Biology subjects

Arnold, W. D.

Publications and source records attributed to Arnold, W. D..

7 recordsLinked to original sources

bioRxiv2026

Tissue nanotransfection-mediated induction of neurogenic programs promotes myoprotective responses in denervated skeletal muscle

Peripheral nerve injuries often result in prolonged skeletal muscle denervation, leading to progressive atrophy, fibrosis, neuromuscular instability, and loss of regenerative capacity before axons can reinnervate distal targets. Here, we developed a non-viral strategy using tissue nanotransfection (TNT) to deliver the neurogenic transcription factor cocktail Ascl1, Brn2, and Myt1l (ABM) directly to denervated skeletal muscle. In vitro, ABM-transfected myoblasts sustained expression of the reprogramming factors, acquired neuron-like morphologies, upregulated neuronal markers including Tuj1, Map2, and Syp, and exhibited electrophysiological properties consistent with membrane excitability. RNA sequencing confirmed broad activation of neurogenic transcriptional programs, with enrichment of pathways associated with neuronal fate commitment, neuron differentiation, axon guidance, synaptogenesis, and developmental signaling. In a mouse model of sciatic nerve transection, TNT enabled localized ABM expression in denervated gastrocnemius muscle. ABM-TNT treatment accelerated resolution of denervation-associated fibrillation potentials and showed trends toward improved twitch and tetanic torque, compound muscle action potential amplitudes, and muscle mass preservation. Transcriptomic profiling of treated muscles 5 weeks after injury revealed distinct gene expression programs enriched for muscle regeneration, neuromuscular organization, trophic support, extracellular matrix remodeling, angiogenesis, myogenesis, and metabolic adaptation. Network analyses further identified activation of neurogenic regulators, neurotrophic signaling, and vascular-support pathways. These findings establish TNT-mediated ABM delivery as a non-viral platform for inducing neurogenic and myoprotective programs in denervated muscle, suggesting a potential strategy to preserve muscle viability during the prolonged interval required for peripheral nerve regeneration.

bioengineering↗

bioRxiv2025

Behavioral Thermoceptive Responses and Morphologic Correlates in Mouse Models of CMT1A, HNPP and Aging

BackgroundThermoceptive dysfunction is a frequent but understudied feature of peripheral neuropathies and aging. Patients often report abnormal heat perception, yet the underlying sensory mechanisms remain unclear. This study evaluated thermoceptive behavior and corresponding structural changes in mouse models of inherited dysmyelinating neuropathy and natural aging to identify shared and divergent mechanisms. MethodsThermal preference was assessed using a user-independent gradient apparatus spanning physiological to noxious temperatures, with automated quantification of time in zone, distance traveled, and velocity. Nocifensive responses were evaluated by hot plate latency. Intraepidermal nerve fiber density (IENFD) was measured in paw pads, and TRPV1-positive dorsal root ganglion (DRG) neurons were analyzed by immunofluorescence and confocal imaging. ResultsThermal gradient testing revealed preserved temperature preference in CMT1A and HNPP mice but significantly altered behavior in aged animals, which spent less time in warmer zones. Hot plate testing showed prolonged times to nocifensive behavior in aged and CMT1A mice, whereas HNPP mice exhibited variable responses. IENFD was markedly reduced in aged mice but preserved in CMT1A and HNPP. DRG analysis revealed smaller soma diameters and reduced proportions of TRPV1-positive A{delta} neurons in aged mice, while CMT1A animals maintained normal morphology. InterpretationAging produces thermoceptive deficits through axonal degeneration and selective A{delta}-fiber vulnerability, whereas CMT1A mice display conduction-related impairment due to dysmyelination. Both models reproduce key human sensory phenotypes and provide translational platforms for studying small-fiber dysfunction and therapeutic interventions in peripheral neuropathies. Key PointsO_LIThermoceptive dysfunction is a common but understudied feature of peripheral neuropathy and aging. C_LIO_LIIn aged mice, altered thermal preference, reduced intraepidermal nerve fiber density, and smaller TRPV1-positive A{delta} neurons indicate a degenerative mechanism of sensory loss. C_LIO_LIIn CMT1A mice, delayed noxious heat responses occur despite preserved epidermal and DRG morphology, consistent with dysmyelination rather than axonal degeneration. C_LIO_LIHNPP mice showed inconsistent nocifensive responses without structural fiber loss, suggesting strain- or injury-dependent effects. C_LIO_LIThermal gradient and hot plate assays demonstrate strong reproducibility and provide relevant outcome measures for preclinical modeling of thermoceptive dysfunction. C_LI

neuroscience↗

bioRxiv2025

Hindlimb Immobilization Impairs Neuromuscular Junction Transmission in Young Rats

BackgroundImmobilization, bed rest, or illness rapidly lead to weakness out of proportion to muscle atrophy. Although the contribution of muscle wasting to weakness is well described, the role of neuromuscular junction (NMJ) dysfunction in early disuse-related weakness is not well understood. ObjectiveWe investigated whether short-term unilateral hindlimb immobilization (HLI) in rats impairs NMJ transmission and contributes to muscle weakness out of proportion to atrophy. MethodsFour-month-old male Fischer-344/Brown Norway rats underwent 10 days of unilateral HLI (n=6) or remained mobile (n=6). Neuromuscular excitability and transmission were assessed using compound muscle action potentials (CMAP), repetitive nerve stimulation (RNS), and single-fiber electromyography (SFEMG). Muscle contractility testing quantified tetanic torque, and post-mortem analysis measured muscle mass. ResultsTen days of HLI reduced gastrocnemius, soleus, and plantaris muscle mass by [~]20-35%. Plantarflexion peak tetanic torque normalized to body weight declined by 23%, and torque-time integral was reduced by 36%, indicating disproportionate functional loss (muscle size versus contractile output) and supporting underlying neural impairment. CMAP amplitude decreased from 69 mV to 53.23 mV (a 22.9% reduction; p = 0.0109), indicating a loss of summated neuromuscular excitability. Furthermore, both RNS and SFEMG indicated features consistent with NMJ transmission defects. RNS revealed CMAP decrement from [~]0% pre-HLI to -9.15% at 40 Hz stimulation and -8.63% at 50 Hz post-HLI. Similarly, SFEMG confirmed marked NMJ transmission defects, with jitter increasing 103% and blocking increased from <1% to >11% of fibers. ConclusionsOur findings suggest that short-term immobilization produces rapid and pronounced impairments in NMJ transmission that contribute to weakness beyond the degree of muscle atrophy. These findings identify the NMJ as an early and vulnerable site of disuse-induced dysfunction and highlight the potential for synaptic-targeted therapies to preserve muscle performance during immobilization and recovery.

physiology↗

bioRxiv2025

Novel neurofilament light (Nefl) E397K mouse models of Charcot-Marie-Tooth type 2E (CMT2E) present early and chronic axonal neuropathy

Charcot-Marie-Tooth (CMT) is the most common hereditary peripheral neuropathy with an incidence of 1:2,500. CMT2 clinical symptoms include distal muscle weakness and atrophy, sensory loss, toe and foot deformities, with some patients presenting with reduced nerve conduction velocity. Mutations in the neurofilament light chain (NEFL) gene result in a specific form of CMT2 disease, CMT2E. NEFL encodes the protein, NF-L, one of the core intermediate filament proteins that contribute to the maintenance and stability of the axonal cytoskeleton. To better understand the underlying biology of CMT2E disease and advance the development of therapeutics, we generated a Nefl+/E397K mouse model. While the Nefl+/E397K mutation is inherited in a dominant manner, we also characterized NeflE397K/E397K mice to determine whether disease onset, progression or severity would be impacted. Consistent with CMT2E, lifespan was not altered in these novel mouse models. A longitudinal electrophysiology study demonstrated significant in vivo functional abnormalities as early as P21 in distal latency, compound muscle action potential (CMAP) amplitude and negative area. A significant reduction in the sciatic nerve axon area, diameter, and G-ratio was also present as early as P21. Evidence of axon sprouting was observed with disease progression. Through the twelve months measured, disease became more evident in all assessments. Collectively, these results demonstrate an early and robust in vivo electrophysiological phenotype and axonal pathology, making Nefl+/E397K and NeflE397K/E397Kmice ideal for the evaluation of therapeutic approaches.

neuroscience↗

bioRxiv2023

The MuSK-BMP pathway regulates synaptic Nav1.4 localization and muscle excitability

The neuromuscular junction (NMJ) is the linchpin of nerve-evoked muscle contraction. Broadly considered, the function of the NMJ is to transduce a nerve action potential into a muscle fiber action potential (MFAP). Efficient information transfer requires both cholinergic signaling, responsible for the generation of endplate potentials (EPPs), and excitation, the activation of postsynaptic voltage-gated sodium channels (Nav1.4) to trigger MFAPs. In contrast to the cholinergic apparatus, the signaling pathways that organize Nav1.4 and muscle fiber excitability are poorly characterized. Muscle-specific kinase (MuSK), in addition to its Ig1 domain-dependent role as an agrin-LRP4 receptor, is also a BMP co-receptor that binds BMPs via its Ig3 domain and shapes BMP-induced signaling and transcriptional output. Here we probed the function of the MuSK-BMP pathway at the NMJ using mice lacking the MuSK Ig3 domain ( {Delta}Ig3-MuSK). Synapses formed normally in {Delta}Ig3-MuSK animals, but the postsynaptic apparatus was fragmented from the first weeks of life. Anatomical denervation was not observed at any age examined. Moreover, spontaneous and nerve-evoked acetylcholine release, AChR density, and endplate currents were comparable to WT. However, trains of nerve-evoked MFAPs in {Delta}Ig3-MuSK muscle were abnormal as revealed by increased jitter and blocking in single fiber electromyography. Further, nerve-evoked compound muscle action potentials (CMAPs), as well as twitch and tetanic muscle torque force production, were also diminished. Finally, Nav1.4 levels were reduced at {Delta}Ig3-MuSK synapses but not at the extrajunctional sarcolemma, indicating that the observed excitability defects are the result of impaired localization of this voltage-gated ion channel at the NMJ. We propose that MuSK plays two distinct roles at the NMJ: as an agrin-LRP4 receptor necessary for establishing and maintaining cholinergic signaling, and as a BMP co-receptor required for maintaining proper Nav1.4 density, nerve-evoked muscle excitability and force production. The MuSK-BMP pathway thus emerges as a target for modulating excitability and functional innervation, which are defective in conditions such as congenital myasthenic syndromes and aging. Significance StatementThe neuromuscular junction (NMJ) is required for nerve-evoked muscle contraction and movement, and its function is compromised during aging and disease. Although the mechanisms underlying neurotransmitter release and cholinergic response at this synapse have been studied extensively, the machinery necessary for nerve-evoked muscle excitation are incompletely characterized. We show that MuSK (Muscle-specific kinase), in its role as a BMP co-receptor, regulates NMJ structure as well as the localization of the voltage-gated sodium channels necessary for full nerve-evoked muscle fiber excitation and force production. This novel function of MuSK is structurally and mechanistically distinct from its role in organizing cholinergic machinery. The MuSK-BMP pathway thus presents a new opportunity to understand mechanisms that may preserve or enhance neuromuscular excitability in the face of aging and disease.

neuroscience↗

bioRxiv2021

Biological Aging of CNS-Resident Cells Alters the Clinical Course and Immunopathology of Autoimmune Demyelinating Disease

Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing remitting MS (RRMS) typically presents in the third or fourth decade, while the mean age of presentation of progressive MS (pMS) is 45 years old. Here we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4+ Th17 cells, is more severe, and less like to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils are more abundant, while B cells are relatively sparse, in central nervous system (CNS) infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrate that radio-resistant, non-hematopoietic cells play a dominant role in shaping age-dependent features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of pMS, EAE in middle-aged adoptive transfer recipients is characterized by widespread microglial activation. Microglia from older mice express a distinctive transcriptomic profile, suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.

neuroscience↗

bioRxiv2020

Efficient precise in vivo base editing in adult dystrophic mice

ABSTRACTBackground Recent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.Methods We employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.Results We showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.Conclusions This study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.Competing Interest StatementThe authors have submitted a patent application based on the results reported in this paper.View Full Text

bioengineering↗