Therapeutic functions of astrocytes to treat α-synuclein pathology in Parkinson's disease.

Intraneuronal inclusions of misfolded α-synuclein (α-syn) and prion-like spread of the pathologic α-syn contribute to progressive neuronal death in Parkinson's disease (PD). Despite the pathologic significance, no efficient therapeutic intervention targeting α-synucleinopathy has been developed. In this study, we provide evidence that astrocytes, especially those cultured from the ventral midbrain (VM), show therapeutic potential to alleviate α-syn pathology in multiple in vitro and in vivo α-synucleinopathic models. Regulation of neuronal α-syn proteostasis underlies the therapeutic function of astrocytes. Specifically, VM-derived astrocytes inhibited neuronal α-syn aggregation and transmission in a paracrine manner by correcting not only intraneuronal oxidative and mitochondrial stresses but also extracellular inflammatory environments, in which α-syn proteins are prone to pathologic misfolding. The astrocyte-derived paracrine factors also promoted disassembly of extracellular α-syn aggregates. In addition to the aggregated form of α-syn, VM astrocytes reduced total α-syn protein loads both by actively scavenging extracellular α-syn fibrils and by a paracrine stimulation of neuronal autophagic clearance of α-syn. Transplantation of VM astrocytes into the midbrain of PD model mice alleviated α-syn pathology and protected the midbrain dopamine neurons from neurodegeneration. We further showed that cografting of VM astrocytes could be exploited in stem cell-based therapy for PD, in which host-to-graft transmission of α-syn pathology remains a critical concern for long-term cell therapeutic effects.

Interleukin-6 signaling requires EHD1-mediated alteration of membrane rafts.

Interleukin-6 (IL-6) is involved in many inflammatory diseases. IL-6 binds to membrane-bound IL-6 receptor α (IL-6Rα) (classic signaling) or soluble IL-6Rα (trans-signaling); this complex then associates with the signal-transducing membrane protein gp130. IL-6Rα and gp130 float on membrane (i.e., lipid) rafts; however, how membrane rafts regulate IL-6 signaling remains unclear. Here, we demonstrate that both IL-6 classic signaling and trans-signaling depend on membrane cholesterol, an essential raft component. Super-resolution fluorescence imaging using perfringolysin O D4 fragments that selectively bind to high cholesterol concentrations revealed that IL-6 and hyper-IL-6, a fusion protein of IL-6 and soluble IL-6Rα, induce the alteration of membrane rafts. IL-6 and hyper-IL-6 induced D4-positive raft (D4 raft) formation without affecting cholera toxin subunit B (CTB)-positive rafts (CTB rafts). Receptor clustering of IL-6Rα and gp130 and STAT3 phosphorylation occurred in D4 rafts. These results indicate that D4 rafts serve as platforms for the assembly of functional IL-6 receptor complexes. We found that Eps15 homology domain-containing protein 1 (EHD1) mediates the formation of functional IL-6 receptor complexes through D4 rafts. Overall, we uncover a novel regulatory mechanism of the EHD1-mediated alteration of membrane raft in IL-6 signaling.

Age-related increase in caveolin-1 expression facilitates cell-to-cell transmission of α-synuclein in neurons.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, with aging being considered the greatest risk factor for developing PD. Caveolin-1 (Cav-1) is known to participate in the aging process. Recent evidence indicates that prion-like propagation of misfolded α-synuclein (α-syn) released from neurons to neighboring neurons plays an important role in PD progression. In the present study, we demonstrated that cav-1 expression in the brain increased with age, and considerably increased in the brain of A53T α-syn transgenic mice. Cav-1 overexpression facilitated the uptake of α-syn into neurons and formation of additional Lewy body-like inclusion bodies, phosphorylation of cav-1 at tyrosine 14 was found to be crucial for this process. This study demonstrates the relationship between age and α-syn spread and will facilitate our understanding of the molecular mechanism of the cell-to-cell transmission of α-syn.

Neural stem cells derived from human midbrain organoids as a stable source for treating Parkinson's disease: Midbrain organoid-NSCs (Og-NSC) as a stable source for PD treatment.

Successful clinical translation of stem cell-based therapy largely relies on the scalable and reproducible preparation of donor cells with potent therapeutic capacities. In this study, midbrain organoids were yielded from human pluripotent stem cells (hPSCs) to prepare cells for Parkinson's disease (PD) therapy. Neural stem/precursor cells (NSCs) isolated from midbrain organoids (Og-NSCs) expanded stably and differentiated into midbrain-type dopamine(mDA) neurons, and an unprecedentedly high proportion expressed midbrain-specific factors, with relatively low cell line and batch-to-batch variations. Single cell transcriptome analysis followed by in vitro assays indicated that the majority of cells in the Og-NSC cultures are ventral midbrain (VM)-patterned with low levels of cellular senescence/aging and mitochondrial stress, compared to those derived from 2D-culture environments. Notably, in contrast to current methods yielding mDA neurons without astrocyte differentiation, mDA neurons that differentiated from Og-NSCs were interspersed with astrocytes as in the physiologic brain environment. Thus, the Og-NSC-derived mDA neurons exhibited improved synaptic maturity, functionality, resistance to toxic insults, and faithful expressions of the midbrain-specific factors, in vitro and in vivo long after transplantation. Consequently, Og-NSC transplantation yielded potent therapeutic outcomes that are reproducible in PD model animals. Collectively, our observations demonstrate that the organoid-based method may satisfy the demands needed in the clinical setting of PD cell therapy.

A Parkinson's disease gene, DJ-1, regulates anti-inflammatory roles of astrocytes through prostaglandin D2 synthase expression.

Dysfunctional regulation of inflammation may contribute to the progression of neurodegenerative diseases. The results of this study revealed that DJ-1, a Parkinson's disease (PD) gene, regulated expression of prostaglandin D2 synthase (PTGDS) and production of prostaglandin D2 (PGD2), by which DJ-1 enhanced anti-inflammatory function of astrocytes. In injured DJ-1 knockout (KO) brain, expression of tumor necrosis factor-alpha (TNF-α) was more increased, but that of anti-inflammatory heme oxygenase-1 (HO-1) was less increased compared with that in injured wild-type (WT) brain. Similarly, astrocyte-conditioned media (ACM) prepared from DJ-1-KO astrocytes less induced HO-1 expression and less inhibited expression of inflammatory mediators in microglia. With respect to the underlying mechanism, we found that PTGDS that induced expression of HO-1 was lower in DJ-1 KO astrocytes and brains compared with their WT counterparts. In addition, PTGDS levels increased in the injured brain of WT mice, but barely in that of KO mice. We also found that DJ-1 regulated PTGDS expression through Sox9. Thus, Sox9 siRNAs reduced PTGDS expression in WT astrocytes, and Sox9 overexpression rescued PTGDS expression in DJ-1 KO astrocytes. In agreement with these results, ACM from Sox9 siRNA-treated astrocytes and that from Sox9-overexpression astrocytes exerted opposite effects on HO-1 expression and anti-inflammation. These findings suggest that DJ-1 positively regulates anti-inflammatory functions of astrocytes, and that DJ-1 dysfunction contributes to the excessive inflammatory response in PD development.

Prion-like Propagation of α-Synuclein Is Regulated by the FcγRIIB-SHP-1/2 Signaling Pathway in Neurons.

Recent evidence of prion-like propagation of α-synuclein (α-syn) into neighboring neurons set up a paradigm to elucidate the mechanism of progression of Parkinson's disease (PD) and to develop therapeutic strategies. Here, we show that FcγRIIB expressed in neurons functions as a receptor for α-syn fibrils and mediates cell-to-cell transmission of α-syn. SHP-1 and 2 are activated downstream by α-syn fibrils through FcγRIIB and play an important role in cell-to-cell transmission of α-syn. Also, taking advantage of a co-culture system, we show that cell-to-cell transmission of α-syn induces intracellular Lewy body-like inclusion body formation and that the FcγRIIB/SHP-1/2 signaling pathway is involved in it. Therefore, the FcγRIIB-SHP-1/-2 signaling pathway may be a therapeutic target for the progression of PD. The in vitro system is an efficient tool for further high-throughput screening that can be used for developing a therapeutic intervention in PD.

Montelukast treatment protects nigral dopaminergic neurons against microglial activation in the 6-hydroxydopamine mouse model of Parkinson's disease.

Although the main cause of degeneration of the nigrostriatal dopaminergic (DA) projection in Parkinson's disease (PD) is still controversial, many reports suggest that excessive inflammatory responses mediated by activated microglia can induce neurotoxicity in the nigrostriatal DA system in vivo. Montelukast, which plays an anti-inflammatory role, is used to treat patients with asthma. In addition, recent studies have reported that its administration could reduce neuroinflammatory activities, showing beneficial effects against various neuropathological conditions. These results suggest that montelukast may be a useful drug to alleviate inflammatory responses in PD, even though there are no reports showing its beneficial effects against neurotoxicity in the nigrostriatal DA system. In the present study, our results showed that treatment with montelukast could protect DA neurons against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity and its administration significantly attenuated the production of neurotoxic cytokines such as tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß) from activated microglia in the substantia nigra (SN) and striatum following 6-OHDA treatment. Therefore, we suggest that montelukast can be used as a potential inhibitor of microglial activation to protect DA neurons in the adult brain against PD.

FcγRIIB mediates the inhibitory effect of aggregated α-synuclein on microglial phagocytosis.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. Although the etiology of PD has not yet been fully understood, accumulating evidence indicates that neuroinflammation plays a critical role in the progression of PD. α-Synuclein (α-Syn) has been considered to be a key player of the pathogenesis of PD, and recent reports that prion-like propagation of misfolded α-syn released from neurons may play an important role in the progression of PD have led to increased attention to the studies elucidating the roles of extracellular α-syn in the CNS. Extracellular α-syn has also been reported to regulate microglial inflammatory response. In this study, we demonstrated that aggregated α-syn inhibited microglial phagocytosis by activating SHP-1. SHP-1 activation was also observed in A53T α-syn transgenic mice. In addition, aggregated α-syn bound to FcγRIIB on microglia, inducing SHP-1 activation, further inhibiting microglial phagocytosis. Aggregated α-syn upregulated FcγRIIB expression in microglia and upregulated FcγRIIB was also observed in A53T α-syn transgenic mice. These data suggest that aggregated α-syn released from neurons dysregulates microglial immune response through inhibiting microglial phagocytosis, further causing neurodegeneration observed in PD. The interaction of aggregated α-syn and FcγRIIB and further SHP-1 activation can be a new therapeutic target against PD.

MAP kinase phosphatase-1 expression is regulated by 15-deoxy-Δ12,14-prostaglandin J2 via a HuR-dependent post-transcriptional mechanism.

In the present study, we demonstrate a mechanism through which 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) induces MKP-1 expression in rat primary astrocytes, leading to the regulation of inflammatory responses. We show that 15d-PGJ2 enhances the efficiency of MKP-1 pre-mRNA processing (constitutive splicing and 3'-end processing) and increases the stability of the mature mRNA. We further report that this occurs via the RNA-binding protein, Hu antigen R (HuR). Our experiments show that HuR knockdown abrogates the 15d-PGJ2-induced increases in the pre-mRNA processing and mature mRNA stability of MKP-1, whereas HuR overexpression further enhances the 15d-PGJ2-induced increases in these parameters. Using cysteine (Cys)-mutated HuR proteins, we show that the Cys-245 residue of HuR (but not Cys-13 or Cys-284) is critical for the direct binding of HuR with 15d-PGJ2 and the effects downstream of this interaction. Collectively, our data show that HuR is a novel target of 15d-PGJ2 and reveal HuR-mediated pre-mRNA processing and mature mRNA stabilization as important regulatory steps in the 15d-PGJ2-induced expression of MKP-1. The potential to use a small molecule such as 15d-PGJ2 to regulate the induction of MKP-1 at multiple levels of gene expression could be exploited as a novel therapeutic strategy aimed at combating a diverse range of MKP-1-associated pathologies.

Role of cysteinyl leukotriene signaling in a mouse model of noise-induced cochlear injury.

Noise-induced hearing loss is one of the most common types of sensorineural hearing loss. In this study, we examined the expression and localization of leukotriene receptors and their respective changes in the cochlea after hazardous noise exposure. We found that the expression of cysteinyl leukotriene type 1 receptor (CysLTR1) was increased until 3 d after noise exposure and enhanced CysLTR1 expression was mainly observed in the spiral ligament and the organ of Corti. Expression of 5-lipoxygenase was increased similar to that of CysLTR1, and there was an accompanying elevation of CysLT concentration. Posttreatment with leukotriene receptor antagonist (LTRA), montelukast, for 4 consecutive days after noise exposure significantly decreased the permanent threshold shift and also reduced the hair cell death in the cochlea. Using RNA-sequencing, we found that the expression of matrix metalloproteinase-3 (MMP-3) was up-regulated after noise exposure, and it was significantly inhibited by montelukast. Posttreatment with a MMP-3 inhibitor also protected the hair cells and reduced the permanent threshold shift. These findings suggest that acoustic injury up-regulated CysLT signaling in the cochlea and cochlear injury could be attenuated by LTRA through regulation of MMP-3 expression. This study provides mechanistic insights into the role of CysLTs signaling in noise-induced hearing loss and the therapeutic benefit of LTRA.

The association between airflow obstruction and radiologic change by tuberculosis.

INTRODUCTION: Cigarette smoking is the most commonly encountered risk factor for chronic obstructive pulmonary disease (COPD). However, it is not the only one and there is consistent evidence from epidemiologic studies that nonsmokers may develop chronic airflow limitation. A history of tuberculosis has recently been found to be associated with airflow obstruction in adults older than 40 years. The aim of this study was to evaluate the association between the radiologic changes by tuberculosis and airflow obstruction in a population based sample. METHODS: A nationwide COPD prevalence survey was conducted. We compared the prevalence of airflow obstruction according to the presence of the radiologic change by the tuberculosis. RESULTS: We analyzed 1,384 subjects who participated in the nationwide Korean COPD survey. All subjects were older than 40 years and took the spirometry and simple chest radiography. We defined the airflow obstruction as FEV1/FVC <0.7. A total of 149 (10.8%) subjects showed airflow obstruction. A total of 167 (12.1%) subjects showed radiologic change by tuberculosis. Among these 167 subjects, 44 (26.3%) had airflow obstruction. For the subjects without radiologic change by tuberculosis, the prevalence of airflow obstruction was only 8.6%. The unadjusted odds ratio for airflow obstruction according to the radiologic change was 3.788 (95% CI: 2.544-5.642). CONCLUSIONS: The radiologic change by tuberculosis was associated with airflow obstruction.

DJ-1 facilitates the interaction between STAT1 and its phosphatase, SHP-1, in brain microglia and astrocytes: A novel anti-inflammatory function of DJ-1.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra. Importantly, altered astrocyte and microglial functions could contribute to neuronal death in PD. In this study, we demonstrate a novel mechanism by which DJ-1 (PARK7), an early onset autosomal-recessive PD gene, negatively regulates inflammatory responses of astrocytes and microglia by facilitating the interaction between STAT1 and its phosphatase, SHP-1 (Src-homology 2-domain containing protein tyrosine phosphatase-1). Astrocytes and microglia cultured from DJ-1-knockout (KO) mice exhibited increased expression of inflammatory mediators and phosphorylation levels of STAT1 (p-STAT1) in response to interferon-gamma (IFN-γ) compared to cells from wild-type (WT) mice. DJ-1 deficiency also attenuated IFN-γ-induced interactions of SHP-1 with p-STAT1 and STAT1, measured 1 and 12h after IFN-γ treatment, respectively. Subsequent experiments showed that DJ-1 directly interacts with SHP-1, p-STAT1, and STAT1. Notably, DJ-1 bound to SHP-1 independently of IFN-γ, whereas the interactions of DJ-1 with p-STAT1 and STAT1 were dependent on IFN-γ. Similar results were obtained in brain slice cultures, where IFN-γ induced much stronger STAT1 phosphorylation and inflammatory responses in KO slices than in WT slices. Moreover, IFN-γ treatment induced neuronal damage in KO slices. Collectively, these findings suggest that DJ-1 may function as a scaffold protein that facilitates SHP-1 interactions with p-STAT1 and STAT1, thereby preventing extensive and prolonged STAT1 activation. Thus, the loss of DJ-1 function may increase the risk of PD by enhancing brain inflammation.

DJ-1 associates with lipid rafts by palmitoylation and regulates lipid rafts-dependent endocytosis in astrocytes.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disease. Several genes have been associated with familial type PD, providing tremendous insights into the pathogenesis of PD. Gathering evidence supports the view that these gene products may operate through common molecular pathways. Recent reports suggest that many PD-associated gene products, such as α-synuclein, LRRK2, parkin and PINK1, associate with lipid rafts and lipid rafts may be associated with neurodegeneration. Here, we observed that DJ-1 protein also associated with lipid rafts. Palmitoylation of three cysteine residues (C46/53/106) and C-terminal region of DJ-1 were required for this association. Lipopolysaccharide (LPS) induced the localization of DJ-1 into lipid rafts in astrocytes. The LPS-TLR4 signaling was more augmented in DJ-1 knock-out astrocytes by the impairment of TLR4 endocytosis. Furthermore, lipid rafts-dependent endocytosis including the endocytosis of CD14, which play a major role in regulating TLR4 endocytosis was also impaired, but clathrin-dependent endocytosis was not. This study provides a novel function of DJ-1 in lipid rafts, which may contribute the pathogenesis of PD. Moreover, it also provides the possibility that many PD-related proteins may operate through common molecular pathways in lipid rafts.

PINK1 deficiency attenuates astrocyte proliferation through mitochondrial dysfunction, reduced AKT and increased p38 MAPK activation, and downregulation of EGFR.

PINK1 (PTEN induced putative kinase 1), a familial Parkinson's disease (PD)-related gene, is expressed in astrocytes, but little is known about its role in this cell type. Here, we found that astrocytes cultured from PINK1-knockout (KO) mice exhibit defective proliferative responses to epidermal growth factor (EGF) and fetal bovine serum. In PINK1-KO astrocytes, basal and EGF-induced p38 activation (phosphorylation) were increased whereas EGF receptor (EGFR) expression and AKT activation were decreased. p38 inhibition (SB203580) or knockdown with small interfering RNA (siRNA) rescued EGFR expression and AKT activation in PINK1-KO astrocytes. Proliferation defects in PINK1-KO astrocytes appeared to be linked to mitochondrial defects, manifesting as decreased mitochondrial mass and membrane potential, increased intracellular reactive oxygen species level, decreased glucose-uptake capacity, and decreased ATP production. Mitochondrial toxin (oligomycin) and a glucose-uptake inhibitor (phloretin) mimicked the PINK1-deficiency phenotype, decreasing astrocyte proliferation, EGFR expression and AKT activation, and increasing p38 activation. In addition, the proliferation defect in PINK1-KO astrocytes resulted in a delay in the wound healing process. Taken together, these results suggest that PINK1 deficiency causes astrocytes dysfunction, which may contribute to the development of PD due to delayed astrocytes-mediated repair of microenvironment in the brain.

Role of vitamin D-binding protein in isocyanate-induced occupational asthma.

The development of a serological marker for early diagnosis of isocyanate-induced occupational asthma (isocyanate-OA) may improve clinical outcome. Our previous proteomic study found that expression of vitamin D-binding protein (VDBP) was upregulated in the patients with isocyanate-OA. In the present study, we evaluated the clinical relevance of VDBP as a serological marker in screening for isocyanate-OA among exposed workers and its role in the pathogenesis of isocyanate-OA. Three study groups including 61 patients with isocyanate-OA (group I), 180 asymptomatic exposed controls (AECs, group II), 58 unexposed healthy controls (NCs, group III) were enrolled in this study. The baseline serum VDBP level was significantly higher in group I compared with groups II and III. The sensitivity and specificity for predicting the phenotype of isocyanate-OA with VDBP were 69% and 81%, respectively. The group I subjects with high VDBP (≥311 µg/ml) had significantly lower PC(20) methacholine levels than did subjects with low VDBP. The in vitro studies showed that TDI suppressed the uptake of VDBP into RLE-6TN cells, which was mediated by the downregulation of megalin, an endocytic receptor of the 25-hydroxycholecalciferol-VDBP complex. Furthermore, toluene diisocyanate (TDI) increased VEGF production and secretion from this epithelial cells by suppression of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] production. The findings of this study suggest that the serum VDBP level may be used as a serological marker for the detection of isocyanate-OA among workers exposed to isocyanate. The TDI-induced VEGF production/ secretion was reversed by 1,25(OH)(2)D(3) treatment, which may provide a potential therapeutic strategy for patients with isocyanate-OA.

Differential SUMOylation of LXRalpha and LXRbeta mediates transrepression of STAT1 inflammatory signaling in IFN-gamma-stimulated brain astrocytes.

To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-gamma-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFalpha, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRbeta formed a trimer with PIAS1-pSTAT1, whereas LXRalpha formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRbeta and LXRalpha were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFalpha expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent inflammatory responses by LXRs in IFN-gamma-stimulated brain astrocytes.

Functional implication of BAFF synthesis and release in gangliosides-stimulated microglia.

BAFF is a recently identified member of the TNF ligand superfamily that plays a critical role in B cell differentiation, survival, and regulation of Ig production. In the present study, we examined whether BAFF is expressed in microglia, and the expression and release of BAFF are regulated by gangliosides. The results showed that BAFF was expressed and released in rat primary microglia as well as in BV-2 cells. Furthermore, its expression and release were increased by gangliosides stimulation and regulated by JAK-STAT, especially the STAT1- and STAT3-dependent signaling pathways. It was of particular interest to observe that SP600125 and SB203580, specific inhibitors of JNK and p38, did not inhibit BAFF synthesis but inhibited the release of sBAFF in gangliosides-treated cells by regulating furin expression, suggesting that the JNK and p38 signaling pathways regulate the release but not the synthesis of BAFF. Moreover, BV-2 cells expressed BAFF-R on their cell surface, and rat primary microglia expressed BAFF-R and TACI on their cell surface. rBAFF increased the release of cytokines, especially IL-6, TNF-alpha, and IL-10, in rat primary microglia as well as in BV-2 cells. These findings imply that BAFF secreted by microglia may play important roles in CNS inflammation by regulating microglia as well as infiltrated B cells.

On the mechanism of internalization of alpha-synuclein into microglia: roles of ganglioside GM1 and lipid raft.

ALpha-synuclein (alpha-syn) has been known to be a key player of the pathogenesis of Parkinson's disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of alpha-syn into cells has also been observed. In this study, we observed that dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric alpha-syn into BV-2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of alpha-syn internalization in dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-treated BV-2 cells. Furthermore, inhibition of clathrin-, caveolae-, and dynamin-dependent endocytosis did not prevent the internalization of alpha-syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of alpha-syn. In addition, we further confirmed these observations by co-culture system of BV-2 cells and alpha-syn-over-expressing SH-SY5Y cells. These findings suggest that extracellular alpha-syn is internalized into microglia via GM1 as well as hitherto-unknown protein receptors in clathrin-, caveolae-, and dynamin-independent, but lipid raft-dependent manner. Elucidation of the mechanism involved in internalization of alpha-syn should be greatly helpful in the development of new treatments of alpha-syn-related neurodegenerative diseases.

Identification of the amino acid sequence motif of alpha-synuclein responsible for macrophage activation.

Alpha-synuclein (Syn) is implicated in the pathogenesis of PD and related neurodegenerative disorders. Recent studies have also shown that alpha-synuclein can activate microglia and enhance dopaminergic neurodegeneration. The mechanisms of microglia activation by alpha-synuclein, however, are not well understood. In this study, we found that not only alpha-synuclein but also beta- and gamma-synucleins activated macrophages (RAW 264.7) in vitro. Macrophages treated with synuclein proteins secreted TNF-alpha in a dose-dependent manner. Synuclein family proteins also increased mRNA transcription of COX-2 and iNOS. Two alpha-synuclein deletion mutants, SynDeltaNAC and Syn61-140, activated macrophages, while deletion mutants Syn1-60 and Syn96-140 did not significantly activate them. Finally, we demonstrated that macrophage activation by alpha-synuclein was accompanied by phosphorylation of ERK. These results suggest that synuclein family proteins can activate macrophages, and that macrophage activation needs both the N-terminal and C-terminal domains of alpha-synuclein, but not the central NAC region.

Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins.

The acidic tail of alpha-synuclein (ATSalpha) has been shown to protect the glutathione S-transferase (GST)-ATSalpha fusion protein from environmental stresses, such as heat, pH and metal ions. In this study, we further demonstrated that the introduction of ATSalpha into other proteins, such as dehydrofolate reductase and adiponectin, renders the fusion proteins resistant to heat-induced aggregation and that the acidic tail of beta- or gamma-synuclein can also protect the fusion proteins from heat-induced aggregation. Interestingly, the heat resistance of GST-ATSalpha deletion mutants, which contain shorter peptides derived from the highly charged regions of ATSalpha, was approximately proportional to the number of added Glu/Asp residues. However, the negative charges in the ATSalpha-derived peptides appear insufficient to explain the extreme heat resistance of the fusion proteins, since polyglutamates appeared to be much less effective than the ATSalpha-derived peptides in conferring heat resistance on the fusion proteins. These results suggest that not only the negatively charged residues but also the specific amino acid sequence of ATSalpha play an important role in conferring extreme heat resistance on the fusion proteins. Furthermore, the heat-induced secondary structural changes and thermal inactivation curves of GST-ATSalpha deletion mutants indicated that the introduction of ATSalpha-derived peptides does not significantly affect the intrinsic stability of the fusion proteins.