Hao Chiang

Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons

Significance Degeneration of demyelinated axons is a major cause of permanent neurological disability in primary diseases of myelin. In addition to myelin loss, dysfunction of axonal mitochondria may be an essential requisite for degeneration of demyelinated axons. Here we describe a significant increase in axonal mitochondrial volume following demyelination of CNS axons. A deficiency in tethering mitochondria to the cytoskeleton prevented this volume increase in demyelinated axons and increased axonal degeneration, which was rescued by pharmacological inhibition of nerve transmission along the demyelinated axon. These results demonstrate that (i) increasing mitochondrial volume in CNS demyelinated axons protects against axonal degeneration and (ii) immobilization of mitochondria is required for the increase in axonal mitochondria volume following CNS demyelination. Axonal degeneration is a primary cause of permanent neurological disability in individuals with the CNS demyelinating disease multiple sclerosis. Dysfunction of axonal mitochondria and imbalanced energy demand and supply are implicated in degeneration of chronically demyelinated axons. The purpose of this study was to define the roles of mitochondrial volume and distribution in axonal degeneration following acute CNS demyelination. We show that the axonal mitochondrial volume increase following acute demyelination of WT CNS axons does not occur in demyelinated axons deficient in syntaphilin, an axonal molecule that immobilizes stationary mitochondria to microtubules. These findings were supported by time-lapse imaging of WT and syntaphilin-deficient axons in vitro. When demyelinated, axons deficient in syntaphilin degenerate at a significantly greater rate than WT axons, and this degeneration can be rescued by reducing axonal electrical activity with the Na+ channel blocker flecainide. These results support the concept that syntaphilin-mediated immobilization of mitochondria to microtubules is required for the volume increase of axonal mitochondria following acute demyelination and protects against axonal degeneration in the CNS.

Enhancement of cutaneous nerve regeneration by 4-methylcatechol in resiniferatoxin-induced neuropathy.

To generate an experimental neuropathy model in which small-diameter sensory nerves are specifically affected and to test a potential treatment, adult mice were given a single injection (50 &mgr;g/kg, i.p.) of the capsaicin analog resiniferatoxin (RTX). On Day 7 after RTX treatment, there was a 53% reduction in unmyelinated nerve density in the medial plantar nerve (p = 0.0067) and a 66% reduction in epidermal nerve density of hind paw skin (p = 0.0004) compared with vehicle-treated controls. Substance P-immunoreactive dorsal root ganglion neurons were also markedly depleted (p = 0.0001). These effects were associated with the functional deficit of prolonged withdrawal latencies to heat stimuli (p = 0.0007) on a hot plate test. The potential therapeutic effects of 4-methylcatechol (4MC) on this neuropathy were then tested by daily injections of 4MC (10 &mgr;g/kg, i.p.) from Days 7 to 35 after neuropathy induction. On Day 35, 4MC-treated mice had an increase in unmyelinated (p = 0.014) and epidermal nerve (p = 0.0013) densities and a reduction in thermal withdrawal latency (p = 0.0091) compared with RTX-only controls. These results indicate that 4MC promoted regeneration of unmyelinated nerves in experimental RTX-induced neuropathy and enhanced function.

P2X3-mediated peripheral sensitization of neuropathic pain in resiniferatoxin-induced neuropathy

Patients suffering from sensory neuropathy due to skin denervation frequently have paradoxical manifestations of reduced nociception and neuropathic pain. However, there is a lack of satisfactory animal models to investigate these phenomena and underlying mechanisms. We developed a mouse system of neuropathy induced by resiniferatoxin (RTX), a capsaicin analog, and examined the functional significance of P2X3 receptor in neuropathic pain. From day 7 of RTX neuropathy, mice displayed mechanical allodynia (p<0.0001) and thermal hypoalgesia (p<0.0001). After RTX treatment, dorsal root ganglion (DRG) neurons of the peripherin type were depleted (p=0.012), while neurofilament (+) DRG neurons were not affected (p=0.62). In addition, RTX caused a shift in neuronal profiles of DRG: (1) increased in P2X3 receptor (p=0.0002) and ATF3 (p=0.0006) but (2) reduced TRPV1 (p=0.036) and CGRP (p=0.015). The number of P2X3(+)/ATF3(+) neurons was linearly correlated with mechanical thresholds (p=0.0017). The peripheral expression of P2X3 receptor in dermal nerves was accordingly increased (p=0.016), and an intraplantar injection of the P2X3 antagonists, A-317491 and TNP-ATP, relieved mechanical allodynia in a dose-dependent manner. In conclusion, RTX-induced sensory neuropathy with upregulation of P2X3 receptor for peripheral sensitization of mechanical allodynia, which provides a new therapeutic target for neuropathic pain after skin denervation.

Sudomotor innervation in transthyretin amyloid neuropathy: Pathology and functional correlates

Autonomic neuropathy is a major component of familial amyloid polyneuropathy (FAP) due to mutated transthyretin, with sudomotor failure as a common manifestation. This study aimed to investigate the pathology and clinical significance of sudomotor denervation.

Role of Peptidergic Nerve Terminals in the Skin: Reversal of Thermal Sensation by Calcitonin Gene-Related Peptide in TRPV1-Depleted Neuropathy

To investigate the contribution of peptidergic intraepidermal nerve fibers (IENFs) to nociceptive responses after depletion of the thermal-sensitive receptor, transient receptor potential vanilloid subtype 1 (TRPV1), we took advantage of a resiniferatoxin (RTX)-induced neuropathy which specifically affected small-diameter dorsal root ganglion (DRG) neurons and their corresponding nerve terminals in the skin. Thermal hypoalgesia (p<0.001) developed from RTX-treatment day 7 (RTXd7) and became normalized from RTXd56 to RTXd84. Substance P (SP)(+) and TRPV1(+) neurons were completely depleted (p = 0.0001 and p<0.0001, respectively), but RTX had a relatively minor effect on calcitonin gene-related peptide (CGRP)(+) neurons (p = 0.029). Accordingly, SP(+) (p<0.0001) and TRPV1(+) (p = 0.0008) IENFs were permanently depleted, but CGRP(+) IENFs (p = 0.012) were only transiently reduced and had recovered by RTXd84 (p = 0.83). The different effects of RTX on peptidergic neurons were attributed to the higher co-localization ratio of TRPV1/SP than of TRPV1/CGRP (p = 0.029). Thermal hypoalgesia (p = 0.0018) reappeared with an intraplantar injection of botulinum toxin type A (botox), and the temporal course of withdrawal latencies in the hot-plate test paralleled the innervation of CGRP(+) IENFs (p = 0.0003) and CGRP contents in skin (p = 0.01). In summary, this study demonstrated the preferential effects of RTX on depletion of SP(+) IENFs which caused thermal hypoalgesia. In contrast, the skin was reinnervated by CGRP(+) IENFs, which resulted in a normalization of nociceptive functions.

Depletion of peptidergic innervation in the gastric mucosa of streptozotocin-induced diabetic rats

Autonomic neuropathy affecting the gastrointestinal system is a major presentation of diabetic neuropathy. Changes in the innervation of gastric mucosa or muscle layers can contribute to gastrointestinal symptoms. The present study investigated this issue by quantitatively analyzing the immunohistochemical patterns of the gastric innervation in rats with streptozotocin (STZ)-induced diabetes. In control rats, calcitonin gene-related peptide (CGRP) and substance P (SP) (+) nerve fibers appeared in the gastric mucosa and muscle layers. Double immunohistochemical staining showed that immunoreactivities for SP and CGRP were co-localized with a pan-neuronal marker protein gene product 9.5. Both SP (+) nerve fibers (p<0.001) and CGRP (+) nerve fibers (p<0.005) were decreased in the gastric mucosa within 4 weeks of diabetes; the reduction persisted throughout 24 weeks. Diabetic rats treated with insulin did not show decrease of SP or CGRP (+) fibers in the mucosa 4 weeks after STZ injection (p>0.05). There was no significant change in SP (+) nerve fibers (p>0.05) or CGRP (+) nerve fibers (p>0.05) of the gastric muscle layers. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression levels of SP and CGRP mRNA in the thoracic dorsal root ganglia were similar between diabetic and control animals (p>0.05). Qualitative and quantitative ultrastructural examinations on the gastric mucosa documented unmyelinated nerve degeneration. These results suggest the existence of gastric sensory neuropathy in STZ-induced diabetes, and this pathology provides a foundation for understanding diabetic gastropathy.

Patterns of target tissue reinnervation and trophic factor expression after nerve grafting.

Background: Reinnervation of target tissues determines functional outcomes after nerve grafting, which is important in traumatic injury caused by accidents or consequences resulting from surgical removal of tumors. Previous studies documented the influences of nerve repair mainly based on nerve morphometry but rarely compared the final outcomes according to target reinnervation patterns by nerve fibers of different categories. Methods: In a mouse model of nerve grafting, the authors analyzed the innervation indexes of different target tissues after transection-reimplantation on the sciatic nerve, which were defined as a parameter on the operated side normalized to that on the control side. Results: Muscle reinnervation appeared to be the best compared with skin reinnervation (p < 0.0001) and sweat gland reinnervation (p < 0.0001) at postoperative month 3. The sudomotor reinnervation was relatively higher than the cutaneous reinnervation (p = 0.014). The abundance of trophin transcripts for brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), and neurotrophin 3 (NT3) was higher in plantar muscles on the operated side than those on the control side. In contrast, transcripts of BDNF, GDNF, nerve growth factor, and NT3 were all similar in the footpad skin between the operated and control sides. Conclusions: The results suggested that, compared with the skin, muscles achieved the best reinnervation after nerve grafting, which was related to higher expression of BDNF, GDNF, and NT3 in muscles than in the skin.

Mitochondrial fission augments capsaicin-induced axonal degeneration

Capsaicin, an agonist of transient receptor potential vanilloid receptor 1, induces axonal degeneration of peripheral sensory nerves and is commonly used to treat painful sensory neuropathies. In this study, we investigated the role of mitochondrial dynamics in capsaicin-induced axonal degeneration. In capsaicin-treated rodent sensory axons, axonal swellings, decreased mitochondrial stationary site length and reduced mitochondrial transport preceded axonal degeneration. Increased axoplasmic Ca2+ mediated the alterations in mitochondrial length and transport. While sustaining mitochondrial transport did not reduce axonal swellings in capsaicin-treated axons, preventing mitochondrial fission by overexpression of mutant dynamin-related protein 1 increased mitochondrial length, retained mitochondrial membrane potentials and reduced axonal loss upon capsaicin treatment. These results establish that mitochondrial stationary site size significantly affects axonal integrity and suggest that inhibition of Ca2+-dependent mitochondrial fission facilitates mitochondrial function and axonal survival following activation of axonal cationic channels.

Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.

Distinct TrkA and Ret modulated negative and positive neuropathic behaviors in a mouse model of resiniferatoxin-induced small fiber neuropathy

ABSTRACT Neurotrophic factors and their corresponding receptors play key roles in the maintenance of different phenotypic dorsal root ganglion (DRG) neurons, the axons of which degenerate in small fiber neuropathy, leading to various neuropathic manifestations. Mechanisms underlying positive and negative symptoms of small fiber neuropathy have not been systematically explored. This study investigated the molecular basis of these seemingly paradoxical neuropathic behaviors according to the profiles of TrkA and Ret with immunohistochemical and pharmacological interventions in a mouse model of resiniferatoxin (RTX)‐induced small fiber neuropathy. Mice with RTX neuropathy exhibited thermal hypoalgesia and mechanical allodynia, reduced skin innervation, and altered DRG expression profiles with decreased TrkA(+) neurons and increased Ret(+) neurons. RTX neuropathy induced the expression of activating transcription factor 3 (ATF3), and ATF3(+) neurons were colocalized with Ret but not with TrkA (P < 0.001). As a neuroprotectant, 4‐Methylcatechol (4MC) promoted skin reinnervation partially with correlated reversal of the neuropathic behaviors and altered neurochemical expression. Gambogic amide, a selective TrkA agonist, normalized thermal hypoalgesia, and GW441756, a TrkA kinase inhibitor, induced thermal hypoalgesia, which was already reversed by 4MC. Mechanical allodynia was reversed by a Ret kinase inhibitor, AST487, which induced thermal hyperalgesia in naïve mice. The activation of Ret signaling by XIB4035 induced mechanical allodynia and thermal hypoalgesia in RTX neuropathy mice in which the neuropathic behaviors were previously normalized by 4MC. Distinct neurotrophic factor receptors, TrkA and Ret, accounted for negative and positive neuropathic behaviors in RTX‐induced small fiber neuropathy, respectively: TrkA for thermal hypoalgesia and Ret for mechanical allodynia and thermal hypoalgesia. HighlightsMechanisms of neuropathic manifestations in resiniferatoxin neuropathy remain unclear.TrkA and Ret signaling accounted for the thermal and mechanical symptoms, respectively.Ret(+):ATF3(+) small‐sized neurons were responsible for neuropathic behaviors.4‐Methylcatechol was able to reverse neuropathic behaviors.