Increased EID1 nuclear translocation impairs synaptic plasticity and memory function associated with pathogenesis of Alzheimer's disease.

Though loss of function in CBP/p300, a family of CREB-binding proteins, has been causally associated with a variety of human neurological disorders, such as Rubinstein-Taybi syndrome, Huntington's disease and drug addiction, the role of EP300 interacting inhibitor of differentiation 1 (EID1), a CBP/p300 inhibitory protein, in modulating neurological functions remains completely unknown. Through the examination of EID1 expression and cellular distribution, we discovered that there is a significant increase of EID1 nuclear translocation in the cortical neurons of Alzheimer's disease (AD) patient brains compared to that of control brains. To study the potential effects of EID1 on neurological functions associated with learning and memory, we generated a transgenic mouse model with a neuron-specific expression of human EID1 gene in the brain. Overexpression of EID1 led to an increase in its nuclear localization in neurons mimicking that seen in human AD brains. The transgenic mice had a disrupted neurofilament organization and increase of astrogliosis in the cortex and hippocampus. Furthermore, we demonstrated that overexpression of EID1 reduced hippocampal long-term potentiation and impaired spatial learning and memory function in the transgenic mice. Our results indicated that the negative effects of extra nuclear EID1 in transgenic mouse brains are likely due to its inhibitory function on CBP/p300 mediated histone and p53 acetylation, thus affecting the expression of downstream genes involved in the maintenance of neuronal structure and function. Together, our data raise the possibility that alteration of EID1 expression, particularly the increase of EID1 nuclear localization that inhibits CBP/p300 activity in neuronal cells, may play an important role in AD pathogenesis.

Description of distributed features of the nestin-containing cells in brains of adult mice: a potential source of neural precursor cells.

The distribution of neural precursor cells (NPCs) in adult mice brain has so far not been described. Therefore, we investigated the distribution of NPCs by analyzing the nestin-containing cells (NCCs) in distinct brain regions of adult nestin second-intron enhancer-controlled LacZ reporter transgenic mice through LacZ staining. Results showed that NCCs existed in various regions of adult mouse brain. In cerebellum, the greatest number of NCCs existed in cortex of the simple lobule, followed by cortex of the cerebellar lobule. In olfactory bulb, NCCs were most numerous in the granular cell layer, followed by the mitral cell layer and the internal plexiform, glomerular, and external plexiform layers. In brain nuclei (nu), NCCs were most numerous in the marginal nu, followed by the brainstem and diencephalon nu. NCCs in sensory nu of brainstem were more numerous than in motor nu, and NCCs in the dorsal of sensory nu were more numerous than in the ventral part. In brain ventricle systems, NCCs were largely distributed in the center of and external to the lateral ventricle, the inferior part of the third ventricle, the dorsal and inferior parts of the fourth ventricle, and the gray matter around the cerebral aqueduct. NCCs in the left vs. right brain were not significantly different. These data collectively indicate that NCCs were extensively distributed in the cerebellum and olfactory bulb, the partial nu of the marginal system, the partial brain nu adjacent to brain ventricle systems, the subependymal zone, and the cerebral cortex around the marginal lobe and were a potential source of NPCs.

Caffeine protects against MPTP-induced blood-brain barrier dysfunction in mouse striatum.

The blood-brain barrier (BBB) is important physiologically. Pathologically, BBB disruption has been implicated in a wide spectrum of neurological disorders including Parkinson's disease (PD). Recent studies indicate that caffeine is protective against PD, but by poorly understood mechanisms. Using a MPTP neurotoxin model of PD we tested the hypothesis that the protective actions of caffeine were because of, at least in part, preventing MPTP-induced BBB dysfunction. FVB mice were pre-treated with caffeine (10 mg/kg, i.p.) or saline for 7 days prior to initiation of neurotoxin treatments; during the 7 days of neurotoxin treatment, caffeine or saline continued to be administered 10 min before each dose of MPTP (20 mg/kg, i.p.). Striatum (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, tight junction protein expression levels, integrity of dopaminergic neurons, and activation of astrocytes and microglia using immunostaining, immunoblotting and real-time PCR techniques. We found that caffeine blocked MPTP-induced decreases in numbers of tyrosine hydroxylase-positive dopaminergic neurons, increases in leakage of Evan's blue dye and FITC-albumin in striatum but not in cerebral cortex or hippocampus, decreases in levels of the tight junction proteins occludin and ZO-1, and increases in reactive gliosis. Our results suggest that caffeine might protect against PD and PD-like features in animal models, in part, by stabilizing the BBB.

Nestin-positive cells in the spinal cord: a potential source of neural stem cells.

Some literatures have reported neural precursor cells (NPCs) exist in spinal cord of adult mammal, however, the NPCs distribution feature in spinal cord of adult mice so far is not described in detail. In order to observe and compare the distribution feature of NPCs in various spinal cord regions of adult mice, to research a potential source of neural stem cells (NSCs), we obtained NPCs distribution feature by analyzing the distribution of the nestin-containing cells (NCCs) in spinal cord of adult nestin second-intron enhancer controlled LacZ reporter transgenic mice (pNes-Tg) with LacZ staining and positive cell quantification. The results showed that: NCCs were observed in various regions of spinal cord of adult mice, but amount of NCCs was different in distinct region, the rank order of NCCs amount in various spinal cord regions was dorsal horn region greater than central canal greater than the ventral and lateral horn. NCCs in dorsal horn region mainly distributed in substantia gelatinosa, NCCs in central canal mainly distributed in ependymal zone, on the contrary, NCCs in ventral, lateral horn, medullae, nucleus regions of spinal cord were comparatively less. The rank order of NCCs amount in various spinal cord segments was cervical segment greater than lumbar sacral segment greater than thoracic segment. There was no significantly difference between NCCs amount in the left and right sides, and within cervical 1-7, thoracic 1-12, lumbar 1-5, sacral segment of spinal cord in adult mice. These data collectively indicate that NPCs extensively distribute in various regions of spinal cord of adult mice, especially in substantia gelatinosa and ependymal zone. NPCs in cervical segment are abundant, NPCs in thoracic segment are the least while compared the different spinal cord segment, the NPCs in various regions of spinal cord of adult mice are a potential source of NSCs.

Degeneration of vagal efferent axons and terminals in cardiac ganglia of aged rats.

Baroreflex control of the heart rate is significantly reduced during aging. However, neural mechanisms that underlie such a functional reduction are not fully understood. We injected the tracer DiI into the left nucleus ambiguus (NA), then used confocal microscopy and a Neurolucida Digitization System to examine qualitatively and quantitatively vagal efferent projections to cardiac ganglia of young adult (5-6 months) and aged (24-25 months) rats (Sprague Dawley). Fluoro-Gold was injected intraperitoneally to counterstain cardiac ganglionic principal neurons (PNs). In aged, as in young rats, NA axons projected to all cardiac ganglia and formed numerous basket endings around PNs in the hearts. However, significant structural changes were found in aged rats compared with young rats. Vagal efferent axons contained abnormally swollen axonal segments and exhibited reduced or even absent synaptic-like terminals around PNs, such that the numbers of vagal fibers and basket endings around PNs were substantially reduced (P < 0.01). Furthermore, synaptic-like varicose contacts of vagal cardiac axons with PNs were significantly reduced by approximately 50% (P < 0.01). These findings suggest that vagal efferents continue to maintain homeostatic control over the heart during aging. However, the marked morphological reorganization of vagal efferent axons and terminals in cardiac ganglia may represent the structural substrate for reduced vagal control of the heart rate and attenuated baroreflex function during aging.

Temporal response of neural progenitor cells to disease onset and progression in amyotrophic lateral sclerosis-like transgenic mice.

Regenerative medicine through neural stem cells (NSCs) or neural progenitor cells (NPCs) has been proposed as an alterative avenue for restoring neurological dysfunction in amyotrophic lateral sclerosis (ALS). It is critical to understand the organization and distribution of endogenous adult NPCs in response to motor neuron degeneration before regenerative medicine can be applied for ALS therapy. For this reason, we analyzed the temporal response of NPCs to motor neuron degeneration in the spinal cord and brain using nestin enhancer-driven LacZ reporter transgenic mice (pNes-Tg mice, control) and bi-transgenic mice containing both the nestin enhancer-driven LacZ reporter gene and mutant G93A-SOD1 gene (Bi-Tg mice). We observed an increase of NPCs in the dorsal horns of the spinal cord at the disease onset and progression stages in the Bi-Tg mice compared with that of age-matched pNes-Tg control mice. In contrast, an increase of NPCs in the ventral horns was detected at the disease progression stage. On the other hand, an increase of NPCs in the motor cortex at the disease-onset stage, but not at the disease progression stage, was detected. Furthermore, a decrease of NPCs in the lateral ventricle at the disease progression stage was observed, whereas no difference in the number of NPCs in the hippocampus was detected at the disease onset and progression stages. Some of the NPCs differentiate into neuron-like cells in response to motor neuron degeneration. The organization and distribution of endogenous adult NPCs in the ALS-like transgenic mice at the disease onset and progression stages provide fundamental bases for consideration of regenerative therapy of ALS by increasing de novo neurogenesis.

VEGF-induced activation of the PI3-K/Akt pathway reduces mutant SOD1-mediated motor neuron cell death.

The increased oxidative stress induced by mutant SOD1 is associated with motor neuron degeneration in both human ALS and transgenic mice expressing mutant SOD1. Vascular endothelial growth factor (VEGF) is neurotrophic and also protects from hypoxia-induced neuronal injury. The potential role of VEGF in preventing mutant SOD1-mediated motor neuron cell death was examined using a mouse NSC34 motor neuron-like cell culture system. Infection with adenovirus containing mutant G93A-SOD1, but not vector control or wild-type SOD1, increased cellular oxidative stress and motor neuron-like cell death. However, NSC34 cells pretreated with VEGF displayed a dose-dependent resistance to oxidative damage from hydrogen peroxide, TNF-alpha, and mutant G93A-SOD1. VEGF activated both PI3-K and MAPK activities in mouse NSC34 motor neuron-like cells. Pharmacological inhibitors and constitutively active as well as dominant negative mutants of MAPK and PI3-K revealed that the protective effects of VEGF were mediated via the PI3-K activity, and that MAPK activation was not associated with NSC34 cell survival. Furthermore, VEGF-induced downstream Akt activation promoted motor neuron-like NSC34 cell survival in the presence of mutant G93A-SOD1. Thus, VEGF protected mouse NSC34 motor neuron-like cell death from mutant G93A-SOD1 effects via PI3-K/Akt activation.

GABA(B) receptor activation inhibits neuronal excitability and spatial learning in the entorhinal cortex by activating TREK-2 K+ channels.

The entorhinal cortex (EC) is regarded as the gateway to the hippocampus and thus is essential for learning and memory. Whereas the EC expresses a high density of GABA(B) receptors, the functions of these receptors in this region remain unexplored. Here, we examined the effects of GABA(B) receptor activation on neuronal excitability in the EC and spatial learning. Application of baclofen, a specific GABA(B) receptor agonist, inhibited significantly neuronal excitability in the EC. GABA(B) receptor-mediated inhibition in the EC was mediated via activating TREK-2, a type of two-pore domain K(+) channels, and required the functions of inhibitory G proteins and protein kinase A pathway. Depression of neuronal excitability in the EC underlies GABA(B) receptor-mediated inhibition of spatial learning as assessed by Morris water maze. Our study indicates that GABA(B) receptors exert a tight control over spatial learning by modulating neuronal excitability in the EC.

A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation.

BACKGROUND: Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells. RESULTS: RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis. CONCLUSION: Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

Neurogenic responses to amyloid-beta plaques in the brain of Alzheimer's disease-like transgenic (pPDGF-APPSw,Ind) mice.

Formation and accumulation of amyloid-beta (A beta) plaques are associated with declined memory and other neurocognitive function in Alzheimer's disease (AD) patients. However, the effects of A beta plaques on neural progenitor cells (NPCs) and neurogenesis from NPCs remain largely unknown. The existing data on neurogenesis in AD patients and AD-like animal models remain controversial. For this reason, we utilized the nestin second-intron enhancer controlled LacZ (pNes-LacZ) reporter transgenic mice (pNes-Tg) and Bi-transgenic mice (Bi-Tg) containing both pPDGF-APPSw,Ind and pNes-LacZ transgenes to investigate the effects of A beta plaques on neurogenesis in the hippocampus and other brain regions of the AD-like mice. We chose transgenic mice at 2, 8 and 12 months of age, corresponding to the stages of A beta plaque free, plaque onset and plaque progression to analyze the effects of A beta plaques on the distribution and de novo neurogenesis of (from) NPCs. We demonstrated a slight increase in the number of NPCs in the hippocampal regions at the A beta plaque free stage, while a significant decrease in the number of NPCs at A beta plaque onset and progression stages. On the other hand, we showed that A beta plaques increase neurogenesis, but not gliogenesis from post-mitotic NPCs in the hippocampus of Bi-Tg mice compared with age-matched control pNes-Tg mice. The neurogenic responses of NPCs to A beta plaques suggest that experimental approaches to promote de novo neurogenesis may potentially improve neurocognitive function and provide an effective therapy for AD.

Structural remodeling of nucleus ambiguus projections to cardiac ganglia following chronic intermittent hypoxia in C57BL/6J mice.

The baroreflex control of heart rate (HR) is reduced following chronic intermittent hypoxia (CIH). Since the nucleus ambiguus (NA) plays a key role in baroreflex control of HR, we examined whether CIH remodels vagal efferent projections to cardiac ganglia. C57BL/6J mice (3-4 months of age) were exposed to either room air (RA) or CIH for 3 months. Confocal microscopy was used to examine NA axons and terminals in cardiac ganglia following Fluoro-Gold (FG) injections to label cardiac ganglia, and microinjections of tracer DiI into the left NA to anterogradely label vagal efferents. We found that: 1) Cardiac ganglia were widely distributed on the dorsal surface of the atria. Although the total number of cardiac ganglia did not differ between RA and CIH mice, the size of ganglia and the somatic area of cardiac principal neurons (PNs) were significantly decreased (P < 0.01), and the size of the PN nuclei was increased following CIH (P < 0.01). 2) NA axons entered cardiac ganglia and innervated PNs with dense basket endings in both RA and CIH mice, and the percentage of innervated PNs was similar (RA: 50 +/- 1.0%; CIH: 49 +/- 1.0%; P > 0.10). In CIH mice, however, swollen cardiac axons and terminals without close contacts to PNs were found. Furthermore, varicose endings around PNs appeared swollen and the axonal varicose area around PNs was almost doubled in size (CIH: 163.1 +/- 6.4 microm(2); RA: 88 +/- 3.9 microm(2), P < 0.01). Thus, CIH significantly altered the structure of cardiac ganglia and resulted in reorganized vagal efferent projections to cardiac ganglia. Such remodeling of cardiac ganglia and vagal efferent projections provides new insight into the effects of CIH on the brain-heart circuitry of C57BL/6J mice.

Chronic intermittent hypoxia impairs baroreflex control of heart rate but enhances heart rate responses to vagal efferent stimulation in anesthetized mice.

Chronic intermittent hypoxia (CIH) leads to increased sympathetic nerve activity and arterial hypertension. In this study, we tested the hypothesis that CIH impairs baroreflex (BR) control of heart rate (HR) in mice, and that decreased cardiac chronotropic responsiveness to vagal efferent activity contributes to such impairment. C57BL/6J mice were exposed to either room air (RA) or CIH (6-min alternations of 21% O(2) and 5.7% O(2), 12 h/day) for 90 days. After the treatment period, mice were anesthetized (Avertin) and arterial blood pressure (ABP) was measured from the femoral artery. Mean ABP (MABP) was significantly increased in mice exposed to CIH (98.7 +/- 2.5 vs. RA: 78.9 +/- 1.4 mmHg, P < 0.001). CIH increased HR significantly (584.7 +/- 8.9 beats/min; RA: 518.2 +/- 17.9 beats/min, P < 0.05). Sustained infusion of phenylephrine (PE) at different doses (0.1-0.4 microg/min) significantly increased MABP in both CIH and RA mice, but the ABP-mediated decreases in HR were significantly attenuated in mice exposed to CIH (P < 0.001). In contrast, decreases in HR in response to electrical stimulation of the left vagus nerve (30 microA, 2-ms pulses) were significantly enhanced in mice exposed to CIH compared with RA mice at low frequencies. We conclude that CIH elicits a sustained impairment of baroreflex control of HR in mice. The blunted BR-mediated bradycardia occurs despite enhanced cardiac chronotropic responsiveness to vagal efferent stimulation. This suggests that an afferent and/or a central defect is responsible for the baroreflex impairment following CIH.

Manganese superoxide dismutase protects mouse cortical neurons from chronic intermittent hypoxia-mediated oxidative damage.

Obstructive sleep apnea (OSA) syndrome has been recognized as a highly prevalent public health problem and is associated with major neurobehavioral morbidity. Chronic intermittent hypoxia (CIH), a major pathological component of OSA, increases oxidative damage to the brain cortex and decreases neurocognitive function in rodent models resembling human OSA. We employed in vitro and in vivo approaches to identify the specific phases and subcellular compartments in which enhanced reactive oxygen species (ROS) are generated during CIH. In addition, we utilized the cell culture and animal models to analyze the consequences of enhanced production of ROS on cortical neuronal cell damage and neurocognitive dysfunction. In a primary cortical neuron culture system, we demonstrated that the transition phase from hypoxia to normoxia (NOX) during CIH generates more ROS than the transition phase from NOX to hypoxia or hypoxia alone, all of which generate more ROS than NOX. Using selective inhibitors of the major pathways underlying ROS generation in the cell membrane, cytosol, and mitochondria, we showed that the mitochondria are the predominant source of enhanced ROS generation during CIH in mouse cortical neuronal cells. Furthermore, in both cell culture and transgenic mice, we demonstrated that overexpression of MnSOD-decreased CIH-mediated cortical neuronal apoptosis, and reduced spatial learning deficits measured with the Morris water maze assay. Together, the data from the in vitro and in vivo experiments indicate that CIH-mediated mitochondrial oxidative stress may play a major role in the neuronal cell loss and neurocognitive dysfunction in OSA. Thus, therapeutic strategies aiming at reducing ROS generation from mitochondria may improve the neurobehavioral morbidity in OSA.

Motor neuron degeneration promotes neural progenitor cell proliferation, migration, and neurogenesis in the spinal cords of amyotrophic lateral sclerosis mice.

The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter-controlled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by bromodeoxyurindine incorporation and LacZ staining was restricted to the ependymal zone surrounding the central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALS-like mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS.

Early response of endogenous adult neural progenitor cells to acute spinal cord injury in mice.

Adult neural progenitor cells (NPCs) are an attractive source for functional replacement in neurodegenerative diseases and traumatic injury to the central nervous system (CNS). It has been shown that transplantation of neural stem cells or NPCs into the lesioned region partially restores CNS function. However, the capacity of endogenous NPCs in replacement of neuronal cell loss and functional recovery of spinal cord injury (SCI) is apparently poor. Furthermore, the temporal and spatial response of endogenous adult NPCs to SCI remains largely undefined. To this end, we have analyzed the early organization, distribution, and potential function of NPCs in response to SCI, using nestin enhancer (promoter) controlled LacZ reporter transgenic mice. We showed that there was an increase of NPC proliferation, migration, and neurogenesis in adult spinal cord after traumatic compression SCI. The proliferation of NPCs detected by 5-bromodeoxyuridine incorporation and LacZ staining was restricted to the ependymal zone (EZ) of the central canal. During acute SCI, NPCs in the EZ of the central canal migrated vigorously toward the dorsal direction, where the compression lesion is generated. The optimal NPC migration occurred in the adjacent region close to the epicenter. More significantly, there was an increased de novo neurogenesis from NPCs 24 hours after SCI. The enhanced proliferation, migration, and neurogenesis of (from) endogenous NPCs in the adult spinal cord in response to SCI suggest a potential role for NPCs in attempting to restore SCI-mediated neuronal dysfunction.

Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease-like mice.

Research reports on de novo neurogenesis, particularly dopaminergic (DA) neurogenesis in the adult mammalian substantia nigra (SN), remain very controversial. For this reason, we used the nestin second intron enhancer-controlled LacZ reporter transgenic mouse model coupled with the 1-methyl-4-phyenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion system to investigate whether there are neurogenesis and DA neurogenesis in the SN of the adult normal and Parkinson's disease (PD)-like mice. First, we demonstrated the presence of neural progenitor cells (NPCs), basal levels of neurogenesis, and DA neurogenesis in the normal adult mouse SN. Second, we showed that there is not only a significant increase in the number of NPCs but also a dramatic increase of neurogenesis from the NPCs in the SN and the midline region adjacent to the SN of the PD-like mice compared with that of normal controls. More importantly, we also demonstrated that there is an increase of DA neurogenesis in the SN of the MPTP-lesioned mice. Third, we showed that the increased DA neurogenesis in the MPTP-lesioned mice was derived from the NPCs and 5-bromodeoxyuridine-positive cells, suggesting that multiple stem cell lineages may contribute to the enhanced neurogenesis in the adult SN. Taken together, these results establish that there are basal levels, albeit low, and increased levels of de novo neurogenesis and DA neurogenesis in the SN of the adult normal and PD-like mice, respectively. The increased NPCs in the MPTP-lesioned mice further suggest that experimental approaches to promote de novo neurogenesis may provide an effective therapy for PD by functional replacement of degenerated DA neurons.

Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SOD1-mediated motor neuron cell death and increase amyotrophic lateral sclerosis-like transgenic mouse survival.

The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients. In an in vitro cell culture system, we demonstrated that infection of mouse NSC-34 motor neuron-like cells with adenovirus containing mutant G93A-SOD1 gene increased cellular oxidative stress, mitochondrial dysfunction, cytochrome c release and motor neuron cell death. Cells pretreated with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and synergistically exacerbated motor neuron-like cell death with mutant G93A-SOD1 but not with wild-type SOD1. Similarly, overexpression of mitochondrial antioxidative genes, MnSOD and GPX4 by stable transfection significantly increased NSC-34 motor neuron-like cell resistance to mutant SOD1. Pre-incubation of cells with spin trapping molecule, 5',5'-dimethylpryrroline-N-oxide (DMPO), prevented mutant SOD1-mediated mitochondrial dysfunction and cell death. Furthermore, treatment of mutant G93A-SOD1 transgenic mice with DMPO significantly delayed paralysis and increased survival. These findings suggest a causal relationship between enhanced oxidative stress and mutant SOD1-mediated motor neuron degeneration, considering that enhanced oxygen free radical production results from the SOD1 structural alterations. Molecular approaches aimed at increasing mitochondrial antioxidative activity or effectively blocking oxidative stress propagation can be potentially useful in the clinical management of human ALS disease.

Intermittent hypoxia is associated with oxidative stress and spatial learning deficits in the rat.

In the adult rat, exposure to intermittent hypoxia (IH), such as occurs in sleep-disordered breathing, is associated with neurobehavioral impairments and increased apoptosis in the hippocampal CA1 region and cortex. We hypothesized that the episodic hypoxic-reoxygenation cycles of IH would induce oxidant stress, and the latter may underlie the IH-associated spatial learning and retention deficits. Adult male rats were therefore exposed to IH (90-second alternations of 10% oxygen and 21% oxygen) or room air (RA) for 7 days, and received twice-daily injections of either 3 mg/kg of the antioxidant PNU-101033E (PNU) or vehicle (V). Rats were then trained in a standard place-training task in the water maze. V-IH displayed significant impairments of spatial learning in the water maze, which were attenuated by PNU-101033E. Post hoc analyses further revealed that V-IH had significantly longer latencies and pathlengths to locate the hidden platform than PNU-IH, V-RA, or PNU-RA, indicating that PNU-101033E treatment reduced the behavioral impairments associated with IH. In addition, treatment with PNU-101033E markedly attenuated the increase in lipid peroxidation, and isoprostane concentrations associated with exposure to IH. Collectively, these findings indicate that the IH exposure is associated with increased oxidative stress, which is likely to play an important role in the behavioral impairments observed in a rodent model of sleep-disordered breathing.

Increased susceptibility to intermittent hypoxia in aging rats: changes in proteasomal activity, neuronal apoptosis and spatial function.

Obstructive sleep apnea (OSA) is a frequent medical condition characterized by intermittent hypoxia (IH) during sleep, and is associated with neurodegenerative changes in several brain regions along with learning deficits. We hypothesized that aging rats exposed to IH during sleep would be particularly susceptible. Young (3-4 months) and aging (20-22 months) Sprague-Dawley rats were therefore exposed to either room air or IH for 14 days. Learning and memory was assessed with a standard place-training version of the Morris water maze. Aging rats exposed to room air (RA) or IH displayed significant spatial learning impairments compared with similarly exposed young rats; furthermore, the decrements in performance between RA and IH were markedly greater in aging compared with young rats (p < 0.01), and coincided with the magnitude of IH-induced decreases in cyclic AMP response element binding (CREB) phosphorylation. Furthermore, decreases in proteasomal activity occurred in both young and aging rats exposed to IH, but were substantially greater in the latter (p < 0.001). Neuronal apoptosis, as shown by cleaved caspase 3 expression, was particularly increased in aging rats exposed to IH (p < 0.01 versus young rats exposed to IH). Collectively, these findings indicate unique vulnerability of the aging rodent brain to IH, which is reflected at least in part, by the more prominent decreases in CREB phosphorylation and a marked inability of the ubiquitin-proteasomal pathway to adequately clear degraded proteins.

Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor.

During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. We report that Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent, as ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity.