Impaired peripheral nerve regeneration in type‐2 diabetic mouse model

Peripheral neuropathy is one of the most common and serious complications of type‐2 diabetes. Diabetic neuropathy is characterized by a distal symmetrical sensorimotor polyneuropathy, and its incidence increases in patients 40 years of age or older. In spite of extensive research over decades, there are few effective treatments for diabetic neuropathy besides glucose control and improved lifestyle. The earliest changes in diabetic neuropathy occur in sensory nerve fibers, with initial degeneration and regeneration resulting in pain. To seek its effective treatment, here we prepared a type‐2 diabetic mouse model by giving mice 2 injections of streptozotocin and nicotinamide and examining the ability for nerve regeneration by using a sciatic nerve transection‐regeneration model previously established by us. Seventeen weeks after the last injection, the mice exhibited symptoms of type‐2 diabetes, that is, impaired glucose tolerance, decreased insulin level, mechanical hyperalgesia, and impaired sensory nerve fibers in the plantar skin. These mice showed delayed functional recovery and nerve regeneration by 2 weeks compared with young healthy mice and by 1 week compared with age‐matched non‐diabetic mice after axotomy. Furthermore, type‐2 diabetic mice displayed increased expression of PTEN in their DRG neurons. Administration of a PTEN inhibitor at the cutting site of the nerve for 4 weeks promoted the axonal transport and functional recovery remarkably. This study demonstrates that peripheral nerve regeneration was impaired in type‐2 diabetic model and that its combination with sciatic nerve transection is suitable for the study of the pathogenesis and treatment of early diabetic neuropathy.     

Three-dimensional organisation of mitochondrial clusters in regenerating dorsal root ganglion (DRG) neurons from neonatal rats: evidence for mobile mitochondrial pools

Abstract   We report for the first time the rearrangement of mitochondrial arrays in developing dorsal root ganglion (DRG) neurons isolated from neonatal rats in culture. Neurons were loaded with the mitochondria-specific fluorescent dye JC-1, and three-dimensional (3D) reconstruction of mitochondrial fluorescence was performed by confocal laser sectioning in fresh neurons and neurons kept in culture up to a week. We found that after 24 hours the mitochondria become reorganised to form clusters in the axonal hillocks. Axonal extension and neuronal network formation coincided with a redistribution of the mitochondrial clusters. In the extended axons the mitochondria become spaced along the axonal length; however, they formed clusters in the branch points and growth cones. We conclude that the initial clusters of mitochondria may be storage pools of mobile mitochondria able to be mobilised to provide energy for axonal transport during neuronal regeneration and neuronal outgrowth. These findings may have relevance to the rate of axonal regeneration and axonal transport in adult DRG neurons, and neuronal polarisation and axonal outgrowth regulation in developing DRG neurons.     

Mitochondria in DRG neurons undergo hyperglycemic mediated injury through Bim, Bax and the fission protein Drp1

Dorsal root ganglia (DRG) neurons degenerate in diabetic neuropathy (DN) and exhibit mitochondrial damage. We studied mitochondria of cultured DRG neurons exposed to high glucose as an in vitro model of DN. High glucose sequentially increases the expression, activation and localization of the pro-apoptotic proteins Bim and Bax and the mitochondrial fission protein dynamin-regulated protein 1 (Drp1). High glucose causes association of Drp1/Bax, similar to other apoptotic stimuli. Collectively, these events promote mitochondrial fragmentation and reduce mitochondrial number, suggestive of apoptotic mitochondrial fission. Drp1 is also upregulated in DRG from experimentally diabetic rats, suggesting a role for mitochondrial fission in DN. Insulin-like growth factor-I (IGF-I) protects high glucose-treated DRG neurons by preventing mitochondrial accumulation of Bim and Bax but does not modulate Drp1 expression or localization. We propose that mitochondria are compromised by convergence of Bim/Bax proteins with Drp1, which contributes to high glucose-induced injury in DRG neurons.     

Absence of SOD1 leads to oxidative stress in peripheral nerve and causes a progressive distal motor axonopathy

Oxidative stress is commonly implicated in the pathogenesis of motor neuron disease. However, the cause and effect relationship between oxidative stress and motor neuron degeneration is poorly defined. We recently identified denervation at the neuromuscular junction in mice lacking the antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD1) (Fischer et al., 2011). These mice show a phenotype of progressive muscle atrophy and weakness in the setting of chronic oxidative stress. Here, we investigated further the extent of motor neuron pathology in this model, and the relationship between motor pathology and oxidative stress. We report preferential denervation of fast-twitch muscles beginning between 1 and 4 months of age, with relative sparing of slow-twitch muscle. Motor axon terminals in affected muscles show widespread sprouting and formation of large axonal swellings. We confirmed, as was previously reported, that spinal motor neurons and motor and sensory nerve roots in these mice are preserved, even out to 18 months of age. We also found preservation of distal sensory fibers in the epidermis, illustrating the specificity of pathology in this model for distal motor axons. Using HPLC measurement of the glutathione redox potential, we quantified oxidative stress in peripheral nerve and muscle at the onset of denervation. SOD1 knockout tibial nerve, but not gastrocnemius muscle, showed significant oxidation of the glutathione pool, suggesting that axonal degeneration is a consequence of impaired redox homeostasis in peripheral nerve. We conclude that the SOD1 knockout mouse is a model of oxidative stress-mediated motor axonopathy. Pathology in this model primarily affects motor axon terminals at the neuromuscular junction, demonstrating the vulnerability of this synapse to oxidative injury.     

DNA breaks detected by in situ end-labelling in dorsal root ganglia of patients with AIDS

Distal sensory axonal polyneuropathy (DSP) is the most frequent HIV-associated peripheral neuropathy. DSPs tend to occur in full-blown AIDS and worsen as CD4 cell counts decrease in blood. To assess a possible role for apoptosis in the pathogenesis of the neuropathy, we used in situ end-labelling (ISEL) detecting DNA strand breaks in DRG neurons of 19 HIV-infected patients, of whom nine had axonal polyneuropathy, and 11 controls. Sensory neurons with ISEL-assessed DNA breaks were observed in 9/19 patients with AIDS, 0/3 patients with pre-AIDS, and 1/11 controls. The prevalence of DNA breaks in neurons was higher in AIDS patients than in controls ( P <0.05). Among AIDS patients, DNA breaks in neurons were more abundant in patients with peripheral neuropathy ( P <0.04). It is possible that DNA breaks of DRG neurons induce the axonopathy and consequently play a role in the pathogenesis of DSP. It cannot be excluded, however, that DNA breaks could represent the result rather than the cause of axonopathy. We suggest that ISEL may detect neurons that were primed to apoptosis before death in relation with the HIV infection, and undergo DNA fragmentation at time of death, rather than neurons that underwent pre-mortem both priming and triggering steps of the apoptotic process. This hypothesis could explain why most ISEL-positive neurons lack typical apoptotic morphology and why normal controls do not show ISEL positive cells.     

Excitotoxicity mediated by non-NMDA receptors causes distal axonopathy in long-term cultured spinal motor neurons

Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long-term culture. Excitotoxicity, mediated principally via non-N-methyl-D-aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.     

Mutant HSPB1 overexpression in neurons is sufficient to cause age-related motor neuronopathy in mice

The small heat shock protein HSPB1 is a multifunctional, α-crystallin-based protein that has been shown to be neuroprotective in animal models of motor neuron disease and peripheral nerve injury. Missense mutations in HSPB1 result in axonal Charcot-Marie-Tooth disease with minimal sensory involvement (CMT2F) and distal hereditary motor neuropathy type 2 (dHMN-II). These disorders are characterized by a selective loss of motor axons in peripheral nerve resulting in distal muscle weakness and often severe disability. To investigate the pathogenic mechanisms of HSPB1 mutations in motor neurons in vivo, we have developed and characterized transgenic PrP-HSPB1 and PrP-HSPB1(R136W) mice. These mice express the human HSPB1 protein throughout the nervous system including in axons of peripheral nerve. Although both mouse strains lacked obvious motor deficits, the PrP-HSPB1(R136W) mice developed an age-dependent motor axonopathy. Mutant mice showed axonal pathology in spinal cord and peripheral nerve with evidence of impaired neurofilament cytoskeleton, associated with organelle accumulation. Accompanying these findings, increases in the number of Schmidt-Lanterman incisures, as evidence of impaired axon-Schwann cell interactions, were present. These observations suggest that overexpression of HSPB1(R136W) in neurons is sufficient to cause pathological and electrophysiological changes in mice that are seen in patients with hereditary motor neuropathy.     

The pathology of a sensory neuropathy affecting Long Haired Dachshund dogs

Summary The microscopical findings in a sensory neuropathy affecting Long Haired Dachshund dogs are described. This disorder, which may have a genetic basis, results in loss of proprioception, touch and sense and reduction or loss of nociception and urinary control. In the distal cutaneous nerves, there was a severe loss of large myelinated fibers but unmyelinated fiber density was not reduced. Many of the remaining myelinated fibers contained accumulations of axonal organelles and teased fibers showed that some were degenerating whilst others had successive areas of paranodal demyelination. There was marked pathology of unmyelinated fibers with proliferation of tubulo-vesicular profiles resulting in the formation of stacks or lamellar arrays and darkening of the axoplasm. There were numerous regenerating unmyelinated axons. In the spinal cord, axonal degeneration was scen in the distal portion of the dorsal columns.The clinical signs in this distal axonopathy were therefore compatible with the widespread sensory nerve pathology which was found. It is concluded that this neuropathy resulted from progressive nerve degeneration and comparisons between this neuropathy and other sensory neuropathies in animals and man are made.Supported by grants from the Medical Research Council of Canada, the Conseil de Recherche en santé du Québec and the Wellcome Trust     

A proposal for a classification of neuropathies according to their axonal transport abnormalities.

Recent studies on axonal transport in experimental neuropathy are reviewed and the following combinations of pathological changes and underlying axonal transport abnormalities are proposed for a classification of polyneuropathies. Alterations of the anterograde transport of slow component a(SCa) leads to changes of the dimensions of the axon calibre without the occurrence either of overt neuropathy or fibre loss. Thus damming of SCa in beta,beta'-iminodiproprionitrile (IDPN) intoxication results in axonal swelling in nerve roots whereas decrease of SCa leads to atrophy distal to the swellings in IDPN intoxication and in streptozotocin induced diabetes as well. Decrease in the amount of material conveyed within the anterograde fast component (aFC) leads to acute axonal degeneration including break down of axons and fibre loss. This state occurs in acute hypoglycaemia and in doxorubicin intoxication. The most frequent type of polyneuropathy, namely distal axonopathy with accumulation of axon organelles leading to distal fibre loss, is associated with decrease in amount of the retrograde fast component (rFC). The transport is impaired before the appearance of symptoms and electrophysiological signs of neuropathy develop in the intoxications induced by parabromophenylacetylurea, acrylamide and 2.5 hexanedione, and the severity of neuropathy is proportional to the rFC impairment.     

Neurotrophin-3 prevents mitochondrial dysfunction in sensory neurons of streptozotocin-diabetic rats

Sensory neurons from streptozotocin (STZ)-diabetic rats exhibit depolarization of mitochondria and the related induction of reactive oxygen species has been proposed to contribute to the etiology of sensory polyneuropathy in diabetes. There is deficient neurotrophin-3 (NT-3)-dependent neurotrophic support of sensory neurons in diabetes and treatment of STZ-diabetic rats with NT-3 prevents neuropathological alterations in peripheral nerve. Therefore, we hypothesized that loss of NT-3 may contribute to mitochondrial dysfunction in sensory neurons in diabetic sensory neuropathy. The specific aim of this study was to determine whether treatment of STZ-diabetic rats with systemic NT-3 could prevent depolarization of the mitochondrial inner membrane potential (Deltapsi(m)). In vitro studies with cultured DRG neurons from control rats revealed that treatment with 50 ng/ml NT-3 for 6 h enhanced the Deltapsi(m), e.g., a higher polarized membrane potential, compared to untreated neurons (P < 0.05). Studies on DRG sensory neurons from control vs. STZ-diabetic rats demonstrated that NT-3 therapy prevented the diabetes-induced depolarization of Deltapsi(m) (P < 0.05) in parallel with normalization of diabetes-dependent deficits in sensory nerve conduction velocity. Furthermore, alterations in mitochondrial function in vitro and in vivo correlated with the level of activation/expression of Akt in DRG neurons.     

Involvement of alpha7beta1 integrin in the conditioning-lesion effect on sensory axon regeneration

Conditioning lesions of peripheral nerves improve axonal regeneration after injury and involve changes in expression of proteins required for axonal growth. Integrin alpha7beta1 expression in motor and sensory neurons increases following nerve lesions and motor axon regeneration is impaired in alpha7 integrin KO mice (J. Neurosci. 20, 1822-1830). To investigate the role of alpha7beta1 integrin in sensory axon regeneration, dorsal root ganglia of adult mice were cultured in gels of laminin-rich extracellular matrix (Matrigel) or collagen. Normal dorsal root ganglia in Matrigel or collagen supplemented with laminin showed spontaneous axonal outgrowth, which was greatly increased in conditioned preparations, but only in the presence of laminin. Conditioned dorsal root ganglia from normal mice cultured with a blocking antibody to beta1 integrin and from alpha7 integrin KO mice showed reduced axonal growth in both Matrigel- and laminin-supplemented collagen gels. Enhanced axonal regeneration after conditioning lesions therefore involves increased responsiveness to laminin and integrin alpha7beta1 expression.     

Differential effects of HIV infected macrophages on dorsal root ganglia neurons and axons

Human immunodeficiency virus-associated distal-symmetric neuropathy (HIV-DSP) is the most common neurological complication of HIV infection. The pathophysiology of HIV-DSP is poorly understood and no treatment is available for this entity. The dorsal root ganglia (DRG) are the principal sites of neuronal damage and are associated with reactive mononuclear phagocytes as well as HIV-infected macrophages. To determine the role of HIV-infected macrophages in the pathogenesis of HIV-DSP, we developed a technique for culturing human DRG's. When the dissociated DRG neurons were exposed to supernatants from macrophages infected with CXCR4 or CCR5 tropic HIV-1 strains axonal retraction was observed without neuronal cell death but there was mitochondrial dysfunction in the neuronal cell body. Even though CXCR4 and CCR5 were expressed on the DRG neurons, the effects were independent of these receptors. Antioxidants rescued the neuronal cell body but not the axon from the toxic effects of the culture supernatants. Further, peripheral nerves of HIV-infected patients obtained at autopsy did not show evidence of increased oxidative stress. These observations suggest a differential effect on the axon and cell body. Different mechanisms of injury may be operative in these two structures.     

Distal neuropathy in spontaneous diabetes mellitus in the dog

Summary Peripheral nerves of three mature dogs with chronic spontaneous diabetes mellitus were examined using qualitative and quantitative techniques. The principal pathologic findings in single-teased fibers were remyelination and axonal regeneration. It is suggested that these changes which occurred in distal plantar nerves but not in more proximal tibial nerves result from a distal axonopathy. The dog may prove to be another useful spontaneous model of diabetic neuropathy.Supported by funds from Scott-Ritchey Research Program, School of Veterinary Medicine, Auburn University     

Primary sensory neurons in x-linked recessive bulbospinal neuronopathy: Histopathology and androgen receptor gene expression

Pathology of the primary sensory neurons was examined in 7 autopsied patients and 6 biopsied sural nerves from the patients with X-linked recessive bulbospinal neuronopathy (SBMA). Large myelinated fibers in the central rami (L-4 posterior root, L-4, T-7, and C-6 segment of the fasciculus gracilis), and in the peripheral rami (sural nerve) were diminished in a distally accentuated manner, while small myelinated and unmyelinated fibers were well preserved in number. Demyelinating process and axonal atrophy was ubiquitous. The diameter frequency histograms of the dorsal root ganglion (DRG) neurons showed a decrease in the number of large diameter neurons and an increase in the number of small diameter neurons without substantial loss of whole number of neurons, which suggested that neuronal size was atrophied. These data suggested central and peripheral distal axonopathy with neuronal atrophy was the process of sensory neuron involvement. Expression of mutant androgen receptor mRNA with elongated CAG repeat in the DRG and sural nerve supported the view that sensory nerve involvement is the primary process in SBMA.© 1995 John Wiley &Sons, Inc.     

Altered tubulin and neurofilament expression and impaired axonal growth in diabetic nerve regeneration

Cytoskeletal protein expression in sensory neurons and sciatic nerve axonal growth were examined in type 1 diabetic BB/Wor rats after sciatic nerve crush injury. Diabetic male rats were subjected to sciatic nerve crush at 6 wk of diabetes. L4 and L5 dorsal root ganglia (DRG) mRNA expression of low and medium molecular weight neurofilaments (NF-L, NF-M), betaII- and betaIII-tubulin as well as protein expression of NF-L, NF-M, and beta-tubulin were examined at various time points following crush injury and compared with age- and sex-matched non-diabetic BB/Wor rats. Steady state mRNA expression of NF-L, NF-M, betaII- and betaIII-tubulin were decreased in diabetic DRG. NF-L and NF-M proteins were also decreased in DRG of uncrushed diabetic animals. After crush injury, betaII- and betaIII-tubulin mRNA were upregulated in control animals at day 2 and day 6, respectively, and beta-tubulin protein showed similarly increased expression after crush injury, while such upregulations did not occur in diabetic animals. Conversely, mRNA and protein expressions of NF-L, NF-M were downregulated to a lesser extent in diabetic animals compared to control rats. These changes were associated with impaired axonal elongation and caliber growth of regenerating fibers in diabetic rats. We propose that upregulation of tubulin has a negative feedback on NF expression in response to nerve injury, as seen in control rats. The absence of this upregulation in diabetic animals may impair its regulatory effect on NF expression and contribute to perturbed nerve regeneration seen in diabetic nerve.     

Role of the peripheral benzodiazepine receptor in sensory neuron regeneration

Peripheral benzodiazepine receptor (PBR) expression increases in small dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury. To determine the functional significance of this induction, we evaluated the effects of PBR ligands on rodent sensory axon outgrowth. In vitro, Ro5-4864, a PBR agonist, enhanced outgrowth only of small peripherin-positive DRG neurons. When DRG cells were preconditioned into an active growth state by a prior peripheral nerve injury Ro5-4864 augmented and PK 11195, a PBR antagonist, blocked the injury-induced increased outgrowth. In vivo, Ro5-4864 increased the initiation of regeneration after a sciatic nerve crush injury and the number of GAP-43-positive axons in the distal nerve while PK 11195 inhibited the enhanced growth produced by a preconditioning lesion. These results show that PBR has a role in the early regenerative response of small caliber sensory axons, the preconditioning effect, and that PBR agonists enhance sensory axon regeneration.     

Axonal regeneration and sprouting as a potential therapeutic target for nervous system disorders

Nervous system disorders are prevalent health issues that will only continue to increase in frequency as the population ages.Dying-back axonopathy is a hallmark of many neurologic diseases and leads to axonal disconnection from their targets,which in turn leads to functional impairment.During the course of many of neurologic diseases,axons can regenerate or sprout in an attempt to reconnect with the target and restore synapse function.In amyotrophic lateral sclerosis(ALS),distal motor axons retract from neuromuscular junctions early in the disease-course before significant motor neuron death.There is evidence of compensatory motor axon sprouting and reinnervation of neuromuscular junctions in ALS that is usually quickly overtaken by the disease course.Potential drugs that enhance compensatory sprouting and encourage reinnervation may slow symptom progression and retain muscle function for a longer period of time in ALS and in other diseases that exhibit dying-back axonopathy.There remain many outstanding questions as to the impact of distinct disease-causing mutations on axonal outgrowth and regeneration,especially in regards to motor neurons derived from patient induced pluripotent stem cells.Compartmentalized microfluidic chambers are powerful tools for studying the distal axons of human induced pluripotent stem cells-derived motor neurons,and have recently been used to demonstrate striking regeneration defects in human motor neurons harboring ALS disease-causing mutations.Modeling the human neuromuscular circuit with human induced pluripotent stem cells-derived motor neurons will be critical for developing drugs that enhance axonal regeneration,sprouting,and reinnervation of neuromuscular junctions.In this review we will discuss compensatory axonal sprouting as a potential therapeutic target for ALS,and the use of compartmentalized microfluidic devices to find drugs that enhance regeneration and axonal sprouting of motor axons.     

Apoptosis and impaired axonal regeneration of sensory neurons after nerve crush in diabetic rats

We investigated the possible induction of apoptosis of dorsal root ganglion (DRG) neurons and the defect of nerve regeneration after crush injury with reference to the JNK/c-jun and cAMP pathway in streptozocin-induced diabetic rats. In addition, the effects of a PGE1 analogue were tested in diabetic rats. At day 0 (before axonal injury), no TUNEL-positive DRG neurons were observed in any group. From day 1 to 7 after axonal injury, TUNEL-positive DRG neurons were seen in diabetic rats, but not in non-diabetic or PGE1-treated diabetic rats. The regeneration distance at day 7 after crush injury was shorter in diabetic rats than in the other groups of rats. The time course of JNK/c-jun phosphorylation did not parallel apoptosis. At day 7, the cAMP content of DRG was higher than that at day 0 in non-diabetic and PGE1-treated rats, whereas it was not increased after 7 days in diabetic rats. These results indicate that in diabetic rats apoptosis of DRG neurons is induced by axonal injury independently of the JNK/c-jun and cAMP pathway and that PGE1 rescues DRG neurons from apoptosis and improves axonal regeneration in diabetic rats.     

Amelioration of retarded neurite outgrowth of dorsal root ganglion neurons by overexpression of PKCdelta in diabetic rats

To examine which isoform of protein kinase C (PKC) may be associated with impaired nerve regeneration in diabetes, we compared neurite outgrowth of isolated dorsal root ganglion (DRG) neurons in streptozocin (STZ)-induced diabetic and control rats. Neurite outgrowth was significantly retarded in diabetic neurons. Rottlerin, a PKCdelta specific inhibitor, significantly retracted neurite outgrowth whereas G?6976, an inhibitor specific for classical PKCs, had no effect, suggesting a significant role of PKCdelta in neurite outgrowth of DRG neurons. The expression of phosphorylated PKCdelta, but not total PKCdelta, in DRGs was decreased in diabetic rats. When this reduced expression was restored by overexpressing the PKCdelta in isolated DRG neurons, retardation of neurite outgrowth was significantly reversed in diabetic rats. These results suggest that a decrease in phosphorylated PKCdelta is at least in part responsible for impaired neurite outgrowth in diabetes, and that PKCdelta plays a significant role in the pathogenesis of diabetic neuropathy. This observation provides a useful clue for the treatment of diabetic neuropathy.