Masaaki Nakai

The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells

Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1β secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca2+-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.

Plasma levels of DJ-1 as a possible marker for progression of sporadic parkinson's disease

DJ-1 is a multifunctional protein whose loss of function by gene mutations may play a causative role for familial Parkinsons disease (PD). A recent study has shown that the expression of this molecule is upregulated in both brains and cerebrospinal fluids (CSF) in various neurological disorders, including sporadic PD, Alzheimers disease (AD) and stroke, raising a possibility that DJ-1 could be a potential biomarker for these diseases. In this context, the main objective of the present study was to determine if DJ-1 was increased in the plasma of PD patients. For this purpose, blood plasma samples collected from sporadic PD patients, dementia with Lewy bodies (DLB) and healthy age-matched controls were analyzed by immunoblotting and enzyme-linked immunosorbent assay. The results showed that the plasma DJ-1 levels in PD (n=104) were higher than those in control (n=80) (p<0.05). Moreover, the plasma DJ-1 levels in the advanced stage of PD (n=52, Yahr III-IV) were higher than those in the early stage of PD (n=52, Yahr I-II) (p<0.05), demonstrating that the plasma DJ-1 was correlated with the disease severity in PD. Plasma DJ-1 levels were also significantly higher in DLB (n=30) compared with both controls and early stage of PD (p<0.01). Taken together, these results suggest that the plasma DJ-1 could be a useful biomarker for the evaluation of the disease severity in PD and possibly in other Lewy body diseases.

Enhanced Lysosomal Pathology Caused by β-Synuclein Mutants Linked to Dementia with Lewy Bodies

Two missense mutations (P123H and V70M) of β-synuclein (β-syn), the homologue of α-syn, have been recently identified in dementia with Lewy bodies. However, the mechanism through which these mutations influence the pathogenesis of dementia with Lewy bodies is unclear. To investigate the role of the β-syn mutations in neurodegeneration, each mutant was stably transfected into B103 neuroblastoma cells. Cells overexpressing mutated β-syn had eosinophilic cytoplasmic inclusion bodies immunopositive for mutant β-syn, and electron microscopy revealed that these cells were abundant in various cytoplasmic membranous inclusions resembling the histopathology of lysosomal storage disease. Consistent with these findings, the inclusion bodies were immunopositive for lysosomal markers, including cathepsin B, LAMP-2, GM2 ganglioside, and ATP13A2, which has recently been linked to PARK9. Notably, formation of these lysosomal inclusions was greatly stimulated by co-expression of α-syn, was dependent on the phosphorylation of α-syn at Ser-129, and was more efficient with the A53T familial mutant of α-syn compared with wild type. Furthermore, the inclusion formation in cells overexpressing mutant β-syn and transfected with α-syn was significantly suppressed by treatment with autophagy-lysosomal inhibitors, which were associated with impaired clearance of syn proteins and enhanced apoptosis, indicating that formation of lysosomal inclusions might be protective. Collectively, the results demonstrated unambiguously that overexpression of β-syn mutants (P123H and V70M) in neuroblastoma cells results in an enhanced lysosomal pathology. We suggest that these missense mutations of β-syn might play a causative role in stimulating neurodegeneration.

Protective Role of Endogenous Gangliosides for Lysosomal Pathology in a Cellular Model of Synucleinopathies

Gangliosides may be involved in the pathogenesis of Parkinsons disease and related disorders, although the precise mechanisms governing this involvement remain unknown. In this study, we determined whether changes in endogenous ganglioside levels affect lysosomal pathology in a cellular model of synucleinopathy. For this purpose, dementia with Lewy body-linked P123H beta-synuclein (beta-syn) neuroblastoma cells transfected with alpha-synuclein were used as a model system because these cells were characterized as having extensive formation of lysosomal inclusions bodies. Treatment of these cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glycosyl ceramide synthase, resulted in various features of lysosomal pathology, including compromised lysosomal activity, enhanced lysosomal membrane permeabilization, and increased cytotoxicity. Consistent with these findings, expression levels of lysosomal membrane proteins, ATP13A2 and LAMP-2, were significantly decreased, and electron microscopy demonstrated alterations in the lysosomal membrane structures. Furthermore, the accumulation of both P123H beta-syn and alpha-synuclein proteins was significant in PDMP-treated cells because of the suppressive effect of PDMP on the autophagy pathway. Finally, the detrimental effects of PDMP on lysosomal pathology were significantly ameliorated by the addition of gangliosides to the cultured cells. These data suggest that endogenous gangliosides may play protective roles against the lysosomal pathology of synucleinopathies.

α-Synuclein Stimulates Differentiation of Osteosarcoma Cells RELEVANCE TO DOWN-REGULATION OF PROTEASOME ACTIVITY

Because a limited study previously showed that α-synuclein (α-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether α-syn might be involved in the regulation of tumor differentiation. For this purpose, α-syn and its non-amyloidogenic homologue β-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with β-syn-overexpressing and vector-transfected cells, α-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in α-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In α-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in α-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of α-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of α-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of α-syn regulation of tumor differentiation and neuropathological effects of α-syn may considerably overlap with each other.

Chaperone and anti-chaperone: two-faced synuclein as stimulator of synaptic evolution.

Previous studies have shown that β‐synuclein (β‐syn), the homologue of α‐syn, inhibited α‐syn aggregation and stabilized Akt cell survival signaling molecule, suggesting that β‐syn was protective against α‐syn‐related neurodegenerative disorders, such as Parkinson’s disease and diffuse Lewy body disease. However, emerging evidence argues that the situation may be not so simple. Two missense mutations of β‐syn were identified in familial and sporadic diffuse Lewy body disease, and wild type β‐syn was induced to form fibril structures in vitro, while, α‐syn was shown to be protective against neurodegeneration caused by deletion of cysteine‐string protein‐α, the presynaptic cochaperone to Hsc70 in mice. Collectively, α‐ and β‐syn are both, but in varying degrees, featured with two opposite properties, namely normal chaperone and anti‐chaperone. By reviewing recent progress in syn biology with a particular focus on β‐syn, this manuscript refers to the intriguing possibility that the dual syn proteins might have acquired a driving force for synaptic evolution. Hypothetically, the anti‐chaperone syn may provoke stress‐induced diverse responses, whereas, the chaperone syn may provide buffering for them, allowing accumulation of nonlethal phenotypic variations in synapses. Consequently, dual syn proteins may cope with forth‐coming stresses in the brain by stimulating adaptive evolution. In this context, failure to regulate this process due to various causes, such as gene mutations and environmental risk factors, may result in imperfect adaptability against stresses, leading to neurodegenerative disorders.

Molecular mechanism of neurodegeneration caused by familial mutations (P123H and V70M) of β-synuclein

We explored possible involvement of autocrine FGF signaling in ectoderm in neural induction and patterning by employing morpholino-antisense oligonucleotides (MOs) against the five members of FGF family, which are present in ectoderm (FGF-2, 3, 4, 8 and 9). When each of these FGFMOs was injected into a dorso-animal blastomere of 8-cell-stage embryos, which is destined to ectodermal and subsequently neural region, only FGF-2MO and FGF-8MO caused severe head defects on the injected side. The defects were largely rescued by co-injection of cognate FGF-2 or -8 that was mismatched to respective MO. We then examined the expression of position-specific neural markers along the A-P axis (otx2, XHR1, fkh5 and HoxD1) as well as an early pan-neural marker (Sox2) in FGF-2or -8-depleted embryos. The in situ analysis showed that the expression of all the five markers was suppressed in the FGF-2or -8-injected region that was marked by co-injected lineage-tracing GFP mRNA. We propose a novel model for neural induction and patterning in Xenopus, which includes autocrine FGF signaling in ectoderm.