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Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS) is caused by mutation in the SACS gene resulting in loss of function of the protein sacsin. A key feature is the formation of abnormal bundles of neurofilaments (NF) in neurons and vimentin intermediate filaments (IF) in cultured fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs−/− neurons. Having studied the mechanism using NF assembled in vitro from purified NF proteins, we report that the SacsJ domain interacts with NF proteins to disassemble NFL filaments, and to inhibit their initial assembly. A cell-penetrating peptide derived from this domain, SacsJ-myc-TAT was efficient in disassembling NF bundles in cultured Sacs−/− motor neurons, restoring the NF network; however, there was some loss of vimentin IF and NF in cultured Sacs+/+ fibroblasts and motor neurons, respectively. These results suggest that sacsin through its SacsJ domain is a key regulator of NF and vimentin IF networks in cells.

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS), is caused by loss of function mutations in the SACS gene, which encodes sacsin, a giant protein of 520 kDa. A key feature of the absence of sacsin in cells is the formation of abnormal bundles of intermediate filaments (IF) including neurofilaments (NF) in neurons and vimentin IF in fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons, however, its mechanism is still unclear. Here, we focused on the role of SacsJ in NF assembly. We report that the SacsJ domain directly interacts with NF proteins in vitro to disassemble NFL filaments, and to inhibit their initial assembly, in the absence of Hsp70. We generated a cell-penetrating peptide derived from this domain, SacsJ-myc-TAT, which was efficient in disassembling both vimentin IF and NF in cultured fibroblasts and Sacs+/+ motor neurons as well as NF bundles in cultured Sacs-/- motor neurons. Whereas a normal NF network was restored in Sacs-/- neurons treated with the SacsJ peptide, there was some loss of IF networks in Sacs+/+ fibroblasts or neurons. These results suggest that SacsJ is a key regulator of NF and IF networks in cells, with implications for its therapeutic use.

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.

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.

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.

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.

The combination of Ca(2+) influx during neurotransmission and low cytosolic Ca(2+) buffering contributes to the preferential vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS). This study investigated the relationship among Ca(2+) accumulation in intracellular compartments, mitochondrial abnormalities, and protein aggregation in a model of familial ALS (fALS1). Human SOD1, wild type (SOD1(WT)) or with the ALS-causing mutation G93A (SOD1(G93A)), was expressed in motor neurons of dissociated murine spinal cord-dorsal root ganglia (DRG) cultures. Elevation of mitochondrial Ca(2+) ([Ca(2+)](m)), decreased mitochondrial membrane potential (Δψ) and rounding of mitochondria occurred early, followed by increased endoplasmic reticular Ca(2+) ([Ca(2+)](ER)), elevated cytosolic Ca(2+) ([Ca(2+)](c)), and subsequent appearance of SOD1(G93A) inclusions (a consequence of protein aggregation). [Ca(2+)](c) was elevated to a greater extent in neurons with inclusions than in those with diffusely distributed SOD1(G93A) and promoted aggregation of mutant protein, not vice versa: both [Ca(2+)](c) and the percentage of neurons with SOD1(G93A) inclusions were reduced by co-expressing the cytosolic Ca(2+)-buffering protein, calbindin D-28K; treatment with the heat shock protein inducer, geldanamycin, prevented inclusions but not the increase in [Ca(2+)](c), [Ca(2+)](m) or loss of Δψ, and inhibiting proteasome activity with epoxomicin, known to promote aggregation of disease-causing mutant proteins including SOD1(G93A), had no effect on Ca(2+) levels. Both expression of SOD1(G93A) and epoxomicin-induced inhibition of proteasome activity caused mitochondrial rounding, independent of Ca(2+) dysregulation and reduced Δψ. That geldanamycin prevented inclusions and mitochondrial rounding, but not Ca(2+) dysregulation or loss of Δψ indicates that chaperone-based therapies to prevent protein aggregation may require co-therapy to address these other underlying mechanisms of toxicity.

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.

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 are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and alphaB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1.HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins.

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent,N-tert-butyl-α-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.

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.

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.

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.

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.

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.

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.

Motor neurons have a high threshold for induction of heat shock proteins (Hsps) in response to stress, a property associated with impaired ability to activate heat shock transcription factor 1 (Hsf1). Hyperphosphorylation of Hsf1 has been established as a requirement for transactivation of heat shock genes. This study demonstrated that the impaired heat shock response in motor neurons is not due to altered phosphorylation of Hsf1 by kinases previously shown to affect activation of Hsf1 in other cells (PKC, GSK3beta, ERK1, CaMKIIalpha). However, a constitutively active form of CaMKIV induced robust expression of Hsp70, as well as transcription of a GFP reporter gene driven by the human inducible Hsp70 promoter in unstressed motor neurons, but not in mouse embryonic fibroblasts. The results point to novel mechanisms of activation of heat shock genes in motor neurons that have relevance to exploitation of endogenous stress responses therapeutically.

High threshold for stress-induced activation of the heat shock transcription factor, Hsf1, may contribute to vulnerability of motor neurons to disease and limit efficacy of agents promoting expression of neuroprotective heat shock proteins (Hsps) through this transcription factor. Plasmid encoding a constitutively active form of Hsf1, Hsf1act, and chemicals shown to activate Hsf1 in other cells were investigated in a primary culture model of familial amyotrophic lateral sclerosis. Hsf1act and the Hsp90 inhibitor, geldanamycin, induced high expression of multiple Hsps in cultured motor neurons and conferred dramatic neuroprotection against SOD1G93A in comparison to Hsp70 or Hsp25 alone. Two other Hsp90 inhibitors, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and radicicol, and pyrrolidine dithiocarbamate induced robust expression of Hsp70 and Hsp40 in motor neurons, but at cytotoxic concentrations. 17-AAG, which penetrates the blood–brain barrier, has exhibited a higher therapeutic index than geldanamycin, but this may not be the case when activation of Hsf1 in neurons is targeted.

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.

Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.