Targeting TEAD/YAP-transcription-dependent necrosis, TRIAD, ameliorates Huntington's disease pathology.

Neuronal cell death in neurodegenerative diseases is not fully understood. Here we report that mutant huntingtin (Htt), a causative gene product of Huntington's diseases (HD) selectively induces a new form of necrotic cell death, in which endoplasmic reticulum (ER) enlarges and cell body asymmetrically balloons and finally ruptures. Pharmacological and genetic analyses revealed that the necrotic cell death is distinct from the RIP1/3 pathway-dependent necroptosis, but mediated by a functional deficiency of TEAD/YAP-dependent transcription. In addition, we revealed that a cell cycle regulator, Plk1, switches the balance between TEAD/YAP-dependent necrosis and p73/YAP-dependent apoptosis by shifting the interaction partner of YAP from TEAD to p73 through YAP phosphorylation at Thr77. In vivo ER imaging with two-photon microscopy detects similar ER enlargement, and viral vector-mediated delivery of YAP as well as chemical inhibitors of the Hippo pathway such as S1P recover the ER instability and necrosis in HD model mice. Intriguingly S1P completely stops the decline of motor function of HD model mice even after the onset of symptom. Collectively, we suggest approaches targeting the signalling pathway of TEAD/YAP-transcription-dependent necrosis (TRIAD) could lead to a therapeutic development against HD.

Ataxin-7 associates with microtubules and stabilizes the cytoskeletal network.

The spinocerebellar ataxia type 7 (SCA7) gene product, Ataxin-7 (ATXN7), localizes to the nucleus and has been shown to function as a component of the TATA-binding protein-free TAF-containing-SPT3-TAF9-GCN5-acetyltransferase transcription complex, although cytoplasmic localization of ATXN7 in affected neurons of human SCA7 patients has also been detected. Here, we define a physiological function for cytoplasmic ATXN7. Live imaging reveals that the intracellular distribution of ATXN7 dynamically changes and that ATXN7 distribution frequently shifts from the nucleus to the cytoplasm. Immunocytochemistry and immunoprecipitation demonstrate that cytoplasmic ATXN7 associates with microtubules (MTs), and expression of ATXN7 stabilizes MTs against nocodazole treatment, while ATXN7 knockdown enhances MT degradation. Interestingly, normal and mutant ATXN7 similarly associate with and equally stabilize MTs. Taken together, these findings provide a novel physiological function of ATXN7 in the regulation of cytoskeletal dynamics, and suggest that abnormal cytoskeletal regulation may contribute to SCA7 disease pathology.

[Andexanet alfa (ONDEXXYA® for Intravenous Injection 200†mg), a reversal agent for direct factor Xa inhibitors: pharmacological characteristics and clinical evidence].

Andexanet alfa is a modified recombinant human factor Xa (FXa) that was designed to serve as a binding target for FXa inhibitors as decoy protein. It sequesters FXa inhibitors from binding to endogenous FXa, thereby reversing anticoagulant effect of FXa inhibitors. Andexanet alfa has been approved in March 2022 in Japan for patients with life-threatening or uncontrolled bleeding while on treatment with a FXa inhibitor, apixaban, rivaroxaban, or edoxaban tosilate hydrate. It is administered via two dosing regimens, based on the type of FXa inhibitor, dose, and time since the last dose. In nonclinical studies, andexanet alfa significantly inhibited bleeding induced by FXa inhibitors in animal bleeding models. In the development for Japanese patients, the following two clinical studies have been conducted to confirm the efficacy and safety. First, safety and the reversal effect of andexanet alfa on the FXa inhibitor-mediated anticoagulant activity in healthy adults were confirmed in the overseas phase 2 study including Japanese subjects. Next, the reversal effect of andexanet alfa on the anticoagulation activity and the hemostasis were demonstrated in patients with acute major bleeding while on FXa inhibitor treatment in the global phase 3b/4 study (ANNEXA-4 study). The subgroup analysis of Japanese population showed that the efficacy and safety results were consistent with those of overall population. Andexanet alfa is the first approved reversal agent for FXa inhibitors in Japan and is expected to contribute to the improvement of prognosis in patients with fatal and/or uncontrolled bleeding by timely reversing anticoagulant effect of FXa inhibitors.

Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D2.

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.

Association analysis of the dopamine receptor D2 (DRD2) SNP rs1076560 in alcoholic patients.

The dopamine system plays a well-established role in alcoholism. In this study, we examined the association between the single-nucleotide polymorphism (SNP) rs1076560 of the dopamine receptor D2 (DRD2) gene and susceptibility to alcoholism. SNP rs1076560 (C/A) is located in intron 6 of DRD2, where it is 1.4 kb downstream from alternative exon 6 and 83 bp upstream from exon 7. A total of 248 alcoholic patients and 322 healthy controls, all Japanese males, were genotyped for rs1076560 polymorphism by direct sequencing and allele-specific PCR. Data were analyzed using standard chi(2) statistics and a backwards logistic regression approach to adjust for the contribution of aldehyde dehydrogenase-2 (ALDH2) genotype status. The DRD2 risk allele A was more prevalent in the alcoholic patients (40.1%) than in the healthy controls (34.0%) (P=0.034, odds ratio=1.300, 95% confidence interval=1.020-1.657). These data identify SNP rs1076560 as a potentially important variable in the development of alcoholism.

HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice.

Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset.

A functional deficiency of TERA/VCP/p97 contributes to impaired DNA repair in multiple polyglutamine diseases.

It is hypothesized that a common underlying mechanism links multiple neurodegenerative disorders. Here we show that transitional endoplasmic reticulum ATPase (TERA)/valosin-containing protein (VCP)/p97 directly binds to multiple polyglutamine disease proteins (huntingtin, ataxin-1, ataxin-7 and androgen receptor) via polyglutamine sequence. Although normal and mutant polyglutamine proteins interact with TERA/VCP/p97, only mutant proteins affect dynamism of TERA/VCP/p97. Among multiple functions of TERA/VCP/p97, we reveal that functional defect of TERA/VCP/p97 in DNA double-stranded break repair is critical for the pathology of neurons in which TERA/VCP/p97 is located dominantly in the nucleus in vivo. Mutant polyglutamine proteins impair accumulation of TERA/VCP/p97 and interaction of related double-stranded break repair proteins, finally causing the increase of unrepaired double-stranded break. Consistently, the recovery of lifespan in polyglutamine disease fly models by TERA/VCP/p97 corresponds well to the improvement of double-stranded break in neurons. Taken together, our results provide a novel common pathomechanism in multiple polyglutamine diseases that is mediated by DNA repair function of TERA/VCP/p97.

Alcoholism and alternative splicing of candidate genes.

Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.