Takashi Ayaki

Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells

Anacardic acid attenuates mutant TDP-43–associated abnormalities in motor neurons derived from ALS patient–specific induced pluripotent stem cells. A Stepping Stone to ALS Drug Screening Amyotrophic lateral sclerosis (ALS) is an untreatable disorder in which the motor neurons degenerate, resulting in paralysis and death. Induced pluripotent stem cell (iPSC) technology makes it possible to analyze motor neurons from patients with ALS and to use them for screening new candidate drugs. In new work, Egawa et al. obtained motor neurons by inducing differentiation of iPSC lines derived from several patients with familial ALS. These patients carried disease-causing mutations in the gene encoding Tar DNA binding protein-43 (TDP-43). The ALS motor neurons in culture recapitulated cellular and molecular abnormalities associated with ALS. For example, the authors found that mutant TDP-43 in the ALS motor neurons perturbed RNA metabolism and that the motor neurons were more vulnerable to cellular stressors such as arsenite. The researchers then used the ALS motor neurons in a drug screening assay and identified a compound called anacardic acid, a histone acetyltransferase inhibitor, that could reverse some of the ALS phenotypes observed in the motor neurons. The new work provides an encouraging step toward using motor neurons generated from iPSCs derived from ALS patients to learn more about what triggers the death of motor neurons in this disease and to identify new candidate drugs that may be able to slow or reverse the devastating loss of motor neurons. Amyotrophic lateral sclerosis (ALS) is a late-onset, fatal disorder in which the motor neurons degenerate. The discovery of new drugs for treating ALS has been hampered by a lack of access to motor neurons from ALS patients and appropriate disease models. We generate motor neurons from induced pluripotent stem cells (iPSCs) from familial ALS patients, who carry mutations in Tar DNA binding protein-43 (TDP-43). ALS patient–specific iPSC–derived motor neurons formed cytosolic aggregates similar to those seen in postmortem tissue from ALS patients and exhibited shorter neurites as seen in a zebrafish model of ALS. The ALS motor neurons were characterized by increased mutant TDP-43 protein in a detergent-insoluble form bound to a spliceosomal factor SNRPB2. Expression array analyses detected small increases in the expression of genes involved in RNA metabolism and decreases in the expression of genes encoding cytoskeletal proteins. We examined four chemical compounds and found that a histone acetyltransferase inhibitor called anacardic acid rescued the abnormal ALS motor neuron phenotype. These findings suggest that motor neurons generated from ALS patient–derived iPSCs may provide a useful tool for elucidating ALS disease pathogenesis and for screening drug candidates.

Clinicopathologic study on an ALS family with a heterozygous E478G optineurin mutation

We investigated a family manifesting amyotrophic lateral sclerosis (ALS) with a heterozygous E478G mutation in the optineurin (OPTN) gene. Clinically, slow deterioration of motor function, mood and personality changes, temporal lobe atrophy on neuroimaging, and bizarre finger deformity were noted. Neuropathologically, TAR DNA-binding protein 43 (TDP-43)-positive neuronal intracytoplasmic inclusions were observed in the spinal and medullary motor neurons. In these cells, the immunoreactivity of nuclear TDP-43 was reduced. Consecutive sections revealed that the inclusions were also reactive with anti-ubiquitin and anti-p62 antibodies, but noticeably negative for OPTN. In addition, TDP-43/p62-positive glial cytoplasmic inclusions (GCIs) were scattered throughout the spinal cord and the medullary motor nuclei. Furthermore, Golgi fragmentation was identified in 70% of the anterior horn cells (AHCs). The presence of AHCs with preserved nuclear TDP-43 and a fragmented Golgi apparatus, which are unrecognizable in sporadic ALS, indicates that patients with the E4787G OPTN mutation would manifest Golgi fragmentation before loss of nuclear TDP-43. In the neocortex, GCIs were sparsely scattered among the primary motor and temporal cortices, but no neuronal TDP-43-positive inclusions were detected. In the amygdala and the ambient gyrus, argyrophilic grains and ballooned neurons were seen. The thorough neuropathologic investigations performed in this work demonstrated that OPTN-positive inclusion bodies, if any, were not prominent. We postulate that optineurinopathy is closely linked with TDP-proteinopathy and speculate that this heterozygous E478G mutation would cause ALS by acting through a dominant-negative mechanism.

Clinicopathologic features of autosomal recessive amyotrophic lateral sclerosis associated with optineurin mutation.

We performed clinicopathological analyses of two amyotrophic lateral sclerosis (ALS) patients with homozygous Q398X optineurin (OPTN) mutation. Clinically, both patients presented signs of upper and lower motor neuron degeneration, but only Patient 1 showed gradual frontal dysfunction and extrapyramidal signs, and temporal lobe and motor cortex atrophy. Neuropathological examination of Patient 1 revealed extensive cortical and spinal motor neuron degeneration and widespread degeneration of the basal ganglia. Bilateral corticospinal tracts exhibited degeneration. Loss of spinal anterior horn cells (AHCs) and gliosis were observed, whereas posterior columns, Clarkes columns, intermediate lateral columns, and the Onufs nucleus were spared. In the brainstem, moderate neuronal loss and gliosis were noted in the hypoglossal and facial motor nuclei. No Bunina bodies were found in the surviving spinal and brainstem motor neurons. Transactivation response (TAR) DNA‐binding protein 43 (TDP‐43)‐positive neuronal and glial cytoplasmic inclusions were observed throughout the central nervous system. The Golgi apparatus in motor neurons of the brainstem and spinal cord was often fragmented. Immunoreactivity for OPTN was not observed in the brain and spinal cord, consistent with nonsense‐mediated mRNA decay of OPTN. The TDP‐43 pathology of Q398X was similar to that of an autosomal dominant E478G mutation. This result suggests that the loss‐of‐function, but not the proteinopathy itself, of OPTN results in TDP‐43 deposits in neuronal and glial cytoplasm and Golgi apparatus fragmentation, leading to multisystem neurodegeneration.

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Analysis of ALS patient iPSC-derived motor neurons indicates that Src/c-Abl inhibitors may have potential for treating ALS. A stepping stone to ALS drug discovery ALS is a heterogeneous motor neuron disease for which there is no treatment and for which a common therapeutic target has yet to be identified. In a new study, Imamura et al. developed a drug screen using motor neurons generated from ALS patient induced pluripotent stem cells (iPSCs). They screened existing drugs and showed that inhibitors of Src/c-Abl kinases promoted autophagy and rescued ALS motor neurons from degeneration. One of the drugs was effective for promoting survival of motor neurons derived from ALS patients with different genetic mutations. The Src/c-Abl pathway may be a potential therapeutic target for developing new drugs to treat ALS. Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis

Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-κB suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-κB suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-α-mediated NF-κB activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-α-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-κB are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-κB activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS.

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Background: The role of RRM1 in the pathogenesis of TDP-43 proteinopathy is unclear. Results: RRM1 was aggregate-prone, mediated by a self-assembly at newly identified amyloidogenic β-strands containing cysteines; cysteine substitution(s) replicated diverse cytopathologies of TDP-43 in ALS. Conclusion: RRM1 misfolding may underlie TDP-43 proteinopathy. Significance: This study proposes a novel mechanism and a new in vitro model for TDP-43 proteinopathy. 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 β-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.

Type of Gradient Recalled-Echo Sequence Results in Size and Number Change of Cerebral Microbleeds

Microbleeds (MBs) are thought to be clusters of hemosiderin-containing macrophages and are more commonly observed in hypertensive microangiopathy (lacunar infarct or intracerebral hemorrhage), cerebral amyloid angiopathy, or cerebral autosomal dominant arteriopathy with subcortical infarcts and

Gradual cerebral hypoperfusion in spontaneously hypertensive rats induces slowly evolving white matter abnormalities and impairs working memory

Rats subjected to bilateral common carotid arteries (CCAs) occlusion or 2-vessel occlusion (2VO) have been used as animal models of subcortical ischemic vascular dementia (SIVD). However, these models possess an inherent limitation in that cerebral blood flow (CBF) drops sharply and substantially after ligation of CCAs without vascular risk factors and causative small vessel changes. We previously reported a novel rat model of 2-vessel gradual occlusion (2VGO) in which ameroid constrictors (ACs) were placed bilaterally in the CCAs of Wistar-Kyoto rats. To simulate SIVD pathology more closely, we applied ACs in spontaneously hypertensive rats (SHRs), which naturally develop small vessel pathology, and compared their phenotypes with SHR-2VO and sham-operated rats. The mortality rate of the SHR-2VGO was 0% while that of the SHR-2VO was 56.5%. The CBF of the SHR-2VO dropped to 50% of the baseline level at 3 h, whereas the SHR-2VGO showed a gradual CBF reduction reaching only 68% of the baseline level at seven days. The SHR-2VGO showed slowly evolving white matter abnormalities and subsequent spatial working memory impairments of a similar magnitude to the remaining SHR-2VO at 28 days. We suggest the SHR-2VGO robustly replicates selective aspects of the pathophysiology of SIVD with low mortality rate.

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‐1β, 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.

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.