Hirokazu Ohtaki

Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses

Human mesenchymal stromal cells (hMSCs) were injected into the hippocampus of adult mice 1 day after transient global ischemia. The hMSCs both improved neurologic function and markedly decreased neuronal cell death of the hippocampus. Microarray assays indicated that ischemia up-regulated 586 mouse genes. The hMSCs persisted for <7 days, but they down-regulated >10% of the ischemia-induced genes, most of which were involved in inflammatory and immune responses. The hMSCs also up-regulated three mouse genes, including the neuroprotective gene Ym1 that is expressed by activated microglia/macrophages. In addition, the transcriptomes of the hMSC changed with up-regulation of 170 human genes and down-regulation of 54 human genes. Protein assays of the hippocampus demonstrated increased expression in microglia/macrophages of Ym1, the cell survival factor insulin-like growth factor 1, galectin-3, cytokines reflective of a type 2 T cell immune bias, and the major histocompatibility complex II. The observed beneficial effects of hMSCs were largely explained by their modulation of inflammatory and immune responses, apparently by alternative activation of microglia and/or macrophages.

Lipopolysaccharide‐induced microglial activation induces learning and memory deficits without neuronal cell deathin rats

We used lipopolysaccharide (LPS) to activate microglia that play an important role in the brain immune system. LPS injected into the rat hippocampus CA1 region activated microglial cells resulting in an increased production of interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α in the hippocampus during the initial stage of treatment. Immunostaining for IL‐1β was increased at 6 hr after LPS injection. IL‐1β‐immunopositive cells were co‐localized with immunostaining for CD11b. Subacute treatment with LPS by the same route for 5 days caused long‐term activation of microglia and induced learning and memory deficits in animals when examined with a step‐through passive avoidance test, but histochemical analysis showed that neuronal cell death was not observed under these experimental conditions. The increased expression of the heme oxygenase‐1 (HO‐1) gene, an oxidative stress maker, was observed. However, the genetic expression of brain‐derived neurotrophic factor (BDNF) and its receptor, TrkB, decreased during the course of LPS treatment. We found decreases in [3H]MK801 binding in the hippocampus CA1 region by LPS‐treatment for 5 days. The data shows that glutamatergic transmission was attenuated in the LPS‐treated rats. These results suggest that long‐term activation of microglia induced by LPS results in a decrease of glutamatergic transmission that leads to learning and memory deficits without neuronal cell death. The physiologic significance of these findings is discussed.

Pleiotropic Functions of PACAP in the CNS

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen‐activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia‐reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1‐R). Reactive astrocytes increased their expression of PAC1‐R, released interleukin‐6 (IL‐6) that is a proinflammatory cytokine with both differentiation and growth‐promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP‐stimulated secretion of IL‐6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1‐R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1‐R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1‐R antagonist (PACAP6‐38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1‐R in mouse neural stem cells and its signal is transmitted to the PAC1‐R‐coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

BackgroundWe hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.MethodsUnilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.ResultsGp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.ConclusionsClassical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Role of PACAP in Ischemic Neural Death

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that was first isolated from an ovine hypothalamus in 1989. Since its discovery, more than 2,000 papers have reported on the tissue and cellular distribution and functional significance of PACAP. A number of papers have reported that PACAP but not the vasoactive intestinal peptide suppressed neuronal cell death or decreased infarct volume after global and focal ischemia in rodents, even if PACAP was administered several hours after ischemia induction. In addition, recent studies using PACAP gene-deficient mice demonstrated that endogenous PACAP also contributes greatly to neuroprotection similarly to exogenously administered PACAP. The studies suggest that neuroprotection by PACAP might extend the therapeutic time window for treatment of ischemia-related conditions, such as stroke. This review summarizes the effects of PACAP on ischemic neuronal cell death, and the mechanism clarified in vivo ischemic studies. In addition, the prospective mechanism of PACAP on ischemic neuroprotection from in vitro neuronal and neuronal-like cell cultures with injured stress model is reviewed. Finally, the development of PACAP and/or receptor agonists for human therapy is discussed.

Activation of microglia induces symptoms of Parkinson’s disease in wild-type, but not in IL-1 knockout mice

BackgroundParkinson’s disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice.MethodsLipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection.ResultsLPS treatment induced the activation of microglia, as demonstrated by production of IL-1β and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice.ConclusionsThe subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment.

Isolation of Peptide Transport System-6 from Brain Endothelial Cells: Therapeutic Effects with Antisense Inhibition in Alzheimer and Stroke Models:

By isolating for the first time ever a peptide transporter from the blood—brain barrier (BBB) and developing an antisense that selectively targets the brain-to-blood efflux component, we were able to deliver a therapeutic concentration of the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) 27 to brain in animal models of Alzheimers and stroke. Efflux pumps at the BBB are major causes of BBB impermeability to peptides. PACAP is neuroprotective in vitro in femtomole amounts, but brain uptake of PACAP27 is limited by an efflux component of peptide transport system-6 (PTS-6). Here, we characterized, isolated, and sequenced this component of PTS-6, identifying it as β-F1 ATPase, and colocalized it with PACAP27 on BBB endothelial cells. Antisenses targeting the BBB inhibited PACAP27 efflux, thus increasing brain uptake of PACAP27. Treatment with antisense +PACAP27 improved cognition in a mouse model of Alzheimers disease and reduced infarct size after cerebral ischemia. This represents the first isolation from BBB tissue of a peptide transporter and shows that inhibition of peptide efflux pumps is a potential strategy for drug delivery to brain.

Reduced postischemic apoptosis in the hippocampus of mice deficient in interleukin-1

The cytokine interleukin‐1 (IL‐1) has been implicated in ischemic brain damage, because the IL‐1 receptor antagonist markedly inhibits experimentally induced neuronal loss. However, to date, no studies have demonstrated the involvement of endogenous IL‐1α and IL‐ 1β in neurodegeneration. We report here, for the first time, that mice lacking IL‐1α/β (double knockout) exhibit markedly reduced neuronal loss and apoptotic cell death when exposed to transient cardiac arrest. Furthermore, we show that, despite the reduced neuronal loss, phosphorylation of JNK/SAPK (c‐Jun NH2‐ terminal protein kinase/stress activated protein kinase) and p38 enzymes remain elevated in IL‐1 knockout mice. In contrast, the inducible nitric oxide (iNOS) immunoreactivity after global ischemia was reduced in IL‐1 knockout mice as compared with wild‐type mice. The levels of nitrite (NO2−) and nitrate (NO3−) in the hippocampus of wild‐type mice were increased with time after ischemia‐reperfusion, whereas the increase was significantly inhibited in IL‐1 knockout mice. These observations strongly suggest that endogenous IL‐1 contributes to ischemic brain damage, and this influence may act through the release of nitric oxide by iNOS. J. Comp. Neurol. 448:203–216, 2002.

Pituitary adenylate cyclase-activating polypeptide (PACAP) prevents hippocampal neurons from apoptosis by inhibiting JNK/SAPK and p38 signal transduction pathways

We have demonstrated that ischemic neuronal death (apoptosis) of rat CA1 region of the hippocampus was prevented by infusing pituitary adenylate cyclase-activating polypeptide (PACAP) either intracerebroventricularly or intravenously. We have also demonstrated that the activity of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, was increased in the hippocampus within 1-6 h after brain ischemia. The molecular mechanisms underlying the PACAP anti-apoptotic effect were demonstrated in this study. Ischemic stress had a strong influence on MAP kinase family, especially on JNK/SAPK and p38. PACAP inhibited the activation of JNK/SAPK and p38 after ischemic stress, while ERK is not suppressed. These findings suggest that PACAP inhibits the JNK/SAPK and p38 signaling pathways, thereby protecting neurons against apoptosis.

Stroke upregulates TNFα transport across the blood–brain barrier

Abstract To determine how cytokine transport systems at the blood–brain barrier (BBB) participate in stroke progression and recovery, we generated a mouse model of transient middle cerebral artery occlusion (tMCAO). After 1 h of occlusion followed by nearly complete reperfusion, the neurological deficits lasted more than a week as shown by several behavioral tests. Despite the prominent infarct area indicated by reduced cerebral perfusion and confirmed by vital staining, the volume of distribution of 131 I-albumin in various brain regions was not significantly altered over time (12 h to 14 days). In sharp contrast, the blood-to-brain permeation of 125 I-TNFα was significantly increased 5 days after tMCAO. Furthermore, excess unlabeled TNFα abolished this enhanced 125 I-TNFα uptake. Thus, not only did the known saturable transport system for TNFα persist, but it functioned at a higher capacity in tMCAO mice. Upregulation of TNFR1 and TNFR2 partially explains the increased transport, as mRNA for both receptors showed the most pronounced increase (15-fold and 30-fold, respectively) in the ischemic hemisphere 5–7 days after tMCAO. However, even in the hemisphere contralateral to the ischemia induced by stroke, there was increased TNFα transport. The bilateral increase in 125 I-TNFα entry from blood to brain suggests that TNFα trafficking in cerebral endothelial cells is influenced by global mediators in addition to the transporting receptors. Given the known multiple modulatory effects of TNFα after stroke, the results indicate that the TNFα transport system at the BBB facilitates neuroplasticity and plays an important role in stroke recovery.