Sandra Tenreiro

Protein phosphorylation in neurodegeneration: friend or foe?

Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimers disease (AD), Parkinsons disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system.

SNCA (α-synuclein)-induced toxicity in yeast cells is dependent on sirtuin 2 (Sir2)-mediated mitophagy

SNCA (α-synuclein) misfolding and aggregation is strongly associated with both idiopathic and familial forms of Parkinson disease (PD). Evidence suggests that SNCA has an impact on cell clearance routes and protein quality control systems such as the ubiquitin-proteasome system (UPS) and autophagy. Recent advances in the key role of the autosomal recessive PARK2/PARKIN and PINK1 genes in mitophagy, highlighted this process as a prominent new pathogenic mechanism. Nevertheless, the role of autophagy/mitophagy in the pathogenesis of sporadic and autosomal dominant familial forms of PD is still enigmatic. The yeast Saccharomyces cerevisiae is a powerful “empty room” model that has been exploited to clarify different molecular aspects associated with SNCA toxicity, which combines the advantage of being an established system for aging research. The contribution of autophagy/mitophagy for the toxicity induced by the heterologous expression of the human wild-type SNCA gene and the clinical A53T mutant during yeast chronological life span (CLS) was explored. A reduced CLS together with an increase of autophagy and mitophagy activities were observed in cells expressing both forms of SNCA. Impairment of mitophagy by deletion of ATG11 or ATG32 resulted in a CLS extension, further implicating mitophagy in the SNCA toxicity. Deletion of SIR2, essential for SNCA toxicity, abolished autophagy and mitophagy, thereby rescuing cells. These data show that Sir2 functions as a regulator of autophagy, like its mammalian homolog, SIRT1, but also of mitophagy. Our work highlights that increased mitophagy activity, mediated by the regulation of ATG32 by Sir2, is an important phenomenon linked to SNCA-induced toxicity during aging.

Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.

Harnessing the power of yeast to unravel the molecular basis of neurodegeneration

Several neurodegenerative diseases, such as Parkinsons disease (PD), Alzheimers disease (AD), Huntingtons disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration has been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well‐characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders.

Simple is good: yeast models of neurodegeneration

The budding yeast, Saccharomyces cerevisiae, is the best-studied eukaryotic cell, at both genetic and physiological levels. As a eukaryote, yeast shares highly conserved molecular and cellular mechanisms with human cells. Thus, this simple fungus is an invaluable model to study the fundamental molecular mechanisms involved in several human diseases. In the particular case of neurodegenerative disorders, yeast models have been able to recapitulate several important features of complex and devastating disorders, such as Huntingtons and Parkinsons diseases. Once validated, these models have also been used to accelerate the identification of both novel therapeutic targets and compounds with therapeutic potential. Here, we review the recent contributions of this simple, but powerful model organism toward our understanding of neurodegeneration.

Visualization of cell-to-cell transmission of mutant huntingtin oligomers.

We developed a new cell model for the visualization of toxic huntingtin oligomers in living cells. Huntingtin exon 1 (25Q or 103Q) was fused to non-fluorescent halves of the Venus protein. When huntingtin dimerizes inside the cells, Venus becomes functionally reconstituted and emits fluorescence. Oligomerization, aggregation and toxicity of mutant huntingtin were assessed by several procedures. We also present evidence that the transmission of huntingtin between cells can be determined in a quantitative manner with our model. Thus, this model can be a powerful screening tool for the identification of modifiers of oligomerization and cell-to-cell traffic of mutant huntingtin.

DJ-1 interactions with α -synuclein attenuate aggregation and cellular toxicity in models of Parkinson’s disease

Parkinson’s disease (PD) is a devastating neurodegenerative disorder characterized by the loss of neurons in the substantia nigra pars compacta and the presence of Lewy bodies in surviving neurons. These intracellular protein inclusions are primarily composed of misfolded α-synuclein (aSyn), which has also been genetically linked to familial and sporadic forms of PD. DJ-1 is a small ubiquitously expressed protein implicated in several pathways associated with PD pathogenesis. Although mutations in the gene encoding DJ-1 lead to familial early-onset PD, the exact mechanisms responsible for its role in PD pathogenesis are still elusive. Previous work has found that DJ-1 – which has protein chaperone-like activity – modulates aSyn aggregation. Here, we investigated possible physical interactions between aSyn and DJ-1 and any consequent functional and pathological relevance. We found that DJ-1 interacts directly with aSyn monomers and oligomers in vitro, and that this also occurs in living cells. Notably, several PD-causing mutations in DJ-1 constrain this interaction. In addition, we found that overexpression of DJ-1 reduces aSyn dimerization, whereas mutant forms of DJ-1 impair this process. Finally, we found that human DJ-1 as well as yeast orthologs of DJ-1 reversed aSyn-dependent cellular toxicity in Saccharomyces cerevisiae. Taken together, these data suggest that direct interactions between DJ-1 and aSyn constitute the basis for a neuroprotective mechanism and that familial mutations in DJ-1 may contribute to PD by disrupting these interactions.

(Poly)phenols protect from α-synuclein toxicity by reducing oxidative stress and promoting autophagy

Parkinsons disease (PD) is the most common movement neurodegenerative disorder and is associated with the aggregation of α-synuclein (αSyn) and oxidative stress, hallmarks of the disease. Although the precise molecular events underlying αSyn aggregation are still unclear, oxidative stress is known to contribute to this process. Therefore, agents that either prevent oxidative stress or inhibit αSyn toxicity are expected to constitute potential drug leads for PD. Both pre-clinical and clinical studies provided evidence that (poly)phenols, pure or in extracts, might protect against neurodegenerative disorders associated with oxidative stress in the brain. In this study, we analyzed, for the first time, a (poly)phenol-enriched fraction (PEF) from leaves of Corema album, and used in vitro and cellular models to evaluate its effects on αSyn toxicity and aggregation. Interestingly, the PEF promoted the formation of non-toxic αSyn species in vitro, and inhibited its toxicity and aggregation in cells, by promoting the autophagic flux and reducing oxidative stress. Thus, C. album (poly)phenols appear as promising cytoprotective compounds, modulating central events in the pathogenesis of PD, such as αSyn aggregation and the impairment of autophagy. Ultimately, the understanding of the molecular effects of (poly)phenols will open novel opportunities for the exploitation of their beneficial effects and for drug development.

Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines, in Yeast, and in Vivo

Background: Little is known about the effect of PD-linked mutations on α-synuclein (α-syn) phosphorylation. Results: The E46K mutation increases α-syn phosphorylation at Ser-129 and influences its subcellular localization in vivo. Conclusion: The E46K mutation alters α-syn localization and post-translational modifications. Significance: PD-linked α-syn mutations may contribute to PD via different mechanisms. Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.

PLK2 modulates α-synuclein aggregation in yeast and mammalian cells.

Phosphorylation of α-synuclein (aSyn) on serine 129 is one of the major post-translation modifications found in Lewy bodies, the typical pathological hallmark of Parkinson’s disease. Here, we found that both PLK2 and PLK3 phosphorylate aSyn on serine 129 in yeast. However, only PLK2 increased aSyn cytotoxicity and the percentage of cells presenting cytoplasmic foci. Consistently, in mammalian cells, PLK2 induced aSyn phosphorylation on serine 129 and induced an increase in the size of the inclusions. Our study supports a role for PLK2 in the generation of aSyn inclusions by a mechanism that does not depend directly on serine 129 phosphorylation.