Abstract
Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKβ to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 μM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 μM) or short hairpin RNA to CaMKKβ. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 μM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKβ-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.
Highlights
The field of innervation of intraepidermal nerve fibers (IENFs) within the skin is plastic and maintained through a combination of collateral sprouting and regeneration that is regulated partly by neurotrophic factors [1, 2]
Distal dyingback of nerve fibers is observed in many peripheral neuropathies including those associated with diabetes, chemotherapyinduced peripheral neuropathy (CIPN), Friedreich’s ataxia, Charcot-Marie-Tooth disease type 2, and human immunodeficiency virus (HIV)
Neurons were grown in defined Hams F-12 medium (Life Technologies, Burlington, ON, Canada) with modified Bottenstein and Sato’s N2 medium containing 0.1 mg/ml transferrin, 20 nM progesterone, 100 μM putrescine, 30 nM sodium selenite, and 1 mg/ml BSA, and supplemented with the following neurotrophic factors (NTFs): 0.1 ng/ml nerve growth factor (NGF), 0.1 ng/ml neurotrophin-3 (NT-3), and 1 ng/ml glial cell line–derived neurotrophic factor (GDNF)
Summary
The field of innervation of intraepidermal nerve fibers (IENFs) within the skin is plastic and maintained through a combination of collateral sprouting and regeneration that is regulated partly by neurotrophic factors [1, 2]. Distal dyingback of nerve fibers is observed in many peripheral neuropathies including those associated with diabetes, chemotherapyinduced peripheral neuropathy (CIPN), Friedreich’s ataxia, Charcot-Marie-Tooth disease type 2, and human immunodeficiency virus (HIV). The most common form of diabetic neuropathy in type 1 and type 2 patients, symmetrical sensorimotor polyneuropathy, exhibits pathological changes in both large and small fibers of peripheral nerves. Defective axon sprouting and regeneration impedes tissue re-innervation [11]. These indices of nerve pathology are observed in rodent models of both type 1 and type 2 diabetes [12, 13], allowing for investigation of both putative pathogenic mechanisms and potential therapies
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