Akio Kikuchi

Severe olfactory dysfunction is a prodromal symptom of dementia associated with Parkinson's disease: a 3 year longitudinal study

Dementia is one of the most debilitating symptoms of Parkinsons disease. A recent longitudinal study suggests that up to 80% of patients with Parkinsons disease will eventually develop dementia. Despite its clinical importance, the development of dementia is still difficult to predict at early stages. We previously identified olfactory dysfunction as one of the most important indicators of cortical hypometabolism in Parkinsons disease. In this study, we investigated the possible associations between olfactory dysfunction and the risk of developing dementia within a 3-year observation period. Forty-four patients with Parkinsons disease without dementia underwent the odour stick identification test for Japanese, memory and visuoperceptual assessments, (18)F-fluorodeoxyglucose positron emission tomography scans and magnetic resonance imaging scans at baseline and 3 years later. A subgroup of patients with Parkinsons disease who exhibited severe hyposmia at baseline showed more pronounced cognitive decline at the follow-up survey. By the end of the study, 10 of 44 patients with Parkinsons disease had developed dementia, all of whom had severe hyposmia at baseline. The multivariate logistic analysis identified severe hyposmia and visuoperceptual impairment as independent risk factors for subsequent dementia within 3 years. The patients with severe hyposmia had an 18.7-fold increase in their risk of dementia for each 1 SD (2.8) decrease in the score of odour stick identification test for Japanese. We also found an association between severe hyposmia and a characteristic distribution of cerebral metabolic decline, which was identical to that of dementia associated with Parkinsons disease. Furthermore, volumetric magnetic resonance imaging analyses demonstrated close relationships between olfactory dysfunction and the atrophy of focal brain structures, including the amygdala and other limbic structures. Together, our findings suggest that brain regions related to olfactory function are closely associated with cognitive decline and that severe hyposmia is a prominent clinical feature that predicts the subsequent development of Parkinsons disease dementia.

A case of NMO seropositive for aquaporin-4 antibody more than 10 years before onset

Neuromyelitis optica (NMO) is characterized by severe optic neuritis and longitudinally extended transverse myelitis (LETM). The clinical and laboratory features of NMO are different from multiple sclerosis (MS).1 An autoantibody to aquaporin-4 (AQP4) has been detected exclusively in the NMO sera.1 Moreover, we demonstrated an extensive loss of AQP4 and glial fibrillary acidic protein immunoreactivities in the perivascular regions with complement and immunoglobulin deposition in NMO that suggests astrocytic impairment. However, when AQP4 antibody is produced in patients with NMO is unknown, and thus it remains unresolved whether there are long-term asymptomatic AQP4 antibody–positive carriers, whether AQP4 antibody alone can be pathogenic, and whether AQP4 antibody is produced secondarily as a result of tissue destruction in attacks of NMO. We herein report a case of NMO in which AQP4 antibody was detected years before the NMO onset. ### Case report. A 34-year-old healthy woman without previous history of inflammatory or neurologic diseases noticed temporary skin eruptions on her chest and shoulders in June 2007. A dermatologist made the diagnosis of eczema. Three weeks later, when the skin eruptions subsided, she noted progressive paresthesia in the chest and toes. Within a few days, she could not walk well due to right leg weakness, and then she was hospitalized. Neurologic examination on …

Plasma membrane ion permeability induced by mutant α-synuclein contributes to the degeneration of neural cells

Mutations in α‐synuclein cause some cases of familial Parkinsons disease (PD), but the mechanism by which α‐synuclein promotes degeneration of dopamine‐producing neurons is unknown. We report that human neural cells expressing mutant α‐synuclein (A30P and A53T) have higher plasma membrane ion permeability. The higher ion permeability caused by mutant α‐synuclein would be because of relatively large pores through which most cations can pass non‐selectively. Both the basal level of [Ca2+]i and the Ca2+ response to membrane depolarization are greater in cells expressing mutant α‐synuclein. The membrane permeable Ca2+ chelator BAPTA‐AM significantly protected the cells against oxidative stress, whereas neither l‐type (nifedipine) nor N‐type (ω‐conotoxin‐GVIA) Ca2+ channel blockers protected the cells. These findings suggest that the high membrane ion permeability caused by mutant α‐synuclein may contribute to the degeneration of neurons in PD.

Serine 129 Phosphorylation of α-Synuclein Induces Unfolded Protein Response-mediated Cell Death

α-Synuclein is a major protein component deposited in Lewy bodies and Lewy neurites that is extensively phosphorylated at Ser129, although its role in neuronal degeneration is still elusive. In this study, several apoptotic pathways were examined in α-synuclein-overexpressing SH-SY5Y cells. Following the treatment with rotenone, a mitochondrial complex I inhibitor, wild type α-synuclein-overexpressing cells demonstrated intracellular aggregations, which shared a number of features with Lewy bodies, although cells overexpressing the S129A mutant, in which phosphorylation at Ser129 was blocked, showed few aggregations. In wild typeα-synuclein cells treated with rotenone, the proportion of phosphorylated α-synuclein was about 1.6 times higher than that of untreated cells. Moreover, induction of unfolded protein response (UPR) markers was evident several hours before the induction of mitochondrial disruption and caspase-3 activation. Eukaryotic initiation factor 2α, a member of the PERK pathway family, was remarkably activated at early phases. On the other hand, the S129A mutant failed to activate UPR. Casein kinase 2 inhibitor, which decreased α-synuclein phosphorylation, also reduced UPR activation. The α-synuclein aggregations were colocalized with a marker for the endoplasmic reticulum-Golgi intermediate compartment. Taken together, it seems plausible that α-synuclein toxicity is dependent on the phosphorylation at Ser129 that induces the UPRs, possibly triggered by the disturbed endoplasmic reticulum-Golgi trafficking.

The AAA-ATPase VPS4 Regulates Extracellular Secretion and Lysosomal Targeting of α-Synuclein

Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like fibrils composed of misfolded proteins. Emerging evidence suggests that these proteins may spread from cell-to-cell and encourage the propagation of neurodegeneration in a prion-like manner. Here, we demonstrated that α-synuclein (αSYN), a principal culprit for Lewy pathology in Parkinsons disease (PD), was present in endosomal compartments and detectably secreted into the extracellular milieu. Unlike prion protein, extracellular αSYN was mainly recovered in the supernatant fraction rather than in exosome-containing pellets from the neuronal culture medium and cerebrospinal fluid. Surprisingly, impaired biogenesis of multivesicular body (MVB), an organelle from which exosomes are derived, by dominant-negative mutant vacuolar protein sorting 4 (VPS4) not only interfered with lysosomal targeting of αSYN but facilitated αSYN secretion. The hypersecretion of αSYN in VPS4-defective cells was efficiently restored by the functional disruption of recycling endosome regulator Rab11a. Furthermore, both brainstem and cortical Lewy bodies in PD were found to be immunoreactive for VPS4. Thus, VPS4, a master regulator of MVB sorting, may serve as a determinant of lysosomal targeting or extracellular secretion of αSYN and thereby contribute to the intercellular propagation of Lewy pathology in PD.

Hypoperfusion in the supplementary motor area, dorsolateral prefrontal cortex and insular cortex in Parkinson's disease.

The changes of regional cerebral blood flow (rCBF) in Parkinsons disease (PD) were investigated. Because of individual differences in brain volume and the extent of brain atrophy, previous functional imaging studies involved potential methodological difficulties. In this study, using the statistical parametric mapping technique, 99mTechnetium-labeled hexamethylpropyleneamineoxime brain single-photon emission computed tomography images from 18 patients with PD were transformed into standard brain-based stereotaxic coordinate spaces and then compared with such images for 11 control subjects matched for age and extent of brain atrophy. A rCBF decrement in the supplementary motor area (SMA) and such decrement in the dorsolateral prefrontal cortex (DLPFC) were observed in the summarized PD images as compared with controls (p<0.005). In a subgroup in the Hoehn-Yahr III/IV stage (11 cases), the rCBF decrement was demonstrated not only in the SMA, but also in the DLPFC and insular cortex (p<0.001). There was a correlation between the degree of the rCBF decrement in the DLPFC or the insular cortex and the score of the unified Parkinsons disease rating scale (p<0.05), while the rCBF decrement in the SMA showed no relationship with the severity of disease. The function of the SMA is closely associated with the nigro-striatal pathway and its impairment can explain the basic akinetic symptoms in PD, which are responsive to L-DOPA treatment. On the other hand, the DLPFC and insular cortex may play key roles in specific symptoms of impairment at advanced stages, such as impaired working memory, postural instability and autonomic dysfunction. We hypothesize that the impairment of the DLPFC and insular function is correlated with the progression of the disease and is related to DOPA-refractory symptoms, which are major problems in the care of patients with advanced PD.

VPS35 dysfunction impairs lysosomal degradation of α-synuclein and exacerbates neurotoxicity in a Drosophila model of Parkinson's disease

Mutations in vacuolar protein sorting 35 (VPS35) have been linked to familial Parkinsons disease (PD). VPS35, a component of the retromer, mediates the retrograde transport of cargo from the endosome to the trans-Golgi network. Here we showed that retromer depletion increases the lysosomal turnover of the mannose 6-phosphate receptor, thereby affecting the trafficking of cathepsin D (CTSD), a lysosome protease involved in α-synuclein (αSYN) degradation. VPS35 knockdown perturbed the maturation step of CTSD in parallel with the accumulation of αSYN in the lysosomes. Furthermore, we found that the knockdown of Drosophila VPS35 not only induced the accumulation of the detergent-insoluble αSYN species in the brain but also exacerbated both locomotor impairments and mild compound eye disorganization and interommatidial bristle loss in flies expressing human αSYN. These findings indicate that the retromer may play a crucial role in αSYN degradation by modulating the maturation of CTSD and might thereby contribute to the pathogenesis of the disease.

In vivo visualization of α-synuclein deposition by carbon-11-labelled 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy]benzoxazole positron emission tomography in multiple system atrophy

The histopathological hallmark of multiple system atrophy is the appearance of intracellular inclusion bodies, named glial cytoplasmic inclusions, which are mainly composed of alpha-synuclein fibrils. In vivo visualization of alpha-synuclein deposition should be used for the diagnosis and assessment of therapy and severity of pathological progression in multiple system atrophy. Because 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy] benzoxazole could stain alpha-synuclein-containing glial cytoplasmic inclusions in post-mortem brains, we compared the carbon-11-labelled 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy] benzoxazole positron emission tomography findings of eight multiple system atrophy cases to those of age-matched normal controls. The positron emission tomography data demonstrated high distribution volumes in the subcortical white matter (uncorrected P < 0.001), putamen and posterior cingulate cortex (uncorrected P < 0.005), globus pallidus, primary motor cortex and anterior cingulate cortex (uncorrected P < 0.01), and substantia nigra (uncorrected P < 0.05) in multiple system atrophy cases compared to the normal controls. They were coincident with glial cytoplasmic inclusion-rich brain areas in multiple system atrophy and thus, carbon-11-labelled 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy] benzoxazole positron emission tomography is a promising surrogate marker for monitoring intracellular alpha-synuclein deposition in living brains.

Accelerated α-synuclein aggregation after differentiation of SH-SY5Y neuroblastoma cells

Abstract α-Synuclein (α-syn) is a major component of inclusion bodies in Parkinsons disease (PD) and other synucleinopathies. To clarify the possible roles of α-syn in the molecular pathogenesis of neurodegenerative diseases, we have established a novel cellular model based on the differentiation of SH-SY5Y cells that overexpress α-syn. In the presence of ferrous iron, differentiation of the cells led to the formation of large perinuclear inclusion bodies, which developed from scattered small aggregates seen in undifferentiated cells. The iron-induced α-syn-positive inclusions co-localized largely with ubiquitin, and some of them were positive for nitrotyrosine, lipid, γ-tubulin and dynein. Notably, treatment with nocodazole, a microtubule depolymerizing agent, interrupted the aggregate formation but led to a concomitant increase of apoptotic cells. Therefore, it appears that an intracellular retrograde transport system via microtubules plays a crucial role in the aggregate formation and also that the aggregates may represent a cytoprotective response against noxious stimuli. This cellular model will enable better understanding of the molecular pathomechanisms of synucleinopathy.

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

Group A saponins in soybean are diversified compounds belonging to a group of triterpene saponins and are causal components for bitterness and astringent aftertastes of soy products. This work describes the identification of Sg-1, a UDP-sugar–dependent glycosyltransferase gene that is responsible for the unpleasant tastes due to allelic variation regulating the terminal sugar species in group A saponins. Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar–dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1a allele encodes the xylosyltransferase UGT73F4, whereas Sg-1b encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1a and Gly-138 in Sg-1b proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-10 is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.