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1.  Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic–ischemic encephalopathy 
The consequences of therapeutic hypothermia for neonatal hypoxic–ischemic encephalopathy are poorly understood. Adverse effects from suboptimal rewarming could diminish neuroprotection from hypothermia. Therefore, we tested whether rewarming is associated with apoptosis. Piglets underwent hypoxia–asphyxia followed by normothermic or hypothermic recovery at 2 hours. Hypothermic groups were divided into those with no rewarming, rewarming at 0.5 °C/hour, or rewarming at 4 °C/hour. Neurodegeneration at 29 hours was assessed by hematoxylin and eosin staining, TUNEL assay, and immunoblotting for cleaved caspase-3. Rewarmed piglets had more apoptosis in motor cortex than did those that remained hypothermic after hypoxia–asphyxia. Apoptosis in piriform cortex was greater in hypoxic–asphyxic, rewarmed piglets than in naive/sham piglets. Caspase-3 inhibitor suppressed apoptosis with rewarming. Rapidly rewarmed piglets had more caspase-3 cleavage in cerebral cortex than did piglets that remained hypothermic or piglets that were rewarmed slowly. We conclude that rewarming from therapeutic hypothermia can adversely affect the newborn brain by inducing apoptosis through caspase mechanisms.
doi:10.1038/jcbfm.2014.245
PMCID: PMC4420851  PMID: 25564240
asphyxia; heart arrest; hypothermia; neuronal cell death; newborn; rewarming
2.  Additive neuroprotection of a 20-HETE inhibitor with delayed therapeutic hypothermia after hypoxia-ischemia in neonatal piglets 
Developmental neuroscience  2015;37(0):376-389.
The severity of perinatal hypoxia-ischemia and the delay in initiating therapeutic hypothermia limit the efficacy of hypothermia. After hypoxia-ischemia in neonatal piglets, the arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), has been found to contribute to oxidative stress at 3 hours of reoxygenation and to eventual neurodegeneration. We tested whether early administration of a 20-HETE-synthesis inhibitor after reoxygenation augments neuroprotection with 3-hour delayed hypothermia. In two hypothermic groups, whole body cooling from 38.5 to 34°C was initiated 3 hours after hypoxia-ischemia. Rewarming occurred from 20 to 24 hours; then anesthesia was discontinued. One hypothermic group received a 20-HETE inhibitor at 5 minutes after reoxygenation. A sham-operated group and another hypoxia-ischemia group remained normothermic. At 10 days of recovery, resuscitated piglets with delayed hypothermia alone had significantly greater viable neuronal density in putamen, caudate nucleus, sensorimotor cortex, CA3 hippocampus, and thalamus than did piglets with normothermic recovery, but the values remained less than those in the sham-operated group. In piglets administered the 20-HETE inhibitor before hypothermia, the density of viable neurons in putamen, cortex, and thalamus was significantly greater than in the group with hypothermia alone. Cytochrome P450 4A, which can synthesize 20-HETE, was expressed in piglet neurons in these regions. We conclude that early treatment with a 20-HETE inhibitor enhances the therapeutic benefit of delayed hypothermia in protecting neurons in brain regions known to be particularly vulnerable to hypoxia-ischemia in term newborns.
doi:10.1159/000369007
PMCID: PMC4514546  PMID: 25721266
asphyxia; cerebral ischemia; cytochrome P450; heart arrest; 20-HETE; hypoxic-ischemic encephalopathy; neonate; neuroprotection
3.  Induced Pluripotent Stem Cells from Familial Alzheimer's Disease Patients Differentiate into Mature Neurons with Amyloidogenic Properties 
Stem Cells and Development  2014;23(24):2996-3010.
Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in β-amyloid (Aβ)42/Aβ40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.
doi:10.1089/scd.2013.0511
PMCID: PMC4267410  PMID: 25027006
4.  Oscillating gradient diffusion MRI reveals unique microstructural information in normal and hypoxia-ischemia injured mouse brains 
Purpose
We investigated whether oscillating gradient diffusion MRI (dMRI) can provide information on brain microstructural changes after formaldehyde fixation and after hypoxic-ischemic (HI) injury beyond that provided by conventional dMRI.
Methods
Pulsed gradient spin echo (PGSE) and oscillating gradient spin echo (OGSE) dMRI of the adult mouse brain was performed in vivo (50-200 Hz, b = 600 mm2/s), and a similar protocol was applied to neonatal mouse brains at 24 hours after unilateral hypoxia-ischemia. Animals were perfusion fixed with 4% paraformaldehyde for ex vivo dMRI and histology.
Results
Apparent diffusion coefficients (ADCs) measured in the live adult mouse brain presented tissue-dependent frequency-dependence. In vivo OGSE-ADC maps at high oscillating frequencies (>100Hz) showed clear contrast between the molecular layer and granule cell layer in the adult mouse cerebellum. Formaldehyde fixation significantly altered the temporal diffusion spectra in several brain regions. In neonatal mouse brains with HI injury, in vivo ADC measurements from edema regions showed diminished edema contrasts at 200 Hz compared to the PGSE results. Histology showed severe tissue swelling and necrosis in the edema regions.
Conclusion
The results demonstrate the unique ability of OGSE-dMRI in delineating tissue microstructures at different spatial scales.
doi:10.1002/mrm.25441
PMCID: PMC4198437  PMID: 25168861
diffusion MRI; oscillating gradient; mouse brain; tissue microstructure; neonatal hypoxia-ischemia
5.  The mitochondrial permeability transition pore regulates Parkinson’s disease development in mutant α-synuclein transgenic mice 
Neurobiology of aging  2013;35(5):1132-1152.
Parkinson’s disease (PD) is a movement disorder caused by neurodegeneration in neocortex, substantia nigra (SN) and brainstem and synucleinopathy. Some inherited PD is caused by mutations in α-synuclein (αSyn), and inherited and idiopathic PD are associated with mitochondrial perturbations. However, the mechanisms of pathogenesis are unresolved. We characterized a human αSyn transgenic mouse model and tested the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the disease mechanisms. C57BL/6 mice expressing human A53T-mutant αSyn driven by a Thy1 promoter develop a severe, age-related, fatal movement disorder involving ataxia, rigidity, and postural instability. These mice develop synucleinopathy and neocortical, SN, and cerebello-rubro-thalamic degeneration involving mitochondriopathy and apoptotic and non-apoptotic neurodegeneration. Interneurons undergo apoptotic degeneration in young mice. Mutant αSyn associated with dysmorphic neuronal mitochondria and bound voltage-dependent anion channels. Genetic ablation of cyclophilin D, an mPTP modulator, delayed disease onset and extended lifespans of mutant αSyn mice. Thus, mutant αSyn transgenic mice on a C57BL/6 background develop PD-like phenotypes, and the mPTP is involved in their disease mechanisms.
doi:10.1016/j.neurobiolaging.2013.11.008
PMCID: PMC3948207  PMID: 24325796
adenine nucleotide translocase; interneuron; porin; ppif; cerebellum; voltage-dependent anion channel
6.  Imaging neurodegeneration in the mouse hippocampus after neonatal hypoxia-ischemia using oscillating gradient diffusion MRI 
Purpose
To investigate if frequency-dependent contrasts using oscillating gradient diffusion MRI (dMRI) can detect hypoxia-ischemia (HI) induced neurodegeneration in the neonatal mouse hippocampus.
Methods
Pulsed- and oscillating-gradient dMR images (at 50, 100, and 150 Hz) were acquired from postmortem fixed brains of mice exposed to neonatal HI using the Rice-Vanucci model. MRI data were acquired at 1, 4, and 8 days following HI, and compared with histological data from the same mice for in situ histological validation of the MRI findings.
Results
The rate of change of apparent diffusion coefficient with gradient frequency (ΔfADC) revealed unique layer-specific contrasts in the neonatal mouse hippocampus. ΔfADC measurements were found to show a significant decrease in response to neonatal HI injury, in the pyramidal (Py) and granule (GrDG) cell layers compared to contralateral regions. The areas of reduced intensity in the ΔfADC maps corresponded to regional neurodegeneration seen with H&E and Fluoro-Jade C stainings, indicating that alterations in ΔfADC contrasts are sensitive to early microstructural changes due to HI-induced neurodegeneration in the studied regions.
Conclusion
The findings show that the frequency-dependence of ADC measurements with oscillating-gradient dMRI can provide a sensitive contrast to detect HI-induced neurodegeneration in neuronal layers of the neonatal mouse hippocampus.
doi:10.1002/mrm.24956
PMCID: PMC3976472  PMID: 24123409
diffusion MRI; oscillating gradient; hypoxia-ischemia; neonatal; mouse hippocampus
7.  Hypoxia-Ischemia and Therapeutic Hypothermia in the Neonatal Mouse Brain – A Longitudinal Study 
PLoS ONE  2015;10(3):e0118889.
Therapeutic hypothermia is standard of care for infants with hypoxic ischemic encephalopathy. Murine models of hypoxic-ischemic injury exist; however, a well-established mouse model of therapeutic hypothermia following hypoxic-ischemic injury is lacking. The goal of this study was to develop a full-term-equivalent murine model of therapeutic hypothermia after hypoxia-ischemia and examine magnetic resonance imaging, behavior, and histology in a region and sex specific manner. Hypoxic-ischemic injury was induced at postnatal day 10 in C57BL6 mice using a modified Vannucci model. Mice were randomized to control, hypothermia (31˚C for 4h), or normothermia (36˚C) following hypoxic-ischemic injury and stratified by sex. T2-weighted magnetic resonance imaging was obtained at postnatal day 18 and 30 and regional and total cerebral and cerebellar volumes measured. Behavioral assessments were performed on postnatal day 14, 21, and 28. On postnatal day 18, normothermic mice had smaller cerebral volumes (p < 0.001 vs. controls and p = 0.009 vs. hypothermia), while at postnatal day 30 both injured groups had smaller volumes than controls. When stratified by sex, only normothermia treated male mice had smaller cerebral volumes (p = 0.001 vs. control; p = 0.008 vs. hypothermia) at postnatal day 18, which persisted at postnatal day 30 (p = 0.001 vs. control). Female mice had similar cerebral volumes between groups at both day 18 and 30. Cerebellar volumes of hypothermia treated male mice differed from control at day 18, but not at 30. Four hours of therapeutic hypothermia in this murine hypoxic-ischemic injury model provides sustained neuroprotection in the cerebrum of male mice. Due to variable degree of injury in female mice, response to therapeutic hypothermia is difficult to discern. Deficits in female behavior tests are not fully explained by imaging measures and likely represent injury not detectable by volume measurements alone.
doi:10.1371/journal.pone.0118889
PMCID: PMC4361713  PMID: 25774892
8.  Sexual dimorphism in BDNF signaling after neonatal hypoxia-ischemia and treatment with necrostatin-1 
Neuroscience  2013;260:106-119.
Brain injury due to neonatal hypoxia-ischemia (HI) is more homogenously severe in male than in female mice. Because, necrostatin-1 (nec-1) prevents injury progression only in male mice, we hypothesized that changes in BDNF signaling after HI and nec-1 are also sex-specific providing differential conditions to promote recovery of those more severely injured. The increased aromatization of testosterone in male mice during early development and the link between 17-β-estradiol (E2) levels and BDNF transcription substantiate this hypothesis. Hence, we aimed to investigate if sexual differences in BDNF signaling existed in forebrain and diencephalon after HI and HI/ nec-1 and their correlation with estrogen receptors (ER). C57B6 mice (p7) received nec-1(0.1 μL[8μM]) or vehicle (veh) intracerebroventricularly after HI. At 24h after HI, BDNF levels increased in both sexes in forebrain without evidence of TrkB activation. At 96h after HI, BDNF levels in forebrain decreased below those seen in control mice of both sexes. Additionally, only in female mice, truncated TrkB (Tc.TrkB) and p75ntr levels increased in forebrain and diencephalon. In both, forebrain and diencephalon, nec-1 treatment increased BDNF levels and TrkB activation in male mice while, prevented Tc.TrkB and p75ntr increases in female mice. While E2 levels were unchanged by HI or HI/ nec-1 in either sex or treatment, ERα: ERβ ratios were increased in diencephalon of nec-1 treated male mice and directly correlated with BDNF levels. Neonatal HI produces sex-specific signaling changes in the BDNF system, that are differentially modulated by nec-1. The regional differences in BDNF levels may be a consequence of injury severity after HI, but sexual differences in response to nec-1 after HI may represent a differential thalamo-cortical preservation or alternatively off-target regional effect of nec-1. The biological significance of ERα predominance and its correlation with BDNF levels is still unclear.
doi:10.1016/j.neuroscience.2013.12.023
PMCID: PMC3950408  PMID: 24361177
cortex; estradiol; p75ntr; plasticity; thalamus; truncated TrkB receptor
9.  The non-human primate striatum undergoes marked prolonged remodeling during postnatal development 
We examined the postnatal ontogeny of the striatum in rhesus monkeys (Macaca mulatta) to identify temporal and spatial patterns of histological and chemical maturation. Our goal was to determine whether this forebrain structure is developmentally static or dynamic in postnatal life. Brains from monkeys at 1 day, 1, 4, 6, 9, and 12 months of age (N = 12) and adult monkeys (N = 4) were analyzed. Nissl staining was used to assess striatal volume, cytoarchitecture, and apoptosis. Immunohistochemistry was used to localize and measure substance P (SP), leucine-enkephalin (LENK), tyrosine hydroxylase (TH), and calbindin D28 (CAL) immunoreactivities. Mature brain to body weight ratio was achieved at 4 months of age, and striatal volume increased from ∼1.2 to ∼1.4 cm3 during the first postnatal year. Nissl staining identified, prominently in the caudate nucleus, developmentally persistent discrete cell islands with neuronal densities greater than the surrounding striatal parenchyma (matrix). Losses in neuronal density were observed in island and matrix regions during maturation, and differential developmental programmed cell death was observed in islands and matrix regions. Immunohistochemistry revealed striking changes occurring postnatally in striatal chemical neuroanatomy. At birth, the immature dopaminergic nigrostriatal innervation was characterized by islands enriched in TH-immunoreactive puncta (putative terminals) in the neuropil; TH-enriched islands aligned completely with areas enriched in SP immunoreactivity but low in LENK immunoreactivity. These areas enriched in SP immunoreactivity but low in LENK immunoreactivity were identified as striosome and matrix areas, respectively, because CAL immunoreactivity clearly delineated these territories. SP, LENK, and CAL immunoreactivities appeared as positive neuronal cell bodies, processes, and puncta. The matrix compartment at birth contained relatively low TH-immunoreactive processes and few SP-positive neurons but was densely populated with LENK-immunoreactive neurons. The nucleus accumbens part of the ventral striatum also showed prominent differences in SP, LENK, and CAL immunoreactivities in shell and core territories. During 12 months of postnatal maturation salient changes occurred in neurotransmitter marker localization: TH-positive afferents densely innervated the matrix to exceed levels of immunoreactivity in the striosomes; SP immunoreactivity levels increased in the matrix; and LENK-immunoreactivity levels decreased in the matrix and increased in the striosomes. At 12 months of age, striatal chemoarchitecture was similar qualitatively to adult patterns, but quantitatively different in LENK and SP in caudate, putamen, and nucleus accumbens. This study shows for the first time that the rhesus monkey striatum requires more than 12 months after birth to develop an adult-like pattern of chemical neuroanatomy and that principal neurons within striosomes and matrix have different developmental programs for neuropeptide expression. We conclude that postnatal maturation of the striatal mosaic in primates is not static but, rather, is a protracted and dynamic process that requires many synchronous and compartment-selective changes in afferent innervation and in the expression of genes that regulate neuronal phenotypes.
doi:10.3389/fncel.2014.00294
PMCID: PMC4170103  PMID: 25294985
striatal mosaic; striosome; rhesus monkey; infant; autism; epigenetics; leucine-enkephalin; nucleus accumbens
10.  GNX-4728, a novel small molecule drug inhibitor of mitochondrial permeability transition, is therapeutic in a mouse model of amyotrophic lateral sclerosis 
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder in humans characterized by progressive degeneration of skeletal muscle and motor neurons in spinal cord, brainstem, and cerebral cortex causing skeletal muscle paralysis, respiratory insufficiency, and death. There are no cures or effective treatments for ALS. ALS can be inherited, but most cases are not associated with a family history of the disease. Mitochondria have been implicated in the pathogenesis but definitive proof of causal mechanisms is lacking. Identification of new clinically translatable disease mechanism-based molecular targets and small molecule drug candidates are needed for ALS patients. We tested the hypothesis in an animal model that drug modulation of the mitochondrial permeability transition pore (mPTP) is therapeutic in ALS. A prospective randomized placebo-controlled drug trial was done in a transgenic (tg) mouse model of ALS. We explored GNX-4728 as a therapeutic drug. GNX-4728 inhibits mPTP opening as evidenced by increased mitochondrial calcium retention capacity (CRC) both in vitro and in vivo. Chronic systemic treatment of G37R-human mutant superoxide dismutase-1 (hSOD1) tg mice with GNX-4728 resulted in major therapeutic benefits. GNX-4728 slowed disease progression and significantly improved motor function. The survival of ALS mice was increased significantly by GNX-4728 treatment as evidence by a nearly 2-fold extension of lifespan (360 days–750 days). GNX-4728 protected against motor neuron degeneration and mitochondrial degeneration, attenuated spinal cord inflammation, and preserved neuromuscular junction (NMJ) innervation in the diaphragm in ALS mice. This work demonstrates that a mPTP-acting drug has major disease-modifying efficacy in a preclinical mouse model of ALS and establishes mitochondrial calcium retention, and indirectly the mPTP, as targets for ALS drug development.
doi:10.3389/fncel.2014.00433
PMCID: PMC4271619  PMID: 25565966
motor neuron disease; therapeutics; motoneuron; mitochondrial permeability transition pore; mitochondria; mitochondrial calcium uptake; neuromuscular junction
11.  Adenosine A2A receptor contributes to ischemic brain damage in newborn piglet 
Pharmacologic inactivation or genetic deletion of adenosine A2A receptors protects ischemic neurons in adult animals, but studies in neonatal hypoxia-ischemia (H-I) are inconclusive. The present study in neonatal piglets examined the hypothesis that A2A receptor signaling after reoxygenation from global H-I contributes to injury in highly vulnerable striatal neurons where A2A receptors are enriched. A2A receptor immunoreactivity was detected in striatopallidal neurons. In nonischemic piglets, direct infusion of the selective A2A receptor agonist CGS 21680 through microdialysis probes into putamen increased phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor NR1 subunit and Na+,K+-ATPase selectively at protein kinase A (PKA)-sensitive sites. In ischemic piglets, posttreatment with SCH 58261, a selective A2A receptor antagonist, improved early neurologic recovery and preferentially protected striatopallidal neurons. SCH 58261 selectively inhibited the ischemia-induced phosphorylation of NR1, Na+,K+-ATPase, and cAMP-regulated phosphoprotein 32 KDa (DARPP32) at PKA-sensitive sites at 3 hours of recovery and improved Na+,K+-ATPase activity. SCH 58261 also suppressed ischemia-induced protein nitration and oxidation. Thus, A2A receptor activation during reoxygenation contributes to the loss of a subpopulation of neonatal putamen neurons after H-I. Its toxic signaling may be related to DARPP32-dependent phosphorylation of PKA-sensitive sites on NR1 and Na+,K+-ATPase, thereby augmenting excitotoxicity-induced oxidative stress after reoxygenation.
doi:10.1038/jcbfm.2013.117
PMCID: PMC3790932  PMID: 23860373
adenosine; dopamine; global ischemia; neonatal ischemia; receptors
12.  Aberrant Regulation of DNA Methylation in Amyotrophic Lateral Sclerosis: A New Target of Disease Mechanisms 
Neurotherapeutics  2013;10(4):722-733.
Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. A diagnosis is fatal owing to degeneration of motor neurons in brain and spinal cord that control swallowing, breathing, and movement. ALS can be inherited, but most cases are not associated with a family history of the disease. The mechanisms causing motor neuron death in ALS are still unknown. Given the suspected complex interplay between multiple genes, the environment, metabolism, and lifestyle in the pathogenesis of ALS, we have hypothesized that the mechanisms of disease in ALS involve epigenetic contributions that can drive motor neuron degeneration. DNA methylation is an epigenetic mechanism for gene regulation engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to carbon-5 in cytosine residues in gene regulatory promoter and nonpromoter regions. Recent genome-wide analyses have found differential gene methylation in human ALS. Neuropathologic assessments have revealed that motor neurons in human ALS show significant abnormalities in Dnmt1, Dnmt3a, and 5-methylcytosine. Similar changes are seen in mice with motor neuron degeneration, and Dnmt3a was found abundantly at synapses and in mitochondria. During apoptosis of cultured motor neuron-like cells, Dnmt1 and Dnmt3a protein levels increase, and 5-methylcytosine accumulates. Enforced expression of Dnmt3a, but not Dnmt1, induces degeneration of cultured neurons. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocks apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with small molecules RG108 and procainamide protects motor neurons from excessive DNA methylation and apoptosis in cell culture and in a mouse model of ALS. Thus, motor neurons can engage epigenetic mechanisms to cause their degeneration, involving Dnmts and increased DNA methylation. Aberrant DNA methylation in vulnerable cells is a new direction for discovering mechanisms of ALS pathogenesis that could be relevant to new disease target identification and therapies for ALS.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0205-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-013-0205-6
PMCID: PMC3805862  PMID: 23900692
Chromatin modification; DNA methyltransferase; 5-methylcytosine; mitochondria; motor neuron; RG108.
13.  Mitochondrial pathobiology in ALS 
Amyotrophic lateral sclerosis (ALS) is the third most common human adult-onset neurodegenerative disease. Some forms of ALS are inherited, and disease-causing genes have been identified. Nevertheless, the mechanisms of neurodegeneration in ALS are unresolved. Genetic, biochemical, and morphological analyses of human ALS as well as cell and animal models of ALS reveal that mitochondria could have roles in this neurodegeneration. The varied functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations. Changes occur in mitochondrial respiratory chain enzymes and mitochondrial programmed cell death proteins in ALS. Transgenic mouse models of ALS reveal possible principles governing the biology of neurodegeneration that implicate mitochondria and the mitochondrial permeability transition pore. This paper reviews how mitochondrial pathobiology might contribute to the mechanisms of neurodegeneration in ALS.
doi:10.1007/s10863-011-9395-y
PMCID: PMC4131252  PMID: 22083126
Apoptosis; Cyclophilin D; Mitochondrial permeability transition pore; Motor neuron; Voltage-dependent anion channel
14.  Nuclear Localization of Human Superoxide Dismutase 1 (SOD1) and Mutant SOD1-Specific Disruption of Survival Motor Neuron Protein Complex in Transgenic Amyotrophic Lateral Sclerosis Mice 
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease that causes degeneration of motor neurons and paralysis. Approximately 20% of familial ALS cases have been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene but it is unclear how mutations in the protein result in motor neuron degeneration. Transgenic (tg) mice expressing mutated forms of human SOD1 (hSOD1) develop clinical and pathological features similar to those of ALS. We used tg mice expressing hSOD1-G93A, hSOD1-G37R, and hSOD1-wild type to investigate a new subcellular pathology involving mutant hSOD1 protein prominently localizing to the nuclear compartment and disruption of the architecture of nuclear gems. We developed methods for extracting relatively pure cell nucleus fractions from mouse CNS tissues and demonstrate low nuclear presence of endogenous SOD1 in mouse brain and spinal cord, but prominent nuclear accumulation of hSOD1-G93A, -G37R and -wild type in tg mice. hSOD1 concentrated in nuclei of spinal cord cells, particularly motor neurons, at a young age. The survival motor neuron protein (SMN) complex is disrupted in motor neuron nuclei prior to disease onset in hSOD1-G93A and -G37R mice; age-matched hSOD1-wild type mice did not show SMN disruption despite a nuclear presence. Our data suggest new mechanisms involving hSOD1 accumulation in the cell nucleus and mutant hSOD1-specific perturbations in SMN localization with disruption of the nuclear SMN complex in the ALS model mice and suggest overlap of pathogenic mechanisms with spinal muscular atrophy.
doi:10.1097/NEN.0b013e318244b635
PMCID: PMC3432922  PMID: 22249462
Cajal body; Gemin 1; Nuclear gems; Snurportin; Spinal muscular atrophy
15.  Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS 
Cytosine methylation is an epigenetic modification of DNA catalyzed by DNA methyltransferases. Cytosine methylation of mitochondrial DNA (mtDNA) is believed to have relative underrepresentation; however, possible tissue and cell differences in mtDNA methylation and relationships to neurodegenerative disease have not been examined. We show by immunoblotting that DNA methyltransferase 3A (Dnmt3a) isoform is present in pure mitochondria of adult mouse CNS, skeletal muscle, and testes, and adult human cerebral cortex. Dnmt1 was not detected in adult mouse CNS or skeletal muscle mitochondria but appeared bound to the outer mitochondrial membrane. Immunofluorescence confirmed the mitochondrial localization of Dnmt3a and showed 5-methylcytosine (5mC) immunoreactivity in mitochondria of neurons and skeletal muscle myofibers. DNA pyrosequencing of two loci (D-loop and 16S rRNA gene) and twelve cytosine-phosphate-guanine (CpG) sites in mtDNA directly showed a tissue differential presence of 5mC. Because mitochondria have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but the disease mechanisms are uncertain, we evaluated mitochondrial Dnmt3a and 5mC levels in human superoxide dismutase-1 (SOD1) transgenic mouse models of ALS. Mitochondrial Dnmt3a protein levels were reduced significantly in skeletal muscle and spinal cord at presymptomatic or early disease. Immunofluorescence showed that 5mC immunoreactivity was present in mitochondria of neurons and skeletal myofibers, and 5mC immunoreactivity became aggregated in motor neurons of ALS mice. DNA pyrosequencing revealed significant abnormalities in 16S rRNA gene methylation in ALS mice. Immunofluorescence showed that 5mC immunoreactivity can be sequestered into autophagosomes and that mitophagy was increased and mitochondrial content was decreased in skeletal muscle in ALS mice. This study reveals a tissue-preferential mitochondrial localization of Dnmt3a and presence of cytosine methylation in mtDNA of nervous tissue and skeletal muscle and demonstrates that mtDNA methylation patterns and mitochondrial Dnmt3a levels are abnormal in skeletal muscle and spinal cord of presymptomatic ALS mice, and these abnormalities occur in parallel with loss of myofiber mitochondria.
doi:10.3389/fncel.2013.00279
PMCID: PMC3872319  PMID: 24399935
ALS; DNA pyrosequencing; Dnmt1; Dnmt3a; mitochondrial DNA; 5-methylcytosine; mitochondria; motor neuron
16.  Inhibitory Synaptic Regulation of Motoneurons: A New Target of Disease Mechanisms in Amyotrophic Lateral Sclerosis 
Molecular neurobiology  2011;45(1):30-42.
Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. It causes the degeneration of motoneurons and is fatal due to paralysis, particularly of respiratory muscles. ALS can be inherited, and specific disease-causing genes have been identified, but the mechanisms causing motoneuron death in ALS are not understood. No effective treatments exist for ALS. One well-studied theory of ALS pathogenesis involves faulty RNA editing and abnormal activation of specific glutamate receptors as well as failure of glutamate transport resulting in glutamate excitotoxicity; however, the excitotoxicity theory is challenged by the inability of anti-glutamate drugs to have major disease-modifying effects clinically. Nevertheless, hyperexcitability of upper and lower motoneurons is a feature of human ALS and transgenic (tg) mouse models of ALS. Motoneuron excitability is strongly modulated by synaptic inhibition mediated by presynaptic glycinergic and GABAergic innervations and postsynaptic glycine receptors (GlyR) and GABAA receptors; yet, the integrity of inhibitory systems regulating motoneurons has been understudied in experimental models, despite findings in human ALS suggesting that they may be affected. We have found in tg mice expressing a mutant form of human superoxide dismutase-1 (hSOD1) with a Gly93 → Ala substitution (G93A-hSOD1), causing familial ALS, that subsets of spinal interneurons degenerate. Inhibitory glycinergic innervation of spinal motoneurons becomes deficient before motoneuron degeneration is evident in G93A-hSOD1 mice. Motoneurons in these ALS mice also have insufficient synaptic inhibition as reflected by smaller GlyR currents, smaller GlyR clusters on their plasma membrane, and lower expression of GlyR1α mRNA compared to wild-type motoneurons. In contrast, GABAergic innervation of ALS mouse motoneurons and GABAA receptor function appear normal. Abnormal synaptic inhibition resulting from dysfunction of interneurons and motoneuron GlyRs is a new direction for unveiling mechanisms of ALS pathogenesis that could be relevant to new therapies for ALS.
doi:10.1007/s12035-011-8217-x
PMCID: PMC3530198  PMID: 22072396
Chloride channel; Glutamate receptor; Glycine receptor; Hb9-eGFP; Excitotoxicity; Hyperexcitability; Interneuron; Renshaw cell
17.  An Approach to Experimental Synaptic Pathology Using Green Fluorescent Protein-Transgenic Mice and Gene Knockout Mice to Show Mitochondrial Permeability Transition Pore-Driven Excitotoxicity in Interneurons and Motoneurons 
Toxicologic pathology  2010;39(1):220-233.
Researchers used transgenic mice expressing enhanced-green fluorescent protein (eGFP) driven by either the glycine transporter-2 gene promoter to specifically visualize glycinergic interneurons or the homeobox-9 (Hb9) gene promoter to visualize motoneurons for assessing their vulnerabilities to excitotoxins in vivo. Stereotaxic excitotoxic lesions were made in adult male and female mouse lumbar spinal cord with the specific N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA) and the non-NMDA ion channel glutamate receptor agonist kainic acid (KA). QA and KA induced large-scale degeneration of glycinergic interneurons in spinal cord. Glycinergic interneurons were more sensitive than motoneurons to NMDA receptor-mediated and non-NMDA glutamate receptor-mediated excitotoxicity. Outcome after spinal cord excitotoxicity was gender-dependent, with males showing greater sensitivity than females. Excitotoxic degeneration of spinal interneurons resembled apoptosis, while motoneuron degeneration appeared non-apoptotic. Perikaryal mitochondrial accumulation was antecedent to both NMDA and non-NMDA receptor-mediated excitotoxic stimulation of interneurons and motoneurons. Genetic ablation of cyclophilin D, a regulator of the mitochondrial permeability transition pore (mPTP), protected both interneurons and motoneurons from excitotoxicity. The results demonstrate in adult mouse spinal cord that glycinergic interneurons are more sensitive than motoneurons to excitotoxicity that stimulates mitochondrial accumulation, and that the mPTP has pro-death functions mediating apoptotic and non-apoptotic neuronal degeneration in vivo.
doi:10.1177/0192623310389475
PMCID: PMC3517994  PMID: 21378209
amyotrophic lateral sclerosis; apoptosis; cyclophilin D; excitotoxicity; green fluorescent protein; mitochondrial fission; synaptic pathology
18.  Neuronal Cell Death in Neonatal Hypoxia-Ischemia 
Annals of neurology  2011;69(5):743-758.
Perinatal hypoxic-ischemic encephalopathy (HIE) is a significant cause of mortality and morbidity in infants and young children. Therapeutic opportunities are very limited for neonatal and pediatric HIE. Specific neural systems and populations of cells are selectively vulnerable in HIE; however, the mechanisms of degeneration are unresolved. These mechanisms involve oxidative stress, excitotoxicity, inflammation, and the activation of several different cell death pathways. Decades ago the structural and mechanistic basis of the cellular degeneration in HIE was thought to be necrosis. Subsequently, largely due to advances in cell biology and to experimental animal studies, emphasis has been switched to apoptosis or autophagy mediated by programmed cell death (PCD) mechanisms as important forms of degeneration in HIE. We have conceptualized based on morphological and biochemical data that this degeneration is better classified according to an apoptosis-necrosis cell death continuum and that programmed cell necrosis has prominent contribution in the neurodegeneration of HIE in animal models. It is likely that neonatal HIE evolves through many cell death chreodes influenced by the dynamic injury landscape. The relevant injury mechanisms remain to be determined in human neonatal HIE, though preliminary work suggests a complexity in the cell death mechanisms greater than that anticipated from experimental animal models. The accurate identification of the various cell death chreodes and their mechanisms unfolding within the immature brain matrix could provide fresh insight for developing meaningful therapies for neonatal and pediatric HIE.
doi:10.1002/ana.22419
PMCID: PMC4000313  PMID: 21520238
19.  Motoneuron subtypes show specificity in glycine receptor channel abnormalities in a transgenic mouse model of amyotrophic lateral sclerosis 
Channels  2011;5(4):299-303.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motoneurons. Recently we studied glycine receptors (GlyRs) in motoneurons in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). Living motoneurons in dissociated spinal cord cultures were identified by using transgenic mice expressing eGFP driven by the Hb9 promoter. We showed that GlyR-mediated currents were reduced in large-sized (diameter >28 µm) Hb9-eGFP+ motoneurons from G93A-SOD1 embryonic mice. Here we analyze GlyR currents in a morphologically distinct subgroup of medium-sized (diameter 10–28 µm) Hb9-eGFP+ motoneurons, presumably gamma or slow-type alpha motoneurons. We find that glycine-induced current densities were not altered in medium-sized G93A-SOD1 motoneurons. No significant differences in glycinergic mIPSCs were observed between G93A-SOD1 and control medium-sized motoneurons. These results indicate that GlyR deficiency early in the disease process of ALS is specific for large alpha motoneurons.
doi:10.4161/chan.5.4.16206
PMCID: PMC3225730  PMID: 21558795
Hb9-eGFP; mutant SOD1; motoneuron culture; patch clamp; mIPSC; gamma motoneuron; alpha motoneuron
20.  Skeletal Muscle DNA Damage Precedes Spinal Motor Neuron DNA Damage in a Mouse Model of Spinal Muscular Atrophy (SMA) 
PLoS ONE  2014;9(3):e93329.
Spinal Muscular Atrophy (SMA) is a hereditary childhood disease that causes paralysis by progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. The mechanisms by which lack of SMN causes SMA pathology are not known, making it very difficult to develop effective therapies. We investigated whether DNA damage is a perinatal pathological event in SMA, and whether DNA damage and cell death first occur in skeletal muscle or spinal cord of SMA mice. We used a mouse model of severe SMA to ascertain the extent of cell death and DNA damage throughout the body of prenatal and newborn mice. SMA mice at birth (postnatal day 0) exhibited internucleosomal fragmentation in genomic DNA from hindlimb skeletal muscle, but not in genomic DNA from spinal cord. SMA mice at postnatal day 5, compared with littermate controls, exhibited increased apoptotic cell death profiles in skeletal muscle, by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling, and electron microscopy. SMA mice had no increased cell death, no loss of choline acetyl transferase (ChAT)-positive motor neurons, and no overt pathology in the ventral horn of the spinal cord. At embryonic days 13 and 15.5, SMA mice did not exhibit statistically significant increases in cell death profiles in spinal cord or skeletal muscle. Motor neuron numbers in the ventral horn, as identified by ChAT immunoreactivity, were comparable in SMA mice and control littermates at embryonic day 15.5 and postnatal day 5. These observations demonstrate that in SMA, disease in skeletal muscle emerges before pathology in spinal cord, including loss of motor neurons. Overall, this work identifies DNA damage and cell death in skeletal muscle as therapeutic targets for SMA.
doi:10.1371/journal.pone.0093329
PMCID: PMC3965546  PMID: 24667816
21.  FAILURE TO COMPLETE APOPTOSIS FOLLOWING NEONATAL HYPOXIA-ISCHEMIA MANIFESTS AS ‘CONTINUUM’ PHENOTYPE OF CELL DEATH AND OCCURS WITH MULTIPLE MANEFESTATIONS OF MITOCHONDRIAL DYSFUNCTION IN RODENT FOREBRAIN 
Neuroscience  2007;149(4):822-833.
Controversy surrounds proper classification of neurodegeneration occurring acutely following neonatal hypoxia-ischemia. By ultrastructural classification, in the first 24 hours after neonatal hypoxia-ischemia in the p7 rat, the majority of striatal cells die having both apoptotic and necrotic features. There is formation of a functional apoptosome, and activation of caspases 9 and 3 occurring simultaneously with loss of structurally intact mitochondria to 34.7±25% and loss of mitochondrial cytochrome C oxidase activity to 34.7±12.7% of control levels by 3 hours after hypoxia-ischemia. There is also loss of the mitochondrial motor protein, kinesin. This combination of activation of apoptosis pathways simultaneous with significant mitochondrial dysfunction may cause incomplete packaging of nuclear and cytoplasmic contents and a hybrid of necrotic and apoptotic features. Evidence for an intermediate biochemistry of cell death including expression of the 17 kDa isoform of caspase-3 in dying neurons lacking a classic apoptotic morphology and degradation of the neuronal cytoskeletal protein spectrin by caspase-3 and calcium-activated calpains yielding 120 kDa and 145/150 kDa fragments, respectively, is also found. In summary, neonatal hypoxia-ischemia triggers apoptotic cascades, and simultaneously causes mitochondrial structural and functional failure. The presence of a ‘continuum phenotype’ of cell death that varies on a cell-by-cell basis suggests that the phenotype of cell death is dependent on the energy available to drive the apoptotic pathways to completion.
doi:10.1016/j.neuroscience.2007.06.060
PMCID: PMC3947608  PMID: 17961929
Continuum cell death; neonatal brain injury; mitochondria; neurodegeneration; ultrastructure
22.  Epigenetic Regulation of Motor Neuron Cell Death Through DNA Methylation 
DNA methylation is an epigenetic mechanism for gene silencing engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to cytosine residues in gene regulatory regions. It is unknown if aberrant DNA methylation can cause neurodegeneration. We tested the hypothesis that Dnmts can mediate neuronal cell death. Enforced expression of Dnmt3a induced degeneration of cultured NSC34 cells. During apoptosis of NSC34 cells induced by camptothecin, levels of Dnmt1 and Dnmt3a increased five-fold and two-fold, respectively, and 5-methylcytosine accumulated in nuclei. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocked apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with RG108 and procainamide protected cultured neurons from excessive DNA methylation and apoptosis. In vivo, Dnmt1 and Dnmt3a are expressed differentially during mouse brain and spinal cord maturation and in adulthood when Dnmt3a is abundant in synapses and mitochondria. Dnmt1 and Dnmt3a are expressed in motor neurons of adult mouse spinal cord, and, during their apoptosis induced by sciatic nerve avulsion, nuclear and cytoplasmic 5-methylcytosine immunoreactivity, Dnmt3a protein levels, and Dnmt enzyme activity increased preapoptotically. Inhibition of Dnmts with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice. In human amyotrophic lateral sclerosis (ALS), motor neurons showed changes in Dnmt1, Dnmt3a, and 5-methylcytosine similar to experimental models. Thus, motor neurons can engage epigenetic mechanisms to drive apoptosis, involving Dnmt upregulation and increased DNA methylation. These cellular mechanisms could be relevant to human ALS pathobiology and disease treatment.
doi:10.1523/JNEUROSCI.1639-11.2011
PMCID: PMC3238138  PMID: 22090490
Apoptosis; axotomy; epigenetics; 5-methylcytosine; DNA methylation; Hb9-eGFP; mitochondria; NSC34 cells; DNA methyltransferase; Dnmt-GFP; RG108
23.  The Olfactory Bulb in Newborn Piglet Is a Reservoir of Neural Stem and Progenitor Cells 
PLoS ONE  2013;8(11):e81105.
The olfactory bulb (OB) periventricular zone is an extension of the forebrain subventricular zone (SVZ) and thus is a source of neuroprogenitor cells and neural stem cells. While considerable information is available on the SVZ-OB neural stem cell (NSC)/neuroprogenitor cell (NPC) niche in rodents, less work has been done on this system in large animals. The newborn piglet is used as a preclinical translational model of neonatal hypoxic-ischemic brain damage, but information about the endogenous sources of NSCs/NPCs in piglet is needed to implement endogenous or autologous cell-based therapies in this model. We characterized NSC/NPC niches in piglet forebrain and OB-SVZ using western blotting, histological, and cell culture methods. Immunoblotting revealed nestin, a NSC/NPC marker, in forebrain-SVZ and OB-SVZ in newborn piglet. Several progenitor or newborn neuron markers, including Dlx2, musashi, doublecortin, and polysialated neural cell adhesion molecule were also detected in OB-SVZ by immunoblotting. Immunohistochemistry confirmed the presence of nestin, musashi, and doublecortin in forebrain-SVZ and OB-SVZ. Bromodeoxyuridine (BrdU) labeling showed that the forebrain-SVZ and OB-SVZ accumulate newly replicated cells. BrdU-positive cells were immunolabeled for astroglial, oligodendroglial, and neuronal markers. A lateral migratory pathway for newly born neuron migration to primary olfactory cortex was revealed by BrdU labeling and co-labeling for doublecortin and class III β tubulin. Isolated and cultured forebrain-SVZ and OB-SVZ cells from newborn piglet had the capacity to generate numerous neurospheres. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Neurosphere-derived cells differentiated into neurons, astrocytes, and oligodendrocytes and were amenable to permanent genetic tagging with lentivirus encoding green fluorescent protein. We conclude that the piglet OB-SVZ is a reservoir of NSCs and NPCs suitable to use in autologous cell therapy in preclinical models of neonatal/pediatric brain injury.
doi:10.1371/journal.pone.0081105
PMCID: PMC3836747  PMID: 24278384
24.  Glycine Receptor Channels in Spinal Motoneurons are Abnormal in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis 
Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). In this study we have examined, using whole-cell patch clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing eGFP driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP+ neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP+ motoneurons prepared from G93A-SOD1 embryos and from their non-transgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell RT-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.
doi:10.1523/JNEUROSCI.2475-10.2011
PMCID: PMC3081715  PMID: 21414903
Hb9-eGFP; mutant SOD1; motoneuron culture; GABAA receptor; patch clamp; single-cell RT-PCR
25.  The Mitochondrial Permeability Transition Pore Regulates Nitric Oxide-Mediated Apoptosis of Neurons Induced by Target Deprivation 
Ablation of mouse occipital cortex induces precisely timed and uniform p53-modulated and Bax-dependent apoptosis of thalamocortical projection neurons in the dorsal lateral geniculate nucleus (LGN) by 7 days postlesion. We tested the hypothesis that this neuronal apoptosis is initiated by oxidative stress and the mitochondrial permeability transition pore (mPTP). Pre-apoptotic LGN neurons accumulate mitochondria, Zn2+ and Ca2+, and generate higher levels of reactive oxygen species (ROS), including superoxide, nitric oxide (NO) and peroxynitrite, than LGN neurons with an intact cortical target. Pre-apoptosis of LGN neurons is associated with increased formation of protein carbonyls, protein nitration, and protein S-nitrosylation. Genetic deletion of nitric oxide synthase 1 (nos1) and inhibition of NOS1 with nitroindazole protected LGN neurons from apoptosis, revealing NO as a mediator. Putative components of the mPTP are expressed in mouse LGN, including the voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (CyPD). Nitration of CyPD and ANT in LGN mitochondria occurs by 2 days after cortical injury. Chemical cross-linking showed that LGN neuron pre-apoptosis is associated with formation of CyPD and VDAC oligomers, consistent with mPTP formation. Mice without CyPD are rescued from neuron apoptosis as are mice treated with the mPTP inhibitors TRO-19622 and TAT-Bcl-XL-BH4. Manipulation of the mPTP markedly attenuated the early pre-apoptotic production of reactive oxygen/nitrogen species in target-deprived neurons. Our results demonstrate in adult mouse brain neurons that the mPTP functions to enhance ROS production and the mPTP and NO trigger apoptosis; thus, the mPTP is a target for neuroprotection in vivo.
doi:10.1523/JNEUROSCI.2225-10.2010
PMCID: PMC3078575  PMID: 21209222
Alzheimer’s disease; amyotrophic lateral sclerosis; axotomy; cell death; DNA damage; Parkinson’s disease; target deprivation; traumatic brain injury

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