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Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI; however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI; however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12h following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Following traumatic brain injury (TBI), mitochondrial function becomes compromised. Mitochondrial dysfunction is characterized by intra-mitochondrial Ca(2+) accumulation, induction of oxidative damage, and mitochondrial permeability transition (mPT). Experimental studies show that cyclosporin A (CsA) inhibits mPT. However, CsA also inhibits calcineurin. In the present study, we conducted a dose-response analysis of NIM811, a non-calcineurin inhibitory CsA analog, on mitochondrial dysfunction following TBI in mice, and compared the effects of the optimal dose of NIM811 (10 mg/kg i.p.) against an optimized dose of CsA (20 mg/kg i.p.). Male CF-1 mice were subjected to severe TBI utilizing the controlled cortical impact model. Mitochondrial respiration was assessed from animals treated with either NIM811, CsA, or vehicle 15 min post-injury. The respiratory control ratio (RCR) of mitochondria from vehicle-treated animals was significantly (p<0.01) lower at 3 or 12 h post-TBI, relative to shams. Treatment of animals with either NIM811 or CsA significantly (p<0.03) attenuated this reduction. Consistent with this finding, both NIM811 and CsA significantly reduced lipid peroxidative and protein nitrative damage to mitochondria at 12 h post-TBI. These results showing the ability of NIM811 to fully duplicate the mitochondrial protective efficacy of CsA supports the conclusion that inhibition of the mPT may be sufficient to explain CsA's protective effects.

This study examined the role of glial cell line-derived neurotrophic factor (GDNF) in synaptic plasticity at the developing neuromuscular junction. Transgenic mice overexpressing GDNF in skeletal muscle under the myosin light chain-1 promoter were isolated. Northern blot and ELISA at 6 weeks of age indicated that GDNF mRNA and protein levels were elevated threefold in the lateral gastrocnemius muscle (LGM) of the GDNF-transgenic animals. Histochemical examination of LGM tissue sections at 6 weeks of age revealed a 70% increase in the number of cholinesterase-positive end plates without changes in end-plate area. Multiple end plates on a single muscle fiber were also observed, in addition to multiple axonal processes terminating on individual end plates. No change in the number of spinal motoneurons, overall LGM size, or muscle type composition was observed. Finally, overexpression of GDNF in muscle caused hypertrophy of neuronal somata in dorsal root ganglia without affecting their number. These findings demonstrate that overexpression of a single neurotrophic factor in skeletal muscle induces multiple end-plate formation and maintains hyperinnervation well beyond the normal developmental period. We suggest that GDNF, a muscle-derived motoneuron neurotrophic factor, serves an important role in the regulation of synaptic plasticity in the developing and adult neuromuscular junction., (Copyright 2001 Academic Press.)

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral-caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury., (Copyright 1998 Academic Press.)

In this study we report the localization and expression of FAC1 protein in developing, normal adult and amyotrophic lateral sclerosis (ALS) lumbar spinal cord. High levels of FAC1 protein were detected in cells throughout all areas (gray and white matter) of the developing lumbar spinal cord. FAC1 protein was localized predominately in nuclei and the cell body of motor neurons during early stages of spinal cord development. In contrast, low levels of FAC1 protein were observed in the adult lumbar spinal cord, localized only in the cell body of large alpha motor neurons found in lamina IX. Interestingly, FAC1 protein expression was elevated in surviving motor neurons of ALS spinal cord compared to the controls and was located both in the nucleus and throughout the cytoplasm of motor neurons. FAC1 protein was also observed in white matter cells and fibers in ALS spinal cord. In support of the immunocytochemical results, in situ hybridization studies demonstrated that FAC1 mRNA is also elevated in ALS spinal cord motor neurons. These data describe the developmental regulation of FAC1 protein in the spinal cord by immunocytochemical techniques and provide evidence that this protein is reexpressed in ALS motor neurons.

Total RNA from rat Schwann cells grown in culture and adult rat skeletal muscle was reverse transcribed, amplified for glial cell line-derived neurotrophic factor (GDNF) messenger RNA (mRNA) using the polymerase chain reaction (PCR), and the PCR products sequenced. Two forms of GDNF were detected in the PCR step, one of a predicted size (GDNF633) and a second smaller form missing a 78-base pair sequence (GDNF555). Sequence analysis demonstrated that GDNF633 is similar to the published sequence of GDNF differing only at three nucleotides. Southern and Northern blot analyses reveal that the two forms are probably derived from a single RNA species that is alternatively spliced. Interestingly, GDNF633 mRNA was found to be selectively upregulated in denervated rat skeletal muscle at 1-2 weeks following axotomy, providing evidence that the innervation status of the muscle may determine the expression profile of the two alternatively spliced forms. Given these findings, we suggest that GDNF may function as a target-derived trophic factor for neuronal populations innervating skeletal muscle, including sensory neurons and spinal cord motoneurons.

A recently cloned neurotrophic factor, termed glial cell line-derived neurotrophic factor (GDNF), has been reported to exhibit selective neurotrophic properties on ventral mesencephalon dopaminergic neurons, which degenerate in patients with Parkinson's disease. In the present study, we used reverse transcriptase followed by polymerase chain reaction (PCR) and in situ hybridization to study the expression of GDNF messenger RNA (mRNA) in the adult rat and human central nervous system (CNS). GDNF transcripts were identified using PCR in all regions of the rat CNS analyzed including striatum, hippocampus, cortex, cerebellum, and spinal cord. Interestingly, the rat hippocampal formation contained two transcripts, i.e., a larger form in addition to the amplified GDNF cDNA found in all other areas analyzed. GDNF PCR products also were observed in human striatum, hippocampus, cortex, and spinal cord, but not cerebellum, and both the striatum and hippocampal formation contained two GDNF transcripts. Finally, GDNF transcripts were detected in a rat Schwann cell line previously shown to secrete a factor that exerts a neurotrophic effect on dopaminergic neurons. In situ hybridization experiments using a cRNA probe hybridized to adult rat brain sections demonstrated no positive GDNF mRNA signal. However, intense GDNF mRNA hybridization signal was found to be associated with dorsal root ganglia in Postnatal Day 1 rats. These findings provide evidence that GDNF is detectable using PCR in a number of nervous system structures and, in some areas, GDNF is expressed in more than one form.

Current therapeutic efforts to treat chronic and progressive neurodegenerative disease include, for the first time, attempts to regenerate affected nervous tissue using neurotrophic factors. The rationale for using trophic factors includes the understanding that they support neuronal survival and regrowth processes. The potential benefits of trophic factor therapy will be no more realized in the near future than in the treatment of amyotrophic lateral sclerosis (ALS). ALS is pathologically characterized by the selective degeneration of specific populations of cranial and spinal motoneurons. Evidence for the existence of factors that support motoneurons has come from studies demonstrating that motoneurons receive trophic influences from various tissues, both central and peripheral, within their local environment. Although the identity of these putative tissue-derived factors has remained enigmatic, recent studies have demonstrated that several previously characterized trophic factors exhibit trophic influences on motoneurons. Among these are several members of the neurotrophin family, most notably brain-derived neurotrophic factor. These neurotrophins meet most of the criteria to be considered motoneuron trophic factors: they are locally available to motoneurons in vivo; motoneurons express specific receptors for these factors; and exogenous application of these factors mimicks the effects of the uncharacterized endogenous agents. The clinical use of these factors for the treatment of ALS, therefore, appears to be scientifically justified.

In the hippocampal formation, nerve growth factor (NGF) is produced in granule cells of the dentate gyrus and a few pyramidal cells of Ammon's horn. Both neuronal populations express N-methyl-D-aspartate (NMDA) receptors and receive putative glutamatergic afferents originating in the entorhinal cortex and projecting via the perforant path. We report in this study that intra-hippocampal or intraventricular injections of NMDA increase NGF mRNA levels in dentate gyrus granule cells as determined using in situ hybridization histochemistry and a solution hybridization assay. NGF mRNA induction is detected within 2 h following NMDA treatment and returns to control levels within 24 h. This NMDA effect is dose-dependent and blocked by pretreatment with 2-amino-5-phosphonopentanoic acid, a competitive NMDA antagonist. Finally, the induction of NGF mRNA is observed in the absence of detectable neurotoxicity or seizure activity. We postulate that normal physiological events associated with the activation of hippocampal NMDA receptors may regulate mRNA expression of this neurotrophic factor.

We have recently demonstrated that a diffusible factor(s) derived from explanted adult rat sciatic nerve can increase the number and neurite outgrowth of embryonic rat dopamine (DA) neurons in culture. The present study extends this finding to compare DA neuron-sciatic nerve co-grafts to grafts of DA-rich neural tissue alone for behavioral and morphological effects in rats with unilateral nigrostriatal lesions of the DA pathway. Our results indicate that the presence of a co-grafted segment of sciatic nerve increased the likelihood of rapid behavioral recovery and promoted complete recovery mediated by small grafts that yielded only modest behavioral changes in the absence of co-grafted nerve. These behavioral effects were accompanied by a modest increase in survival of grafted tyrosine hydroxylase-positive neurons in the striatum and a more pronounced increase in the area and density of striatal reinnervation provided by grafted DA neurons in co-grafted animals. This evidence supports the view that a diffusible product of explanted peripheral nerve acts as a growth-promoting factor for embryonic DA neurons and that the presence of this factor augments the behavioral efficacy of grafted DA neurons.

Nerve growth factor (NGF) is well known to be involved in the development, survival, and maintenance of sympathetic and neural crest-derived sensory neurons in the peripheral nervous system. Over the last 10-15 years, however, the role of NGF as a necessary trophic substrate for magnocellular cholinergic neurons in the central nervous system (CNS) has emerged. Because the trophic effects of NGF are initiated by its interaction with membrane-bound receptors, the characterization, localization, and function of these specific NGF receptors are essential to understanding the many actions of NGF. The first part of this review will summarize briefly the presence and possible role of NGF in the CNS, with the remainder of the review focusing on what is known about the receptor to NGF.