Masaru Kurosawa

Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such as Huntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survival in a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreased polyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse model of Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutamines and stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacy upon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutamine diseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutamine diseases.

beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and γ-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that β subunits of voltage-gated sodium channels are sequentially processed by BACE1 and γ-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity.

A hallmark of polyglutamine diseases, including Huntington disease (HD), is the formation of β-sheet-rich aggregates, called amyloid, of causative proteins with expanded polyglutamines. However, it has remained unclear whether the polyglutamine amyloid is a direct cause or simply a secondary manifestation of the pathology. Here we show that huntingtin-exon1 (thtt) with expanded polyglutamines remarkably misfolds into distinct amyloid conformations under different temperatures, such as 4 °C and 37 °C. The 4 °C amyloid has loop/turn structures together with mostly β-sheets, including exposed polyglutamines, whereas the 37 °C amyloid has more extended and buried β-sheets. By developing a method to efficiently introduce amyloid into mammalian cells, we found that the formation of the 4 °C amyloid led to substantial toxicity, whereas the toxic effects of the 37 °C amyloid were very small. Importantly, thtt amyloids in different brain regions of HD mice also had distinct conformations. The thermolabile thtt amyloid with loop/turn structures in the striatum showed higher toxicity, whereas the rigid thtt amyloid with more extended β-sheets in the hippocampus and cerebellum had only mild toxic effects. These studies show that the thtt protein with expanded polyglutamines can misfold into distinct amyloid conformations and, depending on the conformations, the amyloids can be either toxic or nontoxic. Thus, the amyloid conformation of thtt may be a critical determinant of cytotoxicity in HD.

Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein

Huntingtons Disease (HD) is a dominantly inherited pathology caused by the accumulation of mutant huntingtin protein (HTT) containing an expanded polyglutamine (polyQ) tract. As the polyglutamine binding peptide 1 (QBP1) is known to bind an expanded polyQ tract but not the polyQ motif found in normal HTT, we selectively targeted mutant HTT for degradation by expressing a fusion molecule comprising two copies of QBP1 and copies of two different heat shock cognate protein 70 (HSC70)–binding motifs in cellular and mouse models of HD. Chaperone-mediated autophagy contributed to the specific degradation of mutant HTT in cultured cells expressing the construct. Intrastriatal delivery of a virus expressing the fusion molecule ameliorated the disease phenotype in the R6/2 mouse model of HD. Similar adaptor molecules comprising HSC70–binding motifs fused to an appropriate structure-specific binding agent(s) may have therapeutic potential for treating diseases caused by misfolded proteins other than those with expanded polyQ tracts.

Intracellular localization and splicing regulation of FUS/TLS are variably affected by amyotrophic lateral sclerosis-linked mutations

TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma), is an RNA/DNA-binding protein that plays regulatory roles in transcription, pre-mRNA splicing and mRNA transport. Mutations in TLS are responsible for familial amyotrophic lateral sclerosis (ALS) type 6. Furthermore, TLS-containing intracellular inclusions are found in polyglutamine diseases, sporadic ALS, non-SOD1 familial ALS and a subset of frontotemporal lobar degeneration, indicating a pathological significance of TLS in a wide variety of neurodegenerative diseases. Here, we identified TLS domains that determine intracellular localization of the murine TLS. Among them, PY-NLS located in the C-terminus is a strong determinant of intracellular localization as well as splicing regulation of an E1A-derived minigene. Disruption of PY-NLS promoted the formation of cytoplasmic granules that were partially overlapped with stress granules and P-bodies. Some of the ALS-linked mutations altered both intracellular localization and splicing regulation of TLS, while most mutations alone did not affect splicing regulation. However, phospho-mimetic substitution of Ser505 (or Ser513 in human) could enhance the effects of ALS mutations, highlighting interplay between post-translational modification and ALS-linked mutations. These results demonstrate that ALS-linked mutations can variably cause loss of nuclear functions of TLS depending on the degree of impairment in nuclear localization.

RNA-binding Protein TLS Is a Major Nuclear Aggregate-interacting Protein in Huntingtin Exon 1 with Expanded Polyglutamine-expressing Cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.

Blocking acid-sensing ion channel 1 alleviates Huntington's disease pathology via an ubiquitin-proteasome system-dependent mechanism

Huntingtons disease (HD) is a fatal neurodegenerative disorder. Despite a tremendous effort to develop therapeutic tools in several HD models, there is no effective cure at present. Acidosis has been observed previously in cellular and in in vivo models as well as in the brains of HD patients. Here we challenged HD models with amiloride (Ami) derivative benzamil (Ben), a chemical agent used to rescue acid-sensing ion channel (ASIC)-dependent acidotoxicity, to examine whether chronic acidosis is an important part of the HD pathomechanism and whether these drugs could be used as novel therapeutic agents. Ben markedly reduced the huntingtin-polyglutamine (htt-polyQ) aggregation in an inducible cellular system, and the therapeutic value of Ben was successfully recapitulated in the R6/2 animal model of HD. To reveal the mechanism of action, Ben was found to be able to alleviate the inhibition of the ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. More importantly, we were able to demonstrate that blocking the expression of a specific isoform of ASIC (asic1a), one of the many molecular targets of Ben, led to an enhancement of UPS activity and this blockade also decreased htt-polyQ aggregation in the striatum of R6/2 mice. In conclusion, we believe that chemical compounds that target ASIC1a or pharmacological alleviation of UPS inhibition would be an effective and promising approach to combat HD and other polyQ-related disorders.

Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor

In Huntingtons disease (HD), mutant Huntingtin, which contains expanded polyglutamine stretches, forms nuclear aggregates in neurons. The interactions of several transcriptional factors with mutant Huntingtin, as well as altered expression of many genes in HD models, imply the involvement of transcriptional dysregulation in the HD pathological process. The precise mechanism remains obscure, however. Here, we show that mutant Huntingtin aggregates interact with the components of the NF‐Y transcriptional factor in vitro and in HD model mouse brain. An electrophoretic mobility shift assay using HD model mouse brain lysates showed reduction in NF‐Y binding to the promoter region of HSP70, one of the NF‐Y targets. RT–PCR analysis revealed reduced HSP70 expression in these brains. We further clarified the importance of NF‐Y for HSP70 transcription in cultured neurons. These data indicate that mutant Huntingtin sequesters NF‐Y, leading to the reduction of HSP70 gene expression in HD model mice brain. Because suppressive roles of HSP70 on the HD pathological process have been shown in several HD models, NF‐Y could be an important target of mutant Huntingtin.

Identification of ubiquitin-interacting proteins in purified polyglutamine aggregates

Nuclear aggregates of enhanced green fluorescent protein and nuclear localization signal‐fused truncated N‐terminal huntingtin containing 150 repeats of glutamine residue were purified from ecdysine‐inducible mutant neuro2A cell line by sequential extraction of nuclear soluble proteins. To analyze the aggregate‐interacting proteins, we subjected the nuclear aggregates to high performance liquid chromatography–mass spectrometry analysis. The resulting data revealed the presence of three new putative aggregate‐interacting proteins: ubiquilin 1, ubiquilin 2 and Tollip. These proteins also associated with neuronal intranuclear inclusions in a mouse model of Huntington disease (HD). These aggregate‐interacting proteins contain ubiquitin‐interacting motifs, suggesting that they are recruited to the aggregates where they may lose their normal function.

Sodium channel β4 subunit : down-regulation and possible involvement in neuritic degeneration in Huntington's disease transgenic mice

Sodium channel β4 is a very recently identified auxiliary subunit of the voltage‐gated sodium channels. To find the primarily affected gene in Huntingtons disease (HD) pathogenesis, we profiled HD transgenic mice using a high‐density oligonucleotide array and identified β4 as an expressed sequence tag (EST) that was significantly down‐regulated in the striatum of HD model mice and patients. Reduction in β4 started at a presymptomatic stage in HD mice, whereas other voltage‐gated ion channel subunits were decreased later. In contrast, spinal cord neurons, which generate only negligible levels of expanded polyglutamine aggregates, maintained normal levels of β4 expression even at the symptomatic stage. Overexpression of β4 induced neurite outgrowth in Neuro2a cells, and caused a thickening of dendrites and increased density of dendritic spines in hippocampal primary neurons, indicating that β4 modulates neurite outgrowth activities. These results suggest that down‐regulation of β4 may lead to abnormalities of sodium channel and neurite degeneration in the striatum of HD transgenic mice and patients with HD.