Depending on the stress, histone deacetylase inhibitors act as heat shock protein co-inducers in motor neurons and potentiate arimoclomol, exerting neuroprotection through multiple mechanisms in ALS models.

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.

Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis.

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.

Neurofilament dynamics and involvement in neurological disorders.

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.

Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1.

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.

Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations.

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.