Katrin Beyer

α-Synuclein structure, posttranslational modification and alternative splicing as aggregation enhancers

Abstractα-Synuclein aggregation is thought to be a key event in the pathogenesis of synucleinopathies. Although different α-synuclein alterations and modifications have been proposed to be responsible for early aggregation steps, the mechanisms underlying these events remain unclarified. α-Synuclein is a small protein localized to synaptic terminals and its intrinsic structure has been claimed to be an important factor for self-oligomerization and self-aggregation. α-Synuclein expression studies in cell cultures have demonstrated that posttranslational modifications, such as phosphorylation, oxidation, and sumoylation, are primarily involved in α-synuclein aggregation. Furthermore, in the last few years accumulating evidence has pointed to alternative splicing as a crucial mechanism in the development of neurodegenerative disorders. At least three different α-synuclein isoforms have been described as products of alternative splicing. Two of these isoforms (α-synuclein 112 and α-synuclein 126) are shorter proteins with probably altered functions and aggregation propensity. The present review attempts to summarize the data so far available on α-synuclein structure, posttranslational modifications, and alternative splicing as possible enhancers of aggregation.

Alpha-Synuclein Posttranslational Modification and Alternative Splicing as a Trigger for Neurodegeneration

Lewy body diseases include Parkinson disease and dementia with Lewy bodies and are characterized by the widespread distribution of Lewy bodies in virtually every brain area. The main component of Lewy bodies is alpha-synuclein (AS). Accumulating evidence suggests that AS oligomerization and aggregation are strongly associated with the pathogenesis of Lewy body diseases. AS is a small soluble protein with aggregation-prone properties under certain conditions. These properties are enhanced by posttranslational modifications such as phosphorylation, ubiquitination, nitration, and truncation. Accordingly, Lewy bodies contain abundant phosphorylated, nitrated, and monoubiquitinated AS. However, alternative splicing of the AS gene is also known to modify AS aggregation propensities. Splicing gives rise to four related forms of the protein, the main transcript and those that lack exon 4, exon 6, or both. Since AS structure and properties have been extensively studied, it is possible to predict the consequences of the splicing out of the two aforesaid exons. The present review discusses the latest insights on the mechanisms of AS posttranslational modifications and intends to depict their role in the pathogenesis of Lewy body diseases. The implications of deregulated alternative splicing are examined as well, and a hypothesis for the development of the pure form of dementia with Lewy bodies is proposed.

Alzheimer’s disease and the cystatin C gene polymorphism: an association study

Cystatin C is an amyloidogenic protein that colocalizes with beta-amyloid (Abeta) within arteriolar walls in Alzheimer disease (AD) brains. Recently, a coding polymorphism in the cystatin C gene (CST3) has been claimed to confer risk for the development of late-onset AD. In the present work we have tested the frequencies of CST3-A and CST3-G alleles and used chi-square and logistic regression analyses to assess the association among the CST3 polymorphism, apolipoprotein E4 (APOE4), and AD in a series of 159 AD patients and 155 controls. The CST3-A allele was seen to be an accumulation risk factor for early-onset AD. Furthermore, a synergistic association among the CST3-A allele, APOE4 and AD was found in AD patients whose ages were between 60 and 74 years.

Differential expression of alpha-synuclein, parkin, and synphilin-1 isoforms in Lewy body disease

Alpha-synuclein, parkin, and synphilin-1 are proteins mainly involved in the pathogenesis of Lewy body (LB) diseases. mRNAs of all three undergo alternative splicing, so that the existence of various isoforms has been described. Since increasing evidence supports the importance of differential isoform-expression changes in disease development, we have established isoform-expression profiles in frontal cortices of LB disease brains in comparison with those of Alzheimer disease (AD) and control frontal cortices. The differential expression of four alpha-synuclein, seven parkin, and four synphilin-1 isoforms was ascertained by the use of isoform-specific primers and relative expression analysis with SybrGreen and beta-actin as an internal standard. The establishment of isoform-expression profiles revealed that these are disease specific. Moreover, isoform-expression deregulation of mainly one gene in each disease could be observed. All four alpha-synuclein isoforms were affected in the case of the pure form of dementia with LB, most parkin transcript variants in common LB disease, and all synphilin-1 isoforms in Parkinson disease. Only minor involvement was detected in AD. Finally, the existence of a proprietary isoform-expression profile in common LB disease indicates that this disease develops as a result of its own molecular mechanisms, and so, at the molecular level, it does not exactly share changes found in pure dementia with LB and AD. In conclusion, isoform-expression profiles in LB diseases represent additional evidence for the direct involvement of isoform-expression deregulation in the development of neurodegenerative disorders.

Identification and characterization of a new alpha-synuclein isoform and its role in Lewy body diseases

Alternative splicing is an important mechanism to generate a large number of mRNAs, thus increasing proteome diversity and tissue specificity. Three transcript variants of alpha-synuclein, a neuronal protein mainly involved in synapses, have been described so far. Whereas alpha-synuclein 140 is the whole and main transcript, alpha-synuclein 112 and 126 are short proteins that result from in-frame deletions of exons 3 and 5, respectively. Because the aforesaid alpha-synuclein isoforms show differential expression changes in Lewy body diseases (LBDs), in the present work, we searched for a fourth alpha-synuclein isoform and studied its expression levels in LBD brains. By using isoform-specific primers, isoform co-amplification and direct sequencing, we identified alpha-synuclein 98, which lacks exons 3 and 5. mRNA expression analyses in non-neuronal tissue revealed that alpha-synuclein 98 is a brain-specific splice variant with varying expression levels in different areas of fetal and adult brain. Additionally, we studied alpha-synuclein 98 expression levels by real-time semi-quantitative RT-PCR in the frontal cortices of LBD patients and compared them with those of Alzheimer disease (AD) patients and control subjects. Overexpression of alpha-synuclein 98 in LBD and AD brains would indicate its specific involvement in the pathogenesis of these neurodegenerative disorders.

Protein aggregation mechanisms in synucleinopathies : Commonalities and differences

Synucleinopathies are characterized by the presence of different types of α-synuclein (AS)-positive inclusion in the brain. Thus, whereas Lewy bodies are the hallmark of Parkinson disease and dementia with Lewy bodies, glial and neuronal cytoplasmic inclusions are shown by multiple system atrophy. Because the main component of all these inclusions is conformationally modified AS, aggregation of the latter is thought to be a key pathogenic event in these diseases. Although very little information has been available on AS function and aggregation mechanisms until 2 years ago, recent investigations have greatly improved our understanding of the steps involved in the pathogenesis of synucleinopathies. Additionally, important insights into the specific molecular events (e.g. differential posttranslational modifications or isoform expression profiles) underlying each of these conditions have been gained. The present review summarizes our current knowledge of the commonalities and differences shown by protein aggregation mechanisms in the various synucleinopathies.

Differential expression of α-synuclein isoforms in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is characterized by the widespread presence of Lewy bodies (LBs) in the brain. α‐Synuclein, the main component of LBs, is expressed as two main isoforms (112 and 140), but little is known about their differential expression in the brain. We compared α‐synuclein 112 and α‐synuclein 140 expression levels in the prefrontal cortices of six DLB patients, eight Alzheimer disease (AD) patients, and six control subjects. Relative α‐synuclein 112 and α‐synuclein 140 expression levels were determined by real‐time polymerase chain reaction with competimer technology using a LightCycler System. Whereas total α‐synuclein levels were just marginally elevated in DLB in comparison with the other groups, α‐synuclein 112 was seen to be markedly increased in DLB compared with AD cases and controls. In contrast, α‐synuclein 140 levels were significantly diminished in both neurodegenerative disorders in comparison with controls. These results show differential overexpression of α‐synuclein 112 in DLB, a finding that could be of importance in DLB pathogenesis.

Methionine synthase polymorphism is a risk factor for Alzheimer disease.

&NA; Alzheimer disease (AD) patients show increased plasma levels of homocysteine, whose conversion to methionine is catalyzed by methionine synthase (MS). Although altered MS activity may result from the MS A2756G polymorphism, the latters possible association with AD remains unexplored. To assess whether the MS A2756G polymorphism holds any influence on AD risk, we have analyzed 172 AD patients and 166 controls. We have also investigated whether the MS‐A or MS‐G allele interacts with the APOE4 allele. Our results indicate that association with the MS‐AA genotype is an APOE4 allele‐independent risk factor for AD. These findings provide novel evidence implicating genetic enzymatic alterations of homocysteine metabolic pathways in the pathogenesis of AD.

Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles' characteristics compared to precipitating agents.

Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR, and their total protein content, NTA and Cryo-electron microscopy profiles, and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs.

Age at onset: an essential variable for the definition of genetic risk factors for sporadic Alzheimer's disease.

The aim of our work was to detect minor loci acting as Alzheimers disease (AD) genetic markers. We divided 206 AD patients and 186 individuals as controls into six age at onset/age‐dependent groups. We studied polymorphisms of the genes of apolipoprotein E (APOE) and its promoter, cathepsin D, butyrylcholinesterase, cystatin C, methionine synthase, and cystathionine beta‐synthase. Our results demonstrated that data analysis according to age at onset allows the detection of minor genetic risk factors for AD. Thus, the Th1/E47cs‐G allele was an independent AD risk factor after 80 years, whereas the catD‐T, BChE‐K, CBS‐844ins68, and CBS‐VNTR 19 alleles are independent AD risk factors after 75 years. On the other hand, the CST3‐A allele was an independent AD risk factor before 60 years while the CBS‐VNTR allele 21 was an independent AD risk factor before 64 years. In contrast, the MS‐AA genotype was an AD risk factor unrelated to age at onset. In conclusion, two main tasks remain to be accomplished to facilitate early detection of people at risk of developing AD: (1) the establishment of common criteria to carry out association studies for different genetic markers, including the introduction of AD age at onset as a crucial variable in each study, and (2) the definition of global and population‐specific genetic markers for each age at onset AD subgroup.