Long-term body-weight-supported treadmill training for incomplete cervical spinal cord injury: a case report.

[Purpose] To investigate the effects of long-term body-weight-supported treadmill training on walking ability and physical function in an elderly individual with incomplete cervical spinal cord injury. [Participant and Methods] The patient was a 68 year-old male with an incomplete spinal cord injury at the C3/C4 level, incurred when he was 56 years old. He initiated home-based body-weight-supported treadmill training using a body-weight-supported treadmill installed at his home. His walking ability was measured as the percentage of body weight load reduction, and his physical function was evaluated using manual muscle testing and measuement of the range of motion of his lower limbs. [Results] The physical function of the lower limbs was improved, maintained, or showed delayed decline until 9.5 years post-injury. [Conclusion] Long-term body-weight-supported treadmill training may improve, maintain, or at least delay the decline of the physical function of participants for several years, without causing any remarkable complications.

Mouse Model of Chromosome 15q13.3 Microdeletion Syndrome Demonstrates Features Related to Autism Spectrum Disorder.

The chromosome 15q13.3 microdeletion is a pathogenic copy number variation conferring epilepsy, intellectual disability, schizophrenia, and autism spectrum disorder (ASD). We generated mice carrying a deletion of 1.2 Mb homologous to the 15q13.3 microdeletion in human patients. Here, we report that mice with a heterozygous deletion on a C57BL/6 background (D/+ mice) demonstrated phenotypes including enlarged/heavier brains (macrocephaly) with enlarged lateral ventricles, decreased social interactions, increased repetitive grooming behavior, reduced ultrasonic vocalizations, decreased auditory-evoked gamma band EEG, and reduced event-related potentials. D/+ mice had normal body weight, activity levels, sensory gating, and cognitive abilities and no signs of epilepsy/seizures. Our results demonstrate that D/+ mice represent ASD-related phenotypes associated with 15q13.3 microdeletion syndrome. Further investigations using this chromosome-engineered mouse model may uncover the common mechanism(s) underlying ASD and other neurodevelopmental/psychiatric disorders representing the 15q13.3 microdeletion syndrome, including epilepsy, intellectual disability, and schizophrenia. SIGNIFICANCE STATEMENT: Recently discovered pathologic copy number variations (CNVs) from patients with neurodevelopmental/psychiatric disorders show very strong penetrance and thus are excellent candidates for mouse models of disease that can mirror the human genetic conditions with high fidelity. A 15q13.3 microdeletion in humans results in a range of neurodevelopmental/psychiatric disorders, including epilepsy, intellectual disability, schizophrenia, and autism spectrum disorder (ASD). The disorders conferred by a 15q13.3 microdeletion also have overlapping genetic architectures and comorbidity in other patient populations such as those with epilepsy and schizophrenia/psychosis, as well as schizophrenia and ASD. We generated mice carrying a deletion of 1.2 Mb homologous to the 15q13.3 microdeletion in human patients, which allowed us to investigate the potential causes of neurodevelopmental/psychiatric disorders associated with the CNV.

Hypophosphorylation of ribosomal protein S6 is a molecular mechanism underlying ischemic tolerance induced by either hibernation or preconditioning.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) have an extraordinary capacity to withstand prolonged and profound reductions in blood flow and oxygen delivery to the brain without incurring any cellular damage. As such, the hibernation torpor of I. tridecemlineatus provides a valuable model of tolerance to ischemic stress. Herein, we report that during hibernation torpor, a marked reduction in the phosphorylation of the ribosomal protein S6 (rpS6) occurs within the brains of I. tridecemlineatus. Of note, rpS6 phosphorylation was shown to increase in the brains of rats that underwent an occlusion of the middle cerebral artery. However, such an increase was attenuated after the implementation of an ischemic preconditioning paradigm. In addition, cultured cortical neurons treated with the rpS6 kinase (S6K) inhibitors, D-glucosamine or PF4708671, displayed a decrease in rpS6 phosphorylation and a subsequent increase in tolerance to oxygen/glucose deprivation, an in vitro model of ischemic stroke. Collectively, such evidence suggests that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning. Further identification and characterization of the mechanisms used by hibernating species to increase ischemic tolerance may eventually clarify how the loss of homeostatic control that occurs during and after cerebral ischemia in the clinic can ultimately be minimized and/or prevented. Mammalian hibernation provides a valuable model of tolerance to ischemic stress. Herein, we demonstrate that marked reductions in the phosphorylation of ribosomal protein S6 (rpS6), extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinase p44/42 (p44/42MAPK) and ribosomal protein S6 kinase (S6K) occur within the brains of both hibernating squirrels and rats, which have undergone an ischemic preconditioning paradigm. We therefore propose that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning, via a suppression of protein synthesis and/or energy consumption.

Mossy fiber mis-pathfinding and semaphorin reduction in the hippocampus of α-CaMKII hKO mice.

Recent evidence indicates that alpha-isoform of calcium/calmodulin-dependent protein kinase II (α-CaMKII) deficiency in adult mice induces phenotypic immaturity of dentate granule cells, defined by dendritic disturbance and aberrant maturational neuron marker expression. Mice possessing a heterozygous inactivation of α-CaMKII display abnormal behavioral phenotypes, including working memory deficits similar to those observed in psychiatric patients. Currently, how the guidance of mossy fibers, the axonal projections of granule cells, are topologically regulated in the dentate gyrus of α-CaMKII deficient animals is not well understood, even though axonal morphogenesis is a key factor for modulating neuronal transmission and animal behavior. In the present study, we explored the involvement of semaphorin signaling, a well-studied guidance factor in mossy fiber pathfinding, in α-CaMKII heterozygous knock-out mice (α-CaMKII hKO mice). Using immunohistochemical characterization, we found mossy fibers invade not only the CA3 stratum lucidum region, but also stratum oriens region where mossy fibers do not usually bundle. Furthermore, α-CaMKII hKO mice have decreased semaphorin-3A expression. These results suggest mossy fiber mis-guidance, possibly regulated by semaphorin-3A, is one of the biomarkers reflecting immaturity in dentate granule cells, possibly contributing to abnormal behavioral phenotypes.

Gastrin-releasing peptide contributes to the regulation of adult hippocampal neurogenesis and neuronal development.

In the postnatal hippocampus, newly generated neurons contribute to learning and memory. Disruptions in neurogenesis and neuronal development have been linked to cognitive impairment and are implicated in a broad variety of neurological and psychiatric disorders. To identify putative factors involved in this process, we examined hippocampal gene expression alterations in mice possessing a heterozygous knockout of the calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout gene (CaMK2α-hKO), an established model of cognitive impairment that also displays altered neurogenesis and neuronal development. Using this approach, we identified gastrin-releasing peptide (GRP) as the most dysregulated gene. In wild-type mice, GRP labels NeuN-positive neurons, the lone exception being GRP-positive, NeuN-negative cells in the subgranular zone, suggesting GRP expression may be relevant to neurogenesis and/or neuronal development. Using a model of in vitro hippocampal neurogenesis, we determined that GRP signaling is essential for the continued survival and development of newborn neurons, both of which are blocked by transient knockdown of GRP's cognate receptor (GRPR). Furthermore, GRP appears to negatively regulate neurogenesis-associated proliferation in neural stem cells both in vitro and in vivo. Intracerebroventricular infusion of GRP resulted in a decrease in immature neuronal markers, increased cAMP response element-binding protein (CREB) phosphorylation, and decreased neurogenesis. Despite increased levels of GRP mRNA, CaMK2α-hKO mutant mice expressed reduced levels of GRP peptide. This lack of GRP may contribute to the elevated neurogenesis and impaired neuronal development, which are reversed following exogenous GRP infusion. Based on these findings, we hypothesize that GRP modulates neurogenesis and neuronal development and may contribute to hippocampus-associated cognitive impairment.

The immature dentate gyrus represents a shared phenotype of mouse models of epilepsy and psychiatric disease.

OBJECTIVES: There is accumulating evidence to suggest psychiatric disorders, such as bipolar disorder and schizophrenia, share common etiologies, pathophysiologies, genetics, and drug responses with many of the epilepsies. Here, we explored overlaps in cellular/molecular, electrophysiological, and behavioral phenotypes between putative mouse models of bipolar disorder/schizophrenia and epilepsy. We tested the hypothesis that an immature dentate gyrus (iDG), whose association with psychosis in patients has recently been reported, represents a common phenotype of both diseases. METHODS: Behaviors of calcium/calmodulin-dependent protein kinase II alpha (α-CaMKII) heterozygous knock-out (KO) mice, which are a representative bipolar disorder/schizophrenia model displaying iDG, and pilocarpine-treated mice, which are a representative epilepsy model, were tested followed by quantitative polymerase chain reaction (qPCR)/immunohistochemistry for mRNA/protein expression associated with an iDG phenotype. In vitro electrophysiology of dentate gyrus granule cells (DG GCs) was examined in pilocarpine-treated epileptic mice. RESULTS: The two disease models demonstrated similar behavioral deficits, such as hyperactivity, poor working memory performance, and social withdrawal. Significant reductions in mRNA expression and immunoreactivity of the mature neuronal marker calbindin and concomitant increases in mRNA expression and immunoreactivity of the immature neuronal marker calretinin represent iDG signatures that are present in both mice models. Electrophysiologically, we have confirmed that DG GCs from pilocarpine-treated mice represent an immature state. A significant decrease in hippocampal α-CaMKII protein levels was also found in both models. CONCLUSIONS: Our data have shown iDG signatures from mouse models of both bipolar disorder/schizophrenia and epilepsy. The evidence suggests that the iDG may, in part, be responsible for the abnormal behavioral phenotype, and that the underlying pathophysiologies in epilepsy and bipolar disorder/schizophrenia are strikingly similar.

SREB2/GPR85, a schizophrenia risk factor, negatively regulates hippocampal adult neurogenesis and neurogenesis-dependent learning and memory.

SREB2/GPR85, a member of the super-conserved receptor expressed in brain (SREB) family, is the most conserved G-protein-coupled receptor in vertebrate evolution. Previous human and mouse genetic studies have indicated a possible link between SREB2 and schizophrenia. SREB2 is robustly expressed in the hippocampal formation, especially in the dentate gyrus, a structure with an established involvement in psychiatric disorders and cognition. However, the function of SREB2 in the hippocampus remains elusive. Here we show that SREB2 regulates hippocampal adult neurogenesis, which impacts on cognitive function. Bromodeoxyuridine incorporation and immunohistochemistry were conducted in SREB2 transgenic (Tg, over-expression) and knockout (KO, null-mutant) mice to quantitatively assay adult neurogenesis and newborn neuron dendritic morphology. Cognitive responses associated with adult neurogenesis alteration were evaluated in SREB2 mutant mice. In SREB2 Tg mice, both new cell proliferation and new neuron survival were decreased in the dentate gyrus, whereas an enhancement of new neuron survival occurred in SREB2 KO mouse dentate gyrus. Doublecortin staining revealed dendritic morphology deficits of newly generated neurons in SREB2 Tg mice. In a spatial pattern separation task, SREB2 Tg mice displayed a decreased ability to discriminate spatial relationships, whereas SREB2 KO mice had enhanced abilities in this task. Additionally, SREB2 Tg and KO mice had reciprocal phenotypes in a Y-maze working memory task. Our results indicate that SREB2 is a negative regulator of adult neurogenesis and consequential cognitive functions. Inhibition of SREB2 function may be a novel approach to enhance hippocampal adult neurogenesis and cognitive abilities to ameliorate core symptoms of psychiatric patients.

Adult neurogenesis transiently generates oxidative stress.

An increasing body of evidence suggests that alterations in neurogenesis and oxidative stress are associated with a wide variety of CNS diseases, including Alzheimer's disease, schizophrenia and Parkinson's disease, as well as routine loss of function accompanying aging. Interestingly, the association between neurogenesis and the production of reactive oxidative species (ROS) remains largely unexamined. The adult CNS harbors two regions of persistent lifelong neurogenesis: the subventricular zone and the dentate gyrus (DG). These regions contain populations of quiescent neural stem cells (NSCs) that generate mature progeny via rapidly-dividing progenitor cells. We hypothesized that the energetic demands of highly proliferative progenitors generates localized oxidative stress that contributes to ROS-mediated damage within the neuropoietic microenvironment. In vivo examination of germinal niches in adult rodents revealed increases in oxidized DNA and lipid markers, particularly in the subgranular zone (SGZ) of the dentate gyrus. To further pinpoint the cell types responsible for oxidative stress, we employed an in vitro cell culture model allowing for the synchronous terminal differentiation of primary hippocampal NSCs. Inducing differentiation in primary NSCs resulted in an immediate increase in total mitochondria number and overall ROS production, suggesting oxidative stress is generated during a transient window of elevated neurogenesis accompanying normal neurogenesis. To confirm these findings in vivo, we identified a set of oxidation-responsive genes, which respond to antioxidant administration and are significantly elevated in genetic- and exercise-induced model of hyperactive hippocampal neurogenesis. While no direct evidence exists coupling neurogenesis-associated stress to CNS disease, our data suggest that oxidative stress is produced as a result of routine adult neurogenesis.

Spatial and temporal activation of brain regions in hibernation: c-fos expression during the hibernation bout in thirteen-lined ground squirrel.

Hibernation results in dramatic changes in body temperature and metabolism; however, the central nervous system remains active during deep torpor. By cloning c-fos cDNA from the 13-lined ground squirrel (Spermophilus tridecemlineatus) and using squirrel c-fos mRNA probe for in situ hybridization histochemistry, we systematically analyzed and identified specific brain regions that were activated during six different phases of the hibernation bout. During entrance into torpor, we detected activation of the ventrolateral subdivision of the medial preoptic area ('thermoregulatory center'), and the reticular thalamic nucleus, which is known to inhibit the somatomotor cortex. During torpor, c-fos expression in the cortex was suppressed while the reticular thalamic nucleus remained uniformly active. Throughout torpor the suprachiasmatic nucleus ('biological clock') showed increasing activity, likely participating in phase-change regulation of the hibernation bout. Interestingly, during torpor very strong c-fos activation was seen in the epithelial cells of the choroid plexus and in tanycytes at the third ventricle, both peaking near the beginning of arousal. In arousal, activity of the suprachiasmatic and reticular thalamic nuclei and choroid epithelial cells diminished, while ependymal cells in the lateral and fourth ventricles showed stronger activity. Increasing body temperature during arousal was driven by the activation of neurons in the medial part of the preoptic area. In interbout awake animals, we demonstrated the activation of hypothalamic neurons located in the arcuate nucleus and the dorsolateral hypothalamus, areas involved in food intake. Our observations indicate that the hibernation bout is closely regulated and orchestrated by specific regions of the central nervous system. J. Comp. Neurol. 505:443-458, 2007. (c) 2007 Wiley-Liss, Inc.

Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells.

Hibernation torpor provides an excellent natural model of tolerance to profound reductions in blood flow to the brain and other organs. Here, we report that during torpor of 13-lined ground squirrels, massive SUMOylation occurs in the brain, liver, and kidney. The level of small ubiquitin-related modifier (SUMO) conjugation coincides with the expression level of Ubc9, the SUMO specific E2-conjugating enzyme. Hypothermia alone also increased SUMO conjugation, but not as markedly as hibernation torpor. Increased SUMO conjugation (induced by Ubc9 overexpression, ischemic preconditioning (PC)+/-hypothermia) was necessary and sufficient for tolerance of SHSY5Y neuroblastoma cells to oxygen/glucose deprivation (OGD) ('in vitro ischemia'); decreased SUMO conjugation (induced by a dominant-negative Ubc9) severely reduced tolerance to OGD in these cells. These data indicate that post-translational modification of proteins by SUMOylation is a prominent feature of hibernation torpor and is critical for cytoprotection by ischemic PC+/-hypothermia in SHSY5Y cells subjected to OGD.

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai.

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.

The influences of L(+)-lactate and pH on contractile performance in rabbit glycerinated skeletal muscle.

To know whether L(+)-lactate directly induces the decrease in muscle contractile performance, several parameters of cross-bridge function were measured at various concentrations of lactate and pH in glycerinated rabbit psoas and soleus muscles at three different temperatures (5, 20, 28 degrees C). At all pHs studied (pH 7.0, 6.5, 6.0, and 5.5), isometric tension, unloaded velocity of shortening, and stiffness of a fiber during active and resting state in the presence of 50 mM lactate were not virtually different from those in the absence of lactate, but pH had remarkable effects on these parameters. The active stiffness decreased only slightly, and the small resting stiffness appeared at low pH; they were not affected by the presence of lactate. The present results indicate that the lactate anions may not have marked influence on the interaction between actin and myosin, and the concomitant change in pH with the production of lactate may remarkably affect it, as far as they were examined under the existing conditions of the experimental solutions.

Development of an apparatus to control load by electromagnet for a motility system in vitro.

We developed an electromagnet to perform quick changes in load in the motility system consisting of myosin molecules attached to a magnetizable bead and actin filaments The electromagnet was combined with an inverted microscope and load could be quickly changed under optical observation. The magnetic field was generated by high electric current (6V, 0-125A) and the maximum field was 8,000 Oe. The maximum force exerted on a bead was 80pN at 2.5mm distance from a magnet. The change in force was 0.48% at the distance of 5.0mm from the magnet when a bead moved longitudinally for 30microm. The time to change load was about 20ms. The movements of a bead in water were recorded by video when step changes in magnetic field were applied and it was shown that a bead exactly followed the change in force. This apparatus is very much useful to analyze the transient changes in the movement of a bead, if the movement is relatively slow as in the interaction between actin and myosin from molluscan smooth muscle.

Identification and characterization of a novel mitochondrial tricarboxylate carrier.

Here we report the identification and functional characterization of a novel mitochondrial tricarboxylate carrier protein, designated BBG-TCC, in rat brain. The cDNA encodes the predicted protein of 342-amino acid residues with five putative membrane-spanning domains. The protein has apparent similarity with a mitochondrial tricarboxylate carrier TCC, but is distinct from the other mitochondria anion transporters. BBG-TCC shows a citrate transport activity. It is specifically expressed in the brain and localizes in the mitochondria of Bergmann glial cells. In contrast, the expression of TCC is rather ubiquitous and strong in neuronal cells in the brain. This new family of proteins may contribute to biosynthesis and bioenergetics in the brain.

Expression of mitochondrial tricarboxylate carrier TCC mRNA and protein in the rat brain.

The tricarboxylate carrier protein catalyzes an electroneutral exchange across the mitochondrial inner membrane of tricarboxylate, dicarboxylate or phosphoenolpyruvate. We examined expression and localization of mitochondrial tricarboxylate carrier TCC mRNA and protein in the rat brain. TCC mRNA was ubiquitously expressed in all rat tissues examined and was abundant in brain, liver and kidney. TCC protein as well as mRNA was widely expressed in brain, and the protein expression was strong in neuronal cells in the hippocampus, the olfactory bulb, the corpus mamillare and the cerebellum. Our results suggest that this tricarboxylate carrier protein may contribute to biosynthesis and bioenergetics in neuronal cells in brain.

Peg3/Pw1 is involved in p53-mediated cell death pathway in brain ischemia/hypoxia.

Emerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia.