Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages.

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized ß-transducin repeat-containing protein 1 (ßTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

NF-κB disinhibition contributes to dendrite defects in fly models of neurodegenerative diseases.

Dendrite pathology is frequently observed in various neurodegenerative diseases (NDs). Although previous studies identified several pathogenic mediators of dendrite defects that act through loss of function in NDs, the underlying pathogenic mechanisms remain largely unexplored. Here, our search for additional pathogenic contributors to dendrite defects in NDs identifies Relish/NF-κB as a novel gain-of-toxicity-based mediator of dendrite defects in animal models for polyglutamine (polyQ) diseases and amyotrophic lateral sclerosis (ALS). In a Drosophila model for polyQ diseases, polyQ-induced dendrite defects require Dredd/Caspase-8-mediated endoproteolytic cleavage of Relish to generate the N-terminal fragment, Rel68, and subsequent Charon-mediated nuclear localization of Rel68. Rel68 alone induced neuronal toxicity causing dendrite and behavioral defects, and we identify two novel transcriptional targets, Tup and Pros, that mediate Rel68-induced neuronal toxicity. Finally, we show that Rel68-induced toxicity also contributes to dendrite and behavioral defects in a Drosophila model for ALS. Collectively, our data propose disinhibition of latent toxicity of Relish/NF-κB as a novel pathogenic mechanism underlying dendrite pathology in NDs.

Coiled-coil structure-dependent interactions between polyQ proteins and Foxo lead to dendrite pathology and behavioral defects.

Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and ß-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of ß-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.

Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons.

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.

Epigenetic Changes in Neurodegenerative Diseases.

Afflicted neurons in various neurodegenerative diseases generally display diverse and complex pathological features before catastrophic occurrence of massive neuronal loss at the late stages of the diseases. This complex nature of neuronal pathophysiology inevitably implicates systemwide changes in basic cellular activities such as transcriptional controls and signal cascades, and so on, as a cause. Recently, as one of these systemwide cellular changes associated with neurodegenerative diseases, epigenetic changes caused by protein toxicity have begun to be highlighted. Notably, recent advances in related techniques including next-generation sequencing (NGS) and mass spectrometry enable us to monitor changes in the post-translational modifications (PTMs) of histone proteins and to link these changes in histone PTMs to the specific transcriptional changes. Indeed, epigenetic alterations and consequent changes in neuronal transcriptome are now begun to be extensively studied in neurodegenerative diseases including Alzheimer's disease (AD). In this review, we will discuss details of our current understandings on epigenetic changes associated with two representative neurodegenerative diseases [AD and polyglutamine (polyQ) diseases] and further discuss possible future development of pharmaceutical treatment of the diseases through modulating these epigenetic changes.