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Abstract Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.

Abstract Intrinsically disordered proteins (IDPs) have been in the spotlight for their unique properties, such as their lack of secondary structures and low sequence complexity. Alpha-synuclein and tau are representative disease-related IDPs with low complexity regions in their sequences, accumulating in the brains of patients with Parkinson disease and Alzheimer disease, respectively. Their heat resistance in particular was what attracted our attention. We assumed that there exist many other unidentified proteins that are resistant to heat-treatment, referred to as heat-stable proteins, which would also have low sequence complexity. In this study, we performed proteomic analysis of heat-stable proteins of mouse brains and found that proteins with compositionally biased regions are abundant in the heat-stable proteins. The proteins related to neurodegeneration are known to undergo different types of post-translational modifications (PTMs) such as phosphorylation and ubiquitination. We then investigated the heat-stability and aggregation properties of phosphorylated synuclein and tau with different phosphorylation sites. We suggest that PTMs can be important factors that determine the heat-stability and aggregation properties of a protein. IDPs identified in the heat-stable proteins of mouse brains would be candidates for the pathogenic proteins for neurodegeneration.

Abstract A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles. Analysis of the literature on cellular and animal models reviewed here supports the conclusion that P56S-VAPB, which is aggregation-prone, non-functional and unstable, is expressed at levels that are insufficient to support toxic gain-of-function or dominant negative effects within motor neurons. Instead, insufficient levels of the product of the single wild-type allele appear to be required for pathological effects, and may be the main driver of the disease. In light of the multiple interactions of the VAP proteins, we address the consequences of specific VAPB depletion and highlight various affected processes that could contribute to motor neuron degeneration. In the future, distinction of specific roles of each of the two VAP paralogues should help to further elucidate the basis of p.P56S familial ALS, as well as of other more common forms of the disease.

Abstract Accumulating evidence suggests that the lesions of Parkinson’s disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5–10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit–dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state.

The endoplasmic reticulum (ER) is a morphologically dynamic organelle containing different membrane subdomains with distinct cellular functions. Numerous observations have revealed that ER stress response induced by disturbed ER homeostasis is linked to various neurological/neurodegenerative disorders. In contrast, recent findings unveil that ER structural derangements are linked to the progression of several neurological diseases. The derangements involve two distinct, and likely opposing pathways. One is dysfunction of ER dynamics machinery, leading to disruption of ER network organization. Another one is facilitation of pre-existing machinery, leading to generation of markedly-ordered de novo membranous structure. Restoring the ER network can be the effective way toward the cure of ER-deranged neurological disorders.

In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.