Selective molecular alterations in the autophagy pathway in patients with Lewy body disease and in models of alpha-synucleinopathy.
ABSTRACT: BACKGROUND:Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by alpha-synuclein accumulation that includes dementia with Lewy bodies (DLB) and Parkinson's Disease (PD). Recent evidence suggests that impairment of lysosomal pathways (i.e. autophagy) involved in alpha-synuclein clearance might play an important role. For this reason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLB and Alzheimer's Disease (AD) and in alpha-synuclein transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS:By immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor were elevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 and Beclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying alpha-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, and ultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations were observed in the brains of alpha-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injection of a lentiviral vector expressing Atg7 resulted in reduced accumulation of alpha-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations. CONCLUSIONS/SIGNIFICANCE:This study supports the notion that defects in the autophagy pathway and more specifically in mTor and Atg7 are associated with neurodegeneration in DLB cases and alpha-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects.
Project description:Dementia with Lewy bodies (DLB) represents a huge medical need as it accounts for up to 30% of all dementia cases, and there is no cure available. The underyling spectrum of pathology is complex and creates a challenge for targeted molecular therapies. We here tested the hypothesis that leukotrienes are involved in the pathology of DLB and that blocking leukotrienes through Montelukast, a leukotriene receptor antagonist and approved anti-asthmatic drug, might alleviate pathology and restore cognitive functions. Expression of 5-lipoxygenase, the rate-limiting enzyme for leukotriene production, was indeed elevated in brains with DLB. Treatment of cognitively deficient human alpha-synuclein overexpressing transgenic mice with Montelukast restored memory. Montelukast treatment resulted in modulation of beclin-1 expression, a marker for autophagy, and in a reduction in the human alpha-synulcein load in the transgenic mice. Reducing the protein aggregation load in neurodegenerative diseases might be a novel model of action of Montelukast. Moreover, this work presents leukotriene signaling as a potential drug target for DLB and shows that Montelukast might be a promising drug candidate for future DLB therapy development.
Project description:α-Synuclein is a major component of Lewy bodies in Parkinson disease (PD) and dementia with Lewy bodies (DLB). We recently showed that abnormal α-synuclein with resistance to proteinase K (PK) is deposited at presynapses of distinct brain anatomic regions from the early stages of PD and DLB. NUB1, a synphilin-1-binding protein, also accumulates in Lewy bodies, but it is not known whether abnormal α-synuclein is associated with NUB1. Here, we demonstrate that, in the brain of patients with PD and DLB, NUB1 accumulates in the presynapses in the hippocampus, cerebral neocortex, and substantia nigra in which PK-resistant α-synuclein is deposited. Endogenous NUB1 also accumulated with PK-resistant α-synuclein in the presynapses of transgenic mice that express human α-synuclein with an A53T mutation. Immunoelectron microscopy showed that NUB1 is localized to presynaptic nerve terminals where no abnormal filaments are seen. Biochemical analyses showed that NUB1 coexists with abnormal α-synuclein in the brain of DLB patients. These findings suggest that NUB1 along with abnormal α-synuclein is involved in the pathogenesis of Lewy body disease.
Project description:Synucleinopathies like Parkinson disease and dementia with Lewy bodies (DLB) are characterized by ?-synuclein aggregates within neurons (Lewy bodies) and their processes (Lewy neurites). Whereas ?-synuclein has been genetically linked to the disease process, the pathological relevance of ?-synuclein aggregates is still debated. Impaired degradation is considered to result in aggregation of ?-synuclein. In addition to the ubiquitin-proteasome degradation, the autophagy-lysosomal pathway (ALP) is involved in intracellular degradation processes for ?-synuclein. Here, we asked if modulation of ALP affects ?-synuclein aggregation and toxicity. We have identified an induction of the ALP markers LAMP-2A and LC3-II in human brain tissue from DLB patients, in a transgenic mouse model of synucleinopathy, and in a cell culture model for ?-synuclein aggregation. ALP inhibition using bafilomycin A 1 (BafA1) significantly potentiates toxicity of aggregated ?-synuclein species in transgenic mice and in cell culture. Surprisingly, increased toxicity is paralleled by reduced aggregation in both in vivo and in vitro models. The dichotomy of effects on aggregating and nonaggregating species of ?-synuclein was specifically sensitive to BafA1 and could not be reproduced by other ALP inhibitors. The present study expands on the accumulating evidence regarding the function of ALP for ?-synuclein degradation by isolating an aggregation specific, BafA1-sensitive, ALP-related pathway. Our data also suggest that protein aggregation may represent a detoxifying event rather than being causal for cellular toxicity.
Project description:DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of ?-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of ?-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and ?-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in ?-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB.
Project description:Alzheimer's disease (AD), the most prevalent age-related neurodegenerative disorder, is characterized pathologically by the accumulation of beta-amyloid (Abeta) plaques and tau-laden neurofibrillary tangles. Interestingly, up to 50% of AD cases exhibit a third prevalent neuropathology: the aggregation of alpha-synuclein into Lewy bodies. Importantly, the presence of Lewy body pathology in AD is associated with a more aggressive disease course and accelerated cognitive dysfunction. Thus, Abeta, tau, and alpha-synuclein may interact synergistically to promote the accumulation of each other. In this study, we used a genetic approach to generate a model that exhibits the combined pathologies of AD and dementia with Lewy bodies (DLB). To achieve this goal, we introduced a mutant human alpha-synuclein transgene into 3xTg-AD mice. As occurs in human disease, transgenic mice that develop both DLB and AD pathologies (DLB-AD mice) exhibit accelerated cognitive decline associated with a dramatic enhancement of Abeta, tau, and alpha-synuclein pathologies. Our findings also provide additional evidence that the accumulation of alpha-synuclein alone can significantly disrupt cognition. Together, our data support the notion that Abeta, tau, and alpha-synuclein interact in vivo to promote the aggregation and accumulation of each other and accelerate cognitive dysfunction.
Project description:We applied RT-QuIC assay to detect α-synuclein aggregates in cerebrospinal fluid (CSF) of patients with suspected Creutzfeldt-Jakob disease who had a neuropathological diagnosis of dementia with Lewy bodies (DLB) (n = 7), other neurodegenerative diseases with α-synuclein mixed pathology (n = 20), or without Lewy-related pathology (n = 49). The test had a sensitivity of 92.9% and specificity of 95.9% in distinguishing α-synucleinopathies from non-α-synucleinopathies. When performed in the CSF of patients with DLB (n = 36), RT-QuIC was positive in 17/20 with probable DLB, 0/6 with possible DLB, and 0/10 with Alzheimer disease. These results indicate that RT-QuIC for α-synuclein is an accurate test for DLB diagnosis.
Project description:OBJECTIVE:To determine whether mutations in the genes for alpha-synuclein or beta-synuclein are responsible for dementia with Lewy bodies (DLB), a disorder closely related to Parkinson disease (PD). METHODS:The authors ascertained 33 sporadic cases of DLB and 10 kindreds segregating DLB. DNA samples from the 43 index cases were screened for alterations in the genes for alpha-synuclein and beta-synuclein, as alpha-synuclein alterations cause PD and beta-synuclein may modulate alpha-synuclein aggregation and neurotoxicity. RESULTS:Two amino acid alterations were identified in unrelated DLB index cases: a valine to methionine substitution at codon 70 (V70M) and a proline to histidine substitution at codon 123 (P123H), both in the beta-synuclein gene. These amino acid substitutions occur at conserved residues in highly conserved regions of the beta-synuclein protein. Screening of at least 660 chromosomes from control subjects matched to the patients' population groups failed to identify another V70M or P123H allele. Cosegregation analysis of an extended pedigree segregating the P123H beta-synuclein alteration suggested that it is a dominant trait with reduced penetrance or a risk factor polymorphism. Histopathology and immunohistochemistry analysis of index case brain sections revealed widespread Lewy body pathology and alpha-synuclein aggregation without evidence of beta-synuclein aggregation. CONCLUSION:Mutations in the beta-synuclein gene may predispose to DLB.
Project description:Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of alpha-synuclein (alpha-Syn), and although it is now recognized that alpha-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of alpha-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a beta-sheet structure. To determine whether calpain-cleavage of alpha-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to alpha-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved alpha-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved alpha-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved alpha-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of alpha-Syn in various alpha-synucleinopathies.
Project description:BACKGROUND:Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of inclusions known as Lewy bodies in some brain regions. Lewy bodies consist of α-synuclein and many other proteins including chaperones. OBJECTIVE:To learn more about the role of chaperone complexes in PD and a related disorder, i.e., dementia with Lewy bodies (DLB), in this work we analyzed the expression of HSP90 and its two quite recently identified co-chaperones, SGT1 and CHP-1, in selected brain regions from patients suffering from these diseases. METHODS:To fulfill the aim of our study we used human material and applied immunohistochemistry, Western blot analysis and real time/quantitative PCR (RT-qPCR). RESULTS:We have found that HSP90 mRNA level is higher in the temporal cortex of PD and in frontal cortex of DLB brains, even though level of protein does not change significantly. The mRNA level of SGT1 is higher in the frontal and temporal cortex of PD and in substantia nigra of DLB brains while no significant changes in the level of protein were noticed. Similarly, the mRNA level of CHP-1 was found to be higher in the frontal and temporal cortex of PD and in all examined regions i.e. substantia nigra, frontal and temporal cortex of DLB brains. In the case of CHP-1 the protein level was found to be higher in frontal cortex of PD and in all examined areas of DLB patients. CONCLUSIONS:Our data indicate that the level of HSP90, SGT1 and CHP-1 is upregulated in the majority of cases of PD and DLB, which suggests that the examined proteins might be involved in these pathologies.
Project description:Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of ?-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of A?(1-42) and pGlu-A?(3-42) on the aggregation of ?-synuclein in vitro. The aggregation of human recombinant ?-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of ?-synuclein in the presence of minor concentrations of A?(1-42) and pGlu-A?(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of ?-synuclein and A? in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of ?-synuclein and A? and pGlu-A?, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides ?-synuclein and A? species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.