Yu Nagashima

gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment.

Amyloid β protein (Aβ), a pathogenic molecule associated with Alzheimers disease, is produced by γ-secretase, which cleaves the β-carboxyl terminal fragment (βCTF) of β-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that βCTF is cleaved first at the membrane–cytoplasm boundary, producing two long Aβs, Aβ48 and Aβ49, which are processed further by releasing three residues at each step to produce Aβ42 and Aβ40, respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted γ-secretase system, we confirmed that Aβ49 is converted to Aβ43/40 by successively releasing two or three tripeptides and that Aβ48 is converted to Aβ42/38 by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3–4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that Aβ is generated through the stepwise processing of βCTF by γ-secretase.

Intranuclear Degradation of Polyglutamine Aggregates by the Ubiquitin-Proteasome System

Huntington disease and its related autosomal-dominant polyglutamine (pQ) neurodegenerative diseases are characterized by intraneuronal accumulation of protein aggregates. Studies on protein aggregates have revealed the importance of the ubiquitin-proteasome system as the front line of protein quality control (PQC) machinery against aberrant proteins. Recently, we have shown that the autophagy-lysosomal system is also involved in cytoplasmic aggregate degradation, but the nucleus lacked this activity. Consequently, the nucleus relies entirely on the ubiquitin-proteasome system for PQC. According to previous studies, nuclear aggregates possess a higher cellular toxicity than do their cytoplasmic counterparts, however degradation kinetics of nuclear aggregates have been poorly understood. Here we show that nuclear ubiquitin ligases San1p and UHRF-2 each enhance nuclear pQ aggregate degradation and rescued pQ-induced cytotoxicity in cultured cells and primary neurons. Moreover, UHRF-2 is associated with nuclear inclusion bodies in vitro and in vivo. Our data suggest that UHRF-2 is an essential molecule for nuclear pQ degradation as a component of nuclear PQC machinery in mammalian cells.

Altered γ‐secretase activity in mild cognitive impairment and Alzheimer's disease

We investigated why the cerebrospinal fluid (CSF) concentrations of Aβ42 are lower in mild cognitive impairment (MCI) and Alzheimers disease (AD) patients. Because Aβ38/42 and Aβ40/43 are distinct product/precursor pairs, these four species in the CSF together should faithfully reflect the status of brain γ‐secretase activity, and were quantified by specific enzyme‐linked immunosorbent assays in the CSF from controls and MCI/AD patients. Decreases in the levels of the precursors, Aβ42 and 43, in MCI/AD CSF tended to accompany increases in the levels of the products, Aβ38 and 40, respectively. The ratios Aβ40/43 versus Aβ38/42 in CSF (each representing cleavage efficiency of Aβ43 or Aβ42) were largely proportional to each other but generally higher in MCI/AD patients compared to control subjects. These data suggest that γ‐secretase activity in MCI/AD patients is enhanced at the conversion of Aβ43 and 42 to Aβ40 and 38, respectively. Consequently, we measured the in vitro activity of raft‐associated γ‐secretase isolated from control as well as MCI/AD brains and found the same, significant alterations in the γ‐secretase activity in MCI/AD brains.

FAT10 protein binds to polyglutamine proteins and modulates their solubility

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.

Evaluation of spinal and bulbar muscular atrophy by the clustering index method

Introduction: A reliable electrophysiological marker for clinical trials is increasingly needed in spinal and bulbar muscular atrophy (SBMA). We previously developed a quantitative analysis method for surface electromyography (SEMG), the clustering index (CI) method. Our purpose was to test the utility of the CI method for evaluating lower motor neuron involvement in SBMA patients. Methods: Subjects included 29 SBMA patients and 27 healthy controls. The recording electrode was placed over the abductor digiti minimi (ADM) muscle with a proximal reference. The Z‐score, based on the CI method, was compared with compound muscle action potential (CMAP) amplitude and motor unit number estimation (MUNE), with regard to sensitivity. Results: The Z‐scores of the CI method, CMAP amplitude, and MUNE were abnormal in 100%, 72%, and 93% of the patients, respectively. Interrater reliability of the CI method was sufficiently high. Conclusion: The CI method is promising as a non‐invasive electrophysiological marker in SBMA. Muscle Nerve, 2011

Successful treatment of infliximab-associated immune-mediated sensory polyradiculopathy with intravenous immunoglobulin

Infliximab, a tumor necrosis factor-alpha antagonist, is used to treat many inflammatory diseases. Various forms of demyelinating neuropathies have been reported as neurological complications associated with infliximab use. There have been few reports of pure sensory neuropathy associated with infliximab. We report the clinical, electrophysiological, and pathological findings of a patient with subacute sensory polyradiculopathy 1 month after infliximab therapy for psoriasis vulgaris. Immune-mediated pathogenesis was suggested by positive anti-ganglioside antibodies and rapid response to intravenous immunoglobulin. This is the first reported case of sensory polyradiculopathy with positive anti-ganglioside antibodies following infliximab therapy. Our findings suggest the clinical importance of immunological investigations and treatment in demyelinating neuropathies following infliximab therapy.

In vivo molecular labeling of halogenated volatile anesthetics via intrinsic molecular vibrations using nonlinear Raman spectroscopy

Halogenated volatile anesthetics are frequently used for inhaled anesthesia in clinical practice. No appropriate biological method has been available for visualizing their localization in action. Therefore, despite their frequent use, the mechanism of action of these drugs has not been fully investigated. We measured coherent anti-Stokes Raman scattering (CARS) spectra of sevoflurane and isoflurane, two of the most representative volatile anesthetics, and determined the low-frequency vibrational modes without nonresonant background disturbance. Molecular dynamics calculations predict that these modes are associated with multiple halogen atoms. Because halogen atoms rarely appear in biological compounds, the entire spectral landscape of these modes is expected to be a good marker for investigating the spatial localization of these drugs within the intracellular environment. Using live squid giant axons, we could detect the unique CARS spectra of sevoflurane for the first time in a biological setting.

Clinicopathologic features of myositis patients with CD8-MHC-1 complex pathology.

Objective: To determine the clinical features of myositis patients with the histopathologic finding of CD8-positive T cells invading non-necrotic muscle fibers expressing major histocompatibility complex class 1 (CD8-MHC-1 complex), which is shared by polymyositis (PM) and inclusion body myositis (IBM), in relation to the p62 immunostaining pattern of muscle fibers. Methods: All 93 myositis patients with CD8-MHC-1 complex who were referred to our hospital from 1993 to 2015 were classified on the basis of the European Neuromuscular Center (ENMC) diagnostic criteria for IBM (Rose, 2013) or PM (Hoogendijk, 2004) and analyzed. Results: The 93 patients included were 17 patients with PM, 70 patients with IBM, and 6 patients who neither met the criteria for PM nor IBM in terms of muscle weakness distribution (unclassifiable group). For these PM, IBM, and unclassifiable patients, their mean ages at diagnosis were 63, 70, and 64 years; autoimmune disease was present in 7 (41%), 13 (19%), and 4 (67%); hepatitis C virus infection was detected in 0%, 13 (20%), and 2 (33%); and p62 was immunopositive in 0%, 66 (94%), and 2 (33%), respectively. Of the treated patients, 11 of 16 PM patients and 4 of 6 p62-immunonegative patients in the unclassifiable group showed responses to immunotherapy, whereas all 44 patients with IBM and 2 p62-immunopositive patients in the unclassifiable group were unresponsive to immunotherapy. Conclusions: CD8-MHC-1 complex is present in patients with PM, IBM, or unclassifiable group. The data may serve as an argument for a trial of immunosuppressive treatment in p62-immunonegative patients with unclassifiable myositis.

Atypical parkinsonism caused by Pro105Leu mutation of prion protein: A broad clinical spectrum.

Objective: To delineate molecular and clinical characteristics of 3 families with PRNP P105L mutation, a variant of Gerstmann-Sträussler-Scheinker syndrome whose main motor symptoms were parkinsonism and/or involuntary movements. Methods: The causative mutation was first determined in the affected patients of family 1 using whole-exome sequencing, and then mutational analysis was extended to families 2 and 3. The clinical features of the patients of these 3 families were summarized. Haplotype analysis was performed using high-density single nucleotide polymorphism array. Results: The whole-exome sequencing revealed that the heterozygous mutation c.314C>T (p.P105L) in PRNP was the only known pathogenic mutation shared by the 3 patients of the family with autosomal dominant parkinsonism. We further identified the same mutation in patients of the other 2 families with autosomal dominant parkinsonism and/or involuntary movements. The clinical features of our patients with PRNP P105L mutation included various motor symptoms such as parkinsonism and involuntary movements in addition to progressive dementia. The clinical features in part overlapped with those of other forms of inherited prion diseases, such as fatal familial insomnia and Huntington disease-like type 1. The patients with PRNP P105L mutation shared a haplotype spanning 7.1 Mb around PRNP, raising the possibility that the mutations in the patients originated from a common founder. Conclusion: Most of the patients presented with parkinsonism in addition to progressive dementia. Although spastic paraparesis has been emphasized as the main clinical feature, the clinical spectrum of patients with PRNP P105L is broader than expected.

Fludarabine may overcome resistance to rituximab in IgM-related neuropathy

Rituximab, a monoclonal antibody directed against CD20, became widely used for the treatment of immunoglobulin M (IgM)-related neuropathy. However, response rate by rituximab monotherapy is no more than 30%. Previous studies revealed that fludarabine acts synergistically with rituximab in vitro and that fludarabine also ameliorates IgM-related neuropathy in a subset of patients. Here we present two cases of IgM-related neuropathy in the background of Waldenström macroglobulinaemia, including one with rituximab resistance. They showed marginal to high titres of both anti-myelin associated glycoprotein and anti-sulphate-3-glucuronyl paragloboside antibodies, and their symptoms were featured by prominent motor deterioration. The combination therapy with rituximab and fludarabine stabilised or improved neuropathic symptoms with tolerable adverse events. Fludarabine may have a potential to overcome rituximab resistance. In conclusion, combination therapy with rituximab and fludarabine should be considered for IgM-related neuropathy, especially when efficacy of rituximab monotherapy is insufficient.