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Hsp70.1 has dual functions as chaperone protein and lysosomal stabilizer. Previously, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes ischemic neuronal death by inducing lysosomal rupture. Recently, we found that the consecutive injections of vegetable oil-peroxidation product ‘hydroxynonenal’ induces hepatocyte death via the similar cascade. As Hsp70.1 is related also to fatty acid β-oxidation in the liver, its deficiency is known to cause accumulation of fat. Genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing decrease of phosphatidylcholine with the resultant hepatic steatosis. Here, focusing on disorders of Hsp70.1 and BHMT, we studied the mechanism of hepatocyte degeneration and steatosis, using monkeys after the consecutive injections of synthetic hydroxynonenal. As these monkeys showed a significant impairment of liver functions, the liver tissues without and with hydroxynonenal injections were compared by proteomics, immunoblotting, immunohisto-chemical and electron microscopic analyses. Western blotting showed upregulation of neither Hsp70.1 nor BHMT, but an increased cleavage of both proteins. Proteomics showed downregulation of Hsp70.1, while 2-fold increments of carbonylated BHMT. Hsp70.1 carbonylation was negligible, showing a marked contrast to ischemic neurons which were associated with ~10-fold increments. The control liver histologically showed lipid droplets in Ito cells, but lipid depositions within hepatocytes were very little. In contrast, after hydroxynonenal injections, widespread fatty degeneration and focal coagulation necrosis were observed with accumulation of numerous tiny lipid droplets within and around the degenerating/dying hepatocytes. Electron microscopy showed lysosomal membrane permeabilization/rupture, remarkable dissolution of mitochondria and rough ER membrane, and proliferation of abnormal peroxisomes. It is probable that disruptions of rough ER caused impaired synthesis of Hsp70.1 and BHMT proteins, while impairments of mitochondria and peroxisomes presumably contributed to the sustained generation of reactive oxygen species. Since both Hsp70.1 and BHMT are vulnerable to the long-standing oxidative stressor, hydroxynonenal-induced disorders facilitated degeneration and steatosis of hepatocytes, respectively.

Although excessive consumption of deep-fried foods is regarded as 1 of the most important epidemiological factors of lifestyle diseases such as Alzheimer's disease, type 2 diabetes, and obesity, the exact mechanism remains unknown. This review aims to discuss whether heated cooking oil-derived peroxidation products cause cell degeneration/death for the occurrence of lifestyle diseases. Deep-fried foods cooked in ω-6 PUFA-rich vegetable oils such as rapeseed (canola), soybean, sunflower, and corn oils, already contain or intrinsically generate “hydroxynonenal” by peroxidation. As demonstrated previously, hydroxynonenal promotes carbonylation of heat-shock protein 70.1 (Hsp70.1), with the resultant impaired ability of cells to recycle damaged proteins and stabilize the lysosomal membrane. Until now, the implication of lysosomal/autophagy failure due to the daily consumption of ω-6 PUFA-rich vegetable oils in the progression of cell degeneration/death has not been reported. Since the “calpain-cathepsin hypothesis” was formulated as a cause of ischemic neuronal death in 1998, its relevance to Alzheimer's neuronal death has been suggested with particular attention to hydroxynonenal. However, its relevance to cell death of the hypothalamus, liver, and pancreas, especially related to appetite/energy control, is unknown. The hypothalamus senses information from both adipocyte-derived leptin and circulating free fatty acids. Concentrations of circulating fatty acid and its oxidized form, especially hydroxynonenal, are increased in obese and/or aged subjects. As overactivation of the fatty acid receptor G-protein coupled receptor 40 (GPR40) in response to excessive or oxidized fatty acids in these subjects may lead to the disruption of Ca(2+) homeostasis, it should be evaluated whether GPR40 overactivation contributes to diverse cell death. Here, we describe the molecular implication of ω-6 PUFA-rich vegetable oil-derived hydroxynonenal in lysosomal destabilization leading to cell death. By oxidizing Hsp70.1, both the dietary PUFA- (exogenous) and the membrane phospholipid- (intrinsic) peroxidation product “hydroxynonenal,” when combined, may play crucial roles in the occurrence of diverse lifestyle diseases including Alzheimer's disease.

It is well-known that the cornu Ammonis 1 (CA1) sector of hippocampus is vulnerable for the ischemic insult, whereas the dentate gyrus (DG) is resistant. Here, to elucidate its underlying mechanism, alternations of protein oxidation and expression of DG in the monkey hippocampus after ischemia-reperfusion by the proteomic analysis were studied by comparing CA1 data. Oxidative damage to proteins such as protein carbonylation interrupt the protein function. Carbonyl modification of molecular chaperone, heat shock 70 kDa protein 1 (Hsp70.1) was increased remarkably in CA1, but slightly in DG. In addition, expression levels of nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-2 (SIRT2) was significantly increased in DG after ischemia, but decreased in CA1. Accordingly, it is likely that SIRT2 upregulation and negligible changes of carbonylation of Hsp70.1 exert its neuroprotective effect in DG. On the contrary, carbonylation level of dihydropyrimidinase related protein 2 (DRP-2) and l-lactate dehydrogenase B chain (LDHB) were slightly increased in CA1 as shown previously, but remarkably increased in DG after ischemia. It is considered that DRP-2 and LDHB are specific targets of oxidative stress by ischemia insult and high carbonylation levels of DRP-2 may play an important role in modulating ischemic neuronal death.

Introduction: The adrenal gland was first described by Eustachio in 1564, but its physiologic role remained unknown for three centuries. In the late 1890’s, many researchers had directed their attention to the isolation of adrenal extracts, but all failed until the successful efforts of biochemists Jokichi Takamine and Keizo Uenaka. Takamine’s patents on Adrenalin were the first ever awarded for a hormone and he used his royalties to improve international relations between America and Japan. There is considerable controversy, however, in how adrenaline was first crystalized, where the work was done, and to whom the credit for its discovery is given. Sources and Methods: Published contemporary medical literature, newspaper archives, and publicly accessible research archives in Japan, Sweden, and the United States Results: Takamine was a multilingual businessman with a background in western and eastern medicine, a successful biochemist, and importer/exporter before he took on the project of isolating adrenaline. His successful patents and savvy business acumen allowed him to support innovative research in physiology. He hired Keizo Uenaka, who had experience with isolating ephedrine, to develop the critical methods required to crystallize adrenal extracts where other contemporaries, including Takamine, had failed. Takamine applied for and was awarded five immediate US patents for the discovery although he did not include Uenaka in that effort. Takamine co-founded the Nippon Club and the Japanese Society of New York, both of which ultimately survived the anti-Japanese fervor of the 1940s and, in 1912, worked to bring the gift of 3,000 Japanese Cherry Trees to Washington DC. The Japanese Patent Office honored Takamine as one of Japan’s 10 Great Inventors but it was only after Uenaka’s death in 1960, and the discovery of his laboratory manuals, was Uenaka’s full role in the discovery of adrenaline made known. Conclusions: Jokichi Takamine was an international benefactor and biochemist extraordinaire who, with Keizo Uenaka, was the first to crystallize adrenal medullary catechols. Takamine’s efforts saved lives and did much to engender a spirit of Japanese-American goodwill that persisted for decades.

The lipid oxidation is a key factor for damaging hepatocytes and causing cell death. However, the mechanisms underlying hepatocyte death and the role of the most popular lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in nonalcoholic steatohepatitis (NASH) remains unclear.We demonstrated using hepatoma cell lines, a NASH mouse model, HNE-treated monkeys, and biopsy specimens from patients with NASH that HNE induced hepatocyte death by disintegrating the lysosomal limiting membrane.The degree of HNE deposition in human NASH hepatocytes was more severe in cases with high lobular inflammation, ballooning, and fibrosis scores, and was associated with enlargement of the staining of lysosomes in hepatocytes. In in vitro experiments, HNE activated μ-calpain via G-protein coupled receptor (GPR) 120. The resultant rupture/permeabilization of the lysosomal limiting membrane induced the leakage of cathepsins from lysosomes and hepatocyte death. The blockade of G-protein coupled receptor 120 (GPR120) or μ-calpain expression suppressed lysosomal membrane damage and hepatocyte death by HNE. Alda-1, which activates aldehyde dehydrogenase 2 to degrade HNE, prevented HNE-induced hepatocyte death. Intravenous administration of HNE to monkeys for 6 months resulted in hepatocyte death by a mechanism similar to that of cultured cells. In addition, intraperitoneal administration of Alda-1 to choline-deficient, amino-acid defined treated mice for 8 weeks inhibited HNE deposition, decreased liver inflammation, and disrupted lysosomal membranes in hepatocytes, resulting in improvement of liver fibrosis.These results provide novel insights into the mechanism of hepatocyte death in NASH and will contribute to the development of new therapeutic strategies for NASH.

The Zbtb20 gene encodes for a transcription factor that plays an important role in mammalian cortical development. Recently, its expression was reported in the adult mouse subventricular zone (SVZ), a major neurogenic niche containing neural stem cells throughout life. Here, we analyzed its expression in the adult primate anterior SVZ (SVZa) and rostral migratory stream (RMS) using macaque monkeys (Macaca fuscata). We report that the majority of Ki67+ cells, 71.4% in the SVZa and 85.7% in the RMS, co-label for ZBTB20. Nearly all neuroblasts, identified by their Doublecortin expression, were positive for ZBTB20 in both regions. Nearly all GFAP+ neural stem cells/astrocytes were also positive for ZBTB20. Analysis of images derived from a public database of gene expression in control/ischemic monkey SVZa, showed evidence for ZBTB20 upregulation in postischemic monkey SVZa. Furthermore, the co-localization of ZBTB20 with Doublecortin and Ki67 was increased in the postischemic SVZa. Our results suggest that ZBTB20 expression is evolutionarily conserved in the mammalian neurogenic niche and is reactive to ischemia. This opens the possibility for further functional studies on the role of this transcription factor in neurogenesis in primates.

Adrenal gland was first described by the Italian anatomist Eustachio in 1564, but its physiologic role remained unknown for three centuries. Even after the report of Addison’s disease in 1855, nobody knew its function. Oliver and Schäfer first discovered the blood pressure-raising property of adrenal extracts in 1894. Accordingly, in the late 1890’s, highly motivated scientists had directed their attention to isolate active principle for the therapeutic utilization, but all failed. For instance, Abel’s preparation ‘epinephrine’ was an inactive benzoylated derivative. In August 5, 1900 the Japanese industrial chemist Takamine and his young associate Uenaka settled in New York, succeeded to crystallize the adrenal extract by a procedure different from any yet employed. The active principle was isolated in the vacuum pan, crystallized with ammonia, and confirmed by the Vulpian reaction. ‘Uenaka’s Experimental Memorandum’ describes that the novel crystal was coined ‘adrenalin’ (no “e”) on November 7, 1900. Simultaneously, Takamine applied the US patent which was approved on June 2, 1903, and Parke, Davis & Company trademarked the name ‘Adrenalin’ to market worldwide. As a hemostatic during surgery and for treating heart failure, adrenaline has saved numerous lives. There are historical, etymological and practical justifications for using the term ‘adrenaline’. &nbsp

Beyond guarding the cellular proteome the major stress inducible heat shock protein Hsp70 has been shown to interact with lipids. Non-cytosolic Hsp70 stabilizes membranes during stress challenges and, in pathophysiological states, facilitates endocytosis, counteracts apoptotic mechanisms, sustains survival pathways or represents a signal that can be recognized by the immune system. Disease-coupled lipid-associated functions of Hsp70 may be targeted via distinct subcellular localizations of Hsp70 itself or its specific interacting lipids. With a special focus on interacting lipids, here we discuss localization-dependent roles of the membrane-bound Hsp70 in the context of its therapeutic potential, particularly in cancer and neurodegenerative diseases.

G protein-coupled receptor 40 (GPR40), a receptor for diverse non-esterified fatty acids, is expressed predominantly in the wide variety of neurons of the central nervous system and β-cells in the pancreatic islets. Since deorphanization of GPR40 in 2003, the past decade has seen major advances in our understanding of its role in the insulin secretion. However, there is still a great deal to be elucidated about the role of GPR40 in the brain, because the latter shows the most abundant GPR40 mRNA expression among the human tissues. Since a substantial expression of GPR40 is also seen in the hypothalamus, 'brain-lipid sensing' might be involved in the control of insulin secretion and energy balance. The preceding experiments using monkeys after transient global brain ischemia, have highlighted implication of GPR40 for amplifying adult hippocampal neurogenesis. Although GPR40-mediated intracellular signaling was recently found to result in phosphorylation of cAMP response element-binding protein (CREB) necessary for the neuronal differentiation and synaptic plasticity, the signaling cascade is still incompletely understood. Furthermore, in response to conjugated linoleic acids or trans isomers of arachidonic acid, GPR40 was recently demonstrated in rodents to mediate lipotoxicity to β-cells, neurons, or microvessels, which result in diabetes, retinopathy, stroke, etc. However, it still remains undetermined in humans whether and how oxidized, conjugated, or excessive fatty acids evoke lipotoxicity. Although literature about GPR40 is limited especially about the brain or the brain-pancreas interaction, this review aims at summarizing beneficial as well as detrimental effects of this receptor in the brain and pancreas in response to diverse fatty acids.

Cyclon (cytokine-induced protein with coiled-coil domain), also known as CCDC86 (coiled-coil domain-containing protein 86), is a nuclear protein expressed in lymphocytes in mice where it regulates T-cell responses. Here we show that Cyclon/CCDC86 is expressed in the hippocampus of adult macaque monkeys, including both the hippocampus proper (cornu Ammonis) and the dentate gyrus. Cyclon/CCDC86 was localized to neurons and astrocytes in cornu Ammonis, while in the dentate gyrus the protein was also expressed in immature neurons and a very small fraction of proliferating cells. These results point to a novel expression pattern of Cyclon/CCDC86 beyond its currently known immune cell localization.

On average, every four minutes an individual dies from a stroke, accounting for 1 out of every 18 deaths in the United States. Approximately 795,000 Americans have a new or recurrent stroke each year, with just over 600,000 of these being first attack [1]. There have been multiple animal models of stroke demonstrating that novel therapeutics can help improve the clinical outcome. However, these results have failed to show the same outcomes when tested in human clinical trials. This review will discuss the current in vivo animal models of stroke, advantages and limitations, and the rationale for employing these animal models to satisfy translational gating items for examination of neuroprotective, as well as neurorestorative strategies in stroke patients. An emphasis in the present discussion of therapeutics development is given to stem cell therapy for stroke.

Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) has been used for the detection of Alzheimer’s disease, because its diagnostic value is not widely accepted due to lack of supportive imaging data. In this study, we retrospectively evaluated the validity of RBANS by single photon emission computed tomography (SPECT) to evaluate regional cerebral blood flow (rCBF). Methods: We enrolled 87 subjects with complaints of memory impairment who underwent both RBANS and SPECT. The RBANS scores were assessed, correlating with the decrease of rCBF in the crucial areas. Results: Linear regression analysis showed a significant positive correlation between the total and delayed memory scores of RBANS and the rCBF of the posterior cingulate gyrus and the precuneus. In the 2-sample t-test, the rCBF of the posterior cingulate gyrus in the patients with a total RBANS score of less than 34 was significantly lower, compared to those with a score more than 35. Conclusion: These data indicated a significant correlation between the RBANS total score and the perfusion of the posterior cingulate gyrus, and both methods are considered complementarily useful for detecting early Alzheimer’s disease.

It is well-known that heat-shock protein 70.1 (Hsp70.1), a major protein of the human Hsp70 family, plays cytoprotective roles by both its chaperone function and stabilization of lysosomal membranes. Recently, we found that calpain-mediated cleavage of carbonylated Hsp70.1 in the hippocampal cornu Ammonis1 (CA1) contributes to neuronal death after transient global ischemia. This study aims to elucidate the differential neuronal vulnerability between the motor cortex and CA1 sector against ischemia/reperfusion. Fluoro-Jade B staining and terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labeling analysis of the monkey brain undergoing 20min whole brain ischemia followed by reperfusion, showed that the motor cortex is significantly resistant to the ischemic insult compared with CA1. Up-regulation of Hsp70.1 but absence of its cleavage by calpain facilitated its binding with NF-κB p65/IκBα complex to minimize NF-κB p65 activation, which contributed to a neuroprotection in the motor cortex. In contrast, because activated μ-calpain cleaved carbonylated Hsp70.1 in CA1, the resultant Hsp70.1 dysfunction not only destabilized lysosomal membrane but also induced a sustained activation of NF-κB p65, both of which resulted in delayed neuronal death. We propose that the cascades underlying lysosomal stabilization and regulating NF-κB activation by Hsp70.1 may influence neuronal survival/death after the ischemia/reperfusion.

Necrosis has long been considered accidental and uncontrolled, but during the last decade, it became clear that necrosis is also a well-orchestrated form of cell demise, being as well programmed as apoptosis. To explain the mechanism of neuronal necrosis after ischemia/reperfusion, the 'calpain-cathepsin hypothesis' formulated in 1998 postulates that the post-ischemic μ-calpain activation compromises integrity of the lysosomal membrane, thereby leading to cathepsin spillage. Another cause of the lysosomal rupture occurring during reperfusion is reactive oxygen species (ROS) that generate 4-hydroxy-2-nonenal (HNE) by oxidation of membrane fatty acids such as linoleic and arachidonic acids. HNE is an endogenous neurotoxin, because HNE-induced carbonylation of the substrate protein shows loss of its function. However, the molecular mechanisms of lysosomal membrane breakdown are still poorly understood; especially, the biochemical cascade how μ-calpain and ROS work together to disrupt lysosomal membrane has remained unclarified. Three independent proteomic analyses of cerebral ischemia, glaucoma, or mild cognitive impairment in primates have altogether suggested that the common substrate of calpain and/or ROS is heat-shock protein 70.1 (Hsp70.1; simply Hsp70, also called Hsp72 or HSPA1), a major protein of the human Hsp70 family. Hsp70.1 serves cytoprotective roles as a guardian of the lysosomal membrane integrity by assisting sphingomyelin degradation or maintaining proper protein folding and recycling as a chaperone. However, calpain-mediated cleavage of Hsp70.1, especially after its carbonylation because of the oxidative stresses, can induce lysosomal rupture. Furthermore, Hsp70.1 dysfunction activates nuclear factor-kappaB (NF-κB) signaling that can also promote neurodegeneration. By focusing on Hsp70.1 and related lysosomal factors, this review describes rationale of lysosomal destabilization and rupture for executing programmed neuronal necrosis.

Intracellular fatty acid (FA) chaperones known as FA-binding proteins (FABPs) are a group of molecules known to participate in cellular metabolic processes such as lipid storage, membrane synthesis, and β-oxidation or to coordinate transcriptional programs. However, their role in adult neurogenesis still remains obscure. The FABPs expressed in the central nervous system (CNS) are heart-type (FABP3), epidermal-type (FABP5), and brain-type (FABP7). These three FABPs possess a differential affinity for polyunsaturated fatty acids (PUFAs). Recently, we reported that GPR40, a receptor for free FAs and particularly for PUFAs, is expressed in the CNS of adult monkeys and upregulated after transient global brain ischemia in the hippocampal subgranular zone (SGZ), a neurogenic niche in adulthood. The SGZ showed a peak proliferation of progenitor cells and maximal expression of GPR40 during the second week after ischemia. As both FABPs and GPR40 might be closely related to the adult neurogenesis, here, we studied the expression of FABP 3, 5, and 7 in the SGZ, comparing normal and postischemic adult monkeys. Immunoblotting revealed that FABP5 and FABP7, but not FABP3, were significantly increased on day 15 after ischemia when compared with the nonischemic control. Immunohistochemistry showed that FABP5 was almost undetectable in the control SGZ but was abundant on day 15 after ischemia. FABP 3, 5, and 7 were expressed in S-100β-positive astrocytes and nestin-positive neural progenitors. However, only FABP 5 and 7 were found in bromodeoxyuridine (BrdU)-positive newly generated cells. FABPs were most frequently coexpressed with the S-100β-positive astrocytes, whereas βIII-tubulin-or polysialylated neural cell-adhesion molecule (PSA-NCAM)-positive newborn neurons in the vicinity of the astrocytes expressed none of the three FABPs. These results support a role of astrocyte- and/or neural progenitor-derived FABPs as components of the molecular machine regulating the progenitor cell niche in the adult primate brain.

Objective Benzodiazepines (BZDs) have been reported to cause negative impacts on body stability and cognitive functions, which in turn could result in lethal incidents, including falls, especially in the elderly. This fact notwithstanding, no systematic trial has evaluated the feasibility and benefits of discontinuing BZD-derivative hypnotics in this population, which was addressed in this study. Methods In this 8-week open-label study, subjects aged ≥60 living in a nursing home who received BZD as a hypnotic were recruited. The BZD dose was tapered off over 3 weeks. The following assessments were performed 12 h post-dose at baseline and at endpoint: the Clinical Stabilometric Platform (CSP), the Critical Flicker Fusion Test (CFF), the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and the Leeds Sleep Evaluation Questionnaire (LSEQ). Results Thirty subjects were enrolled (mean ± SD age = 79.1 ± 8.9 years, mean ± SD flurazepam equivalent BZD dose = 19.5 ± 10.9 mg/day). Psychiatric diagnoses (DSM-IV) of subjects were as follows: schizophrenia (n = 12), primary insomnia (n = 9), dementia (n = 7), and bipolar disorder (n = 2). In 26 completers, significant changes were found in a total length and a range of trunk motion with eyes closed. Significant improvements were also observed in the CFF and RBANS immediate memory, language, and attention index scores. Subjective worsening in sleep was not reported in those completers, assessed with the LSEQ. Conclusions Our results suggest that discontinuation of BZD hypnotics is feasible in a majority of elderly persons and leads to an improvement in the stability of body and a recovery in cognitive functions during the daytime. Copyright © 2010 John Wiley & Sons, Ltd.

When bone marrow transplantation (BMT) is carried out, survival of the donor hematopoietic stem cells is crucial to maintain donor hematopoiesis in the recipients. We have shown that intra-bone marrow-bone marrow transplantation (IBM-BMT) can induce the rapid recovery of donor hematopoiesis and allow a reduction in radiation doses as a pretreatment for BMT. If IBM-BMT methodology can be further improved, BMT could be carried out more safely and more easily. In this experiment, we attempted to suppress apoptosis of donor hematopoietic cells using a caspase inhibitor, ZVAD-fmk, upon IBM-BMT in mouse allogeneic IBM-BMT. IBM-BMT with ZVAD-fmk induced superior engraftment of donor hematopoietic cells and greater numbers of day-12 colony-forming units of spleen (CFU-S) than IBM-BMT without ZVAD-fmk upon allogeneic BMT (C57BL/6 into BALB/c mice). ZVAD-fmk slightly suppressed apoptosis of whole BMCs, whereas it significantly suppressed apoptosis of c-kit+/Sca-1+/lineage(-) cells (KSL cells) in vitro. These results suggest that ZVAD-fmk can suppress apoptosis of hematopoietic stem cells and/or immature progenitor cells of the donor bone marrow cells, thereby accelerating the donor hematopoiesis.

Necrotic neuronal death is recently known to be mediated by the calpain-cathepsin cascade from simpler organisms to primates. The main event of this cascade is calpain-mediated lysosomal rupture and the resultant release of lysosomal cathepsins into the cytoplasm. However, the in-vivo substrate of calpain for inducing lysosomal destabilization still remains completely unknown. The recent proteomics data using the post-ischemic hippocampal CA1 tissues and glaucoma-suffered retina from the primates suggested that heat shock protein (Hsp) 70.1 might be the in-vivo substrate of activated mu-calpain at the lysosomal membrane of neurons. Hsp70.1 is known to stabilize lysosomal membrane by recycling damaged proteins and protect cells from oxidative stresses. Here, we studied the molecular interaction between activated mu-calpain and the lysosomal Hsp70.1 in the monkey hippocampal CA1 neurons after the ischemia-reperfusion insult. Immunofluorescence histochemistry showed a colocalization of the activated mu-calpain and upregulated Hsp70.1 at the lysosomal membrane of the post-ischemic CA1 neurons. In-vitro cleavage assay of hippocampal Hsp70.1 by Western blotting demonstrated that Hsp70.1 in the CA1 tissue is an in-vivo substrate of activated mu-calpain, and that carbonylated Hsp70.1 in the CA1 tissue by artificial oxidative stressors such as hydroxynonenal (HNE) or hydrogen peroxide is much more vulnerable to the calpain cleavage. These data altogether suggested that Hsp70.1 can become a target of the carbonylation by HNE, and Hsp70.1 is a modulator of calpain-mediated lysosomal rupture/permeabilization after the ischemia-reperfusion injury.

Glaucoma, an optic neuropathy, is the leading cause of world blindness. In this condition, the damage extends from the retina to the visual center in the brain, although the primary region of damage is thought to be the optic nerve head (ONH), with the lateral geniculate nucleus (LGN) being secondarily affected. We investigated time-dependent alterations in the ONH, the optic nerve (ON), and the LGN after intraocular pressure (IOP) elevation in Japanese monkeys (a species more similar to humans than other macaque species). Nine Japanese monkeys, each with an experimental glaucomatous left eye, and two naive monkeys were studied. Ocular-testing sessions (including IOP measurement and fundus photography) were held weekly. Eyes and brains were enucleated at 2-48 weeks after IOP elevation, and alterations in ONs and LGN were evaluated. The IOP of the treated eyes was monitored periodically and found to be elevated continuously throughout the observation period in each monkey. The ONH of the glaucomatous eyes exhibited time-dependent deep cupping and thinning of the rim area from 2 weeks after the IOP elevation. Loss of axons and a decrease in the area of ON were first observed at 4 and 28 weeks, respectively. Neuronal loss was first observed at 2 weeks in layers 1 and 2 of LGN [magnocellular (M)-layer] and at 12 weeks in layers 3-6 of LGN [parvocellular (P)-layer]. Neuronal shrinkage was first observed at 2 weeks in all layers in LGN. These findings indicate that in Japanese monkeys, damage to neurons in LGN can be detected in the early phase (first few weeks) after an IOP elevation, as can damage to ONH.

Polyunsaturated free fatty acids (PUFAs) are known to play critical roles for the development, maintenance, and function of the brain. Recently, we reported that G-protein coupled receptor 40 (GPR40), one type of PUFA receptors, is expressed throughout the adult primate central nervous system including the hippocampus. This opens a possibility that PUFA might act as extracellular signaling molecules at the GPR40 receptor to regulate neuronal function. Here we studied protein expression of GPR40 in the neurogenic niche of the adult monkey hippocampus under normal and postischemic conditions. Confocal laser microscope analysis of immunostained sections revealed GPR40 immunoreactivity in neural progenitors, immature neurons, astrocytes and endothelial cells of the subgranular zone (SGZ) of the dentate gyrus (DG); a well-known neurogenic niche within the adult brain. Immunoblotting analysis showed that the GPR40 protein increased significantly in the second week after global cerebral ischemia as compared with the control. This was compatible with the postischemic increment of GPR40-positive cells in the SGZ as detected by immunofluorescence imaging. Taken together with our previous findings of the SGZ progenitor cell upregulation after ischemia, the present data suggest that PUFA such as docosahexaenoic acid may act via GPR40 to regulate adult hippocampal neurogenesis in primates.

The G-protein coupled receptor 40 (GRP40) is a transmembrane receptor for free fatty acids, and is known for its relation to insulin secretion in the pancreas. Recent studies demonstrated that spatial memory and hippocampal long-term potentiation of rodents and cognitive function of humans are improved by a dietary supplementation with arachidonic and/or docosahexaenoic acids, which are possible ligands for GPR40. While free fatty acid effects on the brain might be related to GPR40 activation, the role of GPR40 in the central nervous system (CNS) is at present not known. Here, we studied expression and distribution of GPR40 in CNS of adult monkeys by immunoblotting and immunohistochemistry. Immunoblotting analysis showed a band of approximately 31 kDa consistent with the size of GPR40 protein. GPR40 immunoreactivity of was observed in the nuclei and/or perikarya of a wide variety of neurons including neurons in the cerebral cortex, hippocampus, amygdala, hypothalamus, cerebellum, spinal cord. In addition, astrocytes of the cerebral white matter, the molecular layer and multiform layer of the cerebral cortex, the subventricular zone along the anterior horn of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus showed GPR40 immunoreactivity. The present data first provide a morphological basis for clarifying the role of GPR40 in the primate CNS.

That tumors lack innervation is dogma in the field of pathology, but the molecular determinants of this phenomenon remain elusive. We studied the effects of conditioned media from Colon 26 and B16 mouse tumor cell lines on the axonal outgrowth and cellular differentiation of embryonic Institute of Cancer Research (ICR) mouse dorsal root ganglion cells. Tumor-conditioned media suppressed dorsal root ganglion axonal extension but had no effect on neuronal or glial differentiation. We found that the tumor cells expressed most of the class 3 semaphorins - axon guidance molecules. Blocking the activity of class 3 semaphorins with the soluble receptor neuropilin-1 significantly counteracted the tumor-induced inhibition of axonal extension. Together, these results suggest a role for tumor-secreted class 3 semaphorins in selectively inhibiting axonal outgrowth of dorsal root ganglion neurons.

The subventricular zone along the anterior horn (SVZa) of the cerebral lateral ventricle of adult mammals contains multipotent progenitor cells, which supposedly exist in an angiogenic niche. Numerous signals are known to modulate the precursor cell proliferation, migration or differentiation, in rodent models. In contrast, the data on signals regulating the primate SVZa precursors in vivo are scarce. We analyzed the expression at protein level of a panel of angiogenic and/or neurotrophic factors and their receptors in SVZa of adult macaque monkeys, under normal condition or after transient global ischemia which enhances endogenous progenitor cell proliferation. We found that fms-like tyrosine kinase 1 (Flt1), a receptor for vascular endothelial cell growth factor, was expressed by over 30% of the proliferating progenitors, and the number of Flt1-positive precursors was significantly increased by the ischemic insult. Smaller fractions of mitotic progenitors were positive for the neurotrophin receptor tropomyosin-related kinase (Trk) B or the hematopoietic receptor Kit, while immature neurons expressed Flt1 and the neurotrophin receptor TrkA. Further, SVZa astroglia, ependymal cells and blood vessels were positive for distinctive sets of ligands/receptors, which we characterized. The presented data provide a molecular phenotypic analysis of cell types comprising adult monkey SVZa, and suggest that a complex network of angiogenic/neurotrophic signals operating in an autocrine or paracrine manner may regulate SVZa neurogenesis in the adult primate brain.

Neurons are highly specialized post-mitotic cells, so their homeostasis and survival depend on the tightly-regulated, continuous protein degradation, synthesis, and turnover. In neurons, autophagy is indispensable to facilitate recycling of long-lived, damaged proteins and organelles in a lysosome-dependent manner. Since lysosomal proteolysis under basal conditions performs an essential housekeeping function, inhibition of the proteolysis exacerbates level of neurodegeneration. The latter is characterized by an accumulation of abnormal proteins or organelles within autophagic vacuoles which reveal as 'granulo-vacuolar degenerations' on microscopy. Heat-shock protein70.1 (Hsp70.1), as a means of molecular chaperone and lysosomal stabilizer, is a potent survival protein that confers neuroprotection against diverse stimuli, but its depletion induces neurodegeneration via autophagy failure. In response to hydroxynonenal generated from linoleic or arachidonic acids by the reactive oxygen species, a specific oxidative injury 'carbonylation' occurs at the key site Arg469 of Hsp70.1. Oxidative stress-induced carbonylation of Hsp70.1, in coordination with the calpain-mediated cleavage, leads to lysosomal destabilization/rupture and release of cathepsins with the resultant neuronal death. Hsp70.1 carbonylation which occurs anywhere in the brain is indispensable for neuronal death, but extent of calpain activation should be more crucial for determining the cell death fate. Importantly, not only acute ischemia during stroke but also chronic ischemia due to ageing may cause calpain activation. Here, role of Hsp70.1-mediated lysosomal rupture is discussed by comparing ischemic and Alzheimer neuronal death. A common neuronal death cascade may exist between cerebral ischemia and Alzheimer's disease.

Molecular signals regulating adult neurogenesis in primates are largely unknown. Here the authors used differential display to analyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and characterized its expression pattern at the protein level. In contrast with approximately threefold upregulation of Dscam gene on days 5 and 7, immunoblotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or βIII-tubulin, while another is astrocytes positive for S100β. Young astrocytes were in intimate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of primate hippocampus upregulated after ischemia. © 2006 Wiley-Liss, Inc.

The purpose of this study was to determine whether minocycline, a semi-synthetic tetracycline derivative, reduces (a) the in vitro neuronal damage occurring after serum deprivation in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus) and/or (b) the in vivo retinal damage induced by N-methyl-D-aspartate (NMDA) intravitreal injection in mice. In addition, we examined minocycline's putative mechanisms of action against oxidative stress and endoplasmic reticulum (ER) stress. In vitro, retinal damage was induced by 24-h serum deprivation, and cell viability was measured by Hoechst 33342 staining or resazurin reduction assay. In cultures of RGC-5 cells maintained in serum-free medium for up to 24 h, the number of cells undergoing cell death was reduced by minocycline (0.2-20 microM). Serum deprivation resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 2 and 20 microM inhibited this ROS production. However, even at 20 microM minocycline did not inhibit the retinal damage induced by tunicamycin (an ER stress inducer). Furthermore, in mice in vivo minocycline at 90 mg/kg intraperitoneally administered 60 min before an NMDA intravitreal injection reduced the NMDA-induced retinal damage. These findings indicate that minocycline has neuroprotective effects against in vitro and in vivo retinal damage, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.

The neuroprotective effects of minocycline-which is broadly protective in neurologic-disease models featuring cell death and is being evaluated in clinical trials-were investigated both in vitro and in vivo. For the in vivo study, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice. Minocycline at 90 mg/kg intraperitoneally administered 60 min before or 30 min after (but not 4 h after) the occlusion reduced infarction, brain swelling, and neurologic deficits at 24 h after the occlusion. For the in vitro studies, we used cortical-neuron cultures from rat fetuses in which neurotoxicity was induced by 24-h exposure to 500 microM glutamate. Furthermore, the effects of minocycline on oxidative stress [such as lipid peroxidation in mouse forebrain homogenates and free radical-scavenging activity against diphenyl-p-picrylhydrazyl (DPPH)] were evaluated to clarify the underlying mechanism. Minocycline significantly inhibited glutamate-induced cell death at 2 microM and lipid peroxidation and free radical scavenging at 0.2 and 2 microM, respectively. These findings indicate that minocycline has neuroprotective effects in vivo against permanent focal cerebral ischemia and in vitro against glutamate-induced cell death and that an inhibition of oxidative stress by minocycline may be partly responsible for these effects.

From rodents to primates, transient global brain ischemia is a well known cause of delayed neuronal death of the vulnerable neurons including cornu Ammonis 1 (CA1) pyramidal cells of the hippocampus. Previous reports using the rodent experimental paradigm indicated that apoptosis is a main contributor to such ischemic neuronal death. In primates, however, the detailed molecular mechanism of ischemic neuronal death still remains obscure. Recent data suggest that necrosis rather than apoptosis appear to be the crucial component of the damage to the nervous system during human ischemic injuries and neurodegenerative diseases. Currently, necrotic neuronal death mediated by Ca2+-dependent cysteine proteases, is becoming accepted to underlie the pathology of neurodegenerative conditions from the nematode Caenorhabditis elegans to primates. This paper reviews the role of cysteine proteases such as caspase, calpain and cathepsin in order to clarify the mechanism of ischemic neuronal death being triggered by the unspecific digestion of lysosomal proteases.

Previously, we reported surgical results of the simplified retrosigmoid approach for vestibular nerve sectioning in the cases with intractable vertigo. Here, we introduce in details surgical procedures of the simplified retrosigmoid (also called "subasterional" here for simplification) approach developed by one of the authors (J.H.L.). This approach is to operate cerebello-pontine (C-P) angle lesions through a small upper lateral window below the transverse-sigmoid junction. The retroauricular 4-5 cm skin incision, triangular craniectomy 2 cm in width, dural opening and closure, special considerations, and reconstruction of the skull defect were outlined. The bony landmarks for triangle craniectomy below asterion were described in detail. The C-P angle was appropriately approached for microvascular decompression, removal of C-P angle tumors, and vestibular nerve resection. This subasterional approach was characterized by short operation time of 1-3 h and less postoperative discomforts compared to the conventional retrosigmoid approach.

To investigate the effect of global cerebral ischemia on brain cell proliferation in young adult macaques, we infused 5-bromo-2'-deoxyuridine (BrdU), a DNA replication indicator, into monkeys subjected to ischemia or sham-operated. Subsequent quantification by BrdU immunohistochemistry revealed a significant postischemic increase in the number of BrdU-labeled cells in the hippocampal dentate gyrus, subventricular zone of the temporal horn of the lateral ventricle, and temporal neocortex. In all animals, 20-40% of the newly generated cells in the dentate gyrus and subventricular zone expressed the neural progenitor cell markers Musashi1 or Nestin. A few BrdU-positive cells in postischemic monkeys were double-stained for markers of neuronal progenitors (class III beta-tubulin, TUC4, doublecortin, or Hu), neurons (NeuN), or glia (S100beta or GFAP). Our results suggest that ischemia activates endogenous neuronal and glial precursors residing in diverse locations of the adult primate central nervous system.

The samurai chemist Jokichi Takamine (1854–1922) crystallized adrenalin, the first hormone to be isolated in the twentieth century, from the adrenal medulla, in the summer of 1900. This paper reviews Takamine's route to the discovery of adrenalin and presents historical photographs and documents collected in Kanazawa, Japan, where he grew up, and the United States, where he made his major discoveries.

We investigated the fate of proliferating cells in the adult monkey brain after global ischemia. We used the thymidine analogue bromodeoxyuridine (BrdU) to label S-phase cells and their progeny in Japanese macaques subjected to global cerebral ischemia for 20 min or to a sham operation. Subsequently, newly generated cells were identified by BrdU immunohistochemistry, and their immunophenotype was determined quantitatively, using specific markers. The ischemic insult significantly increased the number of proliferating cells in the hippocampus and temporal neocortex, where the majority BrdU-labeled cells expressed markers for microglia (Iba1, CD68, and Ham56) or astrocytes (S-100beta and glial fibrillary acidic protein [GFAP]). In contrast, the proliferation level in the parahippocampal region remained unchanged. This discrepancy prompted us to investigate the postischemic response in the olfactory bulb, a well-known site of adult cell generation that is anatomically distant from the above-mentioned regions but that is also subjected to the global ischemic insult. The olfactory bulb contained clusters of proliferating cells expressing markers for neural (Musashi1 and Nestin) and/or neuronal (class III beta-tubulin) progenitors; these were immunophenotypically distinct from other cell types. Their number and distribution were unaltered by ischemia. Our results demonstrate that cell proliferation and differentiation in the adult macaque brain and olfactory bulb are differentially affected by a common insult.

Vitamin B6 derivatives protect the retinal neurons from excitotoxic injury in vitro. However, their in vivo role in a process involving excitotoxicity, such as ischemia, remains unknown. We studied potential protective effects of pyridoxal 5'-phosphate (PLP) and pyridoxal hydrochloride (pyridoxal) on the retinal neurons in a monkey model of transient global ischemia. Daily intravenous injections (15 mg/kg) of pyridoxal and PLP were performed for consecutive 10 days. On the sixth day, whole brain complete ischemia was produced by clipping the innominate and the left subclavian arteries for 20 min. The monkeys were sacrificed 5 days after ischemia and their retinas were processed for histological analysis. The ischemia induced a marked cellular injury in the retina as shown by the loss of ganglion cells and the reduction of thickness of the ganglion cell, inner plexiform, and inner nuclear layers. PLP significantly prevented the ganglion cell loss and the reduction of thickness of the ganglion cell layer. Pyridoxal significantly prevented the ganglion cell loss as well as the reduction of thickness of ganglion cell, inner plexiform and inner nuclear layers. These results suggest that PLP and pyridoxal counteract the postischemic neuronal death in the adult primate retina, offering a potential for a novel pharmacotherapy of retinal ischemic injury.

To clarify the involvement of intracellular signaling pathway and calpain in the brain injury and its protection by mild hypothermia, immunoblotting analyses were performed in the rat brain after global forebrain ischemia and reperfusion. After 30 min of ischemia followed by 60 min of reperfusion, Ca2+/calmodulin-dependent kinase II (CaM kinase II) and protein kinase C (PKC)-alpha, beta, gamma isoforms translocated to the synaptosomal fraction, while mild hypothermia (32 degrees C) inhibited the translocation. The hypothermia also inhibited fodrin proteolysis caused by ischemia-reperfusion, indicating the inhibition of calpain. These effects of hypothermia may explain the mechanism of the protection against brain ischemia-reperfusion injury through modulating synaptosomal function.

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain-induced leakage of lysosomal cathepsins (calpain-cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase-activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post-ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining on days 3–5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post-ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase-3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.

The aim of the present study was to visualize postischemic hippocampal neuronal death in the living monkey brain, using a high-resolution positron emission tomography (PET) and novel radioligands. In preceding papers, we reported on postischemic hippocampal neuronal death in a model of Japanese monkeys (Macaca fuscata) undergoing a 20-min complete whole-brain ischemia. Using the same model here, we investigated the in vivo bindings of two radiotracers, [11C]Ro15-4513 (a type II benzodiazepine receptor ligand) and [11C](+)3-MPB (a muscarinic cholinergic receptor ligand), in the hippocampus on day 7 after ischemia, as compared to the normal hippocampus. A significant decrease in the in vivo binding of [11C]Ro15-4513 and [11C](+)3-MPB was observed in the postischemic monkey hippocampus on day 7 after ischemia compared to controls. Light and electron microscopic analyses of postischemic CA1 neurons showed typical features of coagulation necrosis, as associated with a marked reduction of postsynaptic densities and presynaptic vesicles. These results suggest that semiquantification of hippocampal neuronal death is possible in the living primate brain using PET, and that the same procedures can be applied for evaluating neuronal cell loss in patients with ischemic injuries and/or dementia. Hippocampus 2002;12:109–118. © 2002 Wiley-Liss, Inc.