Walter E. Mushynski

Neurofilaments in Health and Disease

This article reviews current knowledge of neurofilament structure, phosphorylation, and function and neurofilament involvement in disease. Neurofilaments are obligate heteropolymers requiring the NF-L subunit together with either the NF-M or the NF-H subunit for polymer formation. Neurofilaments are very dynamic structures; they contain phosphorylation sites for a large number of protein kinases, including protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent kinase 5 (Cdk5), extracellular signal regulated kinase (ERK), glycogen synthase kinase-3 (GSK-3), and stress-activated protein kinase gamma (SAPK gamma). Most of the neurofilament phosphorylation sites, located in tail regions of NF-M and NF-H, consist of the repeat sequence motif, Lys-Ser-Pro (KSP). In addition to the well-established role of neurofilaments in the control of axon caliber, there is growing evidence based on transgenic mouse studies that neurofilaments can affect the dynamics and perhaps the function of other cytoskeletal elements, such as microtubules and actin filaments. Perturbations in phosphorylation or in metabolism of neurofilaments are frequently observed in neurodegenerative diseases. A down-regulation of mRNA encoding neurofilament proteins and the presence of neurofilament deposits are common features of human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinsons disease, and Alzheimers disease. Although the extent to which neurofilament abnormalities contribute to pathogenesis in these human diseases remains unknown, emerging evidence, based primarily on transgenic mouse studies and on the discovery of deletion mutations in the NF-H gene of some ALS eases, suggests that disorganized neurofilaments can provoke selective degeneration and death of neurons. An interference of axonal transport by disorganized neurofilaments has been proposed as one possible mechanism of neurofilament-induced pathology. Other factors that can potentially lead to the accumulation of neurofilaments will be discussed as well as the emerging evidence for neurofilaments as being possible targets of oxidative damage by mutations in the superoxide dismutase enzyme (SOD1); such mutations are responsible for approximately 20% of familial ALS cases.

Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis.

Abstract : Mutations in the Cu/Zn‐superoxidedismutase (SOD‐1) gene underlie some familial cases of amytotrophic lateral sclerosis, a neurodegenerative disorder charactreized by loss of cortical, brainstem, and spinal motor nrurons. We present evidence that SOD‐1 mutants alter the activity of molecular chaperones that aid in proper protein folding and targeting of abnormal proteins for degradation. In a cultured cell line (NOH 3T3), resistance to mutant SOD‐1 toxicity correlated with increased overall chaperoning activity (measured by the ability of cytosolic extracts to prevent heat denaturation of catalase) as well as with up‐regulation of individual chaperones/stress proteins. In transgenic mice expressing human SOD‐1 with the G93A mutation, chaperoning activity was decreased in lumbar spinal cord but increased or unchanged in clinically unaffected tissues. Increasing the level of the stress‐inducible chaperone 70‐kDa heat shock protein by gene transfer reduced formation of mutant SOD‐containing proteinaceous aggregates in cultured primary motor neurons expressing G93A SOG‐1 and prolonged their survival. We propose that insufficiency of molecular chaperones may be directly involved in loss of motor neurons in this disease.

Cycling at the interface between neurodevelopment and neurodegeneration.

The discovery of cell cycle regulators has directed cell research into uncharted territory. In dividing cells, cell cycle-associated protein kinases, which are referred to as cyclin-dependent-kinases (Cdks), regulate proliferation, differentiation, senescence and apoptosis. In contrast, all Cdks in post-mitotic neurons, with the notable exception of Cdk5, are silenced. Surprisingly, misregulation of Cdks occurs in neurons in a wide diversity of neurological disorders, including Alzheimers disease, Parkinsons disease and amyotrophic lateral sclerosis. Ectopic expression of these proteins in neurons potently induces cell death with hallmarks of apoptosis. Deregulation of the unique, cell cycle-unrelated Cdk5 by its truncated co-activator, p25 and p29, contributes to neurodegeneration by altering the phosphorylation state of non-membrane-associated proteins and possibly through the induction of cell cycle proteins. On the other hand, cycling Cdks such as Cdk2, Cdk4 and Cdk6, initiate death pathways by derepressing E2F-1/Rb-dependent transcription at the neuronal G1/S checkpoint. Thus, Cdk5 and cycling Cdks may have little in common in the healthy CNS, but they likely conspire in leading neurons to their demise.

Aberrant stress-induced phosphorylation of perikaryal neurofilaments.

The aberrant phosphorylation of the neurofilament high molecular weight subunit (NFH) in the neuronal perikaryon is a common feature of several neurological diseases. We demonstrated a strong correlation between hyperphosphorylation of the NFH carboxyl-terminal domain and activation of stress-activated protein kinase (SAPK) -γ in PC12 cells. Agents that activated SAPKγ in PC12 cells also caused the hyperphosphorylation of perikaryal NFH in cultured dorsal root ganglion neurons. The NFH carboxyl-terminal domain was phosphorylated by SAPKγ in vitro, and the use of peptide substrates indicated that this event occurred preferentially at KSPXE motifs. We propose that SAPKγ, perhaps in concert with other SAPKs, is involved in the abnormal phosphorylation of perikaryal NFH. This finding could lead to new insights into the etiology of several neurological diseases.

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.

Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-α

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-α. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.

Sequence and structure of the mouse gene coding for the largest neurofilament subunit

We have determined the complete nucleotide sequence of the mouse gene encoding the neurofilament NF-H protein. The C-terminal domain of NF-H is very rich in charged amino acids (aa) and contains a 3-aa sequence, Lys-Ser-Pro, that is repeated 51 times within a stretch of 368 aa. The location of this serine-rich repeat in the phosphorylated domain of NF-H indicates that it represents the major protein kinase recognition site. The nfh gene shares two common intron positions with the nfl and nfm genes, but has an additional intron that occurs at a location equivalent to one of the introns in non-neuronal intermediate filament-coding genes. This additional nfh intron may have been acquired via duplication of a primordial intermediate filament gene.

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

The molecular mechanisms underlying AMPA (α‐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 Ca2+ 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, α‐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 Ca2+ influx, depletion of glutathione, and activation of JNK, calpain, and caspase‐3.

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.