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Interest in neurofilaments has risen sharply in recent years with recognition of their potential as biomarkers of brain injury or neurodegeneration in CSF and blood. This is in the context of a growing appreciation for the complexity of the neurobiology of neurofilaments, new recognition of specialized roles for neurofilaments in synapses and a developing understanding of mechanisms responsible for their turnover. Here we will review the neurobiology of neurofilament proteins, describing current understanding of their structure and function, including recently discovered evidence for their roles in synapses. We will explore emerging understanding of the mechanisms of neurofilament degradation and clearance and review new methods for future elucidation of the kinetics of their turnover in humans. Primary roles of neurofilaments in the pathogenesis of human diseases will be described. With this background, we then will review critically evidence supporting use of neurofilament concentration measures as biomarkers of neuronal injury or degeneration. Finally, we will reflect on major challenges for studies of the neurobiology of intermediate filaments with specific attention to identifying what needs to be learned for more precise use and confident interpretation of neurofilament measures as biomarkers of neurodegeneration.

Loss of sacsin, a large 520 kDa multidomain protein, causes autosomal recessive spastic ataxia of the Charlevoix-Saguenay, one of the most common childhood-onset recessive ataxias. A prominent feature is abnormal bundling of neurofilaments in many neuronal populations. This study shows the direct involvement of sacsin domains in regulating intermediate filament assembly and dynamics and identifies important domains for alleviating neurofilament bundles in neurons lacking sacsin. Peptides encoding sacsin internal repeat (SIRPT) 1, J-domains, and ubiquitin-like domain modified neurofilament assembly in vivo. The domains with chaperone homology, the SIRPT and the J-domain, had opposite effects, promoting and preventing filament assembly, respectively. In cultured Sacs-/- motor neurons, both the SIRPT1 and J-domain resolved preexisting neurofilament bundles. Increasing expression of heat shock proteins also resolved neurofilament bundles, indicating that this endogenous chaperone system can compensate to some extent for sacsin deficiency.-Gentil, B. J., Lai, G.-T., Menade, M., Lariviere, R., Minotti, S., Gehring, K., Chapple, J.-P., Brais, B., Durham, H. D. Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

Stress granules (SGs) are cytoplasmic foci that form in response to various external stimuli and are essential to cell survival following stress. SGs are studied in several diseases, including ALS and FTD, which involve the degeneration of motor and cortical neurons, respectively, and are now realized to be linked pathogenically by TDP-43, originally discovered as a component of ubiquitin-positive aggregates within patients’ neurons and some glial cells. So far, studies to undercover the role of TDP-43 in SGs have used primarily transformed cell lines, and thus rely on the extrapolation of the mechanisms to cell types affected in ALS/FTD, potentially masking cell specific effects. Here, we investigate SG dynamics in primary motor and cortical neurons as well as astrocytes. Our data suggest a cell and stress specificity and demonstrate a requirement for TDP-43 for efficient SG dynamics. In addition, based on our in vitro approach, our data suggest that aging may be an important modifier of SG dynamics which could have relevance to the initiation and/or progression of age-related neurodegenerative diseases.

Upregulation of heat shock proteins (HSPs) is an approach to treatment of neurodegenerative disorders with impaired proteostasis. Many neurons, including motor neurons affected in amyotrophic lateral sclerosis (ALS), are relatively resistant to stress-induced upregulation of HSPs. This study demonstrated that histone deacetylase (HDAC) inhibitors enable the heat shock response in cultured spinal motor neurons, in a stress-dependent manner, and can improve the efficacy of HSP-inducing drugs in murine spinal cord cultures subjected to thermal or proteotoxic stress. The effect of particular HDAC inhibitors differed with the stress paradigm. The HDAC6 (class IIb) inhibitor, tubastatin A, acted as a co-inducer of Hsp70 (HSPA1A) expression with heat shock, but not with proteotoxic stress induced by expression of mutant SOD1 linked to familial ALS. Certain HDAC class I inhibitors (the pan inhibitor, SAHA, or the HDAC1/3 inhibitor, RGFP109) were HSP co-inducers comparable to the hydroxyamine arimoclomol in response to proteotoxic stress, but not thermal stress. Regardless, stress-induced Hsp70 expression could be enhanced by combining an HDAC inhibitor with either arimoclomol or with an HSP90 inhibitor that constitutively induced HSPs. HDAC inhibition failed to induce Hsp70 in motor neurons expressing ALS-linked mutant FUS, in which the heat shock response was suppressed; yet SAHA, RGFP109, and arimoclomol did reduce loss of nuclear FUS, a disease hallmark, and HDAC inhibition rescued the DNA repair response in iPSC-derived motor neurons carrying the FUSP525Lmutation, pointing to multiple mechanisms of neuroprotection by both HDAC inhibiting drugs and arimoclomol. Electronic supplementary material The online version of this article (10.1007/s12192-019-01064-1) contains supplementary material, which is available to authorized users.

Neurons are extremely polarised cells in which the cytoskeleton, composed of microtubules, microfilaments and neurofilaments, plays a crucial role in maintaining structure and function. Neurofilaments, the 10-nm intermediate filaments of neurons, provide structure and mechanoresistance but also provide a scaffolding for the organization of the nucleus and organelles such as mitochondria and ER. Disruption of neurofilament organization and expression or metabolism of neurofilament proteins is characteristic of certain neurological syndromes including Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth sensorimotor neuropathies and Giant Axonal Neuropathy. Microfluorometric live imaging techniques have been instrumental in revealing the dynamics of neurofilament assembly and transport and their functions in organizing intracellular organelle networks. The insolubility of neurofilament proteins has limited identifying interactors by conventional biochemical techniques but yeast two-hybrid experiments have revealed new roles for oligomeric, nonfilamentous structures including vesicular trafficking. Although having long half-lives, new evidence points to degradation of subunits by the ubiquitin-proteasome system as a mechanism of normal turnover. Although certain E3-ligases ubiquitinating neurofilament proteins have been identified, the overall process of neurofilament degradation is not well understood. We review these mechanisms of neurofilament homeostasis and abnormalities in motor neuron and peripheral nerve disorders. Much remains to discover about the disruption of processes that leads to their pathological aggregation and accumulation and the relevance to pathogenesis. Understanding these mechanisms is crucial for identifying novel therapeutic strategies.

International audience; Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with paralysis resulting from dysfunction and loss of motor neurons. A common neuropathological finding is attrition of motor neuron dendrites, which make central connections vital to motor control. The chromatin remodelling complex, neuronal Brahma-related gene 1 (Brg1)-associated factor complex (nBAF), is critical for neuronal differentiation, dendritic extension and synaptic function. We have identified loss of the crucial nBAF subunits Brg1, Brg1-associated factor 53b and calcium responsive transactivator in cultured motor neurons expressing FUS or TAR-DNA Binding Protein 43 (TDP-43) mutants linked to familial ALS. When plasmids encoding wild-type or mutant human FUS or TDP-43 were expressed in motor neurons of dissociated spinal cord cultures prepared from E13 mice, mutant proteins in particular accumulated in the cytoplasm. Immunolabelling of nBAF subunits was reduced in proportion to loss of nuclear FUS or TDP-43 and depletion of Brg1 was associated with nuclear retention of Brg1 mRNA. Dendritic attrition (loss of intermediate and terminal dendritic branches) occurred in motor neurons expressing mutant, but not wild-type, FUS or TDP-43. This attrition was delayed by ectopic over-expression of Brg1 and was reproduced by inhibiting Brg1 activity either through genetic manipulation or treatment with the chemical inhibitor, (E)-1-(2-Hydroxyphenyl)-3-((1R, 4R)-5-(pyridin-2-yl)-2, 5-diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one, demonstrating the importance of Brg1 to maintenance of dendritic architecture. Loss of nBAF subunits was also documented in spinal motor neurons in autopsy tissue from familial amyotrophic sclerosis (chromosome 9 open reading frame 72 with G4C2 nucleotide expansion) and from sporadic cases with no identified mutation, pointing to dysfunction of nBAF chromatin remodelling in multiple forms of ALS.

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin’s physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin–proteasome system was not impaired. There was evidence for alterations in the autophagy–lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity.

Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUS(R495X) transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 170 SACS mutations have been reported worldwide and are thought to cause loss of function of sacsin, a poorly characterized and massive 520 kDa protein. To establish an animal model and to examine the pathophysiological basis of ARSACS, we generated Sacs knockout (Sacs(-/-)) mice. Null animals displayed an abnormal gait with progressive motor, cerebellar and peripheral nerve dysfunctions highly reminiscent of ARSACS. These clinical features were accompanied by an early onset, progressive loss of cerebellar Purkinje cells followed by spinal motor neuron loss and peripheral neuropathy. Importantly, loss of sacsin function resulted in abnormal accumulation of non-phosphorylated neurofilament (NF) bundles in the somatodendritic regions of vulnerable neuronal populations, a phenotype also observed in an ARSACS brain. Moreover, motor neurons cultured from Sacs(-/-) embryos exhibited a similar NF rearrangement with significant reduction in mitochondrial motility and elongated mitochondria. The data points to alterations in the NF cytoskeleton and defects in mitochondrial dynamics as the underlying pathophysiological basis of ARSACS.

Intermediate filaments serve important structural roles, but other cellular functions are increasingly recognized. This study demonstrated normal function of the low-molecular-weight neurofilament protein (NFL) in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease (CMT) due to mutations in the Nefl gene. In motor neurons of spinal cord cultured from Nefl-knockout mice, mitochondrial length and the rate of fusion were decreased concomitant with increased motility. These parameters were normalized after expression of NFL(wt) on the Nefl(-/-) background, but not by overexpression of the profusion protein, mitofusin 2 (MFN2). The effects of CMT-causing NFL mutants bore similarities to and differences from Nefl knockout. In the early phase of toxicity before disruption of the neurofilament network, NFL(Q333P) and NFL(P8R) integrated into neurofilaments and had effects on mitochondria similar to those with Nefl knockout. The reduction of fusion rate by NFL(Q333P) was partly due to interference with the function of the profusion protein MFN2, which is mutated in CMT2A, functionally linking these forms of CMT. In the later phase of toxicity, mitochondria essentially stopped moving in neurons expressing NFL mutants, probably a consequence of cytoskeletal disruption. Overall, the data point to important functions of neurofilaments in mitochondrial dynamics as well as primary involvement in CMT2E/1F.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, rapidly progressing, fatal disease occurring in both familial and sporadic forms. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) cause ALS through a gain of toxic function. Calpain activity is increased in mutant SOD1 (SOD1(G93A)) transgenic mice and in models of ischemia because of increased cytosolic calcium, which has been documented in motor neurons in rodent models of familial ALS and in sporadic ALS patients. We report that inhibition of calpain activity using calpastatin prevented the toxicity of SOD1(G93A) in motor neurons of dissociated spinal cord cultures, prolonging viability of and reducing the proportion containing SOD1(G93A) inclusions. The data support the central role of calcium dysregulation in ALS and identify a potential therapeutic pathway.

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.

In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death.

One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1). This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp-32 was identified as a normal modification being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes. Mutating Trp-32 to a residue with a slower rate of oxidative modification, phenylalanine, decreased both the cytotoxicity of mutant SOD-1 and its propensity to form cytoplasmic inclusions in motor neurons of dissociated mouse spinal cord cultures.

Mutations in Cu/Zn-superoxide dismutase 1 (SOD1) are responsible for a familial form of amyotrophic lateral sclerosis (FALS). It has been proposed that oxidative stress and abnormal metal homeostasis contribute to death of motor neurons in this disease. Also, inability of motor neurons to upregulate protective proteins under stress may contribute to their preferential vulnerability to toxicity. Metallothioneins (MT) are low molecular weight, metal-binding proteins with established antioxidant capabilities. This study investigated the ability of motor neurons to upregulate MT isoforms in response to expression of mutant SOD1G93A or exposure to other neurotoxicants, and the ability of MT-I gene transfer to protect motor neurons from these stresses. MT isoform-I and -II were expressed constitutively in astrocytes and other non-neuronal cells of dissociated spinal cord cultures, but not in motor neurons. MT-I/II was upregulated in astrocytes, but not motor neurons, following treatment with ZnCl2 or excitotoxic concentrations of glutamate. MT-III expression was restricted to neurons and was unaffected by treatment with ZnCl2, paraquat, or glutamate. Overexpression of MT-I in motor neurons by gene transfer reduced the toxicity of ZnCl2 and paraquat, but failed to protect them against glutamate or SOD1G93A. These data are evidence against metal-catalyzed, oxidative stress being the primary mechanisms of toxicity conferred by disease-causing mutations in SOD1.

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.

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.

BACKGROUND : An increase in reactive oxygen species (ROS) burden and subsequent oxidative damage to nucleic acids, proteins, and lipids, occurs during the normal aging process of cells, including neurons. OBJECTIVE : With this in mind we hypothesize that an oxidative stress which leads to an increased state of oxidation, but is not itself lethal, nonetheless renders the motor neuron more vulnerable to other sublethal stressors. METHOD AND RESULTS : We have combined paraquat-induced oxidative stress with sublethal increased exogenous glutamate, or thermal stress, or a combination of both, and demonstrate a synergistic effect of low-level stressors on the viability of motor neurons. CONCLUSION : This model of low-level oxidative stress reveals the subsequent vulnerability of motor neurons to genetic or environmental stressors and may in part explain the mid-life/late-life symptom onset in both familial and sporadic ALS patients.

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.

Enhanced production of nitrotyrosine and subsequent protein nitration has been proposed as the mechanism by which mutant SODI causes death of motor neurons in a familial form of amyotrophic lateral sclerosis (FALS-I). We have tested this hypothesis in a primary culture model in which mutant human SOD I was expressed in motor neurons of dissociated spinal cord cultures. Preventing formation of nitrotyrosine by inhibiting nitric oxide synthase rescued cultured motor neurons from excitotoxic death induced by adding glutamate to the culture medium, but failed to significantly delay death of motor neurons expressing the G93A mutant SOD I. The results do not support generation of nitrotyrosine being the predominant lethal gain of function conferred by mutations in SOD I.

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene underlie some familial cases of amyotrophic lateral sclerosis, a neurodegenerative disorder characterized by loss of cortical, brainstem, and spinal motor neurons. 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 (NIH 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 SOD-1 and prolonged their survival. We propose that insufficiency of molecular chaperones may be directly involved in loss of motor neurons in this disease.

Monoamine oxidase (MAO; EC 1.4.3.4) is known to have an important role in the regulation of biogenic amines in the brain and peripheral tissues. It is also known that circulating platelets represent an excellent model for an easy assessment of the effect of MAO-B inhibitors in extracerebral tissue. The present study was carried out to determine the effects of methylmercury (MeHg) on the activity of MAO in synaptosomes of different brain regions of male Sprague-Dawley rats as well as in rat blood platelets both in vitro and in vivo. MeHg pretreatment inhibited the activity of MAO in the synaptosomes of the cortex, hypothalamus, hippocampus, striatum, cerebellum, and brain stem in a concentration-dependent (0-10 microM) manner. The threshold concentration of MeHg for such inhibition in different brain synaptosomes was found to be the same (i.e., 1 microM) except for in the rat striatum it was 2.5 microM, and the IC50 value for MeHg was found to be around 2.1 microM. Significant inhibition of the MAO activity was also observed in synaptosomes of the cortex, cerebellum, hypothalamus, and hippocampus as well as in platelets of rats 24 h after treatment by gavage with a total cumulative dose of 35 mg/kg (5 mg/kg/day for 7 days). The decrease of such activity was found to be at maximum in different brain synaptosomes and platelets 24 h following treatment with a cumulative total dose of 75 mg/kg (7.5 mg/kg/day for 10 days); the treated animals showed signs of ataxia under these conditions. The data have further shown that methylmercury is capable of inhibiting the MAO activity in different brain synaptosomes to different degrees but without showing any specificity towards any specific brain region. The present in vivo results suggest that the platelet MAO activity may be used as a potential biomarker of early neurotoxicity due to repeated exposure to MeHg in rats.

Excitotoxicity and disruption of Ca(2+) homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca(2+) entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L- and T-type voltage-gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca(2+) and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca(2+) overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA-binding proteins transactive response DNA-binding protein 43 (TDP-43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP-43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1G93A similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca2+ accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1G93A transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo. Electronic supplementary material The online version of this article (doi:10.1007/s12192-013-0467-2) contains supplementary material, which is available to authorized users.

To promote clinical and patient oriented research, as part of the Second International ALS Conference in Tarrytown, NY, USA, seven pairs of clinicians and scientists were asked to lead discussions with meeting attendees on six major topics (one of which was discussed by two groups); each one the focus of a 90-min Breakout Session. Approximately 25 meeting attendees participated in each session. The Breakout Sessions considered six major themes: 1) Approaches to encourage clinicians to engage in more clinical research to discover the pathogenesis and cause of ALS; 2) Exploring avenues to build more effective partnerships between basic scientists and ALS physicians; 3) Increasing patient interest and commitment to participating in non-trial clinical research; 4) Brainstorming about factors that are most critical to the discovery of the pathogenesis and cause of ALS; 5) Finding ways to incorporate clinical research projects into clinical trials; and 6) Developing state-of-the-art epidemiological studies to solve the mystery of ALS. In this paper, we present the reports from each of the Breakout Sessions; and we provide a wrap-up of the entire conference.

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1(G93A) transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1(G93A) by gene transfer or in presymptomatic SOD1(G93A) transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules. In vivo, in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers [or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100-1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.

The neurotoxicity of styrene and its major metabolites, styrene oxide and styrene glycol, was investigated in dissociated primary cultures of murine spinal cord-dorsal root ganglia (DRG)-skeletal muscle using morphological and electrophysiological endpoints. Styrene and styrene oxide (but not styrene glycol) were acutely cytotoxic to both neuronal and non-neuronal cells in the cultures; concentrations in excess of 2 and 0.2 mM, respectively, induced blebbing, vacuolation, detachment from the substratum and cell death in neuronal and non-neuronal cells within 4 days. No effects on neuronal morphology were observed in cultures treated with sublethal concentrations of styrene or styrene oxide for up to 3 weeks. The results suggest that oxidation of multiple cellular macromolecules that underlies the toxicity of styrene in other organ systems may also be responsible for damage to cells in the nervous system. No changes in action potential production indicative of a 'solvent effect' on membrane electrical properties was apparent in cultures treated with up to 8 mM styrene or 10 mM styrene glycol.

The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS.

We have developed a series of mouse-mouse neural hybrid cell lines by fusing the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells. Of 30 neuroblastoma-spinal cord (NSC) hybrids displaying a multipolar neuron-like phenotype, 10 express choline acetyltransferase, and 4 induce twitching in cocultured mouse myotubules. NSC-19, NSC-34, and their subclones express additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. In addition, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin-neurofilament switch with maturation in culture, similar to that occurring in neuronal development. NSC cell lines appear to model selected aspects of motor neuron development in an immortalized clonal system.

Background Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U) associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival. Results In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months). This is mediated at least in part through an anti-apoptotic mechanism. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. Knockdown of progranulin expression using shRNA technology further reduced cell survival. Conclusion Neurons are among the most long-lived cells in the body and are subject to low levels of toxic challenges throughout life. We have demonstrated that progranulin is abundantly expressed in motor neurons and is cytoprotective over prolonged periods when over-expressed in a neuronal cell line. This work highlights the importance of progranulin as neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including motor neuron disease.

The higher threshold for stress-induced upregulation of heat shock genes in certain populations of neurons, including motor neurons, has implications for their preferential vulnerability to disease and poses challenges for therapeutic intervention. One approach for identifying compounds that are effective in motor neurons involves understanding the mechanisms of heat shock gene regulation and hypothesis-driven therapeutic design. Central to stress-induced activation of heat shock genes is the transcription factor Hsf1, which must be released from Hsp90 complexes, translocate to the nucleus, bind to heat shock elements, and become activated. Most known inducers and co-inducers of the heat shock response promote one or more of these steps, but not all compounds are effective in motor neurons. However, other elements in the promoters of heat shock genes and heat shock transcription factors also contribute to constitutive and stress-induced regulation of heat shock genes and are potential therapeutic targets. Another approach is to screen chemical libraries using a test system that expresses motor neuronal properties, with positive hits being validated in vivo. The most effective therapies will be those that upregulate multiple chaperones and co-chaperones that enable refolding and degradation in addition to sequestering misfolded proteins

With conventional immunocytochemical techniques on fixed tissue, antibodies which recognize highly phosphorylated neurofilament proteins strongly label axons, but often react poorly with perikaryal neurofilaments. The reactivity of one such antibody, SMI31, with neurofilaments in vivo has been investigated by microinjecting purified SMI31 into large neurons in living cultures of embryonic mouse spinal cord. Microinjected SMI31 (SMI31I) labeled perikarya and dendrites in a fibrillar pattern indistinguishable from that of microinjected SMI32 (SMI32I), which labels hypophosphorylated neurofilaments of perikarya and dendrites in fixed tissue. SMI31 also labeled perikarya and dendrites when applied to whole unfixed cultures after extraction with 1% Triton X-100 or to cultures fixed in acetone after Triton-extraction, but prior to exposure to primary antibody. SMI31 labeled mainly axons when applied after fixation with acetone without Triton-extraction. Positive labeling of neurofilaments and various inclusions in neuronal somata with antibodies against highly phosphorylated neurofilaments has been described in a number of neurotoxic and neurodegenerative diseases and after neuronal injury. The results of this study indicate that explanations other than alterations in phosphorylation could account for these observations.

Neuron-glial interactions are important in development of the nervous system and pathogenesis of disease. Primary cell cultures prepared from nervous tissue are often used to study the properties of individual cell types and how they interact with each other. Isolation of pure populations of cells and their culture is challenging, particularly from murine spinal cord. The purpose of this study was to optimize various protocols to achieve efficient, parallel isolation and purification of primary motor neurons, microglia and astrocytes from the same mouse embryonic spinal cord sample. Following dissociation of E12 embryonic spinal cords, motor neurons were isolated at 97% purity by a single step centrifugation of the cell suspension through multiple discontinuous density gradients of NycoPrep. The residual mixed cell pellet was resuspended and cultured for 2 weeks. Mixed cultures were then shaken to release microglia, which were then harvested from the medium and subjected to another round of differential adhesion to achieve 99% purity. The astrocytes remaining in the mixed cultures were culled to 98% purity by treatment with leucine methyl ester and a subsequent vigorous shaking step to remove any remaining microglia and neurons. Furthermore, no cross contamination was observed in the glial cultures. This technique provides a simple, convenient, and reliable method of obtaining highly purified preparations of motor neurons, microglia and astrocytes from embryonic spinal cord for the study of spinal cord cell biology and motor neuron diseases.

Oxidative stress, which results from a complex interplay of pro- and anti-oxidant forces, is generally considered to be the major effector of accumulation of oxidatively modified protein accumulation in cells, although reduced degradation due to impairment of proteolytic activity could also contribute. The discovery that a familial lateral sclerosis (ALS) results from mutations in the gene encoding Cu/Zn superoxide dismutase a anti-oxidant enzyme, stimulated considerable evaluation of reactive oxygen species (ROS) generation and oxidative protein damage in both familial and sporadic forms of the disease. Mutations in SOD1 do not cause disease by compromising dismutating activity, but through some toxic gain of function. Although exacerbation of other copper-catalyzed enzymatic activities has been demonstrated in vitro, there is little evidence substantiating that this property is responsible for toxicity in vivo. Studies of ROS generation and oxidative damage in vivo have produced mixed results, but collectively are consistent with oxidative stress playing a secondary role in pathogenesis of the disease. Studies of post-mortem tissue from sporadic ALS patients has yielded more consistent evidence of accumulation of oxidative damage to proteins, lipids, and DNA, but the time course of accumulation cannot be determined and the initiating causes of the disease have not been identified. The interplay between motor neurons and glial cells is important in the clinical progression of both familial and sporadic motor neuron diseases and release of reactive oxygen and nitrogen species or cytokines from microglia could contribute to the demise of motor neurons. This review describes the general mechanisms of radical-mediated cellular damage followed by the evidence for and against the contribution of oxidative injury to the pathogenesis of motor neuron diseases.

Publisher Summary This chapter discusses the physiological profile of motor neurons that result in the reduced safety factor to survive disease-related stresses. Motor neurons are under a great deal of stress because of their size, high level of glutamatergic input, calcium permeable receptor and ion channels, an extensive and aggregation-prone neurofilament (NF) network, and axons that extend outside the protection of the blood–brain barrier. Despite this stressful existence, motor neurons susceptible in amyotrophic lateral sclerosis (ALS) are deficient in several protective mechanisms, including cytosolic calcium-binding proteins and glutathione, and have a high threshold for inducing expression of protective molecules, such as stress proteins and metallothioneins, when under stress. How a cell responds to the disease-causing stresses may be as important in determining outcome as the identity of initiating factors, and it depends on the protective mechanisms at its disposal given the complement of physiological stresses with which it has to cope.

PC12 cells are well characterized for their ability to differentiate into neuronal-like cells when challenged with nerve growth factor. It has been reported that the calpain and proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (CI) is also able to induce neurite outgrowth in PC12 cells. In this study, we report that the inhibitor of proteasomal chymotrypsin-like activity, carbobenzoxy-Ile-Glu-(O-tert-butyl)-Ala-Leu-aldehyde (PSI), can also induce differentiation of PC12 cells. Induction of neurite outgrowth with PSI, CI, or its close analogue, carbobenzoxy-Leu-Leu-leucinal (MG132), was associated with stress-activated protein kinase (SAPK) activation. Neurite formation induced by protease inhibition was independent of mitogen-activated protein kinase/extracellular signal-regulated kinase, p38/reactivating kinase, or phosphatidylinositol 3-kinase activities. The exact mechanism by which protease inhibition activates SAPKs remains to be elucidated ; however, our results suggest that the SAPK signal transduction cascade may be an alternative and/or parallel pathway in the regulation of neuronal differentiation.

First generation, replication-defective adenoviral vectors are highly effective for gene transfer into the central nervous system, but the host's immune response limits the utility of this vector for possible therapy of neurological disease or long-term gene transfer studies in experimental animals. We have demonstrated the effectiveness of FK506 (tacrolimus), a powerful immunosuppressant that readily crosses the blood-brain barrier, in maintaining adenovirus-mediated reporter gene transfer following stereotaxic injection of the recombinant (AdCMVlacZ) into mouse striatum. After 28 days, beta-galactosidase expression was reduced by 75% relative to day 10 in immunocompetent animals, accompanied by an inflammatory reaction in the region of transduced cells; however, in mice receiving daily s.c. injections of FK506, beta-galactosidase activity was maintained at the 10 days post-injection level.

Research pertaining to gene transfer into cells of the nervous system is one of the fastest growing fields in neuroscience. An important application of gene transfer is gene therapy, which is based on introducing therapeutic genes into cells of the nervous system by ex vivo or in vivo techniques. With the eventual development of efficient and safe vectors, therapeutic genes, under the control of a suitable promoter, can be targeted to the appropriate neurons or glial cells. Gene therapy is not only applicable to the treatment of genetic diseases of the nervous system and the control of malignant neoplasia, but it also has therapeutic potential for acquired degenerative encephalopathies (Alzheimer's disease, Parkinson's disease), as well as for promoting neuronal survival and regeneration in various pathological states.

Characteristic responses of motor neurons to injury include an apparent increase in the phosphorylation of C-terminal domains of neurofilament proteins in the perikaryal and dendritic compartments. This change was induced in dissociated cultures of embryonic spinal cord by activation of protein kinase C (PKC). PKC was activated by : (a) exposure of cultures to 10 nM 12-o-tetradecanoyl phorbol 13-acetate (TPA) ; (b) microinjection of I mM dioctanoylglycerol (diC 8 ) directly into perikarya of motor neurons ; (c) addition of 10 μM diC 8 to the culture medium. Activation of PKC led to different immediate and long term effects on neurofilaments of motor neurons. After 30 minutes (min), fragmentation of the neurofilament network was observed by labeling with antibodies to low and high molecular weight neurofilament proteins ; glial filaments were disassembled after 10 min and reassembled by 1 hour (h). From 4 to 24 h, motor neurons were observed with extensions of perikaryal cytoplasm or massive enlargements of proximal dendritic processes, both containing intact neurofilament networks. Over 1 to 12 days, there was a gradual increase in the number of motor neuronal perikarya immunoreactive with antibodies to neurofilament proteins phosphorylated at KSP sites on the C-terminal domains (SMI31, SMI34). It is proposed that activation of PKC secondary to other injurious events may contribute to the changes in neurofilaments observed in motor neuron diseases.

SUMMARY 1. Clonal cell lines, primary cultured neurones and transgenic animals expressing mutant genes linked to familial forms of neurodegenerative diseases provide models in which to examine the interaction between expression of a predisposing gene and exposure to neurotoxic chemicals. Methods of establishing these models are reviewed. 2. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD-1) have been identified in cases of familial amyotrophic lateral sclerosis linked to chromosome 21. We report that in clonal lines of PC12 cells, the cytotoxicity of a glutathione-depleting epoxide, styrene oxide, varied with SOD activity in a manner similar to that previously demonstrated for redox cycling chemicals. These preliminary data suggest that either low or high SOD-1 activities may be associated with greater toxicity of a variety of neurotoxic chemicals and their metabolites.

It was previously reported that a monoclonal antibody to beta-amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament-associated protein (beta APP-IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60-67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord-dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. beta APP-IFAP was expressed in all non-neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), beta APP-IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that beta APP-IFAP may be the product of a member of the beta APP multigene family expressed developmentally in non-neuronal cells.

The carboxyl-terminal region of both the medium and high molecular weight neurofilament proteins contains repeated sequences that are sites for phosphorylation. The monoclonal antibody SMI31 specifically recognizes the conformation of these multiphosphorylated domains in an intermediate state of phosphorylation. Microinjection of SMI31 into living spinal motor neurons in culture resulted in a gradual collapse of the arrays of neurofilaments in perikarya and dendrites. In some cells, antibody-decorated filaments penetrated the axon and accumulated in proximal axonal segments causing their swelling. In dorsal root ganglion neurons, microinjected SMI31 bound to neurofilaments but did not induce collapse of the network or proximal axonal swelling. This study supports a role for phosphorylation of neurofilament sidearms in control of neurofilament transport and illustrates that interference with these sites has different consequences on neurofilament organization and morphology in different cell types.

Recent findings have established that cutaneous nerves modulate both skin homeostasis and various skin diseases, by influencing cell growth and differentiation, inflammation and wound healing. In order to study the influence of epidermis, hair follicles and capillaries on sensory neurons, and vice-versa, we developed a tissue-engineered model of innervated endothelialized reconstructed skin (MIERS). Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human fibroblasts and human endothelial cells. Keratinocytes or mice newborn immature hair follicle buds were then seeded on the opposite side of the MIERS to study their influence on sensory nerves growth, and vice versa. A vigorous neurite elongation was detected inside the reconstructed dermis after 14 and 31 days of neurons culture. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. We have successfully developed the MIERS that allowed us to study the effects of epidermis and capillaries on nerve growth. This model will be a useful tool to study the modulation of sensory nerves on wound healing, angiogenesis, hair growth and neurogenic inflammation in the skin.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to amplify the heat shock response in cell lines by increasing the binding of heat shock transcription factor–1 to heat shock elements within heat shock gene promoters. Because overexpression of the inducible heat shock protein 70 (Hsp70) was neuroprotective in a culture model of motor neuron disease, this study investigated whether NSAIDs induce Hsp70 and confer cytoprotection in motor neurons of dissociated spinal cord cultures exposed to various stresses. Two NSAIDs, sodium salicylate and niflumic acid, lowered the temperature threshold for induction of Hsp70 in glia but failed to do so in motor neurons. At concentrations that increased Hsp70 in heat shocked glial cells, sodium salicylate failed to delay death of motor neurons exposed to hyperthermia, paraquat-mediated oxidative stress, and glutamate excitotoxicity. Neither sodium salicylate nor the cyclooxygenase-2 inhibitor, niflumic acid, protected motor neurons from the toxicity of mutated Cu/Zn-superoxide dismutase (SOD-1) linked to a familial form of the motor neuron disease, amyotrophic lateral sclerosis. Thus, treatment with 2 types of NSAIDs failed to overcome the high threshold for the activation of heat shock response in motor neurons.

The temporal evolution of changes in the distribution of neurofilaments, vimentin-intermediate filaments (IF) and glial fibrillary acidic protein (GFAP)-IF induced by 2,5-hexanedione (2,5HD) has been investigated in dissociated cultures of fetal mouse spinal cord and dorsal root ganglia (DRG). Aggregates of GFAP-IF, labelled by antibodies against GFAP, appeared in astrocytes approximately one week after axonal swellings filled with neurofilaments were detected in neurons by labelling with antibodies to 68 kD or 200 kD neurofilament proteins. The threshold concentration required for aggregation of GFAP-IF was 1.4 to 2 times that required for neurofilament accumulations. In contrast to findings in cultured human skin fibroblasts, aggregates of vimentin-IF were not found in 2,5HD-treated cultures from mouse tissue (non-neuronal cells of spinal cord-DRG or fibroblasts from skin or muscle). The IF of 3T3 cells, a mouse fibroblast cell line, formed diffuse juxtanuclear aggregates only after high levels of exposure to 2,5HD. The sensitivity of vimentin-IF to aggregation by 2,5HD was proportional to the IF content of the cell type. These differences in the sensitivity of IF to aggregation by 2,5HD among cell types and species explain why IF-aggregates have not been observed previously in non-neuronal cells in human patients with the neuropathy associated with 2,5HD, or in experimental studies with rodents.

Axonal swellings associated with large aggregates of neurofilaments are characteristic of neuropathies caused by chemical neurotoxins (n-hexane, methyl n-butyl ketone, and acrylamide) or giant axonal neuropathy (GAN--an autosomal recessive genetic disease). In GAN, filamentous aggregates have been shown also to occur in other cell types including cultured skin fibroblasts. Therefore, we studied the effects of 2,5-hexanedione (the neurotoxic metabolite of n-hexane and methyl n-butyl ketone) and acrylamide on normal human skin fibroblasts in tissue culture. We show that both neurotoxins induce aggregation of intermediate filaments of the vimentin type in the cultured fibroblasts without disrupting microtubules.

Using a variety of cell types in culture, we have investigated the relevance of intermolecular crosslinking involving intermediate filament proteins (IF-proteins) to the changes in distribution of intermediate filaments induced by the neurotoxicant, 2,5-hexanedione (2,5HD). Aggregation of vimentin-filaments (vimentin-IF), glial fibrillary acidic protein (GFAP)-IF and neurofilaments was preceded by appearance on immunoblots of a high molecular weight complex (IF-complex) labeled by antibodies against the IF-protein of the cell type: pV-170 from human skin fibroblasts and pV-130 from 3T3 mouse fibroblasts, which were labeled by anti-vimentin and, from cultures of dissociated mouse spinal cord and dorsal root ganglia, pNFH-300 labeled by antibody against the 200 kDa neurofilament protein (NF-200 or NF-H) and a doublet labeled by antiserum to GFAP (pGFAP-145).pV-170 was detected in human skin fibroblasts within one hour of exposure to 2,5HD and the amount of both pV-170 and pNFH-300 was related to the concentration of 2,5HD and the duration of exposure. Intermediate filament-complexes were not detected in PtK1 epithelial cells by labeling of transblots with anti-cytokeratin, nor are keratin-IF aggregated by 2,5HD. Intermediate filament-complexes were not detected in fibroblasts with IF-aggregates secondary to disruption of microtubules by colchicine or in fibroblasts from patients with giant axonal neuropathy.