ryanodine and Nerve-Degeneration

Transected axons of the central nervous system fail to regenerate and instead die back away from the lesion site, resulting in permanent disability. Although both intrinsic (eg, microtubule instability, calpain activation) and extrinsic (ie, macrophages) processes are implicated in axonal dieback, the underlying mechanisms remain uncertain. Furthermore, the precise mechanisms that cause delayed "bystander" loss of spinal axons, that is, ones that were not directly damaged by the initial insult, but succumbed to secondary degeneration, remain unclear. Our goal was to evaluate the role of intra-axonal Ca(2+) stores in secondary axonal degeneration following spinal cord injury.. We developed a 2-photon laser-induced spinal cord injury model to follow morphological and Ca(2+) changes in live myelinated spinal axons acutely following injury.. Transected axons "died back" within swollen myelin or underwent synchronous pan-fragmentation associated with robust Ca(2+) increases. Spared fibers underwent delayed secondary bystander degeneration. Reducing Ca(2+) release from axonal stores mediated by ryanodine and inositol triphosphate receptors significantly decreased axonal dieback and bystander injury. Conversely, a gain-of-function ryanodine receptor 2 mutant or pharmacological treatments that promote axonal store Ca(2+) release worsened these events.. Ca(2+) release from intra-axonal Ca(2+) stores, distributed along the length of the axon, contributes significantly to secondary degeneration of axons. This refocuses our approach to protecting spinal white matter tracts, where emphasis has been placed on limiting Ca(2+) entry from the extracellular space across cell membranes, and emphasizes that modulation of axonal Ca(2+) stores may be a key pharmacotherapeutic goal in spinal cord injury.

Topics: Animals; Axons; Bacterial Proteins; Boron Compounds; Caffeine; Calcium; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Inhibitors; Laser Therapy; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Degeneration; Purinergic P1 Receptor Antagonists; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord Injuries; Thapsigargin; Time Factors

Human immunodeficiency virus (HIV)-associated sensory neuropathy (HIV-SN) occurs in a large fraction of patients infected with HIV. Viral components, including the coat protein gp120, are thought to exert toxic actions on dorsal root ganglia (DRG) sensory neurons that can be further exacerbated by treatment of HIV infection with some antiretroviral agents. In a tissue culture model of HIV-SN, we found that gp120-induced axonal degeneration in DRG sensory neurons was prevented by treatment with the immunophilin ligand GPI-1046. Gp120 induced a rapid and large release of endoplasmic reticulum (ER) calcium in DRG neurons that was attenuated by treatment with GPI-1046. Further experiments suggested that GPI-1046 reduced the total ER calcium load by attenuating store-operated calcium (SOC) entry. Together, these results suggest that GPI-1046 protects DRG from gp120-induced axonal damage by decreasing the entry of calcium through SOC, thus reducing the total volume of ER calcium that is available to be released by gp120.

Topics: Analysis of Variance; Animals; Axons; Calcium; Dose-Response Relationship, Drug; Embryo, Mammalian; Endoplasmic Reticulum; Enzyme Inhibitors; Ganglia, Spinal; HIV Envelope Protein gp120; Macrocyclic Compounds; Nerve Degeneration; Neurons; Oxazoles; Pyrrolidines; Rats; Rats, Sprague-Dawley; Ryanodine; Thapsigargin

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited, neurodegenerative disease caused by an expansion of polyglutamine tracts in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are predominantly affected in SCA2. The cause of PC degeneration in SCA2 is unknown. Here we demonstrate that mutant Atx2-58Q, but not wild-type (WT) Atx2-22Q, specifically associates with the cytosolic C-terminal region of type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), an intracellular calcium (Ca(2+)) release channel. Association with Atx2-58Q increased the sensitivity of InsP(3)R1 to activation by InsP(3) in planar lipid bilayer reconstitution experiments. To validate physiological significance of these findings, we performed a series of experiments with an SCA2-58Q transgenic mouse model that expresses human full-length Atx2-58Q protein under the control of a PC-specific promoter. In Ca(2+) imaging experiments, we demonstrated that stimulation with 3,5-dihydroxyphenylglycine (DHPG) resulted in higher Ca(2+) responses in 58Q PC cultures than in WT PC cultures. DHPG-induced Ca(2+) responses in 58Q PC cultures were blocked by the addition of ryanodine, an inhibitor of the ryanodine receptor (RyanR). We further demonstrated that application of glutamate induced more pronounced cell death in 58Q PC cultures than in WT PC cultures. Glutamate-induced cell death of 58Q PC cultures was attenuated by dantrolene, a clinically relevant RyanR inhibitor and Ca(2+) stabilizer. In whole animal experiments, we demonstrated that long-term feeding of SCA1-58Q mice with dantrolene alleviated age-dependent motor deficits (quantified in beam-walk and rotarod assays) and reduced PC loss observed in untreated SCA2-58Q mice by 12 months of age (quantified by stereology). Results of our studies indicate that disturbed neuronal Ca(2+) signaling may play an important role in SCA2 pathology and also suggest that the RyanR constitutes a potential therapeutic target for treatment of SCA2 patients.

Topics: Animals; Ataxins; Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Death; Cells, Cultured; Chlorocebus aethiops; COS Cells; Dantrolene; Excitatory Amino Acid Agents; Glutamic Acid; Glycine; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mice, Transgenic; Motor Activity; Nerve Degeneration; Nerve Tissue Proteins; Purkinje Cells; Resorcinols; Ryanodine; Spinocerebellar Ataxias