Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons.

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.

Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice.

BACKGROUND: Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown. PURPOSE: Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal. RESULTS: We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro. CONCLUSIONS: These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.

The Mas-related G protein-coupled receptor d (Mrgprd) mediates pain hypersensitivity in painful diabetic neuropathy.

ABSTRACT: Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, axonal degeneration, and changes in cutaneous innervation. However, the complete molecular profile underlying the hyperexcitable cellular phenotype of DRG nociceptors in PDN has not been elucidated. This gap in our knowledge is a critical barrier to developing effective, mechanism-based, and disease-modifying therapeutic approaches that are urgently needed to relieve the symptoms of PDN. Using single-cell RNA sequencing of DRGs, we demonstrated an increased expression of the Mas-related G protein-coupled receptor d (Mrgprd) in a subpopulation of DRG neurons in the well-established high-fat diet (HFD) mouse model of PDN. Importantly, limiting Mrgprd signaling reversed mechanical allodynia in the HFD mouse model of PDN. Furthermore, in vivo calcium imaging allowed us to demonstrate that activation of Mrgprd-positive cutaneous afferents that persist in diabetic mice skin resulted in an increased intracellular calcium influx into DRG nociceptors that we assess in vivo as a readout of nociceptors hyperexcitability. Taken together, our data highlight a key role of Mrgprd-mediated DRG neuron excitability in the generation and maintenance of neuropathic pain in a mouse model of PDN. Hence, we propose Mrgprd as a promising and accessible target for developing effective therapeutics currently unavailable for treating neuropathic pain in PDN.

Mitochondrial calcium uniporter deletion prevents painful diabetic neuropathy by restoring mitochondrial morphology and dynamics.

ABSTRACT: Painful diabetic neuropathy (PDN) is an intractable complication affecting 25% of diabetic patients. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, resulting in calcium overload, axonal degeneration, and loss of cutaneous innervation. The molecular pathways underlying these effects are unknown. Using high-throughput and deep-proteome profiling, we found that mitochondrial fission proteins were elevated in DRG neurons from mice with PDN induced by a high-fat diet (HFD). In vivo calcium imaging revealed increased calcium signaling in DRG nociceptors from mice with PDN. Furthermore, using electron microscopy, we showed that mitochondria in DRG nociceptors had fragmented morphology as early as 2 weeks after starting HFD, preceding the onset of mechanical allodynia and small-fiber degeneration. Moreover, preventing calcium entry into the mitochondria, by selectively deleting the mitochondrial calcium uniporter from these neurons, restored normal mitochondrial morphology, prevented axonal degeneration, and reversed mechanical allodynia in the HFD mouse model of PDN. These studies suggest a molecular cascade linking neuropathic pain to axonal degeneration in PDN. In particular, nociceptor hyperexcitability and the associated increased intracellular calcium concentrations could lead to excessive calcium entry into mitochondria mediated by the mitochondrial calcium uniporter, resulting in increased calcium-dependent mitochondrial fission and ultimately contributing to small-fiber degeneration and neuropathic pain in PDN. Hence, we propose that targeting calcium entry into nociceptor mitochondria may represent a promising effective and disease-modifying therapeutic approach for this currently intractable and widespread affliction. Moreover, these results are likely to inform studies of other neurodegenerative disease involving similar underlying events.

Reducing CXCR4-mediated nociceptor hyperexcitability reverses painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.