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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.

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.

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.

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.

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.

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.

金沢大学医薬保健研究域医学系, 金沢大学附属病院脳神経外科, A 16-year-old girl presented with multiple cerebral cavernous angiomas with calcifications due to repeated hemorrhages and a thrombosed cerebral arteriovenous malformation (AVM). Her 18-year-old elder sister also had multiple cerebral cavernous angiomas associated with calcifications, which suggested presence of repeated previous hemorrhages. Surgical removal via a right occipital craniotomy resulted in a good outcome. The histological diagnosis was thrombosed AVM. Evaluation of congenital vascular anomaly needs to take into consideration the combination of other congenital vascular anomalies and their familial occurrence.

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion.

For the past decade evidence has gathered for the implication of lysosomes in the development of programmed cell death. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) not only as a molecular chaperone for damaged/aged proteins but also as a guardian of lysosomal integrity. Hsp70.1-mediated lysosomal stabilization is tightly regulated, but its disorder under extreme conditions results in lysosomal rupture to cause cell death. Comparing with the pathological hallmark ‘granulo-vacuolar degenerations’ in Alzheimer’s disease, the postischemic monkey neurons were carefully observed with microscope. Intriguingly, we found very similar change, and identified it as ‘lysosomal vesiculosis’ by electron microscopy. However, the exact molecular and structural impacts of the Hsp70.1 disorder upon the lysosomal membrane are hardly elucidated in the human brain because of the practical and ethical problems. Accordingly, using the monkey brain tissues after in-vivo and in-vitro oxidative stresses, we studied molecular modifications of Hsp70.1 and its counterpart bis(monoacylglycero) phosphate (BMP), because these molecules are closely related to the lysosomal membrane stability by regulating acid sphingomyelinase. It still remains unelucidated whether ischemia/reperfusion can alter composition or amount of BMP in the brain. Regardless of the brain regions studied, the normal monkey brain tissues showed calpain-mediated cleavage of Hsp70.1 after in-vitro oxidative stress. In the CA1 after in-vivo ischemia/reperfusion, we first found that docosahexaenoic and oleic acids in BMP showed a significant decrease on days 1 and 3, compared to non-ischemic controls. Since Hsp70.1- BMP binding is indispensable for activating acid sphingomyelinase and producing ceramide to stabilize lysosomal membrane, dysfunction of Hsp70.1 and BMP presumably causes storage of sphingomyelin and deficiency of ceramide at the lysosomal membrane, leading to its destabilization in the postischemic CA1 neurons. These data, combined together, suggest that lysosomal destabilization induced by calpain-mediated cleavage of carbonylated Hsp70.1 and the concomitant BMP breakdown causes ‘lysosomal vesiculosis’.

金沢大学医薬保健研究域医学系, A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of μ-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, exhibits mitogenic, motogenic, and morphogenic activities for regeneration of the liver, kidney, and lung. Recently, HGF was clearly shown to enhance neurite outgrowth in vitro. To determine whether HGF has a neuroprotective action against the death of neurons in vivo, we studied the effect of HGF on delayed neuronal death in the hippocampus after 5-minute transient forebrain ischemia in Mongolian gerbils. Continuous postischemic intrastriatal administration of human recombinant HGF (10 or 30 μg) for 7 days potently prevented the delayed death of hippocampal neurons under both anesthetized and awake conditions. Even when HGF infusion started 6 hours after ischemia (i.e., in a delayed manner), HGF exhibited a neuroprotective action. We conclude that HGF, a novel neurotrophic factor, has a profound neuroprotective effect against postischemic delayed neuronal death in the hippocampus, which may have implications for the development of new therapeutic strategies for ischemic neuronal damage in humans.

We examined the effects of peripheral axotomy on the immunoreactivity of E-cadherin and cadherin-associated protein alpha N-catenin in the spinal cord. E-cadherin is known to be exclusively expressed in lamina II of Rexed in the spinal cord dorsal horn. This expression disappeared by day 7 after axotomy and reappeared following nerve ligature (partial axonal regeneration model) on day 63. In contrast, it remained undetectable following nerve clipping (complete degeneration model). Alpha N-catenin was diffusely stained in the gray matter, and the immunoreactivity was specifically intense in the central canal and superficial dorsal horn. The expression of alpha N-catenin in the superficial dorsal horn was similarly reduced by day 7 after axotomy, but recovered by day 63 after nerve ligature. In contrast, it remained at the reduced level after nerve clipping. The alteration of alpha N-catenin immunoreactivity showed a similar pattern consistent with that of E-cadherin. Administration of nerve growth factor (NGF) rescued the immunoreactivity of substance P, which is known to disappear after peripheral axotomy, but not influence that of both E-cadherin or alpha N-catenin. These results clearly showed that peripheral axotomy simultaneously alters the immunoreactivity of E-cadherin and alpha N-catenin in the spinal cord, suggesting a correlation in the expression of both E-cadherin and alpha N-catenin in vivo. E-cadherin-alpha N-catenin complex might be crucial for plasticity of the spinal cord dorsal horn after peripheral axotomy.

Arachnoid villi or granulations are small projections of the arachnoid barrier layer into the venous sinus and its major tributaries. They are closely related to the absorption of cerebrospinal fluid, and are widely accepted to be the origin of human meningiomas. Arachnoid villi and meningiomas show a number of similarities in ultrastructure, cell adhesion mechanisms, and extracellular matrix composition. Ultrastructurally, both arachnoid and meningioma cells are characterized by interdigitations connected with junctional complexes, and extracellular cisterns related to the fluid transport. Extracellular cisterns and the intercellular space reveal abundant membrane-derived multilamellar phospholipids when a conventional ultrastructural fixative supplemented with tannic acid is used. Both arachnoid and meningioma cells are connected by Ca2+-dependent adhesion molecules: epithelial-cadherins which are concentrated at the adherens junctions. Membrane-cytoskeleton interactions by means of merlin and a-catenin molecules are thought to be crucial in signal transduction resulting in contact inhibition of cell growth in normal arachnoid cells. Impairment of these molecules might be related to meningioma-genesis. Glutathione-independent prostaglandin D2 synthase [EC 5.3.99.2] responsible for the biosynthesis of prostaglandin D2 in the central nervous system is also consistently expressed in human arachnoid villi and meningiomas. The multilamellar phospholipids are conceivably related to this arachidonate metabolism.

Alzheimer's disease (AD) is characterized by slowly progressive neuronal death, but its molecular cascade remains elusive for over 100 years. Since accumulation of autophagic vacuoles (also called granulo-vacuolar degenerations) represents one of the pathologic hallmarks of degenerating neurons in AD, a causative connection between autophagy failure and neuronal death should be present. The aim of this perspective review is at considering such underlying mechanism of AD that age-dependent oxidative stresses may affect the autophagic-lysosomal system via carbonylation and cleavage of heat-shock protein 70.1 (Hsp70.1). AD brains exhibit gradual but continual ischemic insults that cause perturbed Ca 2+ homeostasis, calpain activation, amyloid β deposition, and oxidative stresses. Membrane lipids such as linoleic and arachidonic acids are vulnerable to the cumulative oxidative stresses, generating a toxic peroxidation product ‘hydroxynonenal’ that can carbonylate Hsp70.1. Recent data advocate for dual roles of Hsp70.1 as a molecular chaperone for damaged proteins and a guardian of lysosomal integrity. Accordingly, impairments of lysosomal autophagy and stabilization may be driven by the calpain-mediated cleavage of carbonylated Hsp70.1, and this causes lysosomal permeabilization and/or rupture with the resultant release of the cell degradation enzyme, cathepsins ( calpain–cathepsin hypothesis ). Here, the author discusses three topics; (1) how age-related decrease in lysosomal and autophagic activities has a causal connection to programmed neuronal necrosis in sporadic AD, (2) how genetic factors such as apolipoprotein E and presenilin 1 can facilitate lysosomal destabilization in the sequential molecular events, and (3) whether a single cascade can simultaneously account for implications of all players previously reported. In conclusion, Alzheimer neuronal death conceivably occurs by the similar ‘calpain-hydroxynonenal-Hsp70.1-cathepsin cascade’ with ischemic neuronal death. Blockade of calpain and/or extra-lysosomal cathepsins as well as scavenging of hydroxynonenal would become effective AD therapeutic approaches.

Hypoxia is the principal factor that causes angiogenesis. These experiments were conducted to explore how it induces the proliferation of vascular cells, a key step in angiogenesis. Human umbilical vein endothelial cells and bovine retinal pericytes were grown in controlled atmosphere culture chambers containing various concentrations of oxygen. The numbers of both endothelial cells and pericytes increased significantly under hypoxic conditions; the O2 concentrations that achieved maximal growth promotion were 10% for endothelial cells and 2.5% for pericytes. Quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNAs coding for the secretory forms of vascular endothelial growth factor (VEGF), a mitogen specific to endothelial cells, were present in both endothelial cells and pericytes and that their levels increased significantly in the two cell types as the atmospheric O2 concentration decreased. The two genes for VEGF receptors, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase 1 (flt1), were found to be constitutively expressed in endothelial cells, and their relative mRNA levels were ranked in that order. On the other hand, only flt1 mRNA was detected in pericytes under hypoxic conditions. Furthermore, most antisense oligodeoxyribonucleotides complementary to VEGF mRNAs efficiently inhibited DNA synthesis in endothelial cells cultured under hypoxic conditions. These results indicate that autocrine and paracrine VEGFs may take part in the hypoxia-induced proliferation of endothelial cells.

Background and Purpose To determine whether distributional changes of fibronectin, a factor promoting wound healing, occur during the formation and repair of cerebral saccular aneurysms, we performed immunohistochemical analyses in experimental aneurysms. Methods Cerebral aneurysms were induced in rats by both the ligation of the unilateral common carotid artery and induced hypertension. Intimal proliferation in aneurysmal walls was induced by the ligation of the preserved common carotid artery 3 months after the first operation. The distribution of fibronectin was examined by immunohistochemistry in anterior cerebral artery–olfactory artery bifurcations under the following three conditions: normal bifurcations in control rats, early aneurysmal lesions during the aneurysm induction, and aneurysmal lesions with intimal proliferation. Furthermore, the immunohistochemical distributions of type I and IV collagens were examined to evaluate the specificity of fibronectin immunoreactivity. Results In the normal bifurcations, fibronectin was positive in the subintimal space, the surrounding area of the medial smooth muscle cells, and the adventitial fibrous tissue. In early aneurysmal lesions, linear staining of fibronectin and type I and IV collagens in the subendothelial space disappeared with the loss of the internal elastic lamina. In the intimal proliferation of early aneurysmal lesions, fibronectin was strongly immunostained in the subendothelial space and diffusely immunostained in the widened extracellular space surrounding proliferated cells. In contrast, the stainings of type I and IV collagens were sparse or negative. Conclusions Although the present findings regarding dynamic changes of fibronectin distribution do not prove any causality in the process of aneurysm formation and repair, these immunohistochemical changes may constitute the crucial sequela of intimal endothelial damage and its subsequent recovery in cerebral aneurysms.

Гените от фамилията Pax играят важна роля в пролиферацията на множествени клетъчни линии, при формирането на различни органи, включително в развитието и организацията на нервната система. В последно време беше установено значението на два транскрипционни фактора (ТФ) от тази група - pax2 и pax6 в тъканната регенерация и специално за Pax6 гена - нова функция в сегментната организация на задния мозък (Metencephalon). Целта на настоящото изследване беше да проучим присъствието, разпределението и динамиката на тези два ТФ в малкия мозък на новородени, едногодишни и половозрели в млада възраст японски маймуни (Macaca fuscata), при последните и след глобална мoзъчната исхемия. Според възрастта (новородени, едногодишни и възрастни) и срока на преживяемост след исхемията при възрастните (съответно 9 или 15 дни) животните бяха разпределени в няколко контролни и експериментални групи. Приложихме флуоресцентна имунохистохимична техника за оцветяване на замразени срези от малкия мозък за изтъкване на pax2 и pax6. При новородените животни единични Pax2(+) клетки се откриват предимно във вътрешния зърнест слой на малкия мозък и в слоя на клетките на Пуркиние. В по-късна възраст количеството им значително намалява. В групите с исхемия единични Pax2(+) клетки се разполагат в слоя на клетките на Пуркиние и зърнестия слой без разлика в местоположението и количеството в сравнение с контролите. Pax6(+) клетки се откриват в големи количества във всички слоеве на кората на малкия мозък при новородени животни, докато при едногодишните маймуни такива клетки се установяват основно във вътрешния зърнест слой. При възрастните животни от контролната група типичната им локализация е само в зърнестия слой без разлика в сравнение с групите с исхемия. Получените данни потвърждават и допълват достъпната информация относно наличието и типичното разпределение на тези два ТФ в малкия мозък в хода на развитието, без да се установяват промени след глобална мозъчна исхемия.

We investigated a non-human primate (NHP) transient global ischemia (TGI) model which was induced by clipping the arteries originating from the aortic arch. Previously we demonstrated that our TGI model in adult Rhesus macaques (Macaca mulatta) results in marked neuronal cell loss in the hippocampal region, specifically the cornu Ammonis (CA1) region. However, we observed varying degrees of hippocampal cell loss among animals. Here, we report for the first time an anomaly of the aortic arch in some Rhesus macaques that appears as a key surgical factor in ensuring the success of the TGI model in this particular NHP. Eleven adult Rhesus macaques underwent the TGI surgery, which involved 10-15-minute clipping of both innominate and subclavian arteries. Animals were allowed to survive between 1 day and 28 days after TGI. Because of our experience and knowledge that Japanese macaques exhibited only innominate and subclavian arteries arising from the aortic arch, macroscopic visualization of these two arteries alone in the Rhesus macaques initially assured us that clipping both arteries was sufficient to produce TGI. During the course of one TGI operation, however, we detected 3 arterial branches arising from the aortic arch, which prompted us to subsequently search for 3 branches in succeeding TGI surgeries. In addition, we performed post-mortem examination of the heart to confirm the number of arterial branches in the aortic arch. Finally, in order to reveal the pathological effect of the aortic arch anomaly, we compared the hippocampal cell loss between animals found to have 3 arterial branches but had all or only two branches clipped during TGI operation. Post-mortem examination revealed that eight NHPs had the typical two arterial aortic branches, but three NHPs displayed an extra arterial aortic branch, indicating that about 30% of Rhesus macaques had 3 arterial branches arising from the aorta. Histological analyses using Nissl staining showed that in NHPs with the aortic arch anomaly clipping only two of three arterial branches led to a partial cell loss and minimal alteration in number of cell layers in the hippocampal region when compared with clipping all three branches, with the hippocampal cell death in the latter resembling the pathological outcome achieved by clipping the two arterial branches in NHPs displaying the typical two-artery aortic arch. The finding that 3 of 11 NHPs exhibited an extra arterial aortic branch recognizes this aortic arch anomaly in Rhesus macaques that warrants a critical surgical maneuver in order to successfully produce consistent TGI-induced hippocampal cell loss.

Stroke, trauma and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases affect millions of people worldwide. To date, treatment for these disorders is mainly symptomatic. However, the discovery of multipotent neural progenitor cell (NPC) capable of producing not only glia but also neurons in the adult brain has brought revolutionary changes in the field of restorative therapy. Currently, it posits that regeneration of neurons can occur throughout life, opening a door for the development of novel therapies to treat neurological diseases by neuronal regeneration using proliferation, differentiation, and integration of NPC at sites of brain injury. To achieve this goal, we need to strive to better understand the molecular signals involved in the control of NPC fate and the mechanisms inducing their migration and integration into the existing neural networks and synaptic circuits. The functional properties of the newborn neurons and their ability to form appropriate afferent and efferent connections should be determined for optimizing functional recovery in human disorders. In studying adult neurogenesis and adult NPC, the availability of developmental paradigms, such as embryonic neurogenesis and embryonic NPC, stands as a candidate experimental model. During the assembly of neural circuits in the developing brain, embryonic NPC generates functionally distinct neuronal cell types, each at a stereotyped position. In recent years, considerable progress has been made in defining the molecular players controlling embryonic NPC specification [1, 2]. These studies have revealed that neural cell fate depends critically on local environmental signals, which direct cell fates by inducing the expression of intrinsic proteins, notably transcription factors (TFs). In molecular biology, a TF is a protein that regulates transcription of target genes. In particular, TFs regulate the binding of RNA polymerase and the initiation of transcription. TFs act in networks [3] to induce the expression of target genes, which subsequently define the fate of the descendant cells (Fig. 1). In fact, developmental mechanisms may be more than just a model for studying the molecular regulation of adult neurogenesis. Recent laboratory evidence has identified some key embryonic NPC molecular regulators, such as sonic hedgehog and Wnt, as modulators of adult NPC [4–6]. In addition to these secreted signals, some of the intracellular TFs involved in developmental neural patterning were found to be expressed by and to affect aspects of the adult NPC biology (see [7] for a recent review). It is not surprising then that there is an increasing interest on how to employ TFs to manipulate endogenous NPC for the purposes of repairing the injured brain. In the current issue, Scholzke and Schwaninger [8] summarize the present state-of-the-art investigation on the TF involvement in neurogenesis after cerebral ischemia— the most common type of cerebral injury. Their review is unique, as it represents the first attempt to collectively analyze the transcriptional control of adult NPC in the context of reactive neurogenesis after ischemic damage to the brain. At present, the evidence for TF involvement in ischemia-induced adult neurogenesis is rather scarce and reports mainly the association of a certain TF with this process rather than its precise role. To overcome this, the authors review the major molecular players regulating embryonic NPC and then elegantly draw possible J Mol Med DOI 10.1007/s00109-007-0210-5

Клетъчно-тъканните механизми на увреждане и възстановяване след мозъчна исхемия имат фундаментално приложно значение. Целта на настоящото проучването беше да установим промени в клетъчната пролиферация чрез бромодеоксиуридин (BrdU), индикатор за ДНК синтеза, при новородени, едногодишни и половозрели в млада възраст японски маймуни (Macaca fuscata) след глобална мозъчна исхемия на малкия мозък. Според възрастта, схемата на приложение на BrdU (2 дена х 250 mg/kg или 5 дена х 100 mg/kg i.v.) и срока на преживяемост след исхемията (съответно 4, 9 и 15 дни) животните бяха разпределени в няколко контролни и експериментални групи. Приложихме имунохистохимично оцветяване с пероксидаза на замразени срези от малкия мозък на изследваните животни за изтъкване на пролиферативния маркер BrdU. При сравнение на количеството BrdU+ клетки в малкомозъчната кора между групите на новородени, едногодишни и възрастни животни без исхемия се установи, че с увеличаване на възрастта пролиферативните процеси значително намаляват до относително слабо изразено поддържащо ниво. При сравнение между групите на възрастните животни с различна продължителност на исхемия се оказа, че тя е важен фактор, стимулиращ клетъчната пролиферация, като този ефект намалява с времето след исхемията. Предстои фенотипизиране на BrdU+ клетки, за да се определят видовете новообразувани клетки и тяхното съотношение в кората на малкия мозък след исхемията. Получените данни ще допълнят и разширят разбирането за възстановителните процеси след исхемично увреждане на малкия мозък при изследваните примати.

Cerebral cortex оf mammals is mainly generated during the embryonic period by stem cells and their derivative progenitors in the palium of the developing telencephalon. Various genes in complex interactions are involved in the processes of differentiation of the cerebral neurons. Transcriotional factor Sox2 plays a key role for self renewing and sustaining multipotency of embryonic neural stem/progenitor cells. Data about the expression and function of Sox2 in human fetal brain are insufficient and controversial. In the present sudy tissue samples of spontaneously aborted human fetuses aged between 12th to 28th gestational weeks (g. w.) were examined by a standard histological and immunohistochemical technique for paraffin sections. Sox2 expression was followed in the zones of cellular proliferation and migration in the occipital lobe of human fetal telencephalon mainly during 17th g. w. Within ventricular and outer subventricular zones we detected similar amount approximately 45% Sox2+ cells, whereas in the intermediate zone, cortical plate and marginal zone expression of Sox2 was not found. The data obtained on the location and expression dynamics of Sox2 contribute to a more complete understanding of neural stem/progenitor cell biology during embryonic neurogenesis in the human cerebral cortex.

The anterior subventricular zone of the adult mammalian brain contains progenitor cells which are upregulated after cerebral ischemia. We have previously reported that while a part of the progenitors residing in adult monkey anterior subventricular zone travels to the olfactory bulb, many of these cells sustain location in the anterior subventricular zone for months after injury, exhibiting a phenotype of either neural or neuronal precursors. Here we show that ischemia increased the numbers of anterior subventricular zone progenitor cells expressing developmentally regulated transcription factors including Pax6 (paired-box 6), Emx2 (empty spiracles-homeobox 2), Sox 1-3 (sex determining region Y-box 1-3), Ngn1 (neurogenin 1), Dlx1,5 (distalless-homeobox 1,5), Olig1,3 (oligodendrocyte lineage gene 1,3) and Nkx2.2 (Nk-box 2.2), as compared with control brains. Analysis of transcription factor protein expression by sustained neural or neuronal precursors in anterior subventricular zone revealed that these two cell types were positive for characteristic sets of transcription factors. The proteins Pax6, Emx2, Sox2,3 and Olig1 were predominantly localized to dividing neural precursors while the factors Sox1, Ngn1, Dlx1,5, Olig2 and Nkx2.2 were mainly expressed by neuronal precursors. Further, differences between monkeys and non-primate mammals emerged, related to expression patterns of Pax6, Olig2 and Dlx2. Our results suggest that a complex network of developmental signals might be involved in the specification of primate progenitor cells.

金沢大学医薬保健研究域医学系, 金沢大学附属病院脳神経外科, An 81-year-old female presented with a giant aneurysm of the distal anterior cerebral artery (A3) which grew from a small saccular aneurysm to a huge aneurysm within 36 months before manifesting as a mass lesion. The thrombosed portion of the aneurysm showed growth, whereas the aneurysmal cavity did not change in size. Computed tomography and magnetic resonance imaging showed new bleeding in the thrombosed portion. Hemorrhage into the thrombus and/or aneurysmal wall might have caused the aneurysmal growth. She refused surgery and was discharged with no deficits. Distal anterior cerebral artery aneurysm which shows neuroimaging signs of growth requires regular follow up as such lesions may become giant before manifesting clinical symptoms.

金沢大学医薬保健研究域医学系, 金沢大学附属病院脳神経外科, The relationship between the grade of astrocytic tumor and the expression of deoxyribonucleic acid methyltransferase (DNA-MTase) gene was examined. The levels of DNA-MTase messenger ribonucleic acid (mRNA) were measured by semiquantitative reverse transcriptase-polymerase chain reaction in surgical specimens from 12 astrocytic tumors (4 astrocytomas, 6 anaplastic astrocytomas, and 2 glioblastomas) and two normal brain tissues, and in four glioma cell lines. Compared to normal brain tissues, the levels of DNA-MTase mRNA were increased by 16- to 55-fold in low grade astrocytomas, and significantly increased by 200- to 4500-fold in high grade astrocytomas (anaplastic astrocytomas and glioblastomas) and more than 4500-fold in glioma cell lines. In situ hybridization with paraffin-embedded surgical specimens of human astrocytic tumors showed DNA-MTase mRNA was abundantly expressed in high grade astrocytomas. The detection of increased DNA-MTase expression in astrocytic tumor indicates involvement in the tumorigenesis and suggests that blocking of this change with specific inhibitors may offer new therapeutic strategies for malignant astrocytic tumors.

The purposes of this study are, firstly, to define the relationship between volume embolization ratio (VER) and degree of angiographical occlusion in endovascular treatment with Guglielmi detachable coils, and secondly, to examine influences of neck and dome sizes of aneurysms on the VER and the angiographical treatment result, and thirdly, to determine the relationship between the VER and the recanalization of coiled aneurysms. Fifty-two aneurysms in 46 patients were examined. VER ranged 8.1–31.9% (mean 18.5%). The mean VERs of each categories based on angiographical treatment results were 23.1% in complete occlusion, 16.1% in neck remnant and 12.2% in incomplete occlusion, respectively. The VER correlated significantly with both neck and dome size, while the angiographical treatment result was only affected by neck size. Five aneurysms showed aneurysmal recanalization among followed-up 41 aneurysms. All recanalized aneurysms were large, and their VERs were in range of 10.4–17.6%. Measurement of VER is useful to estimate the degree of occlusion objectively and to predict the aneurysmal recanalization. A small aneurysms with a small neck is relatively easy to achieve high VER and angiographical complete occlusion, with the consequence of less recanalization. On the other hand, a large aneurysm is liable to recanalize due to low VER, even if there was little filling of contrast medium in the aneurysmal cavity.

The aim of this study was to investigate the advantages and disadvantages of a two-stage treatment for ruptured cerebral aneurysms; partial embolization in acute stage followed by clipping in chronic stage of subarachnoid hemorrhage. Between April 1997 and August 1999, twenty ruptured cerebral aneurysms were initially treated endovasculary using Guglielmi detachable coils in our institution. Among them, complete embolization could not be achieved in 6 lesions. For these lesions, subsequent clipping was added. The radiological and operative findings, and outcomes of these cases were retrospectively reviewed. In 1 case, rerupture occurred during the endovascular procedure. Rerupture was not observed in any cases in the postembolization period. In 2 cases, complications related to the clipping but not the endovascular procedure occurred. These complications included impaired visual acuity for unverified reasons, and memory disturbance due to sacrifice of a perforator arising from the anterior communicating artery. In 3 cases, coil extraction was needed during the clipping, because the loops of the coil extended into the residual neck. Complications related to coil extraction were not observed in these 3 cases. Acute partial embolization of ruptured aneurysm appears to be effective for the prevention of subsequent rerupture during the subacute period, in which treatment for vasospasm should be performed, and the clipping procedure. However, in the case of relatively large aneurysms, small arteries or other normal structures behind the aneurysm cannot be observed directly during surgery, because of the immovability of the embolized aneurysm. Further, complete clip closure is impossible when loops of coil herniate into the neck. In such situations, coil extraction with or without resection of the aneurysm might be necessary, and care must be taken not to damage parent artery and surrounding vessels.