Sameer B. Shah

NF-M is an essential target for the myelin-directed “outside-in” signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an “outside-in” signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.

Structural and Functional Roles of Desmin in Mouse Skeletal Muscle during Passive Deformation

Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein alpha-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of alpha-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.

Cytoskeletal Disruption After Eccentric Contraction-induced Muscle Injury

Skeletal muscle cytoskeletal proteins are receiving more attention recently based on their importance in maintaining muscle integrity, their role in transmitting force throughout the cell, and their involvement in muscle diseases. In this report, the authors focus on the intermediate filament system of skeletal muscle composed of the protein desmin. Desmin is shown to transmit force from myofibrillar force generators to the muscle surface and to the muscle-tendon junction. This protein is lost rapidly during high-intensity exercise using a rabbit model. Mice were genetically engineered that lack the desmin gene and these muscles were shown to generate lower stress but actually to experience less injury during intense exercise. Finally, direct imaging of muscle cells with fluorescently labeled cytoskeletal proteins shows that lack of the desmin protein results in tremendous disorganization of the myofibrillar lattice which may help to explain desmin myopathies.

Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset.

Mutations in superoxide dismutase (SOD1) are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS.

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila

Amyloid precursor protein (APP) vesicle movement by kinesin-1 and cytoplasmic dynein exhibits kinesin-1–dependent velocity. Our data also suggest that kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and that their direction and velocity are controlled at least in part by dynein IC.

Effects of in vivo injury on the neuromuscular junction in healthy and dystrophic muscles

•  Strength loss induced by lengthening contractions is typically attributed to damaged force‐bearing structures within skeletal muscle. Muscle lacking the structural protein dystrophin, as in Duchenne muscular dystrophy, is particularly susceptible to contraction‐induced injury. •  We tested the hypothesis that changes in neuromuscular junctions (NMJs) contribute to strength loss following lengthening contractions in wild‐type and in dystrophic skeletal muscle. •  NMJs in dystrophic (mdx) mice, the murine model of Duchenne muscular dystrophy, show discontinuous and dispersed motor end‐plate morphology. Following lengthening contractions, mdx quadriceps muscles show a greater loss in force, increased neuromuscular transmission failure and decreased electromyographic measures compared to wild‐type. •  Consistent with NMJ disruption as a mechanism contributing to this force loss, only mdx showed increased motor end‐plate discontinuity and dispersion of acetylcholine receptor aggregates. •  Our results indicate that the NMJ in mdx muscle is particularly susceptible to damage, and might play a role in the exacerbated response to injury in dystrophic muscles.

Phosphorylation of Highly Conserved Neurofilament Medium KSP Repeats Is Not Required for Myelin-Dependent Radial Axonal Growth

Neurofilament medium (NF-M) is essential for the acquisition of normal axonal caliber in response to a myelin-dependent “outside-in” trigger for radial axonal growth. Removal of the tail domain and lysine-serine-proline (KSP) repeats of NF-M, but not neurofilament heavy, produced axons with impaired radial growth and reduced conduction velocities. These earlier findings supported myelin-dependent phosphorylation of NF-M KSP repeats as an essential component of axonal growth. As a direct test of whether phosphorylation of NF-M KSP repeats is the target for the myelin-derived signal, gene replacement has now been used to produce mice in which all serines of NF-Ms KSP repeats have been replaced with phosphorylation-incompetent alanines. This substitution did not alter accumulation of the neurofilaments or their subunits. Axonal caliber and motor neuron conduction velocity of mice expressing KSP phospho-incompetent NF-M were also indistinguishable from wild-type mice. Thus, phosphorylation of NF-M KSP repeats is not an essential component for the acquisition of normal axonal caliber mediated by myelin-dependent outside-in signaling.

Pathophysiology Associated with Traumatic Brain Injury: Current Treatments and Potential Novel Therapeutics

Traumatic brain injury (TBI) is one of the leading causes of death of young people in the developed world. In the United States alone, 1.7 million traumatic events occur annually accounting for 50,000 deaths. The etiology of TBI includes traffic accidents, falls, gunshot wounds, sports, and combat-related events. TBI severity ranges from mild to severe. TBI can induce subtle changes in molecular signaling, alterations in cellular structure and function, and/or primary tissue injury, such as contusion, hemorrhage, and diffuse axonal injury. TBI results in blood–brain barrier (BBB) damage and leakage, which allows for increased extravasation of immune cells (i.e., increased neuroinflammation). BBB dysfunction and impaired homeostasis contribute to secondary injury that occurs from hours to days to months after the initial trauma. This delayed nature of the secondary injury suggests a potential therapeutic window. The focus of this article is on the (1) pathophysiology of TBI and (2) potential therapies that include biologics (stem cells, gene therapy, peptides), pharmacological (anti-inflammatory, antiepileptic, progrowth), and noninvasive (exercise, transcranial magnetic stimulation). In final, the review briefly discusses membrane/lipid rafts (MLR) and the MLR-associated protein caveolin (Cav). Interventions that increase Cav-1, MLR formation, and MLR recruitment of growth-promoting signaling components may augment the efficacy of pharmacologic agents or already existing endogenous neurotransmitters and neurotrophins that converge upon progrowth signaling cascades resulting in improved neuronal function after injury.

Implications of tensile loading for the tissue engineering of nerves.

Nerves and neurons undergo tensile loading, or stretch, in many scenarios, including development and growth, normal joint movement, nerve injury and disease, and orthopedic surgery. The response of the nervous system to such loading is modulated by the conditions of loading. Within an empirically determined range of strains and strain rates, tensile loading holds the potential to accelerate axonal growth. On the other hand, exceeding these limits can damage the nerve, resulting in the dysfunction of sensory and motor systems. Understanding and pushing the limits of nerve stretch holds tremendous potential for tissue engineering efforts to prevent nervous system injury and facilitate nerve repair. This review aims to elucidate the phenomenon of nerve stretch in the peripheral nervous system and in the spinal cord. At the tissue level, we summarize the biomechanical, structural, and functional responses of nerves to tensile loading, in vitro, in situ, and in vivo. Further, we identify a range of strains and strain rates at which the nervous response transits from a regime of growth to injury. At the cell level, we assess the structural and functional plasticity of the neuron under tensile loading conditions that promote growth. We also review extrinsic factors that modulate cellular processes underlying neuronal growth. We propose that these pathways may be exploited during tensile loading to promote axonal growth. Finally, we review recent efforts that examine the tensile loading of nerves in the context of clinical problems such as limb lengthening surgeries and nerve regeneration.

Examination of potential mechanisms of amyloid-induced defects in neuronal transport

Microtubule-based neuronal transport pathways are impaired during the progression of Alzheimers disease and other neurodegenerative conditions. However, mechanisms leading to defects in transport remain to be determined. We quantified morphological changes in neuronal cells following treatment with fibrils and unaggregated peptides of beta-amyloid (Abeta). Abeta fibrils induce axonal and dendritic swellings indicative of impaired transport. In contrast, Abeta peptides induce a necrotic phenotype in both neurons and non-neuronal cells. We tested several popular hypotheses by which aggregated Abeta could disrupt transport. Using fluorescent polystyrene beads, we developed experimental models of physical blockage and localized release of reactive oxygen species (ROS) that reliably induce swellings. Like the beads, Abeta fibrils localize in close proximity to swellings; however, fibril internalization is not required for disrupting transport. ROS and membrane permeability are also unlikely to be responsible for fibril-mediated toxicity. Collectively, our results indicate that multiple initiating factors converge upon pathways of defective transport.