Immunohistochemical study of arginase-1 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis.

Arginase-1, a marker for M2 phenotype alternatively activated macrophages, inhibits inflammation and is associated with phagocytosis of cell debris and apoptotic cells. We analyzed the expression of arginase-1, a competitive enzyme of inducible nitric oxide synthase (iNOS), in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that both arginase-1 and iNOS significantly increased in the spinal cords of rats at the peak stage of EAE compared with the expression level in control animals (p<0.05) and declined thereafter. Immunofluorescent staining demonstrated that increased expression of arginase-1 in EAE spinal cords was confirmed in macrophages as well as in some neurons and astrocytes that were constitutively positive for arginase-1 in normal spinal cords. A semiquantitative analysis by immunofluorescence showed that in EAE lesions, an increased level of arginase-1 immunoreactivity was matched with ED1-positive macrophages, which were also positive for activin A, a marker for the M2 phenotype. Taking all of these findings into consideration, we postulate that the increased level of arginase-1, which is partly from M2 macrophages, contributes to the modulation of neuroinflammation in EAE lesions, possibly through the reduction of nitric oxide in the lesion via competition with iNOS for the use of L-arginine.

Control of neurite outgrowth by RhoA inactivation.

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.

Association of endothelial nitric oxide synthase and mitochondrial dysfunction in the hippocampus of scrapie-infected mice.

The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV-associated dementia (HAD), brain ischemia, Parkinson's disease, and Alzheimer's disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn-SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.

Neurites from PC12 cells are connected to each other by synapse-like structures.

PC12 cells have been used as a model of sympathetic neurons. Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cAMP induce neurite outgrowth from PC12 cells. cAMP induced a greater number of neurites than did NGF. In particular, we attempted to elucidate whether PC12 cell neurites, induced by several factors including NGF, bFGF, and cAMP, form synapses, and whether each neurite has presynaptic and postsynaptic properties. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed that neurites are connected to each other. The connected regions presented dense core vesicles and a clathrin-coated membrane invagination. In addition, typical maker proteins for axon and dendrite were identified by an immuno-staining method. Tau-1, an axonal marker in neurons, was localized at a high concentration in the terminal tips of neurites from PC12 cells, which were connected to neurite processes containing MAP-2, a dendritic marker in neurons. Furthermore, neurites containing SV2 and synaptotagmin, markers of synaptic vesicles, were in contact with neurites harboring drebrin, a marker of the postsynaptic membrane, suggesting that neurites from PC12 cells induced by NGF, bFGF, and cAMP may form synapse-like structures. Tat-C3 toxin, a Rho inhibitor, augmented neurite outgrowth induced by NGF, bFGF, and cAMP. Tat-C3 toxin together with neurotrophins also exhibited synapse-like structures between neurites. However, it remains to be studied whether RhoA inhibition plays a role in the formation of synapse-like structures in PC12 cells.

Reduction of prion infectivity and levels of scrapie prion protein by lithium aluminum hydride: implications for RNA in prion diseases.

Previous studies indicate that RNA may be required for proteinase-resistant prion protein (PrP) amplification and for infectious prion formation in vitro, suggesting that RNA molecules may function as cellular cofactors for abnormal PrP (PrPSc) formation and become part of the structure of the infectious agent. To address this question, we used chemicals that can cleave phosphodiester bonds of RNA and assessed their effects on the infectious agent. Lithium aluminum hydride, a reducing agent that can induce reductive cleavage of oxidized molecules such as carbonyls, carboxyl acids, esters, and phosphodiester bonds, did not affect cellular PrP degradation; however, it destroyed PrPSc, extended the scrapie incubation period, and markedly reduced total RNA concentrations. These results prompted us to investigate whether RNA molecules are cofactors for PrPSc propagation. RNase A treatment of partially purified PrP and of 263K scrapie brain homogenates was sufficient to increase the sensitivity of PrPSc to proteinase K degradation. This is the first evidence that suggests that RNA molecules are a component of PrPSc. Treatment with RNase A alone and PrP degradation by RNase A plus proteinase K in vitro, however, did not result in loss of scrapie infectivity compared with the effects of lithium aluminum hydride. Together, these data suggest that RNA molecules may be important for maintaining the structure of PrPSc and that oxidized molecules can be important in scrapie agent replication and prion infectivity.

Differential regulation of apoptosis by caspase-mediated cleavage of phospholipase D isozymes.

Phospholipase D (PLD) has been implicated in survival and anti-apoptosis, but the molecular mechanism by which it responds to apoptotic stimuli is poorly unknown. Here, we demonstrate that cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site (DDVD(545)S) and PLD2 is cleaved at two or three sites (PTGD(13)ELD(16)S and DEVD(28)T) in the front of N-terminus. Cleavage of PLD was endogenously detected in post-mortem Alzheimer brain together with activated caspase-3, suggesting the physiological relevance. The cleavage of PLD1 but not PLD2 might act as an inactivating process since PLD1 but not PLD2 activity is significantly decreased during apoptosis, suggesting that differential cleavage of PLD isozymes could affect its enzymatic activity. Moreover, caspase-resistant mutant of PLD1 showed more potent anti-apoptotic capacity than that of wild type PLD1, whereas PLD2 maintained anti-apoptotic potency in spite of its cleavage during apoptosis. Moreover, PLD2 showed more potent anti-apoptotic effect than that of PLD1 in overexpression and knockdown experiments, suggesting that difference in anti-apoptotic potency between PLD1 and PLD2 might be due to its intrinsic protein property. Taken together, our results demonstrate that differential cleavage pattern of PLD isozymes by caspase might affect its enzymatic activity and anti-apoptotic function.

Transient impairment of hippocampus-dependent learning and memory in relatively low-dose of acute radiation syndrome is associated with inhibition of hippocampal neurogenesis.

Neurogenesis in the adult hippocampus, which occurs constitutively, is vulnerable to ionizing radiation. In the relatively low-dose exposure of acute radiation syndrome (ARS), the change in the adult hippocampal function is poorly understood. This study analyzed the changes in apoptotic cell death and neurogenesis in the DGs of hippocampi from adult ICR mice with single whole-body gamma-irradiation using the TUNEL method and immunohistochemical markers of neurogenesis, Ki-67 and doublecortin (DCX). In addition, the hippocampus-dependent learning and memory tasks after single whole-body gamma-irradiation were examined in order to evaluate the hippocampus-related behavioral dysfunction in the relatively low-dose exposure of ARS. The number of TUNEL-positive apoptotic nuclei in the dentate gyrus (DG) was increased 6-12 h after acute gamma-irradiation (a single dose of 0.5 to 4 Gy). In contrast, the number of Ki-67- and DCX-positive cells began to decrease significantly 6 h postirradiation, reaching its lowest level 24 h after irradiation. The level of Ki-67 and DCX immunoreactivity decreased in a dose-dependent manner within the range of irradiation applied (0-4 Gy). In passive avoidance and object recognition memory test, the mice trained 1 day after acute irradiation (2 Gy) showed significant memory deficits, compared with the sham controls. In conclusion, the pattern of the hippocampus-dependent memory dysfunction is consistent with the change in neurogenesis after acute irradiation. It is suggested that a relatively low dose of ARS in adult ICR mice is sufficiently detrimental to interrupt the functioning of the hippocampus, including learning and memory, possibly through the inhibition of neurogenesis.

JAK-STAT signaling pathway mediates astrogliosis in brains of scrapie-infected mice.

Scrapie is characterized histologically, in part, by astrogliosis in brain and spinal cord. However, the mechanisms of astrogliosis in brain injury occurring during prion infection are not well understood. In this study, we investigated the expression levels and cellular localization of Janus kinase (JAK) -signal transducers and activators of transcription (STAT) signaling molecules and growth factors such as leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) by western blot analysis and immunohistochemistry. We found that expression levels of LIF and CNTF were increased in scrapie-infected brains and phosphorylated (p)-JAK2, p-STAT1 (Ser727 and Tyr701), p-STAT3 (Tyr705), and glial fibrillary acidic protein were expressed strongly in scrapie-infected brains. Moreover, we found that p-STAT1 and p-STAT3 were found mainly in the nucleus in scrapie-infected brains. Immunohistochemically, p-STAT1 was colocalized with LIF and CNTF and p-JAK2 in many reactive astrocytes in scrapie-infected brains. In contrast, immunostaining for p-STAT3 was found in comparatively few astrocytes in limited regions; p-STAT3 staining merged with p-JAK2 in hippocampus sections of scrapie-infected brains. Taken together, our results suggest that activation of JAK2-STAT1 signaling pathway occurred in reactive astrocytes in hippocampus of scrapie-infected brains.

Alteration of iron regulatory proteins (IRP1 and IRP2) and ferritin in the brains of scrapie-infected mice.

Considerable evidence suggests that oxidative stress may be involved in the pathogenesis of Transmissible Spongiform Encephalopathies (TSEs). To investigate the involvement of iron metabolism in TSEs, we examined the expression levels of iron regulatory proteins (IRPs), ferritins, and binding activities of IRPs to iron-responsive element (IRE) in scrapie-infected mice. We found that the IRPs-IRE-binding activities and ferritins were increased in the astrocytes of hippocampus and cerebral cortex in the brains of scrapie-infected mice. These results suggest that alteration of iron metabolism contributes to development of neurodegeneration and that some protective mechanisms against iron-induced oxidative damage may occur during the pathogenesis of TSEs.

Galectin-3 expression is correlated with abnormal prion protein accumulation in murine scrapie.

To investigate the involvement of galectin-3 in the process of neurodegeneration in prion diseases, the expression and cellular localization of galectin-3 in the brain were studied in scrapie, a mouse model of prion disease. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses showed that the expression of galectin-3 protein and mRNA was induced in scrapie-affected brains, particularly at the time when the abnormal prion protein PrP(Sc) began to accumulate in the brains. Immunohistochemically, immunostaining for galectin-3 was found mainly in B4-isolectin-positive cells (presumably activated microglia/macrophages), but not in astrocytes. Galectin-3 immunoreactivity was localized mainly in areas of PrP(Sc) accumulation and neuronal death in scrapie-infected brains. These findings suggest that the expression of galectin-3 by activated microglia/macrophages in prion disease correlates with abnormal prion protein accumulation.

Phospholipase D1 is associated with amyloid precursor protein in Alzheimer's disease.

Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease (AD). We investigated the involvement of phospholipase D (PLD) in the pathophysiology of AD. We showed dramatic upregulation of PLD1 immunoreactivity in reactive astroglial cells in brain tissue sections from authentic AD patients. Expression and activity of PLD1 were up-regulated in brain tissues from AD patients, especially caveolae membrane fraction, compared with those of control brains. Interestingly, PLD1 physically interacts and colocalizes with APP and caveolin-3. We found that APP was associated with the pleckstrin homology domain of PLD1, and the amyloid region of APP interacted with PLD. Elevated expression of APP stimulated PLD activity in human astroglioma cells. These results suggest that up-regulation of PLD might have a role in the neuronal pathology associated with AD.

Scrapie infection activates the replication of ecotropic, xenotropic, and polytropic murine leukemia virus (MuLV) in brains and spinal cords of senescence-accelerated mice: implication of MuLV in progression of scrapie pathogenesis.

Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.

Enhanced expression of netrin-1 protein in the sciatic nerves of Lewis rats with experimental autoimmune neuritis: possible role of the netrin-1/DCC binding pathway in an autoimmune PNS disorder.

Netrin-1 is a chemotropic factor that plays an important role as a survival factor in the adult nervous system. To investigate whether netrin-1 is involved in autoimmune injury of the peripheral nervous system (PNS), the temporal expression of netrin-1 protein was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis revealed a significant increase in the level of netrin-1 protein in the sciatic nerves of rats on days 11 to 24 post-immunization (p.i.) compared to controls; netrin-1 expression declined by day 30 p.i. Immunohistochemistry revealed that netrin-1 protein was expressed weakly in Schwann cells and vessels in the sciatic nerves of normal and CFA-immunized control rats. In the sciatic nerves of EAN-affected rats, netrin-1 immunoreactivity was increased mainly in the cell membrane and extracellular matrix of OX42-positive macrophages and S100-positive Schwann cells at the peak and recovery phases of EAN. Moreover, the putative netrin-1 receptor, deleted in colorectal cancer (DCC), was expressed mainly in axons, some macrophages, and Schwann cells in EAN-affected sciatic nerves, although the level of protein expression did not change significantly over the course of EAN. We suggest that a significant increase in netrin-1 expression contributes to host cell survival and axon regeneration to counter autoimmune injury and inflammation, which may play a role in recovery from EAN-induced paralysis.

Analysis of the expression of endogenous murine leukemia viruses in the brains of senescence-accelerated mice (SAMP8) and the relationship between expression and brain histopathology.

Many studies have explored the premature aging of accelerated senescence-prone (SAMP8) mice. However, the cause of premature aging in this strain remains unknown. We analyzed the expression of ecotropic, xenotropic, and polytropic murine leukemia viruses (MuLVs) in the brains of accelerated senescence-resistant (SAMR1) and SAMP8 mice. No ecotropic mRNA was detected in SAMR1 mice, and only Akv-type ecotropic MuLV mRNA was detected in SAMP8 mice. Restriction mapping of the full-length infectious E-MuLV genome from SAMP8 confirmed its identity as Akv. mRNAs corresponding to a prototypical polytropic MuLV and to an unusual xenotropic MuLV were detected at equal levels in SAMP8 and SAMR1 mice, but no infectious virus of either host range type was detected. In order to determine the cellular localization of Akv expression in SAMP8 mice, we used immunohistochemistry and electron microscopy to detect expression of the E-MuLV capsid gag (CAgag) gene in striatum, brainstem, hippocampus, and cerebellum of 12-month-old SAMR1 and SAMP8 mice. The CAgag antigen was seen in the neurons, oligodendroglia, and vascular endothelium of these brain regions of SAMP8 mice, but not in SAMR1 mice. To evaluate the correlation between activation of astrocytes and expression of Akv, we performed double-immunohistochemical staining for both glial fibrillary acidic protein (GFAP) and CAgag in SAMR1 and SAMP8 mice. Strong astrocytic activation and extensive vacuolation were observed around CAgag-positive neurons in SAMP8 mice, whereas in SAMR1 mice neither astrocytosis nor vacuolation were present. CAgag antigen was also localized in astrocytes of the hippocampus region of SAMP8 mice. Electron micrography showed that a number of vacuoles were found in the cytoplasm of MuLV-positive neurons and the extracellular space surrounding these neurons showed lytic changes. These results suggest that endogenous Akv provirus is expressed in neurons, astrocytes, vascular endothelium, and oligodendroglia in the brains of SAMP8 and that this virus could play an important role in the brain aging processes in this mouse strain.

Detection of JC virus type 1 in peripheral lymphocytes, brain and cerebrospinal fluid from two Korean AIDS patients with progressive multifocal leukoencephalopathy.

The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating central nervous system disease progressive multifocal leukoencephalopathy (PML), which occurs in 4-7% of AIDS patients. Two Korean AIDS patients with PML were assayed for JCV, and the virus was genotyped by polymerase chain reaction, DNA sequencing and phylogenetic analysis. Using immunohistochemical analysis, we also examined the distribution of JCV antigen in the brains of the patients. The JCV genome was detected in peripheral lymphocytes, brain and cerebrospinal fluid from these Korean PML patients. Although type 2 is the most common genotype in Asia, the genotype of the JCV in these two AIDS patients was characterized as type 1, which is of European origin. We found that JCV antigen was selectively detected in oligodendrocytes and astrocytes of the brains from these patients. Compared to the prototype type 1 (Mad-1), two different nucleotides (G-->C) in the KOR-1 strain identified here were found at positions 2488 and 2490 of the major capsid protein VP1 gene. In summary, this is the first report of PML in Korean AIDS patients; it is also the first isolation of JCV type 1 in PML in East Asians.