downstream "balanced" HSC) proliferation after acute inflammatory stress, indicating that Sema4A enforces myHSC quiescence. Strikingly, aged Sema4A knock-out myHSC expand but almost completely lose reconstitution capacity. The effect of Sema4A is non cell-autonomous, since upon transplantation into Sema4A-deficient environment, wild-type myHSC excessively proliferate but fail to engraft long-term. Sema4A constrains inflammatory signaling in myHSC and acts via a surface receptor Plexin-D1. Our data support a model whereby the most primitive tissue stem cells critically rely on a dedicated signal from the niche for self-renewal and life-long persistence.

cell biology↗