Biology subjects

Yousuf, M. S.

Publications and source records attributed to Yousuf, M. S..

12 recordsLinked to original sources

bioRxiv2026

Progressive neurodegeneration in human dorsal root ganglion from diabetes to painful neuropathy

Diabetic painful neuropathy (DPN) is characterized by neuropathic pain accompanied by loss of sensory function. We hypothesized that neurodegeneration in the dorsal root ganglion (DRG) could underlie DPN progression. To address this question, we performed multi-omic evaluation on DRGs from otherwise healthy organ donors, donors with diabetes but no neuropathy, and donors with clinically diagnosed DPN. We discovered that the first stages of neurodegeneration begin early in diabetes before the onset of DPN, with Nageotte nodule formation accompanied by apoptotic gene expression and decreased proportion of specific populations of A-fibers in DPN with remodeling of non-neuronal cells. Our findings define DPN as a neurodegenerative disorder of the DRG, identify molecular markers of disease stage, and highlight the need for early intervention to prevent irreversible neurodegeneration.

neuroscience↗

bioRxiv2025

Eukaryotic initiation factor 3d Regulates Context-Dependent Pain Hypersensitivity Through the Integrated Stress Response

Eukaryotic translation initiation factor 3 subunit D (eIF3d) is a noncanonical cap binding protein implicated in selective mRNA translation under stress conditions. Here, we investigate the contribution of eIF3d to pain processing using a heterozygous eIF3d knockout (eIF3d+/-) mouse model. We first validated this model, confirming substantial reductions in eIF3d mRNA and protein levels in dorsal root ganglia. Baseline assessments revealed no differences in mechanical, thermal, cold, or spontaneous pain behaviors between eIF3d+/- (HET) and eIF3d+/+ (WT) mice, indicating intact basal nociceptive function. In pain models involving peripheral inflammation and metabolic stress, including methylglyoxal injection, IL-6 administration and paw incision, HET mice displayed significantly reduced mechanical and cold hypersensitivity. In contrast, HET mice exhibited increased second phase nocifensive behavior in the formalin test, possibly indicating enhanced central sensitization. Hyperalgesic priming was comparable between HET and WT mice following IL-6 exposure. Experimental autoimmune encephalomyelitis (EAE) induced mice were unaffected by eIF3d reduction. These findings demonstrate that eIF3d selectively modulates nociceptive plasticity under defined stress conditions and suggests a context dependent role in the regulation of inflammatory and central pain sensitization. HighlightsO_LIBaseline mechanical, thermal, cold and spontaneous pain are intact in eIF3d+/-mice C_LIO_LIMethylglyoxal-evoked ISR activation and mechanical pain is blunted in eIF3d+/-mice C_LIO_LIIL-6-evoked mechanical and cold pain are reduced without altered priming C_LIO_LIMechanical hypersensitivity is reduced in eIF3d+/- mice with paw incision C_LIO_LIEAE pain is unaltered but increased pain in phase II formalin pain in eIF3d+/-mice C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/695844v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@26d9aforg.highwire.dtl.DTLVardef@9d7350org.highwire.dtl.DTLVardef@fdbabeorg.highwire.dtl.DTLVardef@1ef1074_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

A molecular map of the human spinal dorsal and ventral horn defines arrangement of neuronal types and glial sex differences

The spinal cord is the gateway for somatosensory and nociceptive information to the brain and a key locus for sensory-motor integration. Studies in mice have advanced our understanding of spinal cord circuitry, and transcriptomic studies have begun to characterize the human spinal cord; however, major gaps in knowledge persist. We conducted single-nucleus sequencing of lumbar spinal cord tissue from 11 adult organ donors and annotated spinal cord cell types with high resolution spatial transcriptomics. We identified 34 spatially and transcriptionally defined neuronal classes and detected sex-specific cell types and states across multiple glial populations. Electrophysiological recordings from dorsal horn neurons revealed firing patterns for neuronal subtypes and group I mGluR-dependent plasticity. Our work defines previously unknown aspects of human spinal cord molecular anatomy and physiology.

neuroscience↗

bioRxiv2025

Enabling wider access to human molecular neuroscience research in pain: A simple preservation method for human dorsal root ganglion neurons in Hibernate A media

The use of human dorsal root ganglion (DRG) from organ donors opens the door for research into the molecular biology and physiology of human nociceptors; however, there are barriers to working with this tissue including logistical difficulties and limited access. We present an approach using Hibernate media to temporarily store either whole DRGs or dissociated DRG neurons prior to culturing and functional testing. Dissociation of DRGs following temporary storage (4-16hrs) in Hibernate media resulted in similar neuronal and immune cell yield as acutely dissociated DRGs. Neurons derived from DRGs stored in Hibernate media prior to dissociation exhibited similar electrophysiological properties and capsaicin responses as acutely dissociated DRG neurons. Similarly, neurons from acutely dissociated DRGs stored in Hibernate media (>24hrs) and shipped to geographically distant laboratories produced neuronal cultures displaying comparable electrophysiological properties as acutely cultured neurons. This approach overcomes insurmountable logistical burdens and increases access to freshly recovered human DRGs.

neuroscience↗

bioRxiv2025

Spatial transcriptomic profiling of human paravertebral sympathetic chain ganglia reveals diabetes-induced neuroplasticity

The paravertebral sympathetic chain ganglia (SCG) are autonomic ganglia critical for regulating the "fight-or-flight" response. Symptoms of sympathetic dysfunction are prevalent in diabetes, affecting up to 90% of patients. The molecular and cellular composition of the human SCG and its alteration in diabetes remains poorly defined. To address this gap, we performed spatial transcriptomic profiling of lumbar SCGs from diabetic and non-diabetic organ donors. We identified 3 three distinct neuronal populations, two noradrenergic (NA1 and NA2) and one cholinergic (CHO), based on tyrosine hydroxylase (TH) and SLC18A3 expression, respectively. We also characterized 9 non-neuronal populations consisting of Schwann cells, immune cells, fibroblasts, adipocytes, and endothelial cells. In diabetic SCGs, we observed a significant loss of myelinating Schwann cells and a phenotypic shift of cholinergic neurons toward a noradrenergic identity. Additionally, diabetes was associated with a significant reduction in the transcripts of vasodilatory neuropeptides, such as VIP and CALCA, suggesting a mechanism for impaired vascular control. Upstream regulator analysis highlighted altered neurotrophic signaling in diabetes, with enhanced NGF/TRKA and diminished BDNF/TRKB activity, potentially driven by target-derived cues. Comparison between SCG and dorsal root ganglia (DRG) neurons revealed ganglia-specific genes, like SCN3A and NPY (SCG) versus SCN10A and GPX1 (DRG), offering specific therapeutic targets for autonomic dysfunction or pain. Our findings provide a transcriptomic characterization of human SCG, revealing molecular signatures that underlie diabetic autonomic dysfunction. This work lays a foundation for the development of therapies to restore sympathetic function and avoid unintended autonomic effects in the development of analgesics. Significance StatementAutonomic dysfunction affects up to 90% of people with diabetes, yet the human sympathetic nervous system remains poorly molecularly defined. To address this gap, we present a spatial transcriptomic profile of the human sympathetic chain ganglia (SCG), revealing how diabetes affects the human autonomic nervous system. We show that diabetes shifts the cholinergic neuronal population to a noradrenergic phenotype and reduces vasodilation neuropeptide expression, potentially explaining impaired vascular control and thermoregulation. Comparative analysis of sympathetic and sensory ganglia reveals distinct gene profiles that may inform novel therapeutic strategies. These findings offer critical insight into the molecular drivers of diabetic autonomic neuropathy and lay the groundwork for safer, more precise treatments that selectively modulate autonomic or sensory function in chronic disease.

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bioRxiv2025

Neurobiological and Chemical Characterization of the Cyanobacterial Metabolite Veraguamide E

Ver Es structure was validated by {superscript 1}H NMR, HRMS, and molecular networking analyses. Computational docking and NMR titration confirmed direct, saturable, and tight binding of Ver E to the human Sigma-2 receptor/transmembrane protein 97 ({sigma}2R/TMEM97). Functional calcium imaging in primary mouse sensory neurons revealed that Ver E increases intracellular Ca{superscript 2} levels without modulating store-operated calcium entry (SOCE). Multi-well microelectrode array experiments using human induced pluripotent stem cell (hiPSC) derived nociceptors showed that Ver E significantly reduced neuronal activity at physiological temperatures, but not under heat-stress conditions. Ver E exhibited no cytotoxicity at concentrations up to 30 {micro}M in HEK293 cells, and immunocytochemistry confirmed that it does not alter phosphorylated eIF2 (p-eIF2) expression, indicating a mechanism distinct from integrated stress response modulators. Collectively, these findings position Ver E as a non-toxic compound capable of selectively modulating neuronal excitability, thereby advancing the development of novel therapeutics for pain management. SignificanceNatural products have long been recognized as a rich source of therapeutics, accounting for over 60% of currently approved small-molecule drugs and underscoring their pivotal role in drug discovery. Marine cyanobacteria produce structurally diverse secondary metabolites with a wide array of biological activities. Among these are the veraguamides, a family of depsipeptides that have shown promise as future therapeutics in our recent studies. This work presents a detailed biological and chemical characterization of veraguamide E (Ver E), isolated from a Panamanian marine cyanobacterial collection. The {sigma}2R/TMEM97 system has been identified as a promising target to address unmet need for non-opioid therapeutics which can modulate neuronal excitability in the context of chronic pain. Discovery and identification of novel compounds which modulate this system can help us better understand its function as well as allow us to develop future therapeutics targeting this pathway. HighlightsO_LIVeraguamide E specifically binds {sigma}2R/TMEM97 receptor with high affinity. C_LIO_LIComputational docking and NMR confirm a distinct binding mechanism. C_LIO_LIVer E modulates calcium signaling in mouse DRG neurons and human iPSC-derived nociceptors. C_LIO_LIVer E demonstrates no detectable cytotoxicity in human cell lines. C_LI

neuroscience↗

bioRxiv2025

Expansion of OSMR expression and signaling in the human dorsal root ganglion links OSM to neuropathic pain

RNA sequencing studies on human dorsal root ganglion (hDRG) from patients suffering from neuropathic pain show upregulation of OSM, linking this IL-6 family cytokine to pain disorders. In mice, however, OSM signaling causes itch behaviors through a direct effect on its cognate receptor expressed uniquely by pruriceptive sensory neurons. We hypothesized that an expansion in function of OSM-OSM receptor (OSMR) in sensory disorders in humans could be explained by species differences in receptor expression and signaling. Our in situ hybridization and immunohistochemical findings demonstrate broad expression of OSMR in DRG nociceptors and afferent fibers innervating the superficial and deep skin of humans. In patch-clamp electrophysiology, OSM directly activates human sensory neurons engaging MAPK signaling to promote action potential firing. Using CRISPR editing we show that OSM activation of MAPK signaling is dependent on OSMR and not LIFR in hDRG. Bulk, single-nuclei, and single-cell RNA-seq of OSM-treated hDRG cultures reveal expansive similarities in the transcriptomic signature observed in pain DRGs from neuropathic patients, indicating that OSM alone can orchestrate transcriptomic signatures associated with pain. We conclude that OSM-OSMR signaling via MAPKs is a critical signaling factor for DRG plasticity that may underlie neuropathic pain in patients.

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bioRxiv2025

Decreased KCC2 expression in the human spinal dorsal horn associated with chronic pain and long-term opioid use

Loss of GABAergic and glycinergic inhibitory efficacy in the spinal dorsal horn is associated with neuropathic pain and opioid-induced hyperalgesia in rodent models. Downregulation of the KCC2 chloride extrusion transporter is a key mechanism underlying this decreased inhibitory efficacy, but to-date there is no evidence supporting or opposing this hypothesis in humans. Here we demonstrate that KCC2 expression is decreased in superficial dorsal horn neurons of organ donors who died with a documented history of pain, or of long-term opioid use. We show profoundly decreased KCC2 dorsal horn membrane expression in a primary cohort associated with either chronic pain or opioid use, and in a replication cohort of mixed chronic pain and opioid use history. These results show that decreased dorsal horn inhibitory efficacy likely promotes chronic pain in humans and support the development of therapeutics augmenting KCC2 function as a treatment for chronic pain and opioid use disorders.

neuroscience↗

bioRxiv2024

Ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans

Ephrin-B-EphB signaling promotes pain through signaling between dorsal root ganglion (DRG) neurons and spinal cord neurons in the dorsal horn, and through signaling between peripheral cells and EphB receptors expressed by DRG neurons. Previous findings link ephrin-B expression in painful peripheral tissues in patients to chronic pain, suggesting the clinical significance of this signaling, but the direct effects of ephrins on DRG neurons have not been widely studied. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Our work demonstrates that ephrin-B2-EphB2 signaling drives activation of MNK-eIF4E in DRG neurons to cause an enhanced response to inflammatory mediator signaling in both mice and humans and hyperalgesic priming in two models in mice. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2 injection into the same hindpaw. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by the MNK inhibitor eFT508. Similar effects on hyperalgesic priming were seen in a dural injection model. We generated a sensory neuron specific knockout of EphB2 using Pirt-Cre mice and found that these mice lacked responses to ephrin-B2 injection. We used Ca2+-imaging to determine direct effects of ephrin-B2 on DRG neurons and found that ephrin-B2 treatment enhanced Ca2+ transients in response to PGE2 which were absent in DRG neurons from MNK1-/- and EphB2-PirtCre mice. In experiments on human DRG neurons we found that ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+ responses to PGE2 treatment, both of which were blocked by eFT508 treatment. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling. The findings offer insight into how ephrin-B signaling promotes pain, and suggests treatment avenues for prevention or reversal of chronic pain associated with EphB activation in sensory neurons. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/581414v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9ff5c9org.highwire.dtl.DTLVardef@1c17916org.highwire.dtl.DTLVardef@f4a5b0org.highwire.dtl.DTLVardef@1aa00c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Diroximel fumarate acts through Nrf2 to attenuate methylglyoxal-induced nociception in mice and decreases ISR activation in DRG neurons

Diabetic neuropathic pain is associated with elevated plasma levels of methylglyoxal (MGO). MGO is a metabolite of glycolysis that causes mechanical hypersensitivity in mice by inducing the integrated stress response (ISR), which is characterized by phosphorylation of eukaryotic initiation factor 2 (p-eIF2). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of antioxidant proteins that neutralize MGO. We hypothesized that activating Nrf2 using diroximel fumarate (DRF) would alleviate MGO-induced pain hypersensitivity. We pretreated male and female C57BL/6 mice daily with oral DRF prior to intraplantar injection of MGO (20 ng). DRF (100 mg/kg) treated animals were protected from developing MGO-induced mechanical and cold hypersensitivity. Using Nrf2 knockout mice we demonstrate that Nrf2 is necessary for the anti-nociceptive effects of DRF. In cultured mouse and human dorsal root ganglion (DRG) sensory neurons, we found that MGO induced elevated levels of p-eIF2. Co-treatment of MGO (1 {micro}M) with monomethyl fumarate (MMF, 10, 20, 50 {micro}M), the active metabolite of DRF, reduced p-eIF2 levels and prevented aberrant neurite outgrowth in human DRG neurons. Our data show that targeting the Nrf2 antioxidant system with DRF is a strategy to potentially alleviate pain associated with elevated MGO levels. PerspectiveThis study demonstrates that activating Nrf2 with DRF prevents the development of pain caused by MGO in mice and reduces ISR in mouse and human DRG in vitro models. We propose that Nrf2 activators like DRF should be tested to alleviate diabetic neuropathic pain associated with elevated MGO in patients. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/572877v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5e0703org.highwire.dtl.DTLVardef@11e95bforg.highwire.dtl.DTLVardef@f92f2borg.highwire.dtl.DTLVardef@187f12d_HPS_FORMAT_FIGEXP M_FIG C_FIG Article HighlightsO_LIMGO induces mechanical and cold hypersensitivity in mice that is prevented with pre-treatment with DRF. C_LIO_LIDRF pre-treatment does not protect Nrf2-knockout mice from developing pain hypersensitivity suggesting that Nrf2 is necessary for DRFs antinociceptive effects. C_LIO_LIMMF, the active metabolite of DRF, prevents MGO-induced increase in p-eIF2a levels in mouse and human DRG neurons in vitro. C_LIO_LIMMF prevents MGO-induced aberrant neurite outgrowth in human DRG neurons. C_LIO_LINrf2 activators, like the FDA-approved DRF, is an option to alleviate neuropathic pain in patients with diabetes. C_LI

neuroscience↗

bioRxiv2023

Validation of σ2R/TMEM97 as a neuropathic pain target: Specificity, human expression and mechanism of action.

The Sigma 2 receptor ({sigma}2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that {sigma}2R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal anti-nociceptive effect is approximately 24 hours following dosing. We sought to understand this unique anti-neuropathic pain effect by addressing two key questions: do these {sigma}2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout (KO) mice for Tmem97, we find that a new {sigma}2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce anti-nociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion (DRG) neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a {sigma}2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2 levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that {sigma}2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate {sigma}2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain. Significance StatementNeuropathic pain is a major medical problem that is poorly treated with existing therapeutics. Our findings demonstrate that targeting {sigma}2R/TMEM97 with a newly described modulator reduces pain hypersensitivity in a mouse model with exquisite selectivity. We also identify integrated stress response (ISR) inhibition as a potential mechanism of action that links the receptor to cellular signaling events that have preclinical and clinical validation for pain relief. Our work suggests that {sigma}2R/TMEM97 can be selectively engaged by specific small molecules to produce ISR inhibition in a subset of cells that are critical for neuropathic pain. {sigma}2R/TMEM97-targeted therapeutics thus have the potential to offer effective pain relief without engagement of opioid receptors.

neuroscience↗

bioRxiv2020

ER stress in the dorsal root ganglia regulates BK channel physiology and contributes to pain hypersensitivity in a mouse model of multiple sclerosis

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went to assess disease-mediated changes in the functional properties of Ca2+-sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.

neuroscience↗