Cytoplasm Of Cortical Neurons Research Articles

Expression Analyses of Rich2/Arhgap44, a Rho Family GTPase-Activating Protein, during Mouse Brain Development

Rho family small GTPases, such as Rho, Rac, and Cdc42, play essential roles during brain development, by regulating cellular signaling and actin cytoskeletal reorganization. Rich2/Arhgap44, a Rac- and Cdc42-specific GTPase-activating protein, has been reported to be a key regulator for dendritic spine morphology and synaptic function. Given the essential roles of Rac and Cdc42 in brain development, Rich2 is supposed to take part in brain development. However, not only the molecular mechanism involved but also the expression profile of Rich2 during neurodevelopment has not yet been elucidated. In this study, we carried out expression analyses of Rich2 by focusing on mouse brain development. In immunoblotting, Rich2 exhibited a tissue-dependent expression profile in the young adult mouse, and the expression was increased during brain development. In immunohistochemical analyses, Rich2 was observed in the cytoplasm of cortical neurons at postnatal day (P) 0 and then came to be enriched in the nucleus with moderate distribution in neuropils at P7. Later at P30, a complex immunostaining pattern of Rich2 was observed; Rich2 was distributed in the nucleus, cytoplasm, and neuropils in many cortical neurons, whereas other neurons frequently displayed little expression. In the hippocampus at P7, Rich2 was distributed mainly in the cytoplasm of excitatory neurons in the cornu ammonis regions, while it was moderately detected in the nucleus in the dentate granule cells. Notably, Rich2 was distributed in excitatory synapses of the cornu ammonis 1 region at P30. Biochemical fractionation analyses also detected Rich2 in the postsynaptic density. Taken together, Rich2 is found to be expressed in the central nervous system in a developmental stage-dependent manner and may be involved in synapse formation/maintenance in cortical neurons.

Expression Analyses of Cep152, a Responsible Gene Product for Autosomal Recessive Primary Microcephaly, during Mouse Brain Development

Centrosomal protein 152 (Cep152) regulates centriole duplication as a molecular scaffold during the cell cycle. Its gene abnormalities are responsible for autosomal recessive primary microcephaly 9 and Seckel syndrome. In this study, we prepared an antibody against mouse Cep152, anti-Cep152, and performed expression analyses focusing on mouse brain development. Western blotting analyses revealed that Cep152 with a molecular mass of ∼150 kDa was expressed strongly at embryonic day (E)13 and then gradually decreased during the brain development process. Instead, protein bands of ∼80 kDa and ∼60 kDa came to be recognized after postnatal day (P)15 and P30, respectively. In immunohistochemical analyses, Cep152 was enriched in the centrosome of neuronal progenitors in the ventricular zone at E14, whereas it was diffusely distributed mainly in the cytoplasm of cortical neurons at P18. In developing cerebellum at P7, Cep152 was localized at the centrosome in the external granular layer, where neurogenesis takes place. Notably, biochemical analysis revealed that Cep152 was also present in the postsynaptic density fraction. Subsequent immunofluorescent analyses showed co-localization of Cep152 with excitatory synaptic markers, PSD95 and synaptophysin, but not with an inhibitory synaptic marker gephyrin in differentiated primary cultured hippocampal neurons. The obtained results suggest that Cep152 takes part not only in neurogenesis during corticogenesis but also in the regulation of synaptic function in differentiated neurons.

The role of vascular injury within the conditions of choline deficiency in rats with scopolamine-induced alzheimer's type dementia

Background. The relationship between choline deficiency and vascular dysfunction continues to be relevant in the study of Alzheimer's disease. Objective. To study the morphological characteristics of vascular injury within the conditions of choline deficiency in rats with scopolamine-induced Alzheimer's type dementia. Methods. The experiment was performed on 48 WAG population male rats weighing 180-230 gr. Rats from groups Scop-14, Scop-14-SC, Scop-28, Scop-28-SC were injected intraperitoneally with scopolamine (Scop) butylbromide at a dosage of 1 mg/kg of body mass during 14 and 28 days and intravenously with mesenchymal stem cells (SC) at a single dosage of 500000 cells per 1 rat. Control animals (gr.C) were injected with 0.9% sodium chloride. Brain slides were stained with Congo-red and gallocyanine-chromium alum according to Einarson's method for total nucleic acids. The VEGF, E-cadherin expression was immunohistochemically determined in the brain cells cytoplasm. Results. The congophilic staining of the arteries walls, a decrease in endothelial cells with low the E-cadherin expression and an increase in the number of pericytes in the capillary wall was observed in the experimental groups. In gr.Scop-28 VEGF expression in endothelial cells, hippocampal neurons was greater than in gr.Scop-14. It indicated more intensive activation of angiogenesis and acetylcholine synthesis with correspondingly more pronounced vascular damage and choline deficiency. The cytoplasm of cortical neurons was diffusely labeled with VEGF antibodies in response to hypoxia, but the level of expression was almost no different from that in gr.C. In all groups, the optical density of the neuropile of the large hemispheres according to Einarson’s staining was reduced, i.e., the level of RNA in the neuronal processes was reduced. The introduction of stem cells restored the capillary wall due to young endothelial cells, reduced the VEFG synthesis in all studied cells and increased the RNA content in neuronal processes. Conclusion. The relationship between choline deficiency, neuronal process loss and vascular damage has been found. The blood vessels self-repair was occurred by substitution, after the stem cells introduction - by restitution.

5-Lipoxygenase inhibition reduces inflammation and neuronal apoptosis via AKT signaling after subarachnoid hemorrhage in rats.

Early brain injury (EBI) is a major contributor to the high mortality and morbidity after subarachnoid hemorrhage (SAH). Inflammatory responses and neuronal apoptosis are important causes of EBI. Because 5- lipoxygenase (5-LOX) is known to be involved various central nervous system diseases, we investigated the effects of 5-LOX inhibition during EBI after SAH. Zileuton and LY294002 were used to inhibit expression of 5-LOX and Akt, respectively. We found that 5-LOX expression was significantly increased in the cytoplasm of cortical neurons after SAH and was accompanied by upregulated expression of the inflammatory factors LTB4, TNF-α, IL-1β and IL-6; upregulation of the pro-apoptotic factor Bax; downregulation of the anti-apoptotic factor Bcl-2; and an increased apoptosis rate. Gastric Zileuton administration significantly suppressed all of those effects and improved neurological function. Zileuton also upregulated activated (phosphorylated) AKT levels, and these beneficial effects of Zileuton were abolished by intracerebroventricular infusion of the PI3K inhibitor LY294002. Taken together, these findings indicate that 5-LOX mediates pro-inflammatory and pro-apoptotic effects that contribute to EBI after SAH and that those effects are suppressed by activation of PI3K/Akt signaling. This suggests targeting 5-LOX may be an effective approach to treating EBI after SAH.

Bystin (BYSL) as a possible marker of severe hypoxic-ischemic changes in neuropathological examination of forensic cases

Bystin (BYSL) is a 306-amino acid protein encoded in humans by the BYSL gene located on the 6p21.1 chromosome. It is conserved across a wide range of eukaryotes. BYSL was reported to be a sensitive marker for the reactive astrocytes induced by ischemia/reperfusion and chemical hypoxia in vitro and is considered to be one of the common characteristics of astrogliosis. In our study we examined whether BYSL could be used as a marker for hypoxic-ischemic changes in forensic cases. Groups suspected of acute hypoxic-ischemic changes presented strong BYSL expression in the cytoplasm of neocortical neurons especially in layers 3–5, that seemed to be short-lasting. In the hypoxic-ischemic-reperfusion group we did not find BYSL expression. BYSL expression in the cytoplasm of cortical neurons was minimal in the control group (cardiac arrest). BYSL seems to be a promising early marker of severe hypoxic-ischemic changes in neuropathological examination of forensic cases and certainly requires further studies.

Cyclin-dependent kinase 5/Collapsin response mediator protein 2 pathway may mediate sevoflurane-induced dendritic development abnormalities in rat cortical neurons

Sevoflurane has been reported to induce neurotoxicity and cognitive impairment in the developing brains. However, the underlying molecular mechanisms remain poorly understood. Recent studies have demonstrated aberrant cyclin-dependent kinase 5 (CDK5) activity is implicated in inhaled anesthetic-induced neurotoxicity. CDK5/CRMP2 signaling is involved in the cortical and hippocampal dendritic development. The aim of present study is to investigate whether the CDK5/CRMP2 pathway mediates sevoflurane-induced dendritic development abnormalities. Rat primary cortical neurons were treated with 4% sevoflurane for 6h, the CDK5 inhibitor roscovitine or the vehicle (0.3% DMSO) was administered 12h before sevoflurane or carrying gases exposure. Cortical neurons were harvested for further analysis 0h, 12h and 24h after exposure. Sevoflurane exposure for 6h did not reduce cell viability and slightly increased the expression of cleaved caspase-3. Sevoflurane induced abnormal CDK5 activation by increasing the expression of its activator p25 and promoted the phosphorylation of CRMP2 (Ser522). The increased phospho-CRMP2 (Ser522) was mainly distributed in the cytoplasm of cortical neurons. Sevoflurane significantly reduced the number of primary dendrites and the number of branching points; whereas it did not influence the total dendritic length. Suppression of CDK5 activation with roscovitine attenuated neuronal apoptosis, hyperphosphorylation of CRMP2 (Ser522) and dendritic development abnormalities induced by sevoflurane. Our results indicate that activation of the CDK5/CRMP2 pathway may mediate sevoflurane-induced dendritic development abnormalities in the cortical neurons. The physiological significance of these findings remains to be determined.

Abnormal Expression of FBXL20 in Refractory Epilepsy Patients and a Pilocarpine-Induced Rat Model.

E3 ubiquitin ligases are important protein-modifying enzymes involved in the pathogenesis of a variety of neurodegenerative diseases. F-box and leucine-rich repeat protein 20 (FBXL20), an E3 ubiquitin ligase widely expressed in the central nervous system, plays an important role in the ubiquitin-dependent degradation of regulating synaptic membrane exocytosis 1 (RIM1), which is an important factor in the release of synaptic vesicles. FBXL20 has been associated with a variety of neurodegenerative diseases; thus, we hypothesized that FBXL20 is involved in the development of epilepsy. Herein, we used immunofluorescence staining, immunohistochemistry and western blotting to determine the expression pattern of FBXL20 in temporal lobe epilepsy patients and pilocarpine-induced epilepsy animal models. We also injected SD rats with lentivirus-vector mediated overexpression of FBXL20. The results showed that FBXL20 is expressed in the membrane and the cytoplasm of cortical neurons, and overexpression of FBXL20 decreased the onset level of spontaneous seizure, the frequency and duration of seizures. Additionally, FBXL20 protein level was decreased but RIM1 protein level was increased in the epileptic group compared with the LV-FBXL20 and LV-GFP group. These findings in humans were consistent with the results from a pilocarpine-induced animal model of chronic epilepsy. Thus, abnormal expression of FBXL20 might play an important role in the development of epilepsy.

Influence of cerebral global ischemia-reperfusion on succinate dehydrogenase activity in neurons of different neocortical layers

The aim of the study was to investigate changes in activity of succinate dehydrogenase (SDH) in cytoplasm of neurons of different cortical layers in early and late reperfusion period after global cerebral ischemia in rats. Reversible global cerebral ischemia was modeled by occlusion of the brachiocephalic trunk, left subclavian artery and left common carotid artery for 10 minutes and following reperfusion during 2 or 7 days. The SDH activity in cytoplasm of neurons of II, III and V cortical layers was determined histoenzymatically. It is shown that the SDH activity in neurons of the studied cortical layers was characterized by the increased reperfusion period to the 2 days with a subsequent increased activity of the reperfusion period to the 7 days. The change in the SDH activity in cytoplasm of cortical neurons depends on the particular cerebral layer and duration of postischemic reperfusion.

EAAC1 gene deletion alters zinc homeostasis and enhances cortical neuronal injury after transient cerebral ischemia in mice

The excitatory amino acids glutamate and cysteine are actively transported into neurons from the extracellular space by the high affinity glutamate transporter EAAC1. The astrocyte glutamate transporters, GLT1 and GLAST, are the primary mediators of glutamate clearance. EAAC1 has a limited role in this function. However, uptake of cysteine into neurons via EAAC1 contributes to neuronal antioxidant function by providing cysteine substrate for glutathione synthesis. Mice in which the EAAC1 gene has been deleted were seen to have enhanced susceptibility to neuronal oxidative stress and developed brain atrophy and cognitive function decline with aging. The aim of the current study was to evaluate if EAAC1 confers protection against ischemic events. Young adult CD-1 wild-type or EAAC1−/− mice were subjected to 30min of bilateral common carotid artery occlusion and evaluated for neuronal death and zinc translocation. The intensity of TSQ fluorescence in the cytoplasm of cortical neurons in the EAAC1−/− mice was significantly higher than wild-type mice, indicating that the cortical neurons of EAAC1−/− mice contain higher cytoplasmic concentrations of labile (or free) zinc. Zinc translocation into cortical neurons was also enhanced in EAAC1−/− mice. Three days after ischemia, Fluoro-Jade B staining revealed that EAAC1−/− mice had more than twice as many degenerating neurons as wild-type mice. N-acetylcysteine, a membrane-permeant cysteine pro-drug, normalized basal zinc levels, reduced TSQ (+) neurons and reduced ischemic neuronal death in the EAAC1−/− mice when delivered in a pre-treatment fashion. Taken together, this study implicates EAAC1-dependent cysteine uptake as an endogenous source of enhancing antioxidant function and zinc homeostasis in neurons in the ischemic brain.

SIK2 Is a Key Regulator for Neuronal Survival after Ischemia via TORC1-CREB

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.

Retranslocation of active protein kinase C-beta II during calcium overload of cultured neurons

We have performed simultaneous real time recording of the intracellular level of Ca2+ ([Ca2+]i) and intracellular localization of protein kinase C (PKC) in cultured rat cortical neurons. The neurons were transfected with a plasmid that encoded a chimerical protein of PKCβII and green fluorescent protein (GFP) and loaded with fura-2FF. In the resting neurons, PKCβII-GFP was uniformly distributed in the cytoplasm of cortical neurons. The primary [Ca2+]i increase caused by glutamate was accompanied by translocation of PKCβII-GFP from the cytosol to the plasma membrane. A secondary [Ca2+]i elevation (destabilization of calcium homeostasis) was associated with retranslocation of PKCβII-GFP from the plasma membrane to the cytoplasmic organelle-like structures. Similar organelle-like structures were observed after immunohistochemical staining of PKCβII in cultured cerebellar granule cells and cortical neurons subjected to long-term glutamate treatment. It seems that PKCβII was active in these structures because the receptor for activated C kinase RACK1 also translocated into similar structures. Formation of clusters by PKCβII was calcium-dependent because the clusters also formed after treatment of neurons with calcium ionophore ionomycin. No retranslocation of PKC was observed after treatment with the calcium-independent activator of PKCβII phorbol-myristate acetate (PMA); in this case both PKCβII and RACK1 remained on the plasma membrane of neurons. The results obtained suggest that deregulation of neuronal calcium homeostasis during hyperstimulation of glutamate receptors induces specific changes in the localization of active PKCβII.

Parkinson's Disease α-Synuclein Transgenic Mice Develop Neuronal Mitochondrial Degeneration and Cell Death

Alpha-synuclein (alpha-Syn) is enriched in nerve terminals. Two mutations in the alpha-Syn gene (Ala53--> Thr and Ala30--> Pro) occur in autosomal dominant familial Parkinson's disease. Mice overexpressing the human A53T mutant alpha-Syn develop a severe movement disorder, paralysis, and synucleinopathy, but the mechanisms are not understood. We examined whether transgenic mice expressing human wild-type or familial Parkinson's disease-linked A53T or A30P mutant alpha-syn develop neuronal degeneration and cell death. Mutant mice were examined at early- to mid-stage disease and at near end-stage disease. Age-matched nontransgenic littermates were controls. In A53T mice, neurons in brainstem and spinal cord exhibited large axonal swellings, somal chromatolytic changes, and nuclear condensation. Spheroid eosinophilic Lewy body-like inclusions were present in the cytoplasm of cortical neurons and spinal motor neurons. These inclusions contained human alpha-syn and nitrated synuclein. Motor neurons were depleted (approximately 75%) in A53T mice but were affected less in A30P mice. Axonal degeneration was present in many regions. Electron microscopy confirmed the cell and axonal degeneration and revealed cytoplasmic inclusions in dendrites and axons. Some inclusions were degenerating mitochondria and were positive for humanalpha-syn. Mitochondrial complex IV and V proteins were at control levels, but complex IV activity was reduced significantly in spinal cord. Subsets of neurons in neocortex, brainstem, and spinal cord ventral horn were positive for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, cleaved caspase-3, and p53. Mitochondria in neurons had terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive matrices and p53 at the outer membrane. Thus, A53T mutant mice develop intraneuronal inclusions, mitochondrial DNA damage and degeneration, and apoptotic-like death of neocortical, brainstem, and motor neurons.

Reactivation of HSV-1 in the brain of patients with familial Alzheimer's disease.

Herpes simplex virus type 1 (HSV-1) has been proposed as an environmental risk factor for sporadic Alzheimer's disease, although this issue is still in dispute. The involvement of HSV-1 in the pathogenesis of familial Alzheimer's disease, the uncommon type of Alzheimer's disease, has not been addressed yet. We investigated formalin-fixed, paraffin-embedded, postmortem brain tissue sections of three patients with familial Alzheimer's disease for the presence of HSV-1 DNA. The nested polymerase chain reaction (PCR) detected the HSV-1 glycoprotein D gene in the brain of all three patients with familial Alzheimer's disease preferentially in the frontal and temporal cortices, whereas only one case out of six age-matched, non-Alzheimer's disease individuals could disclose the presence of HSV-1 gene. The PCR detected HSV-1 DNA in the frontal cortex of the two patients with sporadic Alzheimer's disease. The presence of HSV-1 was associated with beta-amyloid deposition in the cerebral cortex. To clarify the localization of HSV-1 in the brain tissue of patients with familial Alzheimer's disease, the in situ hybridization of the tyramide signal amplification system was used. It detected the HSV-1-specific signals predominantly in the cytoplasm of cortical neurons in a dot-like staining fashion. In addition, high-sensitivity immunohistochemistry revealed the existence of HSV-1 antigens in the cytoplasm of cortical neurons. This report provides the first evidence of reactivation of HSV-1 in the brain of patients with familial Alzheimer's disease, associated with beta-amyloid deposition, and suggests the possible involvement of HSV-1 together with genetic factors in the pathogenesis of familial Alzheimer's disease.

Expression analysis of the gene encoding for the U-box-type ubiquitin ligase UBE4A in human tissues.

The Ubiquitination Factor E4A gene (UBE4A) encodes for a U-box-type ubiquitin ligase, originally described as an E4 ubiquitination factor. UBE4A is a mammalian homolog of Saccharomyces cerevisiae Ufd2. The UBE4A gene has been mapped on the human chromosome region 11q23.3, a critical region involved in some specific cancers such as neuroblastoma. Northern blots analysis on foetal and adult human tissues revealed a single band of approximately 7.5 kb transcript most abundant in the heart, skeletal muscle and kidney. We generated a polyclonal antibody to UBE4A and performed immunoblot and immunohistochemical analysis. The UBE4A protein appeared as a single band of approximately 125 kDa. UBE4A was present in the skeletal muscle, kidney and liver; a faint band was visible in peripheral blood leukocytes and spleen. We did not reveal expression of UBE4A in whole brain, colon, lung and heart. At the cellular level, UBE4A results predominantly expressed in the nucleus and the cytoplasm of cortical neurons and liver and in the nucleus of tubular kidney cells. In the liver, the nucleus of similar cells appeared to be unstained or stained at different levels suggesting that UBE4A may have a cell cycle dependent expression or a role of in cell cycle control. In conclusion, our results show that UBE4A is expressed in different tissues in a pattern that seems to be dependent from cell type and cell cycle and that UBE4A might have a specific role in different biochemical processes other than ubiquitination, including growth or differentiation.

Excessive expression of synaptojanin in brains with Down syndrome

We investigated the expression of synaptojanin, which has been mapped on 21q22.2 on human chromosome, in the cerebral cortex of patients with Down syndrome (DS), using immunohistochemistry and immunoblotting. Synaptojanin expression was observed in Cajal–Retzius cells, cortical plate neurons, subplate neurons, intermediate neurons, germinal matrix cells and the ventricular neuroepithelium of the fetal cerebrum in both controls and DS. After birth, synaptojanin immunoreactivity was mainly observed in cytoplasm of cortical neurons and neurophils. These expressions of synaptojanin suggest a broader role in not only synaptic vesicle recycling, but also the regulation of neuronal migration and synaptogenesis in the fetal period. In comparison with controls, DS brains clearly showed higher immunoreactivity of synaptojanin in every structure, and most of the large neurons showed immunoreactivity. Western blotting with synaptojanin confirmed the increased expression in DS brains. Although the reason for excessive expression of synaptojanin in DS brains is obscured, one possibility can be explained on the basis of a gene dosage effect. As another possibility, on the assumption that synaptojanin modulates synaptic transmission and plays roles in clathrin-mediated synaptic vesicle endocytosis and signaling, the excessive expression of synaptojanin may be involved in compensatory mechanisms occurring in developing DS brains, such as neuronal loss, atrophic basilar dendrites, decreased spines and abnormal synaptic density and length.

Induction of NADPH cytochrome P450 reductase by the Alzheimer beta-protein. Amyloid as a "foreign body".

A large body of data suggests that the Alzheimer's amyloid peptide (Abeta) causes degeneration and death of neurons by mechanisms that involve reactive oxygen species. The pathways involved in Abeta-mediated oxidative injury are only partially understood. We theorized that abnormal microaggregates and/or pathological conformations of Abeta peptides may behave as xenobiotics and trigger the induction of NADPH cytochrome P450 reductase (CP450r), an enzyme which, if induced by non-physiological substrates (such as xenobiotics like drugs or other 'foreign molecules'), is known to cause oxidative stress. In order to test this hypothesis, i.e. that Abeta can increase the expression of CP450r, SK-N-SH human neuroblastoma cells were exposed to Abeta25-35 and Abeta1-42 and then examined for induction of this enzyme in immunoblots, using specific antibodies. Following exposure to Abeta peptides, neuroblastoma cells showed a clear-cut induction of CP450r. To determine whether this mechanism is operational in vivo, we investigated the expression of CP450r in a transgenic mouse model of Alzheimer's disease (AD) and in brains from patients afflicted with AD, using an immunocytochemical approach. Tissue sections from brains of transgenic mice exhibited strong immunoreactivity for CP450r, surrounding amyloid deposits. The pattern of expression of CP450r was similar to that exhibited by neuritic and oxidative stress markers. Sections from non-transgenic mice showed no detectable immunoreactivity. Immunostaining of sections from four brains with neuropathologically confirmed AD showed a pattern of abnormality different from transgenic mice that was characterized by abnormal immunoreactivity for CP450r within the cytoplasm of cortical neurons. No labeling was seen in sections from aged-matched control brains. The data showed that CP450r is induced by Alzheimer amyloid peptide and that such a response must be considered as one possible mechanism whereby Abeta causes oxidative stress.

C-Jun N-terminal kinase (JNK) and JNK interacting protein response in rat brain after transient middle cerebral artery occlusion

c-Jun response is involved in the development of ischemic brain injury, which is activated by c-Jun N-terminal kinase-1 (JNK-1). The activity of JNK-1 is strictly regulated, and only the phosphorylated form of JNK (phospho-JNK) which is translocated to the nucleus has an ability to activate c-Jun response. There is a protein which inhibits JNK-1 activation, and known as JNK interacting protein-1 (JIP-1). In this study, we investigated change in JNK-1, phospho-JNK, and JIP-1 immunoreactivity in rat brain after transient middle cerebral artery (MCA) occlusion. Immunoreactive JNK-1 was scant in the sham-control brain, but it was induced at 1 h after reperfusion, which was slightly increased at 3 h of reperfusion. By contrast, phospho-JNK remained negative till 3 h. At 8 h, JNK-1 and phospho-JNK became distinctly positive, and nuclei as well as cytoplasm were stained. Thereafter, immunoreactivity for JNK-1 and phospho-JNK became furthermore dense, and most neurons revealed positively stained nuclei. Immunoreactivity for JIP-1 remained negative till 8 h of reperfusion, but at 24 and 72 h, cytoplasm of cortical neurons at the MCA boundary area was positively stained. This JIP-1 induction got behind the JNK-1 activation, and therefore, may be a vain effort for neurons to survive. Inhibition of JNK-1 activation might become an innovative means of therapy for stroke treatment in the future.

Antisera against ganglioside GM2: immunochemical and immunohistological studies.

Antisera against ganglioside GM2 were raised in rabbits and tested by immunodiffusion, complement fixation and immunohistology. The presence of precipitating antibodies was demonstrated by immunodiffusion techniques (Ouchterlony and reversed Mancini). GM2-antibody titres were determined by a quantitative microcomplement fixation assay. The GM2-antibodies were shown to be specifically directed against GM2 and did not cross-react with the major brain gangliosides. Application of GM2-antiserum to brain sections from a case of GM2-gangliosidosis (Sandhoff's disease) in an immunofluorescence study produced a specific fluorescence of the granular GM2-storage material within the cytoplasm of cortical neurons. With immunoperoxidase staining by electron microscopy, labelling of the surface of the membraneous cytoplasmic bodies was obtained.