Yukako Nakajo

Induced Spreading Depression Activates Persistent Neurogenesis in the Subventricular Zone, Generating Cells With Markers for Divided and Early Committed Neurons in the Caudate Putamen and Cortex

Background and Purpose— Status epilepticus and cerebral ischemia stimulate persistent neurogenesis in the adult brain, but both conditions cause neuronal damage. We determined whether spreading depression, a common epiphenomenon of these conditions, stimulates persistent neurogenesis. Methods— We analyzed the effect of KCl-induced spreading depression on persistent neurogenesis and the spatio-temporal distribution of cells exhibiting immunohistochemical markers for divided and early committed neurons (new neurons) in the adult rat brain. Results— After induction of spreading depression for 48 hours, the density of mitotic cells, divided cells, and new neurons in the subventricular zone increased at days 1 to 3, days 3 to 6, and day 6, respectively (P<0.05). The divided cell density in the rostral migratory stream and the stream size increased at day 12 (P<0.001). Vehicle (saline) infusion or induction of spreading depression for 4 hours only did not increase the divided cell density, but the latter increased new neuron density in the subventricular zone (P<0.001). Double-labeled new neuron-like cells also appeared in the caudate putamen or cortex in ectopic fashion at day 3, with dramatic increases at days 6 and 12. Administration of the NMDA receptor antagonist, MK-801, which inhibits the propagation of spreading depression, abolished the increase in new neurons in the subventricular zone and the appearance of ectopic new neuron-like cells after 48-hour KCl infusion. There was no neuronal damage, as evidenced by mature neuron density, neurite density, and apoptotic cell appearance after spreading depression for 48 hours. Conclusions— Spreading depression has the potential to stimulate persistent neurogenesis or to produce ectopic new neuron-like cells.

NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia.

The N-myc downstream-regulated gene (NDRG) family consists of four related proteins, NDRG1-NDRG4, in mammals. We previously generated NDRG1-deficient mice that were unable to maintain myelin sheaths in peripheral nerves. This condition was consistent with human hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D, caused by a nonsense mutation of NDRG1. In contrast, the effects of genetic defects of the other NDRG members remain unknown. In this study, we focused on NDRG4, which is specifically expressed in the brain and heart. In situ mRNA hybridization on the brain revealed that NDRG4 was expressed in neurons of various areas. We generated NDRG4-deficient mice that were born normally with the expected Mendelian frequency. Immunochemical analysis demonstrated that the cortex of the NDRG4-deficient mice contained decreased levels of brain-derived neurotrophic factor (BDNF) and normal levels of glial cell line-derived neurotrophic factor, NGF, neurotrophin-3, and TGF-β1. Consistent with BDNF reduction, NDRG4-deficient mice had impaired spatial learning and memory but normal motor function in the Morris water maze test. When temporary focal ischemia of the brain was induced, the sizes of the infarct lesions were larger, and the neurological deficits were more severe in NDRG4-deficient mice compared with the control mice. These findings indicate that NDRG4 contributes to the maintenance of intracerebral BDNF levels within the normal range, which is necessary for the preservation of spatial learning and the resistance to neuronal cell death caused by ischemic stress.

Genetic increase in brain-derived neurotrophic factor levels enhances learning and memory

Brain-derived neurotrophic factor (BDNF), a neurotrophin, is known to promote neuronal differentiation stimulating neurite outgrowth in the developing CNS, and is also known to modulate synaptic plasticity, thereby contributing to learning and memory in the mature brain. Here, we investigated the role of increased levels of intracerebral BDNF in learning and memory function. Using genetically engineered transgenic BDNF overexpressing mice (RTG-BDNF), young adult, homozygous (+/+), heterozygous (+/-), or wild-type (-/-) littermates, we analyzed escape latency to a hidden-platform and swimming velocity in the Morris Water Maze test (MWM) with modifications for the mice. The MWM comprised 4 trials per day over 5 consecutive days (sessions) without prior or subsequent training. In a separate set of animals, BDNF protein levels in the cortex, thalamostriatum and the hippocampus were measured quantitatively using ELISA. In the BDNF (+/-) mice, the BDNF levels in the cortex, the thalamostriatum and the hippocampus were significantly high, compared to the wild-type littermates; 238%, 158%, and 171%, respectively (P<0.01, one-way ANOVA and a post-hoc test in each region). The BDNF levels in the BDNF (+/+) mice were not elevated. The BDNF (+/-), but not the (+/+) mice, demonstrated significantly shorter escape latency, shorter total path length in the MWM, and more frequent arrivals at the location where the platform had been placed previously in the probe trial, compared with the wild-type littermates (P<0.05, at each time pint). Because the maximum swimming velocity was not affected in the BDNF-transgenic mice, increased BDNF levels in the brain were found to enhance spatial learning and memory function. Although it has been postulated that excessive BDNF is deteriorating for neuronal survival or neurite outgrowth, further investigations are needed to clarify the mechanism of paradoxical lack of increase in BDNF levels in the (+/+) mouse brain.

Alogliptin, a dipeptidylpeptidase-4 inhibitor, for patients with diabetes mellitus type 2, induces tolerance to focal cerebral ischemia in non-diabetic, normal mice.

Effective interventions that provide obvious neuroprotection are currently fairly limited. Glucagon-like peptide-1 (GLP-1), an enhancer of insulin production with a trophic effect on β cells in the islets, has been found to be trophic for neuronal cells. Alogliptin benzoate (AGL), a selective inhibitor of dipeptidylpeptidase-4 (DPP-4) functioning as a long-acting agonist of GLP-1, is in clinical use worldwide for patients with diabetes mellitus type 2. To clarify whether administration of AGL, independent of the insulinotropic effect, protects the brain against focal ischemia, we investigated the effect of AGL on the development of cerebral infarction in non-diabetic normal mice. Male C57BL/6J mice were administered AGL (7.5, 15, or 30μg) once a day for three weeks by intragastric gavage. After the induction of temporary focal ischemia, volumes of infarcted lesions and neurological deficits were analyzed at 24h (acute phase) and seven days (chronic phase). In the acute phase, significant reductions were observed in the volumes of infarcted lesions (p=0.009), and in the severity of neurological deficits (p=0.004), in the group treated with 15μg of alogliptin benzoate, but not the 7.5 or 30μg-treated groups. This significant reduction in volumes of infarcted lesions persisted into the chronic phase. At the end of the AGL treatment; before the induction of ischemia, the levels of brain-derived neurotrophic factor (BDNF), a potent neuroprotectant in the brain, were elevated in the cortex (p=0.008), or in the whole forebrain (p=0.023). AGL could be used as a daily neuroprotectant or an enhancer of BDNF production aiming to attenuate cerebral injuries, for the growing number of people who have the risk of ischemic stroke.

Induced Spreading Depression Evokes Cell Division of Astrocytes in the Subpial Zone, Generating Neural Precursor-Like Cells and New Immature Neurons in the Adult Cerebral Cortex

Background and Purpose— New immature neurons appear out of the germinative zone, in cortical Layers V to VI, after induced spreading depression in the adult rat brain. Because neural progenitors have been isolated in the cortex, we set out to determine whether a subgroup of mature cells in the adult cortex has the potential to divide and generate neural precursors. Methods— We examined the expression of endogenous markers of mitotic activity, proliferating cell nuclear antigen, and vimentin as a marker for neuronal progenitor cells, if any, in the adult rat cortex after spreading depression stimulation. Immunohistochemical analysis was also performed using antibodies for proliferating cell nuclear antigen, for vimentin, and for nestin. Nestin is a marker for activity dividing neural precursors. Results— At the end of spreading depression (Day 0), glial fibrillary acidic protein-positive cells in the subpial zone and cortical Layer I demonstrated increased mitotic activity, expressing vimentin and nestin. On Day 1, nestin+ cells were found spreading in deeper cortical layers. On Day 3, vimentin−/nestin+, neural precursor-like cells appeared in cortical Layers V to VI. On Day 6, new immature neurons appeared in cortical Layers V to VI. Induced spreading depression evokes cell division of astrocytes residing in the subpial zone, generating neural precursor-like cells. Conclusions— Although neural precursor-like cells found in cortical Layers V to VI might have been transferred from the germinative zone rather than the cortical subpial zone, astrocytic cells in the subpial zone may be potent neural progenitors that can help to reconstruct impaired central nervous system tissue. Special caution is required when observing or treating spreading depression waves accompanying pathological conditions in the brain.

Repeated application of an electric field increases BDNF in the brain, enhances spatial learning, and induces infarct tolerance.

Development of a safe method to increase brain-derived neurotrophic factor (BDNF) in the brain is expected to have utility in enhancing learning and memory, in protecting the brain, and in suppressing appetite. We investigated the effects of whole-body exposure to high voltage electric potential (HELP), which generates an electric field and current density in the body, on BDNF levels in the brain, spatial learning, or resistance to cerebral infarction development after focal ischemia. Adult mice (C57BL/6J) were exposed to 3.5 kV, or 5.8 kV for 5 h a day, making indirect contact with the ground via room air, over 1, 3, 6 or 12 consecutive weeks. After treatment, BDNF levels, performances in the Morris water maze task (MWM), or development of infarct lesion after focal ischemia was analyzed. Treatment with 3.5 kV for 1, 3, 6 or 12 weeks, or with 5.8 kV for 1, 3 or 12 weeks increased BDNF levels in the cortex (P<0.05, one-way ANOVA). Every HELP treatment differentially improved escape latency in the MWM, compared with the corresponding untreated controls (P<0.05, one-way ANOVA). Treatment with 3.5 kV for 6 or 12 weeks, but not with 5.8 kV protected the brain suppressing cerebral infarction development (P<0.05). The HELP treatment with 3.5 kV for 6 or 12 weeks improves spatial learning, gently suppressing body weight gain, and protects the brain against cerebral infarction.

The Role of the Host Defense System in the Development of Cerebral Vasospasm: Analogies between Atherosclerosis and Subarachnoid Hemorrhage

Similar to atherosclerosis, platelet-derived growth factor (PDGF)-BB, a major growth factor for vascular smooth muscle cells, is produced in arterial walls to repair arteries after subarachnoid hemorrhage (SAH). On review of a series of research articles that focus on defensive host responses to SAH, PDGF-BB is identified as a spasmogen, based on the following findings: (1) foreign substances injected into the subarachnoid space cause persistent constriction of cerebral arteries with a time course and histological features almost identical to those seen after SAH; (2) persistent constriction induced by SAH or a foreign substance is dependent on the complement system; (3) the complement system, which stimulates platelets, macrophages and endothelial cells to secrete PDGF-BB, is activated in both the cerebrospinal fluid (CSF) and plasma immediately after SAH; (4) PDGF-BB levels in the CSF are significantly elevated in patients with delayed cerebral ischemia; (5) the immunodensity of PDGF-BB in the arterial walls correlates well with the severity of cerebral vasospasm; (6) intracisternal injection of PDGF-BB induces persistent constriction of cerebral arteries in a dose-dependent manner; (7) prolonged contact with blood clots promotes the contractile response of cerebral arteries to PDGF-BB, and (8) administration of an antagonist of PDGF-BB function suppresses the development of cerebral vasospasm.

Platelet-derived growth factor-induced severe and chronic vasoconstriction of cerebral arteries: proposed growth factor explanation of cerebral vasospasm.

OBJECTIVEAfter subarachnoid hemorrhage (SAH), platelet-derived growth factor-BB (PDGF-BB) is secreted in and around the cerebral arteries. To clarify the role of PDGF-BB in the development of vasospasm after SAH, we determined whether PDGF-BB alone can cause long-lasting vasoconstriction of a severity similar to that of vasospasm. In addition, the anti-vasospastic effect of trapidil, an antagonist of PDGF-BB function, was investigated. METHODSWe infused recombinant PDGF-BB (10 μg/mL saline as the vehicle) (n = 14) into the subarachnoid space of rabbits and analyzed alterations in the caliber of the basilar artery using repeated angiography. To study the role of PDGF-BB on the development of vasospasm, trapidil was administered continuously starting 1 hour after SAH, on day 0 (0.63–1.25 mg/kg /h or vehicle) for 47 hours (n = 24), or after the full development of cerebral vasospasm on day 2 (3.0 mg/kg/h or vehicle) for 0.5 hours (n = 17), and alterations in the caliber of the basilar artery were monitored. RESULTSPDGF-BB caused long-lasting vasoconstriction, with maximum constriction of 56% (P < .001) of the control value (= 100%) on day 2, resembling vasospasm seen after SAH. Prolonged administration of intravenous trapidil, starting soon after SAH, prevented the development of vasospasm in a dose-dependent manner (P < .05, .01, or .001). Intravenous or intra-arterial administration of trapidil significantly dilated vasospasm (P < .01) on day 2, at least transiently. CONCLUSIONPDGF-BB, a growth factor synthesized in the subarachnoid space after SAH, can cause severe and long-lasting vasoconstriction. Significant prevention and resolution of vasospasm can be achieved by the PDGF-BB antagonist trapidil. We propose that excessive production of PDGF-BB, essentially aiming to repair injured arteries, causes cerebral vasospasm. Although the half-life of trapidil in serum may be shorter than that of PDGFG-BB–derived spasmogenic signaling, trapidil is a candidate drug for constructing a new therapeutic modality for preventing and resolving vasospasm.

Possible signaling pathways mediating neuronal calcium sensor-1-dependent spatial learning and memory in Mice

Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/-) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production.