Ahmet Hoke

Injury-Induced Proteoglycans Inhibit the Potential for Laminin-Mediated Axon Growth on Astrocytic Scars

Following injury to the adult CNS, the expression of a number of extracellular matrix molecules increases in regions of reactive gliosis. This glial matrix includes certain chondroitin sulfate proteoglycans (CS-PGs) which have been correlated with an inhibition of axon outgrowth. In order to test the influence of glial associated CS-PGs on neurite elongation directly, we sought to determine whether enzymatic modification of injury-induced CS-PGs could enhance neurite outgrowth across the surface of intact glial scars formed in vivo after implanting nitrocellulose filters into the cortex of adult rats. This gliotic tissue was subsequently explanted in vitro and used as a substrate for growing embryonic retinal neurons. Treatment of adult explants with chondroitinase ABC led to a significant increase in mean neurite length over the scar surface. Heparitinase treatment caused a much smaller, although significant, increase in neurite outgrowth. This suggested that more than one type of PG was present or that a single PG with both CS and HS side chains was upregulated. Western analysis revealed that a PG(s) with a core protein between 180 and 400 kDa was found to be relatively more abundant in areas of reactive gliosis induced to form in adult rather than neonatal animals. Simultaneous treatment of adult glial scars with chondroitinase and antibodies to the beta 1, beta 2 chain of laminin partially reversed the growth-enhancing effect of enzymatic digestion alone. These data demonstrate that the increase in neurite outgrowth along the surface of reactive astrocytes following enzymatic modification of injury-induced PGs was due, in part, to the presence of laminin. Thus, in this model of gliosis, particular PGs may act as inhibitors of neurite outgrowth by attenuating the potential for axon elongation that could occur due to the concomitant expression of growth-promoting molecules in regions of reactive gliosis.

Schwann Cells Express Motor and Sensory Phenotypes That Regulate Axon Regeneration

Schwann cell phenotype is classified as either myelinating or nonmyelinating. Additional phenotypic specialization is suggested, however, by the preferential reinnervation of muscle pathways by motoneurons. To explore potential differences in growth factor expression between sensory and motor nerve, grafts of cutaneous nerve or ventral root were denervated, reinnervated with cutaneous axons, or reinnervated with motor axons. Competitive reverse transcription-PCR was performed on normal cutaneous nerve and ventral root and on graft preparations 5, 15, and 30 d after surgery. mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 was expressed vigorously by denervated and reinnervated cutaneous nerve but minimally by ventral root. In contrast, mRNA for pleiotrophin (PTN) and glial cell line-derived neurotrophic factor was upregulated to a greater degree in ventral root. ELISA confirmed that NGF and BDNF protein were significantly more abundant in denervated cutaneous nerve than in denervated ventral root, but that PTN protein was more abundant in denervated ventral root. The motor phenotype was not immutable and could be modified toward the sensory phenotype by prolonged reinnervation of ventral root by cutaneous axons. Retrograde labeling to quantify regenerating neurons demonstrated that cutaneous nerve preferentially supported cutaneous axon regeneration, whereas ventral root preferentially supported motor axon regeneration. Schwann cells thus express distinct sensory and motor phenotypes that are associated with the support of regeneration in a phenotype-specific manner. These findings suggest that current techniques of bridging gaps in motor and mixed nerve with cutaneous graft could be improved by matching axon and Schwann cell properties.

Recovery from paralysis in adult rats using embryonic stem cells

We explored the potential of embryonic stem cell–derived motor neurons to functionally replace those cells destroyed in paralyzed adult rats.

A framework for diagnosing and classifying intensive care unit-acquired weakness

Neuromuscular dysfunction is prevalent in critically ill patients, is associated with worse short-term outcomes, and is a determinant of long-term disability in intensive care unit survivors. Diagnosis is made with the help of clinical, electrophysiological, and morphological observations; however, the lack of a consistent nomenclature remains a barrier to research. We propose a simple framework for diagnosing and classifying neuromuscular disorders acquired in critical illness.

Mechanisms of Disease: what factors limit the success of peripheral nerve regeneration in humans?

Functional recovery after repair of peripheral nerve injury in humans is often suboptimal. Over the past quarter of a century, there have been significant advances in human nerve repair, but most of the developments have been in the optimization of surgical techniques. Despite extensive research, there are no current therapies directed at the molecular mechanisms of nerve regeneration. Multiple interventions have been shown to improve nerve regeneration in small animal models, but have not yet translated into clinical therapies for human nerve injuries. In many rodent models, regeneration occurs over relatively short distances, so the duration of denervation is short. By contrast, in humans, nerves often have to regrow over long distances, and the distal portion of the nerve progressively loses its ability to support regeneration during this process. This can be largely attributed to atrophy of Schwann cells and loss of a Schwann cell basal lamina tube, which results in an extracellular environment that is inhibitory to nerve regeneration. To develop successful molecular therapies for nerve regeneration, we need to generate animal models that can be used to address the following issues: improving the intrinsic ability of neurons to regenerate to increase the speed of axonal outgrowth; preventing loss of basal lamina and chronic denervation changes in the denervated Schwann cells; and overcoming inhibitory cues in the extracellular matrix.

A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation.

In the peripheral nervous system, regeneration of motor and sensory axons into chronically denervated distal nerve segments is impaired compared to regeneration into acutely denervated nerves. In order to find possible causes for this phenomenon we examined the changes in the expression pattern of the glial cell-line-derived neurotrophic factor (GDNF) family of growth factors and their receptors in chronically denervated rat sciatic nerves as a function of time with or without regeneration. Among the GDNF family of growth factors, only GDNF mRNA expression was rapidly upregulated in Schwann cells as early as 48 h after denervation. This upregulation peaked at 1 week and then declined to minimal levels by 6 months of denervation. The changes in the protein expression paralleled the changes in the expression of the GDNF mRNA. The mRNAs for receptors GFRalpha-1 and GFRalpha-2 were upregulated only after maximal GDNF upregulation and remained elevated as late as 6 months. There were no significant changes in the expression of GFRalpha-3 or the tyrosine kinase coreceptor, RET. When we examined the expression of GDNF in a delayed regeneration paradigm, there was no upregulation in the distal chronically denervated tibial nerve even when the freshly axotomized peroneal branch of the sciatic nerve was sutured to the distal tibial nerve. This study suggests that one of the reasons for impaired regeneration into chronically denervated peripheral nerves may be the inability of Schwann cells to maintain important trophic support for both motor and sensory neurons.

HIV-associated sensory neuropathies

Peripheral neuropathy has emerged as the most common neurological complication of HIV infection [1– 4]. There are several discrete types of HIV-associated neuropathy, which can be classified according to the timing of their appearance during HIV infection, their etiology and whether they are primarily axonal or demyelinating (Table 1). Some represent a consequence of HIV infection producing neuropathological damage [e.g., distal symmetrical polyneuropathy (DSP)], while others are related to opportunistic pathogens [e.g., cytomegalovirus (CMV) polyradiculitis]. An increasingly common group is that which occurs as a result of treatment toxicity [e.g., toxic neuropathy from antiretroviral drugs (TNA) and lactic acidosis syndrome].

Advances in peripheral nerve regeneration

Rodent models of nerve injury have increased our understanding of peripheral nerve regeneration, but clinical applications have been scarce, partly because such models do not adequately recapitulate the situation in humans. In human injuries, axons are often required to extend over much longer distances than in mice, and injury leaves distal nerve fibres and target tissues without axonal contact for extended amounts of time. Distal Schwann cells undergo atrophy owing to the lack of contact with proximal neurons, which results in reduced expression of neurotrophic growth factors, changes in the extracellular matrix and loss of Schwann cell basal lamina, all of which hamper axonal extension. Furthermore, atrophy and denervation-related changes in target tissues make good functional recovery difficult to achieve even when axons regenerate all the way to the target tissue. To improve functional outcomes in humans, strategies to increase the speed of axonal growth, maintain Schwann cells in a healthy, repair-capable state and keep target tissues receptive to reinnervation are needed. Use of rodent models of chronic denervation will facilitate our understanding of the molecular mechanisms of peripheral nerve regeneration and create the potential to test therapeutic advances.

Transplanted neural stem cells promote axonal regeneration through chronically denervated peripheral nerves

Regeneration in the peripheral nervous system is impaired after prolonged periods of denervation. Currently, no interventions exist to alter the outcome after prolonged denervation. To examine the role of transplanted neural stem cells (NSC), we prepared chronically denervated distal tibial nerve segments. After 6 months of chronic denervation, we transplanted vehicle, C17.2 mouse NSCs, or C17.2 mouse NSCs engineered to overexpress GDNF to the distal tibial nerve and performed a peroneal nerve cross-suture. In animals transplanted with the NSCs, there was better regeneration of the peroneal axons into the tibial nerve as measured by counting the number of axons and by the emergence of compound motor action potentials in the tibial innervated foot muscles. Improved regeneration correlated with a reduction of chondroitin sulphate proteoglycan (CSPG) immunoreactivity in the extracellular matrix (ECM). In vitro, supernatant from C17.2 NSCs contained large quantities of secreted matrix metalloprotease-2 (MMP-2), degraded the CSPGs on chronically denervated tibial nerve sections, and reversed the CSPG-induced inhibition of neuritic outgrowth of DRG neurons. This reversal was inhibited by selective MMP-2 inhibitors. This is the first successful demonstration of regeneration through a chronically denervated nerve. These findings suggest that improved regeneration in the PNS can be accomplished by combining neurotrophic factor support and removal of axon growth inhibitory components in the extracellular matrix.

Motor Neuron Disease Occurring in a Mutant Dynactin Mouse Model Is Characterized by Defects in Vesicular Trafficking

Amyotrophic lateral sclerosis (ALS), a fatal and progressive neurodegenerative disorder characterized by weakness, muscle atrophy, and spasticity, is the most common adult-onset motor neuron disease. Although the majority of ALS cases are sporadic, ∼5–10% are familial, including those linked to mutations in SOD1 (Cu/Zn superoxide dismutase). Missense mutations in a dynactin gene (DCTN1) encoding the p150Glued subunit of dynactin have been linked to both familial and sporadic ALS. To determine the molecular mechanism whereby mutant dynactin p150Glued causes selective degeneration of motor neurons, we generated and characterized mice expressing either wild-type or mutant human dynactin p150Glued. Neuronal expression of mutant, but not wild type, dynactin p150Glued causes motor neuron disease in these animals that are characterized by defects in vesicular transport in cell bodies of motor neurons, axonal swelling and axo-terminal degeneration. Importantly, we provide evidence that autophagic cell death is implicated in the pathogenesis of mutant p150Glued mice. This novel mouse model will be instrumental for not only clarifying disease mechanisms in ALS, but also for testing therapeutic strategies to ameliorate this devastating disease.