Biology subjects

Siddiq, M. M.

Publications and source records attributed to Siddiq, M. M..

9 recordsLinked to original sources

bioRxiv2026

Epidural Thin Film Implants of Polymerized Curcumin Downregulate Inflammatory Markers and Improve Functional Recovery After Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a notoriously intractable medical problem for which there is no neuroprotective pharmacotherapy, despite four decades of clinical trials on systemically administered small molecules that target plausible injury mechanisms. The shared failure mode of those trials is delivery: oxidative and inflammatory injury evolves locally at the lesion site over days to weeks, a timescale that far exceeds the half-life of systemically administered drugs. Furthermore, systemically administered drugs often cannot be dosed at high enough levels to achieve efficacious concentrations locally at the lesion site, without off-target effects. In this study, we show the first example of a locally implantable drug depot that exerts long-lasting activity at the lesion site. We demonstrate in vivo efficacy of a poly(pro-drug) thin film deployed locally, to address delivery problem in SCI. The films are composed of a copolyester in which curcumin, a potent natural antioxidant, is incorporated as a backbone repeat unit alongside poly(ethylene glycol) sebacate segments. The soft, thin film ([~]100 {micro}m) is employed as an epidural implant that quenches reactive oxygen species on-contact and can sustain activity for over two months, which is orders of magnitude longer than free curcumin (half-life of [~]10 min in buffer). A single epidural P50 film, placed on the exposed dura immediately after a moderate-to-severe T9 contusion in mice, produced a statistically significant improvement in Basso Mouse Scale locomotor recovery at 14 days post-injury, relative to a non-antioxidant control polymer of same architecture. The film preserved myelinated white matter, attenuated reactive gliosis (GFAP, Iba1), and produced coherent downregulation of neuroinflammatory gene-expression programs. These effects were spatially restricted to tissue rostral to the lesion, consistent with on-contact, locally confined polymer activity. Because curcumin is covalently enchained within the polymer itself, rather than blended as a payload in a carrier matrix, release is governed by backbone hydrolysis and the architecture can generalize to other phenolic bioactives, potentially establishing epidural poly(pro-drug) thin films as a mechanistically grounded, surgical platform for sustained local pharmacotherapy of CNS injury.

bioengineering↗

bioRxiv2023

Muscle-restricted Nox4 knockout partially corrects muscle contractility following spinal cord injury in mice

Spinal cord injury (SCI) results in severe atrophy of skeletal muscle in paralyzed regions, and a decrease in the force generated by muscle per unit of cross-sectional area. Oxidation of skeletal muscle ryanodine 1 receptors (RyR1) reduces contractile force due to reduced binding of calstabin 1 to RyR1 together with altered gating of RyR1. One cause of RyR1 oxidation is NADPH oxidase 4 (Nox4). We have previously shown that in rats, RyR1 was oxidized and bound less calstabin 1 at 56 days after spinal cord injury (SCI) by transection. Here, we used a conditional knock-out mouse model of Nox4 in muscle to investigate the role of Nox4 in reduced muscle specific force after SCI. Peak twitch force in control mice after SCI was reduced by 42% compared to sham-operated controls but was increased by approximately 43% in SCI Nox4 conditional KO mice compared to SCI controls although it remained less than that for sham-operated controls. Unlike what observed in rats, after SCI the expression of Nox4 was not increased in gastrocnemius muscle and binding of calstabin 1 to RyR1 was not reduced in this muscle. The results suggest a link between Nox4 expression in muscle tissue and reduction in muscle twitch force, however further studies are needed to understand the mechanistic basis for this linkage.

physiology↗

bioRxiv2023

Spinal Cord Injury regulates circular RNA expression in axons

IntroductionNeurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. MethodsWe obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that were then sequenced. We used bioinformatics analyses to identify DEGS and map them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. ResultsThe supernatant fraction was highly enriched for axonal mRNA. Using Gene Ontology, the second most significant pathway for all differentially expressed genes (DEGs) was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth. Focusing on Rims2 we used Circular RNA Interactome to predict that several of the miRNAs that bind to circRims2 also bind to the 3UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. DiscussionBy using our method for enrichment of axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 a known regulator of axonal regeneration indicating that axonal mRNA contribute to regeneration.

neuroscience↗

bioRxiv2023

Boldine modulates glial transcription and functional recovery in a murine model of contusion spinal cord injury

Membrane channels such as connexins (Cx), pannexins (Panx) and P2X7 receptors (P2X7R) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X7R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing (of the spinal cord revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

neuroscience↗

bioRxiv2022

Single Nucleus RNA Sequencing of Human Pancreatic Islets In Vitro and In Vivo Identifies New Gene Sets and Three β-Cell Subpopulations with Different Transcriptional Profile

Single-cell RNA sequencing (scRNA-seq) has provided valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo and provides limited information on the active transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and affords enhanced information from intronic sequences that can be leveraged to identify actively transcribing genes in islet cell populations. Here, we first sought to compare scRNA-seq and snRNA-seq analysis of human islets in vitro using exon reads or combined exon and intron reads, respectively. Datasets reveal similar human islet cell clusters using both approaches. In the snRNA-seq data, however, the top differentially expressed genes in human islet endocrine cells are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT ({beta} cells), PTPRT, FAP, PDK4, LOXL4 ( cells), LRFN5, ADARB2, ERBB4, KCNT2 ({delta} cells) and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 ({gamma} cells). Notably, these markers also accurately define endocrine cell populations in human islet grafts in vivo. Further, by integrating the information from nuclear and cytoplasmic transcriptomes, we identify three {beta}-cell sub-clusters: an active INS mRNA transcribing cluster ({beta}1), an intermediate INS mRNA-transcribing cluster ({beta}2), and a mature INS mRNA rich cluster ({beta}3). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA rich cluster ({beta}3) becomes the predominant sub-cluster in vivo. In summary, snRNA-seq analysis of human islet cells is a previously unrecognized tool that can be accurately employed for improved identification of human islet cell types and their transcriptional status in vivo.

cell biology↗

bioRxiv2020

The human ApoE4 variant reduces functional recovery and neuronal sprouting after incomplete spinal cord injury in male mice

Spinal cord injury (SCI) is a devastating form of neurotrauma. Patients who carry one or two ApoE4 alleles show worse functional outcomes and longer hospital stays after SCI but the cellular and molecular underpinnings for this genetic link remain poorly understood. Thus, there is a great need to generate animal models to accurately replicate the genetic determinants of outcomes after SCI to spur development of treatments that improve physical function. Here, we examined outcomes after a moderate contusion SCI of transgenic mice expressing human ApoE3 or ApoE4. ApoE4 mice have worse locomotor function and coordination after SCI. Histological examination revealed greater glial staining in ApoE4 mice after SCI associated with reduced levels of neuronal sprouting markers. Bulk RNA sequencing revealed that subcellular processes (SCPs), such as extracellular matrix organization and inflammatory responses, were highly-ranked among upregulated genes at 7 days after SCI in ApoE4 variants. Conversely, SCPs related to neuronal action potential and neuron projection development were increased in ApoE3 mice at 21 days. In summary, our results reveal a clinically relevant SCI mouse model that recapitulates the influence of ApoE genotypes on post-SCI function in individuals who carry these alleles and suggest that the mechanisms underlying worse recovery for ApoE4 animals involve glial activation and loss of sprouting and synaptic activity.

neuroscience↗

bioRxiv2018

Cannabinoid 1-Receptor regulation of deep and distributed subcellular processes enables neurite outgrowth

Neurite outgrowth is an integrated whole cell process that represents the earliest steps in neuronal differentiation. We studied cannabinoid-1 receptor regulated neurite outgrowth to identify the different subcellular processes (SCPs) involved and how they come together to produce a whole cell response. We identified the genes that were differentially expressed after CB1 receptor stimulation. From the differentially expressed components we identified the SCPs involved and how the SCPs connect to one another both horizontally and vertically. We used these SCP networks to identify genes, which when knocked down would validate the involvement of the SCP in neurite outgrowth. We identified a varied list of SCPs including those involved in pyrimidine metabolism, lipid biosynthesis, mitochondrial transport function, mRNA splicing and stability in addition to those involved in membrane vesicle production, transport and fusion and microtubule dynamics. Overall these data and computational analyses show that the downstream SCPs required for neurite outgrowth are widely distributed and involve a range of normal cellular functions. Additionally, several of these processes can be considered distal in terms of their proximity to cell signaling pathways and the microtubule and membrane vesicle dynamics, the proximal SCPs required for neurite outgrowth. Hence we conclude that receptor regulation of neurite outgrowth is distributed and deep.

cell biology↗

bioRxiv2018

Activated Protein C blocks the inhibitory effect on neurite outgrowth by extracellular histones that mediates its inhibition through a retrograde YB-1 signal

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors we have detected elevated levels of histone H3 in human cerebrospinal fluid (CSF) 24 hours after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor YB-1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C (APC) in vitro, and APC treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS. One sentence summaryProteins typically associated with chromatin structure play an unexpected role in limiting axonal regeneration after injury.

neuroscience↗

bioRxiv2017

Systems Therapeutics for Axonal Regeneration in the Central Nervous System

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axon using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Morphology experiments show that the four-drug combination promotes axonal regrowth to the optic chiasm and beyond. Physiologically, drug treatment restored pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. We conclude that spatially targeted drug treatment can promote robust axonal regeneration and can restore limited functional recovery.

neuroscience↗