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1.
Exp Neurol ; 285(Pt A): 96-107, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27567739

ABSTRACT

In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400µM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p<0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the non-infused side (p<0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1µM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1-G93A mice, even at late stages of disease.


Subject(s)
Amyotrophic Lateral Sclerosis/complications , Enzyme Inhibitors/administration & dosage , Methylene Blue/administration & dosage , Neuromuscular Junction Diseases/drug therapy , Neuromuscular Junction Diseases/etiology , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Bungarotoxins/pharmacokinetics , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Delivery Systems , Enzyme Inhibitors/therapeutic use , Fluorescent Antibody Technique , Humans , Methylene Blue/therapeutic use , Mice , Mice, Transgenic , Motor Endplate/drug effects , Motor Endplate/physiology , Muscle Contraction/drug effects
2.
J Cereb Blood Flow Metab ; 34(4): 621-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24398937

ABSTRACT

The central nervous system (CNS) is an active participant in the innate immune response to infection and injury. In these studies, we show embryonic cortical neurons express a functional, deoxyribonucleic acid (DNA)-responsive, absent in melanoma 2 (AIM2) inflammasome that activates caspase-1. Neurons undergo pyroptosis, a proinflammatory cell death mechanism characterized by the following: (a) oligomerization of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC); (b) caspase-1 dependency; (c) formation of discrete pores in the plasma membrane; and (d) release of the inflammatory cytokine interleukin-1ß (IL-1ß). Probenecid and Brilliant Blue FCF, inhibitors of the pannexin1 channel, prevent AIM2 inflammasome-mediated cell death, identifying pannexin1 as a cell death effector during pyroptosis and probenecid as a novel pyroptosis inhibitor. Furthermore, we show activation of the AIM2 inflammasome in neurons by cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients and oligomerization of ASC. These findings suggest neuronal pyroptosis is an important cell death mechanism during CNS infection and injury that may be attenuated by probenecid.


Subject(s)
Apoptosis , Inflammasomes/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , Adolescent , Adult , Aged , Animals , Apoptosis/drug effects , Apoptosis/immunology , Apoptosis Regulatory Proteins/metabolism , Brain Injuries/cerebrospinal fluid , Brain Injuries/immunology , Brain Injuries/metabolism , Brain Injuries/pathology , Caspase 1/metabolism , Cell Culture Techniques , Cell Death , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , DNA-Binding Proteins , Female , Humans , Immunity, Innate/drug effects , Inflammasomes/immunology , Male , Middle Aged , Neurons/drug effects , Neurons/immunology , Neurons/pathology , Poly dA-dT/pharmacology , Probenecid/pharmacology , Rats , Rats, Sprague-Dawley , Young Adult
3.
J Neurophysiol ; 108(8): 2203-14, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22832569

ABSTRACT

Previous work demonstrated that hyperthermia (43°C for 2 h) results in delayed, apoptotic-like death in striatal neuronal cultures. We investigated early changes in mitochondrial function induced by this heat stress. Partial depolarization of the mitochondrial membrane potential (ΔΨ(m)) began about 1 h after the onset of hyperthermia and increased as the stress continued. When the heat stress ended, there was a partial recovery of ΔΨ(m), followed hours later by a progressive, irreversible depolarization of ΔΨ(m). During the heat stress, O(2) consumption initially increased but after 20-30 min began a progressive, irreversible decline to about one-half the initial rate by the end of the stress. The percentage of oligomycin-insensitive respiration increased during the heat stress, suggesting an increased mitochondrial leak conductance. Analysis using inhibitors and substrates for specific respiratory chain complexes indicated hyperthermia-induced dysfunction at or upstream of complex I. ATP levels remained near normal for ∼4 h after the heat stress. Mitochondrial movement along neurites was markedly slowed during and just after the heat stress. The early, persisting mitochondrial dysfunction described here likely contributes to the later (>10 h) caspase activation and neuronal death produced by this heat stress. Consistent with this idea, proton carrier-induced ΔΨ(m) depolarizations comparable in duration to those produced by the heat stress also reduced neuronal viability. Post-stress ΔΨ(m) depolarization and/or delayed neuronal death were modestly reduced/postponed by nicotinamide adenine dinucleotide, a calpain inhibitor, and increased expression of Bcl-xL.


Subject(s)
Heat-Shock Response , Mitochondria/metabolism , Neurons/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis , Brain/cytology , Brain/metabolism , Calcium/metabolism , Membrane Potential, Mitochondrial , Mitochondrial Proteins/metabolism , Oligomycins , Oxygen/metabolism , Protons , Rats , Respiration
4.
Neurosci Lett ; 465(3): 226-9, 2009 Nov 20.
Article in English | MEDLINE | ID: mdl-19765637

ABSTRACT

Bone morphogenetic protein 7 (BMP7) has been shown to ameliorate reduced dendritic growth induced by glutamate excitotoxicity in neuronal tissue cultures and/or provide an enhancement of functional recovery in central nervous system (CNS) injury. BMP7 expression is modulated by spinal cord injury (SCI), but the molecular mechanisms involved in neuroprotection have not been clearly defined. Here, we show that BMP7 treatment of rats subjected to mild cervical SCI significantly increased the pro-survival mitogen-activated protein kinase-38 (MAPK-38) pathway and levels of N-methyl-D-aspartate receptor 1 (NMDAR-1) resulting in a significant increase in neuronal sparing in the ventral horn of the spinal cord. Moreover, BMP7 was neuroprotective against glutamate-mediated excitotoxicity in cultured cortical neurons. These studies show that BMP7 administration may be used as a therapeutic strategy to reduce the damaging excitotoxic effects following SCI.


Subject(s)
Bone Morphogenetic Protein 7/administration & dosage , MAP Kinase Signaling System/drug effects , Neurons/cytology , Neurons/drug effects , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/metabolism , Animals , Cell Survival/drug effects , Cervical Vertebrae/injuries , Dose-Response Relationship, Drug , Female , Neuroprotective Agents/administration & dosage , Rats , Rats, Inbred F344 , Receptors, N-Methyl-D-Aspartate/metabolism , Treatment Outcome
5.
Neurochem Res ; 33(9): 1852-8, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18351461

ABSTRACT

Activation of cyclin dependent kinases (Cdks) contributes to neuronal death following ischemia. We used oxygen-glucose deprivation (OGD) in septal neuronal cultures to test for possible roles of cell cycle proteins in neuronal survival. Increased cdc2-immunoreactive neurons were observed at 24 h after the end of 5 h OGD. Green fluorescent protein (GFP) or GFP along with a wild type or dominant negative form of the retinoblastoma protein (Rb), or cyclin-dependent kinase5 (Cdk5), were overexpressed using plasmid constructs. Following OGD, when compared to controls, neurons expressing both GFP and dominant negative Rb, RbDeltaK11, showed significantly less damage using microscopy imaging. Overexpression of Rb-wt did not affect survival. Surprisingly, overexpression of Cdk5-wild type significantly protected neurons from process disintegration but Cdk5T33, a dominant negative Cdk5, gave little or no protection. Thus phosphorylation of the cell cycle regulator, Rb, contributes to death in OGD in septal neurons but Cdk5 can have a protective role.


Subject(s)
Cyclin-Dependent Kinase 5/metabolism , Glucose/metabolism , Neurons/metabolism , Oxygen/metabolism , Retinoblastoma Protein/metabolism , Septum of Brain/cytology , Animals , CDC2 Protein Kinase/metabolism , Cell Cycle/physiology , Cyclin-Dependent Kinase 5/genetics , Neurons/cytology , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinoblastoma Protein/genetics
6.
Prog Brain Res ; 162: 347-71, 2007.
Article in English | MEDLINE | ID: mdl-17645927

ABSTRACT

Hyperthermia can cause brain damage and also exacerbate the brain damage produced by stroke and amphetamines. The developing brain is especially sensitive to hyperthermia. The severity of, and mechanisms underlying, hyperthermia-induced neuronal death depend on both temperature and duration of exposure. Severe hyperthermia can produce necrotic neuronal death. For a window of less severe heat stresses, cultured neurons exhibit a delayed death with apoptotic characteristics including cytochrome c release and caspase activation. Little is known about mechanisms of hyperthermia-induced damage upstream of these late apoptotic effects. This chapter considers several possible upstream mechanisms, drawing on both in vivo and in vitro studies of the nervous system and other tissues. Hyperthermia-induced damage in some non-neuronal cells includes endoplasmic reticular stress due to denaturing of nascent polypeptide chains, as well as nuclear and cytoskeletal damage. Evidence is presented that hyperthermia produces mitochondrial damage, including depolarization, in cultured mammalian neurons.


Subject(s)
Apoptosis/physiology , Fever/pathology , Neurons/pathology , Animals , Caspases/metabolism , Humans , Mitochondria/metabolism , Mitochondria/pathology , Neurons/metabolism , Neurons/ultrastructure
7.
J Neurochem ; 91(1): 77-87, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15379889

ABSTRACT

Cultures of embryonic rat septum were exposed for 24-48 h to 2-5 nm okadaic acid (OA), an inhibitor of pp1A and pp2A phosphatases. This stress killed approximately 75% of neurons. A neurotrophin (NT) combination (nerve growth factor and brain-derived neurotrophic factor, each 100 ng/mL) plus a bone morphogenetic protein (BMP6 or BMP7, 5 nm) reduced the death of both cholinergic and non-cholinergic neurons, and preserved choline acetyltransferase (ChAT) activity assayed 2-6 days post-stress. This NT + BMP combination preserved ChAT activity better than either NTs or BMPs alone, and was effective even if trophic factor addition was delayed until 12 h after stress onset. A general caspase inhibitor (qVD-OPH, 10 micro g/mL) also increased survival of stressed cholinergic neurons, but its protection of ChAT activity was shorter lived than that produced by the NT + BMP combination. Neither the NT + BMP combination nor the caspase inhibitor reduced the OA-induced increase in tau phosphorylation. These findings indicate that NTs and BMPs have synergistic protective effects against an OA stress, and suggest that at least some of these protective effects occur upstream of caspase activation.


Subject(s)
Acetylcholinesterase/metabolism , Bone Morphogenetic Proteins/therapeutic use , Nerve Growth Factors/therapeutic use , Septal Nuclei/drug effects , Stress, Physiological/prevention & control , Animals , Caspase 3 , Caspases/metabolism , Cell Death/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Dose-Response Relationship, Drug , Drug Combinations , Embryo, Mammalian , Enzyme Inhibitors/toxicity , Fluoresceins/metabolism , Glial Fibrillary Acidic Protein/metabolism , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins , Immunohistochemistry/methods , Marine Toxins , Nuclear Proteins/metabolism , Okadaic Acid/toxicity , Oxazoles/pharmacology , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphorylation/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Septal Nuclei/cytology , Stress, Physiological/chemically induced , Time Factors , Transfection/methods , bcl-X Protein , tau Proteins/metabolism
8.
J Neurochem ; 87(4): 958-68, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14622126

ABSTRACT

Hyperthermia can contribute to brain damage both during development and post-natally. We used rat embryonic striatal neurons in culture to study mechanisms underlying hyperthermia-induced neuronal death. Heat stress at 43 degrees C for 2 h produced no obvious signs of damage during the first 12 h after the stress, but more than 50% of the neurons died during the next 3 days. More than 40% of the neurons had activated caspases 24 h following the heat stress. Caspase-3 activity increased with a delay of more than 10 h following cessation of the heat stress, reaching a peak at approximately 18 h. Neuronal death measured 1-3 days after the stress was reduced by the general caspase inhibitors qVD-OPH (10-20 microm) and zVAD-fmk (50-100 microm). These inhibitors were protective even when added 9 h after cessation of the heat stress, consistent with the delayed activation of caspases. In contrast, blockers of Na+ channels and ionotropic glutamate receptors did not reduce the heat-induced death, indicating that glutamate excitotoxicity was not required for this neuronal death. These results show that the neuronal death produced by heat stress has characteristics of apoptosis, and that caspase inhibitors can delay this death.


Subject(s)
Caspases/metabolism , Corpus Striatum , Heat Stress Disorders/enzymology , Neurons/physiology , Animals , Caspase Inhibitors , Cell Death/drug effects , Cell Death/physiology , Cells, Cultured , Corpus Striatum/cytology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Hot Temperature/adverse effects , Neurons/cytology , Neurons/drug effects , Rats , Sodium Channel Blockers/pharmacology , Time Factors
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