Biology subjects

Cox, J. J.

Publications and source records attributed to Cox, J. J..

9 recordsLinked to original sources

bioRxiv2025

The study of differential expressions of MCPH and Seckel syndrome genes and their paralogues

Seckel syndrome and MCPH are single-gene neurodevelopmental disorders, with or without dwarfism. These genes are parts of essential cellular pathways but are regulated differently in both forms of microcephaly. These genes have diverged from single ancestral genes in to related but different gene paralogues during evolution. It is suggested that the paralogues might be redundant in other body organs and might rescue any abnormality in cases of MCPH, but not in Seckel syndrome. This study uncovers the spatio-temporal dynamics of these genes together with their paralogues at the mRNA level where the different tissues of embryos might exhibit different transcript variants or different levels of the various transcript variants to find the complementation in case of the MCPH but divergence in case of the Seckel syndrome. Here, we studied Cdk5Rap2-Pde4dip, Phc1-Phc3 (for MCPH) and Cep63-Ccdc67 (for Seckel) in the embryonic mouse tissues using RT-PCR throughout the peak of neurogenesis from E12.5 to E18.5. We found already known and novel spatio-temporal differential expression suggesting different regulation at the mRNA level. The results of PCR were analyzed on the agarose gel and were quantified by assigning relative intensity percentage scores to each band and plotting the cumulative expression of each gene in to the graphs for spatio-temporal dynamics and plotted each gene separately for temporal dynamics. For MCPH, Cdk5Rap2-Pde4dip had similar profiles and seemed to be redundant whereas Phc1-Phc3 seem to have complementary roles in brain development. The expression profiles of Cep63-Ccdc67 were very different indicating of their divergent and essential roles in development.

genetics↗

bioRxiv2024

Novel therapies for cancer-induced bone pain

1.Cancer pain is a growing problem, especially with the substantial increase in cancer survival. Reports indicate that bone metastasis, whose primary symptom is bone pain, occurs in 65-75% of patients with advanced breast or prostate cancer. We optimized a preclinical in vivo model of cancer-induced bone pain (CIBP) involving the injection of Lewis Lung Carcinoma cells into the intramedullary space of the femur of C57BL/6 mice or transgenic mice on a C57BL/6 background. Mice gradually reduce the use of the affected limb, leading to altered weight bearing. Symptoms of secondary cutaneous heat sensitivity also manifest themselves. Following optimization, three potential analgesic treatments were assessed; 1) single ion channel targets (targeting the voltage-gated sodium channels NaV1.7, NaV1.8, or acid-sensing ion channels), 2) silencing {micro}-opioid receptor-expressing neurons by modified botulinum compounds, and 3) targeting two inflammatory mediators simultaneously (nerve growth factor (NGF) and tumor necrosis factor (TNF)). Unlike global NaV1.8 knockout mice which do not show any reduction in CIBP-related behavior, embryonic conditional NaV1.7 knockout mice in sensory neurons exhibit a mild reduction in CIBP-linked behavior. Modified botulinum compounds also failed to cause a detectable analgesic effect. In contrast, inhibition of NGF and/or TNF resulted in a significant reduction in CIBP-driven weight-bearing alterations and prevented the development of secondary cutaneous heat hyperalgesia. Our results support the inhibition of these inflammatory mediators; and more strongly their dual inhibition to treat CIBP, given the superiority of combination therapies in extending the time needed to reach limb use score zero in our CIBP model.

neuroscience↗

bioRxiv2024

Two distinct mechanisms for Nav1.7 null analgesia

Genetic deletion and pharmacological inhibition are distinct approaches to unravelling pain mechanisms, identifying targets and developing new analgesics. Both approaches have been applied to the voltage-gated sodium channels Nav1.7 and Nav1.8. Genetic deletion of Nav1.8 in mice leads to a loss of pain, and antagonists are effective analgesics. Complete embryonic loss of Nav1.7 in humans or in mouse sensory neurons leads to profound analgesia substantially mediated by endogenous opioid signaling, and anosmia that is opioid independent. Autonomic function appears to be normal. Adult deletion of Nav1.7 in sensory neurons also leads to analgesia with diminished sensory neuron excitability but there is no opioid component of analgesia. Pharmacological inhibition of Nav1.7 leads to dramatic side-effects on the autonomic nervous system. Here we compare and contrast the distinct embryonic and adult null mechanisms of Nav1.7 loss-of-function analgesia. We describe an endogenous opioid mechanism of analgesia that provides new opportunities for therapeutic intervention and pain relief. SummaryIn contrast to Nav1.8, Nav1.7, a genetically validated human pain target is unsuitable for small molecule drug development because of its wide spread expression both centrally and peripherally.

neuroscience↗

bioRxiv2024

Analgesic targets identified in mouse sensory neuron somata and terminal pain translatomes

The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (+ Nerve Growth Factor (NGF)) and pain-free conditions (Nav1.7 null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behaviour. Terminal-enriched transcripts encoding synaptic proteins (e.g. Synaptotagmin), non-coding RNAs, transcription factors (e.g. Znf431), proteins associated with trans-synaptic trafficking (HoxC9), GABA generating enzymes (Gad1 and Gad2) and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons.

neuroscience↗

bioRxiv2023

Chemogenetic silencing of NaV1.8 positive sensory neurons reverses chronic neuropathic and bone cancer pain in FLEx PSAM4-GlyR mice

Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically-activated chloride channel PSAM4 -GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging upon activation of PSAM4 -GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4 -GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4 -GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4 -GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4 -GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4 -GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use. Significance statementChronic pain is a massive problem. Peripheral nerve block is effective in many chronic pain conditions, demonstrating the importance of peripheral drive in chronic pain. We used chemogenetic tools based on the modified ligand-gated chloride channel PSAM4 -GlyR to silence dorsal root ganglion neurons in vitro and in vivo. This approach reduces pain-like behavior in acute and chronic pain models, including resistant pain conditions like neuropathic pain or cancer-induced bone pain. We generated a mouse line that expresses PSAM4 -GlyR in a Cre-dependent manner, providing a useful research tool to address not only the role of nociceptive sensory neurons in pain states but also the function of genetically defined sets of neurons throughout the nervous system in normal and pathological conditions.

neuroscience↗

bioRxiv2023

Transcriptomic profiling reveals a pro-nociceptive role for Angiotensin II in inflammatory bowel disease

ObjectiveVisceral pain is a leading cause of morbidity in inflammatory bowel disease (IBD), contributing significantly to reduced quality of life. Currently available analgesics often lack efficacy or have intolerable side-effects, driving the need for a more complete understanding of the mechanisms causing pain. MethodsWhole transcriptome gene expression analysis was performed by bulk RNA sequencing of colonic biopsies from patients with ulcerative colitis (UC) and Crohns disease (CD) reporting abdominal pain and compared with non-inflamed control biopsies. Putative pro-nociceptive mediators were identified based on pathway analysis of differentially expressed genes in IBD tissue and single cell gene expression in colonic neurons. Pro-nociceptive activity of identified mediators was assessed in assays of sensory neuron and colonic afferent activity. ResultsRNA sequencing analysis highlighted a 7.6-fold increase in the expression of angiotensinogen transcripts, Agt, the precursor to angiotensin II (Ang II), in samples from UC patients (p = 3.2x10-8). Consistent with the marked expression of the angiotensin AT1 receptor in colonic neurons, Ang II elicited an increase in intracellular Ca2+ in capsaicin-sensitive, voltage gated sodium channel subtype NaV1.8-positive sensory neurons. Ang II also evoked action potential discharge in high-threshold colonic nociceptors. These effects were inhibited by the AT1 receptor antagonist valsartan. ConclusionFindings from our study identify AT1 receptor-mediated colonic nociceptor activation as a novel pathway of visceral nociception in IBD patients with UC. This work highlights the potential utility of angiotensin receptor blockers, such as valsartan, as treatments for pain in IBD.

neuroscience↗

bioRxiv2022

Molecular basis of FAAH-OUT-associated human pain insensitivity

Chronic pain affects millions of people worldwide. Studying pain insensitive individuals helps to identify novel analgesic strategies. Here we report how the recently discovered FAAH-OUT lncRNA-encoding gene, which was found from studying a pain insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme. We demonstrate that the disruption in FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression. Furthermore, using transcriptomic analyses we have uncovered a network of genes that are dysregulated from disruption of the FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed and a platform for development of future gene and small molecule therapies.

neuroscience↗

bioRxiv2019

CRISPR interference at the FAAH-OUT genomic region reduces FAAH expression

FAAH-OUT encodes a putative long non-coding RNA which is located next to the FAAH gene on human chromosome 1. Recently an ~8 kb microdeletion, that removes upstream regulatory elements and the first 2 exons of FAAH-OUT, was reported in a pain insensitive patient (PFS) with additional clinical symptoms including a happy, non-anxious disposition, fast wound healing, fear and memory deficits, and significant post-operative nausea and vomiting induced by morphine. PFS also carries a hypomorphic SNP in FAAH that significantly reduces the activity of the encoded fatty-acid amide hydrolase enzyme. The FAAH and FAAH-OUT mutations identified in PFS result in elevated levels of anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamine (OEA) measured in peripheral blood. These bioactive lipids, which are normally degraded by FAAH, have diverse biological functions including known roles in pain pathways.\n\nHere we report the first mechanistic insights into how the FAAH-OUT microdeletion affects FAAH function. Gene editing in a human cell line to mimic the FAAH-OUT microdeletion observed in PFS results in reduced expression of FAAH. Furthermore, CRISPRi experiments targeting the promoter region of FAAH-OUT or a short highly evolutionarily conserved element in the first intron of FAAH-OUT also result in reduced expression of FAAH. These experiments confirm the importance of FAAH-OUT and specific genomic elements within the ~8 kb microdeleted sequence to normal FAAH expression. Our results also highlight the potential of CRISPRi and gene editing strategies that target the FAAH-OUT region for the development of novel FAAH-based analgesic and anxiolytic therapies.

genetics↗

bioRxiv2017

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene targeted mice

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. Besides action potential propagation, NaV1.7 regulates neurotransmitter release, integrates depolarizing inputs over long periods and regulates transcription. In order to better understand these functions, we generated an epitope-tagged NaV1.7 mouse that showed normal pain behavior. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 NaV1.7 associated proteins including known interactors, such as the sodium channel {beta}3 subunit (Scn3b) and collapsin response mediator protein (Crmp2), and novel interactors. Selected novel NaV1.7 protein interactors membrane-trafficking protein synapototagmin-2 (Syt2), G protein-regulated inducer of neurite outgrowth 1 (Gprin1), L-type amino acid transporter 1 (Lat1) and transmembrane P24 trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated using co-immunoprecipitation and functional assays. The information provided with this physiologically normal epitope-tagged mouse should provide useful insights into the pain mechanisms associated with NaV1.7 channel function.

neuroscience↗