Gabriela Fragoso

Amyloid β-Induced Nerve Growth Factor Dysmetabolism in Alzheimer Disease

We previously reported that the precursor form of nerve growth factor (pro-NGF) and not mature NGF is liberated in the CNS in an activity-dependent manner, and that its maturation and degradation occur in the extracellular space by the coordinated action of proteases.Here, we present evidence of diminished conversion of pro-NGF to its mature form and of greater NGF degradation in Alzheimer disease (AD) brain samples compared with controls. These alterations of the NGF metabolic pathway likely resulted in the increased pro-NGF levels. The pro-NGF was largely in a peroxynitrited form in the AD samples. Intrahippocampal injection of amyloid-&bgr; oligomers provoked similar upregulation of pro-NGF in naive rats that wasaccompanied by evidence of microglial activation (CD40), increased levels of inducible nitric oxide synthase, and increased activity of the NGF-degrading enzyme matrix metalloproteinase 9. The elevated inducible nitric oxide synthase provoked the generation of biologically inactive, peroxynitrite-modified pro-NGF in amyloid-&bgr; oligomer-injected rats. These parameters were corrected by minocycline treatment. Minocycline also diminished altered matrix metalloproteinase 9, inducible nitric oxide synthase, and microglial activation (CD40); improved cognitive behavior; and normalized pro-NGF levels in a transgenic mouse AD model. The effects of amyloid-&bgr; amyloid CNS burden on NGF metabolism may explain the paradoxical upregulation of pro-NGF in AD accompanied by atrophy of forebrain cholinergic neurons.

Developmental differences in H2O2-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3

The molecular mechanisms underlying H2O2‐induced toxicity were characterized in rat oligodendrocyte cultures. While progenitor cells were more sensitive than mature oligodendrocytes to H2O2, the antioxidant, N‐acetyl‐l‐cysteine, blocked toxicity at both stages of development. Differentiated oligodendrocytes contained more glutathione than did progenitors and were less susceptible to decreases in glutathione concentration induced by H2O2 stress. As free radicals have been considered to serve as second messengers, we examined the effect of H2O2 on activation of the mitogen‐activated protein kinases (MAPK), extracellular signal‐regulated kinases (ERK) 1/2 and p38. H2O2 caused a time‐ and concentration‐dependent increase in MAPK phosphorylation, an effect that was totally blocked by N‐acetyl‐l‐cysteine. Further exploration of potential mechanisms involved in oligodendrocyte cell death showed that H2O2 treatment caused DNA condensation and fragmentation at both stages of development, whereas caspase 3 activation and poly (ADP‐ribose) polymerase cleavage were significantly increased only in oligodendrocyte progenitors. The pan‐caspase inhibitor, benzyloxycarbonyl‐Val‐Ala‐Asp fluoromethyl ketone, blocked DNA fragmentation in progenitors and produced a small but significant level of protection from H2O2 toxicity in progenitors and mature oligodendrocytes. In contrast, inhibitors of both p38 and MEK reduced H2O2‐induced death most significantly in oligodendrocytes. The poly (ADP‐ribose) polymerase inhibitor, PJ34, reduced H2O2‐induced toxicity on its own but was most effective when combined with benzyloxycarbonyl‐Val‐Ala‐Asp fluoromethyl ketone or PD169316. The finding that molecular mechanisms conferring resistance to reactive oxygen species toxicity are regulated during oligodendrocyte differentiation may be of importance in designing therapies for certain neurological diseases affecting white matter.

Inhibition of p38 mitogen-activated protein kinase interferes with cell shape changes and gene expression associated with Schwann cell myelination.

In the present study we demonstrate that p38, a member of the mitogen-activated protein kinase (MAPK) family, is essential for ascorbate- and laminin-induced myelination in Schwann cell-dorsal root ganglion neuron cocultures. The inhibitory effect of the specific p38 blockers, PD 169316 and SB 203580, on ascorbate-induced myelination was exerted during the early stages (1-2 days) of ascorbate treatment. Inhibition of p38 was further shown to prevent the alignment of Schwann cells along axons in laminin-treated cocultures. The addition of laminin to Schwann cell-dorsal root ganglion neuron cocultures stimulated phosphorylation of p38, thereby demonstrating a link between laminin-induced myelination and p38 activation. Similarly, the small heat shock protein, Hsp27, which is phosphorylated by MAPKAPK2, a downstream substrate of p38, was phosphorylated in response to the addition of laminin to the cocultures. The p38 inhibitors did not affect the proliferation or survival of Schwann cells in the cocultures as assessed by BrdU incorporation and total cell counts. However, p38 inhibition interfered with an early stage in myelination, thereby preventing ascorbate-induced increases in the levels of mRNAs encoding MBP, MAG, and P(0) and reducing laminin deposition. These results indicate that activation of p38 by a signaling pathway(s) involving laminin and appropriate integrin receptor(s) is required for the alignment of Schwann cells with axons that precedes myelination.

p38 Mitogen-Activated Protein Kinase Is Required for Central Nervous System Myelination

The p38 MAPKs are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. Here, we report that p38 regulates oligodendrocyte differentiation. Inhibition of p38 with PD169316 and SB203580 prevented accumulation of protein and mRNA of cell‐stage specific markers characteristic of differentiated oligodendrocytes, including myelin basic protein, myelin‐associated glycoprotein, and the glycosphingolipids, galactosylceramide and sulfatide. In addition, the cell cycle regulator p27kip1 and the transcription factor Sox10 were also significantly reduced. Most significantly, p38 inhibitors completely and irreversibly blocked myelination of dorsal root ganglion neurons by oligodendrocytes and prevented the axolemmal organization of the axo‐glial adhesion molecule Caspr. Our results suggest a role(s) for this kinase in key regulatory steps in the maturation of OLGs and initiation of myelination.

Catecholamine-induced oligodendrocyte cell death in culture is developmentally regulated and involves free radical generation and differential activation of caspase-3.

Oligodendrocyte cultures were used to study the toxic effects of catecholamines. Our results showed that catecholamine‐induced toxicity was dependent on the dose of dopamine or norepinephrine used and on the developmental stage of the cultures, with oligodendrocyte progenitors being more vulnerable. A role for oxidative stress and apoptosis on the mechanism of action of catecholamines on oligodendrocyte cell death was next assessed. Catecholamines caused a reduction in intracellular glutathione levels, an accumulation in reactive oxygen species and in heme oxygenase‐1, the 32 kDa stress‐induced protein. All these changes were prevented by N‐acetyl‐L‐cysteine, a thiocompound with antioxidant activity and a precursor of glutathione, and were more pronounced in progenitors than mature cells, which could contribute to their higher susceptibility. Apoptotic cell death, as assessed by activation of caspase‐9 and –3 and cleavage of poly(ADP‐ribose) polymerase (a substrate of caspase‐3), was only observed in oligodendrocyte progenitors. Pretreatment with zVAD, a general caspase inhibitor, prevented activation of caspase‐9 and ‐3, DNA fragmentation, and decreased progenitors cell death. Furthermore, the expression levels of procaspase‐3 and the ratio of the proapoptotic protein bax to antiapoptotic protein bcl‐xl were several folds higher in immature than mature oligodendrocytes. Taken together, these results strongly suggest that the catecholamine‐induced cytotoxicity in oligodendrocytes is developmentally regulated, mediated by oxidative stress, and have characteristics of apoptosis in progenitor cells. GLIA 40:283–299, 2002.

p38 Mitogen-Activated Protein Kinase Regulates Myelination

The p38 mitogen-activated protein kinase family is emerging as a crucial signaling molecule for a vast number of cellular functions including cell migration, proliferation, and differentiation. The function of p38 in myelination has only been recently addressed. Using pyridinyl imidazole-based p38 α/β selective inhibitors, we have reported a critical role for this kinase in the regulation of myelination, specifically, in controlling the differentiation of Schwann cells, and oligodendrocytes, the myelinating glia of the peripheral and central nervous systems, respectively. These compounds inhibited the accumulation of myelin-cell-specific markers, including myelin-specific glycosphingolipids, myelin-associated glycoprotein, and myelin basic protein. More significantly, myelination of dorsal root ganglia neurons by oligodendrocytes was irreversibly blocked by p38 inhibitors. Our current studies are focusing on the molecular mechanisms by which p38 regulates oligodendrocyte and Schwann cell differentiation and its role in models of myelination and remyelination.

Human coronavirus OC43 infection induces chronic encephalitis leading to disabilities in BALB/C mice

Abstract The notion that an infectious respiratory pathogen can damage the central nervous system (CNS) and lead to neurological disease was tested using a human respiratory coronavirus, the OC43 strain of human coronavirus (HCoV-OC43). First, primary cell cultures were used to determine the susceptibility of each type of neural cells to virus infection. Neurons were the target cells, undergoing degeneration during infection, in part due to apoptosis. Second, neuropathogenicity was investigated in susceptible mice. Intracerebral inoculation of HCoV-OC43 into BALB/c mice led to an acute encephalitis with neuronal cell death by necrosis and apoptosis. Infectious virus was apparently cleared from surviving animals, whereas viral RNA persisted for several months. Some of the animals surviving to acute encephalitis presented an abnormal limb clasping reflex and a decrease in motor activity starting several months post-infection. These results suggest that viral persistence could be associated with an increased neuronal degeneration leading to neuropathology and motor deficits in susceptible individuals.

The QKI-6 and QKI-7 RNA Binding Proteins Block Proliferation and Promote Schwann Cell Myelination

Background The quaking viable (qkv) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qkv mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. Methodology/Principal Findings To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27KIP1 and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR. Conclusions/Significance Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

Regulation of peripheral myelination by Src-like kinases.

Fyn, a nonreceptor Src-like tyrosine kinase (SLK), plays an important role in oligodendrocyte differentiation and myelination in the brain. However, its role in myelination of peripheral nerves remains undefined. Here we report that selective inhibitors of SLKs (PP2 and SU6656) caused a dose-dependent decrease in the accumulation of several myelin proteins, including myelin basic protein (MBP), protein zero (P0) and myelin-associated glycoprotein (MAG) in rat Schwann cell-dorsal root ganglion neuron (SC-DRGN) co-cultures. Interestingly, SLK inhibition was insufficient to completely abrogate myelin synthesis, as removal of PP2 after several days of treatment permitted a partial recovery of myelin proteins expression. Furthermore, fewer and shorter myelinated segments formed in the continuous presence of PP2, although the myelin formed was normally compacted. PP2 also decreased the number of SCs expressing Krox-20, a master-regulatory transcription factor expressed by myelinating SCs, by 50%. These results were corroborated by selective knockdown of Fyn and Lyn kinases using siRNA. Extracellular matrix is important to SC differentiation and peripheral myelination. Using phospho-specific antibodies, we showed that addition of extracellular matrix extracts to SC-DRGN co-cultures resulted in the activation of ERK, Akt and p38 MAPK, three protein kinases involved in SC proliferation, differentiation and peripheral myelination. PP2 blocked the phosphorylation of all three kinases. Our results support a role for SLKs in the initiation of peripheral myelination via the activation of p38, Akt and ERK, which regulate Krox-20 expression and peripheral myelination.