bioRxiv Science⌕ Search

Biology subjects

Tawfik, V. L.

Publications and source records attributed to Tawfik, V. L..

8 recordsLinked to original sources

Library docking for Cannabinoid-2 Receptor ligands

Cannabinoid receptors are therapeutically promising GPCRs that are also interesting test systems for structure-based methods, which have targeted them previously. Here we used the CB2 receptor as a template to explore several topical questions in library docking. Whereas an earlier campaign against the CB1 receptor led to potent but relatively non-selective ligands, here we found that targeting interactions with polar, orthosteric site residues led to subtype-selective ligands. Docking hit rate and especially hit affinity improved in moving from a 7 million to a 2.6 billion molecule library. Similar to earlier studies, docking against active and inactive states of the receptor did not reliably bias toward the discovery of agonists or inverse agonists. Cryo-EM structures of two of the new agonists, each in a different chemotype, superposed well on the docking predictions. Correspondingly, structure-based optimization led to 10- to 140-fold improvements within three different series, also consistent with well-behaved ligand families. Hit rates with a fully enumerated 2.6 billion molecule library resembled those of an implied 11 billion molecule library from a building-block method, consistent with the latters ability to explore this space, though higher affinities were discovered from the fully enumerated set. Overall, eight diverse families of ligands, with potencies <100 nM and mostly unrelated to previously known ligands were found. Implications for future studies are considered.

biochemistry↗

Linking Pain and Delirium via Microglial Activation: A Mouse Study Using BSEEG, Behavioral Assays, Immunohistochemistry, and RNA Sequencing

BackgroundThe rising incidence of delirium in surgical and critical care settings, especially in older patients, calls for improved preventative and management strategies. Chronic pain is increasingly recognized as a key risk factor for delirium, and microglial activation may be the central mediator linking these two conditions. MethodsWe used a spared nerve injury (SNI) model of persistent neuropathic pain in tandem with a postoperative delirium (POD) mouse model. Pain assessments, electroencephalography (EEG) recording, and immunofluorescence were performed to characterize pain and delirium-like states. Microglia were isolated for RNA-seq to elucidate gene expression changes comprehensively. ResultsSNI mice showed persistent mechanical hypersensitivity from Day 7 onwards and demonstrated disrupted sleep-wake patterns in EEG indices. Immunofluorescence revealed sustained microglial activation in both the hippocampus and cortex following SNI. RNA-seq analyses indicated the upregulation of pro-inflammatory pathways (e.g., interleukin-6 production, NF-{kappa}B signaling) in SNI mice that also underwent head-mount surgery. Notably, the coincidence of persistent pain and an acute delirium-like state further exacerbated neuroinflammation. ConclusionOur findings suggest that neuropathic pain-induced microglial activation primes the brain for exaggerated inflammatory responses under additional surgical stress, potentially worsening delirium. Further investigations into microglia-focused therapies may inform novel strategies for mitigating delirium in patients with neuropathic pain.

neuroscience↗

Psilocybin has no immediate or persistent analgesic effect in acute and chronic mouse pain models

The psychedelic psilocybin may have lasting therapeutic effects for patients with chronic pain syndromes. Some clinical and preclinical data suggest these putative benefits derive from direct analgesic effects. However, this possibility has not been comprehensively tested in preclinical models. Here, we show that psilocybin is not analgesic over a range of doses across multiple pain assays and models of acute and chronic inflammatory, neuropathic, or musculoskeletal pain in mice.

neuroscience↗

Ethological profiling of pain and analgesia in a mouse model of complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a form of chronic post-injury pain affecting the extremities. The mouse tibial fracture- cast model was developed to enable preclinical study of CRPS mechanisms and guide condition-specific drug development. Given the inherent limitations of reflex pain measures in mice, we sought to characterize pain-like behaviors in this model more holistically. We evaluated spontaneous and evoked pain and naturalistic behaviors after tibial fracture-cast injury in male mice in neutral and aversive environments using LabGym. Here, we report a unique ethological signature of pain in injured mice characterized by reflexive allodynia, thermal hyperalgesia, frequent grooming and reduced rearing in neutral and aversive environments, and decreased paw withdrawal and increased paw licking in an aversive environment. As proof-of-concept, we also leveraged this holistic behavioral evaluation for drug screening by characterizing the peripheral versus central effects of targeting alpha-2 receptors (2-AR) in the tibial fracture-cast model. We evaluated the impact of systemic delivery of dexmedetomidine (DEX), a selective 2-AR agonist, with or without antagonists, on holistic behavioral metrics in injured male mice. We found that DEX reduced mechanical allodynia primarily via central 2-ARs. DEX also decreased motion metrics, grooming and rearing in an open field, and distinctly affected the quality and quantity of grooming in an aversive environment, and this effect was not suppressed by systemic 2-AR antagonists. Ultimately, this study holistically captures pain-related behaviors and provides a detailed characterization of the relative contributions of peripheral and central 2-ARs to alpha2-mediated analgesia in male mice after tibial fracture-cast injury.

neuroscience↗

Aging and injury drive neuronal senescence in the dorsal root ganglia

Aging negatively impacts central nervous system function; however, the cellular impact of aging in the peripheral nervous system remains poorly understood. Aged individuals are more likely to experience increased pain and slower recovery after trauma. Such injury can damage vulnerable peripheral axons of dorsal root ganglion (DRG) neurons resulting in somatosensory dysfunction. One cellular mechanism common to both aging and injury is cellular senescence, a complex cell state that can contribute to the aged pro-inflammatory environment. We uncovered, for the first time, DRG neuron senescence in the context of aging and pain-inducing peripheral nerve injury in young and aged mice. Aged DRG neurons displayed multiple markers of senescence (SA-{beta}-gal, p21, p16, IL6) when compared to young DRG neurons. Peripheral nerve injury triggered a further accumulation of senescent DRG neurons over time post-injury in young and aged DRG. These senescent neurons were dynamic and heterogeneous in their expression of senescence markers, p16, p21, and senescence-associated secretory phenotype (SASP) expression of IL6, which was influenced by age. An electrophysiological characterization of senescence marker-expressing neurons revealed high-firing and nociceptor-like phenotypes within these populations. In addition, we observed improvement in nociceptive behaviors in young and aged nerve-injured mice after treatment with a senolytic agent that eliminates senescent cells. Finally, we confirmed in human post-mortem DRG samples that neuronal senescence is present and increases with age. Overall, we describe a susceptibility of the peripheral nervous system to neuronal senescence with age or injury that may be a targetable mechanism to treat sensory dysfunction, such as chronic pain, particularly in aged populations.

neuroscience↗

Newly repopulated spinal cord microglia exhibit a unique transcriptome and coincide with sex-independent pain resolution

Microglia contribute to the initiation of pain, however, a translationally viable approach addressing how or when to modulate these cells remains elusive. We used a targeted, inducible, genetic microglial depletion strategy at both acute and acute-to-chronic transition phases in the clinically-relevant tibial fracture/casting pain model to determine the contribution of microglia to the initiation and maintenance of pain. We observed complete resolution of pain after transient microglial depletion at the acute-to-chronic phase, which coincided with the timeframe of full repopulation of microglia. These repopulated microglia were morphologically distinct from control microglia, signifying they may exhibit a unique transcriptome. RNA sequencing of repopulated spinal cord microglia identified genes of interest using weighted gene co-expression network analysis (WGCNA). We intersected these genes with a newly-generated single nuclei microglial dataset from human spinal cord dorsal horn and identified human-relevant genes that may ultimately promote pain resolution after injury. This work presents a novel approach to gene discovery in pain and provides comprehensive datasets for the development of future microglial-targeted therapeutics.

neuroscience↗

Schwann cells promote sensory neuron excitability during development

Excitability--the ability to fire action potentials--is a signature feature of neurons. How neurons become excitable during development, and whether excitability is an intrinsic property of neurons or requires signaling from glial cells, remain unclear. Here we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of voltage-gated sodium channels and fire action potential trains. Inactivating this signaling pathway specifically in Schwann cells selectively impairs the maturation of nociceptor and proprioceptor somatosensory neuron subtypes, leading to corresponding sensory defects in thermoception, inflammatory pain, and proprioception. Our studies thus reveal a cell non-autonomous mechanism by which glia regulate neuronal excitability to enable the development of normal sensory functions.

neuroscience↗

Exercise and angiotensin receptor blockade enhance recovery after orthopaedic trauma in mice by decreasing pain and improving muscle regeneration

Chronic pain and disability after limb injury are major public health problems. One key obstacle to addressing these adverse outcomes is that we do not know when exercise should be initiated or whether the beneficial effects of exercise can be reproduced using pharmacological tools. In these studies, we developed and used a murine model of orthopaedic trauma combining tibia fracture and pin fixation with tibialis anterior muscle damage. Behavioral measures included mechanical nociceptive thresholds and distances run on exercise wheels. Bone healing was quantified using microCT scanning, and muscle fiber size distribution as well as fibrosis were followed using immunohistochemistry. We found that the model provided robust mechanical allodynia, fibrosis and a shift to smaller average muscle fiber size lasting up to 5 weeks from injury. We also observed that allowing \"late\" (weeks 1-2) rather than \"early\" (weeks 0-1) wheel running after injury resulted in greater overall running activity and greater reversal of allodynia. In parallel, the late running paradigm was also associated with lower levels of muscle fibrosis and a return towards normal muscle fiber diameters. Providing the anti-fibrotic angiotensin receptor blocker losartan to mice in drinking water blocked TGFbeta production while reducing both allodynia and muscle fibrosis. Combining losartan and late exercise provided no additional benefit. We conclude that early healing after orthopaedic trauma must be allowed prior to the initiation of exercise to achieve optimal pain, functional and physiological outcomes. Losartan may provide many of the same pain, functional and physiological outcomes by its regulation of TGFbeta signaling and is a viable candidate for translational studies.\n\nKey points summaryO_LIOur tibial fracture orthopaedic injury model in mice recapitulates the major manifestations of complex trauma including nociceptive sensitization, bone fracture, muscle fibrosis and muscle fiber loss.\nC_LIO_LIDelayed exercise after complex orthopaedic trauma results in decreased muscle fibrosis and improved pain\nC_LIO_LILosartan, an angiotensin-receptor blocker with antifibrotic abilities, recapitulates the effect of exercise on post-injury recovery\nC_LI

neuroscience↗