Noriyuki Matsukawa

Dietary supplementation of arachidonic and docosahexaenoic acids improves cognitive dysfunction

Age-dependent increase of peroxidation of membrane fatty acids such as arachidonic acid (ARA) and docosahexaenoic acid (DHA) in neurons was reported to cause a decline of the hippocampal long-term potentiation (LTP) and cognitive dysfunction in rodents. Although supplementation of ARA and DHA can improve LTP and cognitive function in rodents, their effects in humans are unknown. The present work was undertaken to study whether ARA and DHA have beneficial effects in human amnesic patients. The subjects were 21 mild cognitive dysfunction (12 MCI-A with supplementation and 9 MIC-P with placebo), 10 organic brain lesions (organic), and 8 Alzheimers disease (AD). The cognitive functions were evaluated using Japanese version of repeatable battery for assessment of neuropsychological status (RBANS) at two time points: before and 90 days after the supplementation of 240 mg/day ARA and DHA, or 240 mg/day of olive oil, respectively. MCI-A group showed a significant improvement of the immediate memory and attention score. In addition, organic group showed a significant improvement of immediate and delayed memories. However, there were no significant improvements of each score in AD and MCI-P groups. It is suggested from these data that ARA and DHA supplementation can improve the cognitive dysfunction due to organic brain damages or aging.

Transplantation of Human Neural Stem Cells Exerts Neuroprotection in a Rat Model of Parkinson's Disease

Neural stem cells (NSCs) possess high potencies of self-renewal and neuronal differentiation. We explored here whether transplantation of human NSCs cloned by v-myc gene transfer, HB1.F3 cells, is a feasible therapeutic option for Parkinsons disease. In vivo, green fluorescent protein-labeled HB1.F3 cells (200,000 viable cells in 3 μl of PBS) when stereotaxically transplanted (same-day lesion-transplant paradigm) into the 6-hydroxydopamine-lesioned striatum of rats significantly ameliorated parkinsonian behavioral symptoms compared with controls (vehicle, single bolus, or continuous minipump infusion of trophic factor, or killed cell grafts). Such graft-derived functional effects were accompanied by preservation of tyrosine hydroxylase (TH) immunoreactivity along the nigrostriatal pathway. Grafted HB1.F3 cells survived in the lesioned brain with some labeled with neuronal marker mitogen-activated protein 2 and decorated with synaptophysin-positive terminals. Furthermore, endogenous neurogenesis was activated in the subventricular zone of transplanted rats. To further explore the neuroprotective mechanisms underlying HB1.F3 cell transplantation, we performed cell culture studies and found that a modest number of HB1.F3 cells were TH and dopamine and cAMP-regulated phosphoprotein 32 positive, although most cells were nestin positive, suggesting a mixed population of mature and immature cells. Administration of the HB1.F3 supernatant to human derived dopaminergic SH-SY5Y cells and fetal rat ventral mesencephalic dopaminergic neurons protected against 6-hydroxydopamine neurotoxicity by suppressing apoptosis through Bcl-2 upregulation, which was blocked by anti-stem cell factor antibody alone, the phosphatidylinositol 3-kinase/Akt inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one] alone, or a combination of both. These results suggest that HB1.F3 cell transplantation exerts neuroprotective effects against dopaminergic depletion in vitro and in vivo because of trophic factor secretion and neuronal differentiation.

Early Changes in KCC2 Phosphorylation in Response to Neuronal Stress Result in Functional Downregulation

The K+ Cl− cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H2O2) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg2+) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl−]i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.

Mannitol facilitates neurotrophic factor up‐regulation and behavioural recovery in neonatal hypoxic‐ischaemic rats with human umbilical cord blood grafts

We recently demonstrated that blood–brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood–brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic‐ischaemic (HI) injury model. Seven‐day‐old Sprague–Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post‐transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme‐linked immunosorbent assay at day 3 post‐transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up‐regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor–mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood–brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.

Hippocampal cholinergic neurostimulating peptides (HCNP).

Neuronal development and differentiation require a variety of cell interactions. Diffusible molecules from target neurons play an important part in mediating such interactions. Our early studies used explant culture technique to examine the factors that enhance the differentiation of septo-hippocampal cholinergic neurons, and they revealed that several components resident in the hippocampus are involved in the differentiation of presynaptic cholinergic neurons in the medial septal nucleus. One of these components, originally purified from young rat hippocampus, is a novel undecapeptide (hippocampal cholinergic neurostimulating peptide; HCNP); this enhances the production of ChAT, but not of AchE. Later experiments revealed that: (1) a specific receptor appears to mediate this effect; (2) NGF and HCNP act cooperatively to regulate cholinergic phenotype development in the medial septal nucleus in culture; and (3) these two molecules differ both in their mechanism of release from the hippocampus and their mechanism of action on cholinergic neurons. The amino acid sequence deduced from base sequence analysis of cloned HCNP-precursor protein cDNA shows that HCNP is located at the N-terminal domain of its precursor protein. The 21 kDa HCNP precursor protein shows homology with other proteins, and it functions not only as an HCNP precursor, but also as a binding protein for ATP, opioids and phosphatidylethanolamine. The distribution and localization of HCNP-related components and the expression of their mRNAs support the notion that the precursor protein is multifunctional. In keeping with its multiple functions, the multiple enhancers and promoters found in the genomic DNA for HCNP precursor protein may be involved in the regulation of its gene in a variety of cells and at different stages of development. Furthermore, several lines of evidence obtained from studies of humans and animal models suggest that certain types of memory and learning disorders are associated with abnormal accumulation and expression of HCNP analogue peptide and/or its precursor protein mRNA in the hippocampus.

Therapeutic targets and limits of minocycline neuroprotection in experimental ischemic stroke

BackgroundMinocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, has been shown to promote therapeutic benefits in experimental stroke. However, equally compelling evidence demonstrates that the drug exerts variable and even detrimental effects in many neurological disease models. Assessment of the mechanism underlying minocycline neuroprotection should clarify the drugs clinical value in acute stroke setting.ResultsHere, we demonstrate that minocycline attenuates both in vitro (oxygen glucose deprivation) and in vivo (middle cerebral artery occlusion) experimentally induced ischemic deficits by direct inhibition of apoptotic-like neuronal cell death involving the anti-apoptotic Bcl-2/cytochrome c pathway. Such anti-apoptotic effect of minocycline is seen in neurons, but not apparent in astrocytes. Our data further indicate that the neuroprotection is dose-dependent, in that only low dose minocycline inhibits neuronal cell death cascades at the acute stroke phase, whereas the high dose exacerbates the ischemic injury.ConclusionThe present study advises our community to proceed with caution to use the minimally invasive intravenous delivery of low dose minocycline in order to afford neuroprotection that is safe for stroke.

Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not

Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3.4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.

Transplantation of bone marrow-derived stem cells: a promising therapy for stroke.

Stroke remains a major cause of death in the US and around the world. Over the last decade, stem cell therapy has been introduced as an experimental treatment for stroke. Transplantation of stem cells or progenitors into the injured site to replace the nonfunctional cells, and enhancement of proliferation or differentiation of endogenous stem or progenitor cells stand as the two major cell-based strategies. Potential sources of stem/progenitor cells for stroke include fetal neural stem cells, embryonic stem cells, neuroteratocarcinoma cells, umbilical cord blood-derived nonhematopoietic stem cells, and bone marrow-derived stem cells. The goal of this article is to provide an update on the preclinical use of bone marrow-derived stem cells with major emphasis on mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) because they are currently most widely applied in experimental stroke studies and are now being phased into early clinical trials. The phenotypic features of MSCs and MAPCs, as well as their application in stroke, are described.

Behavioral and histological characterization of intrahippocampal grafts of human bone marrow-derived multipotent progenitor cells in neonatal rats with hypoxic-ischemic injury.

Children born with hypoxic-ischemic (HI) brain injury account for a significant number of live births wherein no clinical treatment is available. Limited clinical trials of stem cell therapy have been initiated in a number of neurological disorders, but the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. One major postulated mechanism underlying therapeutic benefits of stem cell therapy involves stimulation of endogenous neurogenesis via transplantation of exogenous stem cells. To this end, transplantation has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents as highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. Here, we investigated the efficacy of intrahippocampal transplantation of multipotent progenitor cells (MPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, which involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI injury, animals received stereotaxic hippocampal injections of vehicle or cryopreserved MPCs (thawed just prior to transplantation) derived either from Sprague-Dawley rats (syngeneic) or Fisher rats (allogeneic). All animals were treated with daily immunosuppression throughout the survival period. Behavioral tests were conducted on posttransplantation days 7 and 14 using the elevated body swing test and the rotarod to reveal general and coordinated motor functions. MPC transplanted animals exhibited reduced motor asymmetry and longer time spent on the rotarod than those that received the vehicle infusion. Both syngeneic and allogeneic MPC transplanted injured animals did not significantly differ in their behavioral improvements at both test periods. Immunohistochemical evaluations of graft survival after behavioral testing at day 14 posttransplantation revealed that syngeneic and allogeneic transplanted MPCs survived in the hippocampal region. These results demonstrate for the first time that transplantation of MPCs ameliorated motor deficits associated with HI injury. In view of comparable behavioral recovery produced by syngeneic and allogeneic MPC grafts, allogeneic transplantation poses as a feasible and efficacious cell replacement strategy with direct clinical application. An equally major finding is the observation lending support to the hippocampus as an excellent target brain region for stem cell therapy in treating HI injury.

Notch-Induced Rat and Human Bone Marrow Stromal Cell Grafts Reduce Ischemic Cell Loss and Ameliorate Behavioral Deficits in Chronic Stroke Animals

Gene transfection with Notch 1 intracellular domain and subsequent growth factor treatment stimulate neuron-like differentiation of bone marrow stromal cells (BMSCs). Here, we examined the potential of transplanting Notch-induced BMSCs to exert therapeutic effects in a rat model of chronic ischemic stroke. In experiment 1, Notch-induced rat BMSCs were intrastriatally transplanted in rats at 1 month after being subjected to transient occlusion of middle cerebral artery (MCAo). Compared to post-stroke/pretransplantation level, significant improvements in locomotor and neurological function were detected in stroke rats that received 100 k and 200 k BMSCs, but not in those that received 40 k BMSCs. Histological results revealed 9%-15% graft survival, which dose-dependently correlated with behavioral recovery. At 5 weeks post-transplantation, some grafted BMSCs were positive for the glial marker GFAP (about 5%), but only a few cells (2-5 cells per brain) were positive for the neuronal marker NeuN. However, at 12 weeks post-transplantation, where the number of GFAP-positive BMSCs was maintained (5%), there was a dramatic increase in NeuN-positive BMSCs (23%). In experiment 2, Notch-induced human BMSCs were intrastriatally transplanted in rats at 1 month following the same MCAo model. Improvements in both locomotor and neurological function were observed from day 7 to day 28 post-transplantation, with the high dose (180 k) displaying significantly better behavioral recovery than the low dose (90 k) or vehicle. There were no observable adverse behavioral effects during this study period that also involved chronic immunosuppression of all animals. Histological analyses revealed a modest 5%-7% graft survival, with few (<1%) cells expressing an intermediate MAP2 neuronal marker, but not glial or oligodendroglial markers. In addition, striatal peri-infarct cell loss was significantly reduced in transplanted stroke animals compared to vehicle-treated stroke animals. The present study demonstrates the potential of Notch-induced BMSC cell therapy for patients presenting with fixed ischemic stroke.