thapsigargin and Nerve-Degeneration

Allele-specific distinctions in the human apolipoprotein E (

Topics: Alleles; Amyloid beta-Peptides; Animals; Animals, Genetically Modified; Apolipoproteins E; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium; Homeostasis; Humans; Larva; Nerve Degeneration; Neuroprotection; Protein Isoforms; Starvation; Survival Analysis; Thapsigargin

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (Dicer

Topics: Aging; Alleles; Animals; Cell Survival; Cells, Cultured; Cellular Senescence; Dopaminergic Neurons; Down-Regulation; Endoplasmic Reticulum Stress; Gene Deletion; Mesencephalon; Mice, Knockout; MicroRNAs; Motor Activity; Nerve Degeneration; Neuroprotection; Ribonuclease III; RNA, Messenger; Thapsigargin

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

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3)R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3)R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3)R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3)R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), increased [Ca(2+)](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3)R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.

Topics: Analysis of Variance; Animals; Calcium; Calcium Signaling; Cell Line, Transformed; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Female; Gene Expression Regulation; Glutamic Acid; Humans; Inositol 1,4,5-Trisphosphate Receptors; Methoxyhydroxyphenylglycol; Microtubule-Associated Proteins; Mutagenesis, Site-Directed; Nerve Degeneration; Neurons; Point Mutation; Pregnancy; Rats; Thapsigargin; Transfection

Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.

Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Cell Death; Endoplasmic Reticulum; Enzyme Inhibitors; HEK293 Cells; Humans; Huntingtin Protein; Mutation; Nerve Degeneration; Nerve Tissue Proteins; Nuclear Proteins; Phenotype; Proteasome Endopeptidase Complex; Thapsigargin; Ubiquitin; Valosin Containing Protein

Although the photoreceptors cell death is the main cause of some retinopathies diseases, the mechanisms involved in this process are poorly understood. The neuroprotective effects of interleukin-4 (IL-4) have been shown in several tissues, including retina. We demonstrate that treatment of rat retinal explants with IL-4 completely inhibited the thapsigargin-induced rod photoreceptor cell death after 24 hr in culture. We also showed that IL-4 receptor alpha subunit (IL-4Ralpha) is abundantly present in retina. Colocalization of IL-4Ralpha and rhodopsin indicate a direct effect of this cytokine in rod photoreceptor cells. Moreover, IL-4 increased the intracellular levels of cAMP in 7.4-fold, indicating that the neuroprotective effect of this cytokine was completely blocked by RpcAMP, an inhibitor of protein kinase (PKA). Our data demonstrate, for the first time, the neuroprotective effect of IL-4 through cAMP/PKA pathway in thapsigargin-induced photoreceptor cell death.

Topics: Animals; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Enzyme Inhibitors; Interleukin-4; Nerve Degeneration; Neuroprotective Agents; Organ Culture Techniques; Rats; Receptors, Interleukin-4; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction; Thapsigargin; Up-Regulation

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

The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca signaling in primary sensory neurons after injury. The authors examined the effect of injury on intracellular Ca stores of the endoplasmic reticulum, which critically regulate the Ca signal and neuronal function.. Intracellular Ca levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats after L5 spinal nerve ligation, compared to neurons from control animals.. Endoplasmic reticulum Ca stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca-free bath solution, which removes the contribution of store-operated membrane Ca channels, and after blockade of the mitochondrial, sarco-endoplasmic Ca-ATPase and the plasma membrane Ca ATPase pathways. Ca released by the sarco-endoplasmic Ca-ATPase blocker thapsigargin and by the Ca-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca stores in axotomized neurons were not expanded by neuronal activation by K depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca-ATPase was normal. Luminal Ca concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation.. Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca stores and a reduced luminal Ca concentration. Depletion of Ca stores may contribute to the pathogenesis of neuropathic pain.

Topics: Animals; Axons; Axotomy; Caffeine; Calcium; Calcium Signaling; Capsaicin; Cells, Cultured; Central Nervous System Stimulants; Endoplasmic Reticulum; Hyperalgesia; Ionomycin; Ligation; Male; Nerve Degeneration; Pain Measurement; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel; Sciatic Nerve; Sensory Receptor Cells; Spinal Nerves; Thapsigargin

The mechanisms of intracellular calcium store depletion and store-related Ca(2+) dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca(2+) by BAPTA/AM (50 microM), or La(3+) (100 microM) enhanced while dantrolene (100 microM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca(2+) stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca(2+) concentrations ([Ca(2+)](i)).

Topics: Animals; Apoptosis; Calcium; Calcium Signaling; Chelating Agents; Dantrolene; Endoplasmic Reticulum; Enzyme Inhibitors; Fura-2; Heat-Shock Proteins; Homeostasis; Intracellular Fluid; Lanthanum; Molecular Chaperones; Muscle Relaxants, Central; Nerve Degeneration; Neurons; PC12 Cells; Rats; Stress, Physiological; Thapsigargin

We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system.

Topics: Animals; Anisomycin; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; Blotting, Western; Caspases; Cells, Cultured; DNA-Binding Proteins; Enzyme Inhibitors; Immunohistochemistry; In Situ Nick-End Labeling; Nerve Degeneration; Protein Synthesis Inhibitors; Protein Transport; Rats; Retinal Ganglion Cells; Thapsigargin; Time Factors; Transcription Factors

Previous work on peripheral sympathetic neurons indicated that a decline in sarco/endoplasmic reticulum calcium ATPase (SERCA) function occurs with advancing age. Therefore, an age-related decline in mechanisms controlling intracellular calcium homeostasis could contribute to altered neuronal function and/or degeneration. In this study we sought to extend the findings on peripheral neurons and to detect possible age-related declines in SERCA function and expression of SERCA3 in central neurons from cerebral cortex from young (6-month) and old (20-month) rats. Functional studies compared ATP-dependent 45Ca(2+)-uptake into microsomes and plasma membrane vesicles (PMVs). We and found no significant difference in 45Ca(2+)-uptake between microsomes or PMVs between young and old animals. On the other hand expression of SERCA3 mRNA in rat cerebral cortex showed a significant decline with advancing age. However, comparison of SERCA3 protein content did not reveal a corresponding decline; implying that SERCA mRNA turnover rates may be greater in the younger group. Although the present work with rat cerebral cortex does not indicate an age-related decline in SERCA function, previous work from our laboratory on sympathetic nerves and by others on the hippocampus indicate such a decline. In light of our previous and current studies, aging may affect calcium homeostatic mechanisms in central and peripheral autonomic neurons differently.

Topics: Aging; Animals; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cerebral Cortex; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Homeostasis; Intracellular Fluid; Male; Microsomes; Nerve Degeneration; Neurons; Protein Isoforms; Rats; Rats, Inbred F344; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Subcellular Fractions; Thapsigargin; Transport Vesicles

In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.

Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium Channels; Calcium Signaling; Calcium-Binding Proteins; Calnexin; Calreticulin; Cell Size; Chromosome Mapping; Endoplasmic Reticulum; Helminth Proteins; Heterotrimeric GTP-Binding Proteins; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate Receptors; Ion Transport; Larva; Membrane Proteins; Molecular Sequence Data; Mutation; Necrosis; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Ribonucleoproteins; Ryanodine Receptor Calcium Release Channel; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thapsigargin; Touch