Transglutaminase 2 accelerates neuroinflammation in amyotrophic lateral sclerosis through interaction with misfolded superoxide dismutase 1.

Although the aberrant assembly of mutant superoxide dismutase 1 (mSOD1) is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), the molecular basis of superoxide dismutase 1 (SOD1) oligomerization remains undetermined. We investigated the roles of transglutaminase 2 (TG2), an endogenous cross-linker in mSOD1-linked ALS. TG2 interacted preferentially with mSOD1 and promoted its oligomerization in transfected cells. Purified TG2 directly oligomerized recombinant mutant SOD1 and the apo-form of the wild-type SOD1 proteins in a calcium-dependent manner, indicating that misfolded SOD1 is a substrate of TG2. Moreover, the non-cell-autonomous effect of extracellular TG2 on the neuroinflammation was suggested, since the TG2-mediated soluble SOD1 oligomers induced tumor necrosis factor-α, interleukin-1ß, and nitric oxide in microglial BV2 cells. TG2 was up-regulated in the spinal cord of pre-symptomatic G93A SOD1 transgenic mice and in the hypoglossal nuclei of mice suffering nerve ligation. Furthermore, inhibition of spinal TG2 by cystamine significantly delayed the progression and reduced SOD1 oligomers and microglial activation. These results indicate a novel role of TG2 in SOD1 oligomer-mediated neuroinflammation, as well as in the involvement in the intracellular aggregation of misfolded SOD1 in ALS.

Aberrant assembly of RNA recognition motif 1 links to pathogenic conversion of TAR DNA-binding protein of 43 kDa (TDP-43).

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant ß-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.

Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

Accumulating evidence suggests that pathogenic TAR DNA-binding protein (TDP)-43 fragments contain a partial RNA-recognition motif domain 2 (RRM2) in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration. However, the molecular basis for how this domain links to the conformation and function of TDP-43 is unclear. Previous crystal analyses have documented that the RRM2-DNA complex dimerizes under acidic and high salt conditions, mediated by the intermolecular hydrogen bonds of Glu246-Ile249 and Asp247-Asp247. The aims of this study were to investigate the roles of Glu246 and Asp247 in the molecular assembly of RRM2 under physiological conditions, and to evaluate their potential use as markers for TDP-43 misfolding due to the aberrantly exposed dimer interface. Unexpectedly, gel filtration analyses showed that, regardless of DNA interaction, the RRM2 domain remained as a stable monomer in phosphate-buffered saline. Studies using substitution mutants revealed that Glu246 and, especially, Asp247 played a crucial role in preserving the functional RRM2 monomers. Substitution to glycine at Glu246 or Asp247 induced the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA-binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. A novel monoclonal antibody against peptides containing Asp247 was found to react with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not with nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play pivotal roles in the proper conformation and function of TDP-43. In particular, Asp247 should be studied as a molecular target with an aberrant conformation related to TDP-43 proteinopathy.

Protein misdirection inside and outside motor neurons in Amyotrophic Lateral Sclerosis (ALS): a possible clue for therapeutic strategies.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive muscle wasting and weakness with no effective cure. Emerging evidence supports the notion that the abnormal conformations of ALS-linked proteins play a central role in triggering the motor neuron degeneration. In particular, mutant types of superoxide dismutase 1 (SOD1) and TAR DNA binding protein 43kDa (TDP-43) are key molecules involved in the pathogenesis of familial and sporadic ALS, respectively. The commonalities of the two proteins include a propensity to aggregate and acquire detrimental conformations through oligomerization, fragmentation, or post-translational modification that may drive abnormal subcellular localizations. Although SOD1 is a major cytosolic protein, mutated SOD1 has been localized to mitochondria, endoplasmic reticulum, and even the extracellular space. The nuclear exclusion of TDP-43 is a pathological hallmark for ALS, although the pathogenic priority remains elusive. Nevertheless, these abnormal behaviors based on the protein misfolding are believed to induce diverse intracellular and extracellular events that may be tightly linked to non-cell-autonomous motor neuron death. The generation of mutant- or misfolded protein-specific antibodies would help to uncover the distribution and propagation of the ALS-linked proteins, and to design a therapeutic strategy to clear such species. Herein we review the literature regarding the mislocalization of ALS-linked proteins, especially mutant SOD1 and TDP-43 species, and discuss the rationale of molecular targeting strategies including immunotherapy.

Induction of protective immunity by vaccination with wild-type apo superoxide dismutase 1 in mutant SOD1 transgenic mice.

Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated TH2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-γ (IFNγ) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFNγ, in the spinal cord implicating IFNγ in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFNγ or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the TH1/TH2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing TH1 immunity. Thus, because of its lower TH1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.