Harutsugu Tatebe

Extracellular neurosin degrades α-synuclein in cultured cells

Neurosin, also called kallikrein 6, is a trypsin-like serine protease predominantly expressed in the central nervous system. Neurosin may degrade alpha-synuclein, a major component of the Lewy bodies commonly observed in dopaminergic neurons of patients with sporadic Parkinsons disease. In the present study, we investigated the localization and proteolytic activity of human neurosin using cultured cells to elucidate the physiological role of this enzyme at the cellular level. Heterologous expression of pre-pro-neurosin was localized to the endoplasmic reticulum and secreted. The proteolytic activity of neurosin was analyzed by zymography and fluorescent substrate, and showed that extracellular neurosin had protease activity but intracellular neurosin did not. We also coexpressed alpha-synuclein with neurosin and demonstrated that alpha-synuclein was not cleaved within cells, but extracellular alpha-synuclein was degraded by secreted neurosin. These findings suggest that neurosin targets the extracellular alpha-synuclein.

p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions

α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson’s disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated in vitro. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.

Differential Expression of Alpha-Synuclein in Hippocampal Neurons

α-Synuclein is the major pathological component of synucleinopathies including Parkinsons disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.

Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinsons disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils.

Serum Levels of Coenzyme Q10 in Patients with Multiple System Atrophy

The COQ2 gene encodes an essential enzyme for biogenesis, coenzyme Q10 (CoQ10). Recessive mutations in this gene have recently been identified in families with multiple system atrophy (MSA). Moreover, specific heterozygous variants in the COQ2 gene have also been reported to confer susceptibility to sporadic MSA in Japanese cohorts. These findings have suggested the potential usefulness of CoQ10 as a blood-based biomarker for diagnosing MSA. This study measured serum levels of CoQ10 in 18 patients with MSA, 20 patients with Parkinson’s disease and 18 control participants. Although differences in total CoQ10 (i.e., total levels of serum CoQ10 and its reduced form) among the three groups were not significant, total CoQ10 level corrected by serum cholesterol was significantly lower in the MSA group than in the Control group. Our findings suggest that serum CoQ10 can be used as a biomarker in the diagnosis of MSA and to provide supportive evidence for the hypothesis that decreased levels of CoQ10 in brain tissue lead to an increased risk of MSA.

Decrease in plasma levels of α-synuclein is evident in patients with Parkinson's disease after elimination of heterophilic antibody interference.

There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfered with ELISA measurements of A-syn and are accordingly considered to be an important confounder in A-syn ELISAs. HA produced falsely exaggerated signals in A-syn ELISAs more prominently in plasma samples than in CSF samples. After elimination of HA interference, it was found that hemolysis did not have a significant effect on the signals obtained using our A-syn ELISA. Furthermore, plasma levels of A-syn were significantly lower in the PD group compared with the control group following elimination of HA interference with an HA inhibitor. Our results demonstrate that HA was a major confounder that should be controlled in A-syn ELISAs, and that plasma A-syn could be a useful biomarker for the diagnosis of PD if adequately quantified following elimination of HA interference.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

The domestic cat as a natural animal model of Alzheimer's disease.

IntroductionAlzheimer’s disease (AD) is the most dominant neurodegenerative disorder that causes dementia, and no effective treatments are available. To study its pathogenesis and develop therapeutics, animal models representing its pathologies are needed. Although many animal species develop senile plaques (SP) composed of amyloid-β (Aβ) proteins that are identical to those found in humans, none of them exhibit neurofibrillary tangles (NFT) and subsequent neurodegeneration, which are integral parts of the pathology of AD.ResultsThe present study shows that Aβ accumulation, NFT formation, and significant neuronal loss all emerge naturally in the hippocampi of aged domestic cats. The NFT that form in the cat brain are identical to those seen in human AD in terms of their spatial distribution, the cells they affect, and the tau isoforms that comprise them. Interestingly, aged cats do not develop mature argyrophilic SP, but instead accumulate intraneuronal Aβ oligomers in their hippocampal pyramidal cells, which might be due to the amino acid sequence of felid Aβ.ConclusionsThese results suggest that Aβ oligomers are more important than SP for NFT formation and the subsequent neurodegeneration. The domestic cat is a unique animal species that naturally replicates various AD pathologies, especially Aβ oligomer accumulation, NFT formation, and neuronal loss.

Monoclonal antibody with conformational specificity for a toxic conformer of amyloid β42 and its application toward the Alzheimer's disease diagnosis.

Amyloid β-protein (Aβ42) oligomerization is an early event in Alzheimer’s disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aβ42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aβ42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aβ42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aβ42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aβ42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aβ42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.

Increased levels of plasma total tau in adult Down syndrome

Down syndrome (DS) is the most prevalent chromosomal abnormality. Early-onset dementia with the pathology of Alzheimer’s disease (AD) frequently develops in DS. Reliable blood biomarkers are needed to support the diagnosis for dementia in DS, since positron emission tomography or cerebrospinal fluid sampling is burdensome, particularly for patients with DS. Plasma t-tau is one of the established biomarkers for the diagnosis of AD, suggesting the potential value of t-tau as a biomarker for dementia in DS. The aim of this study was to assess and compare plasma levels of t-tau in adults with DS and in an age-matched control population. In this study, plasma levels of t-tau in 21 patients with DS and 22 control participants were measured by an ultrasensitive immunoassay technology, the single-molecule immunoarray (Simoa) method. We observed significantly increased plasma t-tau levels in the DS group (mean ± standard deviation (SD) = 0.643±0.493) compared to those in the control group (mean ± SD = 0.470±0.232): P = 0.0050. Moreover, age dependent correlation of plasma t-tau was only found in the DS group, and not in the control group. These findings suggest that elevated plasma t-tau levels reflect AD pathology and therefore have potential as an objective biomarker to detect dementia in adult DS.