Heterogeneity in the properties of NEFL mutants causing Charcot-Marie-Tooth disease results in differential effects on neurofilament assembly and susceptibility to intervention by the chaperone-inducer, celastrol.

Aberrant aggregation of neurofilament proteins is a common feature of neurodegenerative diseases. For example, neurofilament light protein (NEFL) mutants causing Charcot-Marie-Tooth disease induce misassembly of neurofilaments. This study demonstrated that mutations in different functional domains of NEFL have different effects on filament assembly and susceptibility to interventions to restore function. The mouse NEFL mutants, NEFL(Q333P) and NEFL(P8R), exhibited different assembly properties in SW13-cells, cells lacking endogenous intermediate filaments, indicating different consequences of these mutations on the biochemical properties of NEFL. The p.Q333P mutation caused reversible misfolding of the protein. NEFL(Q333P) could be refolded and form coil-coiled dimers, in vitro using chaotropic agent, and in cultured cells by induction of HSPA1 and HSPB1. Celastrol, an inducer of chaperone proteins, induced HSPA1 expression in motor neurons and prevented the formation of neurofilament inclusions and mitochondrial shortening induced by expression of NEFL(Q333P), but not in sensory neurons. Conversely, celastrol had a protective effect against the toxicity of NEFL(P8R), a mutant which is sensitive to HSBP1 but not HSPA1 chaperoning, only in large-sized sensory neurons, not in motor neurons. Importantly, sensory and motor neurons do not respond identically to celastrol and different chaperones are upregulated by the same treatment. Thus, effective therapy of CMT not only depends on the identity of the mutated gene, but the consequences of the specific mutation on the properties of the protein and the neuronal population targeted.

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.

Response of human oligodendrocyte progenitors to growth factors and axon signals.

We examined the effects of growth factors and axonal signals on the differentiation of human fetal and adult oligodendrocyte progenitor cells (OPCs) and determined whether these effects translated into enhanced axonal ensheathment. Only small numbers of fetal OPCs grown in defined medium expressed the oligodendroglial lineage markers Olig2 and O4. The combination of platelet-derived growth factor-AA and basic fibroblast growth factor enhanced proliferation of Olig2-positive and O4-positive cells; a combination of brain-derived neurotrophic factor and insulin-like growth factor 1 promoted O4-positive cell differentiation, galactocerebroside expression, and morphological complexity. Coculturing with rodent dorsal root ganglion neurons in defined medium alone enhanced OPC differentiation and myelin basic protein expression. The addition of brain-derived neurotrophic factor/insulin-like growth factor 1 further enhanced differentiation, axonal attachment and ensheathment, and clustering of the contactin-associated protein Caspr and Na+ channels. By contrast, most adult OPCs were O4 positive and Olig2 positive in defined medium; both brain-derived neurotrophic factor/insulin-like growth factor 1 and platelet-derived growth factor-AA/basic fibroblast growth factor promoted their myelin basic protein expression and membrane sheet formation; coculture with dorsal root ganglion neurons further increased myelin basic protein expression. Growth factors also enhanced attachment of adult OPCs to axons, but their capacity to ensheath axons was lower than that of fetal OPCs. These results demonstrate that fetal and adult OPCs show measurable responses to selected growth factors and axon signals that correlate with their capacity for axon ensheathment. The distinct properties of fetal and adult OPCs may be related to differences in their chronological age and initial differentiation states.

Mitogen-activated protein kinase activated protein kinase 2 (MK2) participates in p38 MAPK regulated control of oligodendrocyte differentiation.

The p38 mitogen-activated protein kinases (p38 MAPKs) are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. We have previously reported a role for p38 MAPK in the regulation of oligodendrocyte (OLG) differentiation and Schwann cell myelination. Here, we extend our previous findings by showing that a p38 substrate, mitogen-activated protein kinase activated protein kinase 2 (MK2) is a downstream element of the p38 signaling pathway responsible for effecting OLG differentiation. Inhibition of MK2 activity in oligodendrocyte progenitors (OLPs) using CMPD1 [4-(2'-fluorobiphenyl-4-yl)-N-(4-hydroxyphenyl)-butyramide] blocked the activation of MK2 and resulted in decreased accumulation of myelin-differentiation markers, including myelin-associated glycoprotein (MAG) and myelin basic protein (MBP). We corroborated these findings using a small-interfering RNA to MK2, which decreased the myelin-specific lipid galactosylceramide and MAG. Treatment of cultures with CMPD1 decreased the steady state levels of mRNA encoding myelin transcription factor 1 (Myt1), MAG, MBP, and Opalin, a transmembrane sialylglycoprotein expressed in oligodendrocytes. In contrast, increases were observed in the mRNA levels of OLG transcriptional repressors, including transcription factor 4 (Tcf4), Notch1, and inhibitor of differentiation 2 (Id2). Furthermore, we found that the predominantly expressed isoform of p38 in OLGs, p38alpha, and MK2 can form coimmunoprecipitable complexes in OLPs and OLGs. Our results demonstrate that the p38-MK2 pathway is a component of the signaling cascade regulating OLG differentiation.

Amyloid beta-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-beta oligomers provoked similar upregulation of pro-NGF in naive rats that was accompanied 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-beta 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-beta amyloid CNS burden on NGF metabolism may explain the paradoxical upregulation of pro-NGF in AD accompanied by atrophy of forebrain cholinergic neurons.

The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

BACKGROUND: The quaking viable (qk(v)) 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 qk(v) 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 p27(KIP1) 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, p27(KIP1) 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.

Mitochondrial and axonal abnormalities precede disruption of the neurofilament network in a model of charcot-marie-tooth disease type 2E and are prevented by heat shock proteins in a mutant-specific fashion.

Mutations in NEFL encoding the light neurofilament subunit (NFL) cause Charcot-Marie-Tooth disease type 2E (CMT2E), which affects both motor and sensory neurons. We expressed the disease-causing mutants NFL and NFL in motor neurons of dissociated spinal cord-dorsal root ganglia and demonstrated that they are incorporated into the preexisting neurofilament network but eventually disrupt neurofilaments without causing significant motor neuron death. Importantly, rounding of mitochondria and reduction in axonal diameter occurred before disruption of the neurofilament network, indicating that mitochondrial dysfunction contributes to the pathogenesis of CMT2E, as well as to CMT caused by mitofusin mutations. Heat shock proteins (HSPs) are involved in the formation of the neurofilament network and in protecting cells from misfolded mutant proteins. Cotransfection of HSPB1 with mutated NEFL maintained the neurofilament network, axonal diameter, and mitochondrial length in motor neurons expressing NFL, but not NFL. Conversely, HSPA1 cotransfection was effective in motor neurons expressing NFL, but not NFL. Thus, there are NFL mutant-specific differences in the ability of individual HSPs to prevent neurofilament abnormalities, reduction in axonal caliber, and disruption of mitochondrial morphology in motor neurons. These results suggest that HSP inducers have therapeutic potential for CMT2E but that their efficacy would depend on the profile of HSPs induced and the type of NEFL mutation.

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 alpha/beta 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.

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 p27(kip1) 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.

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

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.

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

The molecular mechanisms underlying H(2)O(2)-induced toxicity were characterized in rat oligodendrocyte cultures. While progenitor cells were more sensitive than mature oligodendrocytes to H(2)O(2), 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 H(2)O(2) stress. As free radicals have been considered to serve as second messengers, we examined the effect of H(2)O(2) on activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) 1/2 and p38. H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) toxicity in progenitors and mature oligodendrocytes. In contrast, inhibitors of both p38 and MEK reduced H(2)O(2)-induced death most significantly in oligodendrocytes. The poly (ADP-ribose) polymerase inhibitor, PJ34, reduced H(2)O(2)-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.

A neurotoxic peripherin splice variant in a mouse model of ALS.

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.

The environmental toxin arsenite induces tau hyperphosphorylation.

Abnormally hyperphosphorylated tau polymers known as paired helical filaments constitute one of the major characteristic lesions that lead to the demise of neurons in Alzheimer's disease. Here, we demonstrate that the environmental toxin arsenite causes a significant increase in the phosphorylation of several amino acid residues (Thr-181, Ser-202, Thr-205, Thr-231, Ser-262, Ser-356, Ser-396, and Ser-404) in tau, which are also hyperphosphorylated under pathological conditions. Complementary phosphopeptide mapping revealed a dramatic increase in the (32)P-labeling of many peptides in tau following arsenite treatment. Although arsenite activates extracellular-signal regulated kinases-1/-2 and stress-activated protein kinases, these enzymes did not contribute to the arsenite-increased phosphorylation, nor did they appear to normally modify tau in vivo. Tau phosphorylation induced by arsenite did not involve glycogen synthase kinase-3 or protein phosphatase-1 or -2, but the activity responsible for tau hyperphosphorylation could be inhibited with the protein kinase inhibitor roscovitine. The effects of arsenite on the phosphorylation of some tau mutations (DeltaKappa280, V337M, and R406W) associated with frontal-temporal dementia with parkinsonism linked to chromosome 17 was analyzed. The unchallenged and arsenite-induced phosphorylation of some mutant proteins, especially R406W, was altered at several phosphorylation sites, indicating that these mutations can significantly affect the structure of tau in vivo. Although the major kinase(s) involved in aberrant tau phosphorylation remains elusive, these results indicate that environmental factors, such as arsenite, may be involved in the cascade leading to deregulation of tau function associated with neurodegeneration.

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3.

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.