Tiago F. Outeiro

α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells

Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein-containing (α-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology.

Interactions among α-synuclein, dopamine, and biomembranes

Parkinson’s disease (PD) is a neurologic disorder resulting from the loss of dopaminergic neurons in the brain. Two lines of evidence suggest that the protein α-synuclein plays a role in the pathogenesis of PD: Fibrillar α-synuclein is a major component of Lewy bodies in diseased neurons, and two mutations in α-synuclein are linked to early-onset disease. Accordingly, the fibrillization of α-synuclein is proposed to contribute to neurodegeneration in PD. In this report, we provide evidence that oligomeric intermediates of the α-synuclein fibrillization pathway, termed protofibrils, might be neurotoxic. Analyses of protofibrillar α-synuclein by atomic force microscopy and electron microscopy indicate that the oligomers consist of spheres, chains, and rings. α-Synuclein protofibrils permeabilize synthetic vesicles and form pore-like assemblies on the surface of brain-derived vesicles. Dopamine reacts with α-synuclein to form a covalent adduct that slows the conversion of protofibrils to fibrils. This finding suggests that cytosolic dopamine in dopaminergic neurons promotes the accumulation of toxic α-synuclein protofibrils, which might explain why these neurons are most vulnerable to degeneration in PD. Finally, we note that aggregation of α-synuclein likely occurs via different mechanisms in the cell versus the test tube. For example, the binding of α-synuclein to cellular membranes might influence its self-assembly. To address this point, we have developed a yeast model that might enable the selection of random α-synuclein mutants with different membrane-binding affinities. These variants might be useful to test whether membrane binding by α-synuclein is necessary for neurodegeneration in transgenic animal models of PD.Parkinsons disease (PD) is a neurologic disorder resulting from the loss of dopaminergic neurons in the brain. Two lines of evidence suggest that the protein alpha-synuclein plays a role in the pathogenesis of PD: Fibrillar alpha-synuclein is a major component of Lewy bodies in diseased neurons, and two mutations in alpha-synuclein are linked to early-onset disease. Accordingly, the fibrillization of alpha-synuclein is proposed to contribute to neurodegeneration in PD. In this report, we provide evidence that oligomeric intermediates of the alpha-synuclein fibrillization pathway, termed protofibrils, might be neurotoxic. Analyses of protofibrillar alpha-synuclein by atomic force microscopy and electron microscopy indicate that the oligomers consist of spheres, chains, and rings. alpha-Synuclein protofibrils permeabilize synthetic vesicles and form pore-like assemblies on the surface of brain-derived vesicles. Dopamine reacts with alpha-synuclein to form a covalent adduct that slows the conversion of protofibrils to fibrils. This finding suggests that cytosolic dopamine in dopaminergic neurons promotes the accumulation of toxic alpha-synuclein protofibrils, which might explain why these neurons are most vulnerable to degeneration in PD. Finally, we note that aggregation of alpha-synuclein likely occurs via different mechanisms in the cell versus the test tube. For example, the binding of alpha-synuclein to cellular membranes might influence its self-assembly. To address this point, we have developed a yeast model that might enable the selection of random alpha-synuclein mutants with different membrane-binding affinities. These variants might be useful to test whether membrane binding by alpha-synuclein is necessary for neurodegeneration in transgenic animal models of PD.

SIRT1 and SIRT2: emerging targets in neurodegeneration

Sirtuins are NAD‐dependent protein deacetylases known to have protective effects against age‐related diseases such as cancer, diabetes, cardiovascular and neurodegenerative diseases. In mammals, there are seven sirtuins (SIRT1‐7), which display diversity in subcellular localization and function. While SIRT1 has been extensively investigated due to its initial connection with lifespan extension and involvement in calorie restriction, important biological and therapeutic roles of other sirtuins have only recently been recognized. Here, we review the potential roles and effects of SIRT1 and SIRT2 in neurodegenerative diseases. We discuss different functions and targets of SIRT1 and SIRT2 in a variety of neurodegenerative diseases including Alzheimers disease (AD), Parkinsons disease (PD) and Huntingtons Disease (HD). We also cover the role of SIRT1 in neuronal differentiation due to the possible implications in neurodegenerative conditions, and conclude with an outlook on the potential therapeutic value of SIRT1 and SIRT2 in these disorders.

Convergence of miRNA expression profiling, α-synuclein interacton and GWAS in Parkinson's disease.

miRNAs were recently implicated in the pathogenesis of numerous diseases, including neurological disorders such as Parkinsons disease (PD). miRNAs are abundant in the nervous system, essential for efficient brain function and play important roles in neuronal patterning and cell specification. To further investigate their involvement in the etiology of PD, we conducted miRNA expression profiling in peripheral blood mononuclear cells (PBMCs) of 19 patients and 13 controls using microarrays. We found 18 miRNAs differentially expressed, and pathway analysis of 662 predicted target genes of 11 of these miRNAs revealed an over-representation in pathways previously linked to PD as well as novel pathways. To narrow down the genes for further investigations, we undertook a parallel approach using chromatin immunoprecipitation-sequencing (ChIP-seq) analysis to uncover genome-wide interactions of α-synuclein, a molecule with a central role in both monogenic and idiopathic PD. Convergence of ChIP-seq and miRNomics data highlighted the glycosphingolipid biosynthesis and the ubiquitin proteasome system as key players in PD. We then tested the association of target genes belonging to these pathways with PD risk, and identified nine SNPs in USP37 consistently associated with PD susceptibility in three genome-wide association studies (GWAS) datasets (0.46≤OR≤0.63) and highly significant in the meta-dataset (3.36×10−4<p<1.94×10−3). A SNP in ST8SIA4 was also highly associated with PD (p = 6.15×10−3) in the meta-dataset. These findings suggest that several miRNAs may act as regulators of both known and novel biological processes leading to idiopathic PD.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.

SUMMARY α-Synuclein (α-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because α-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson’s disease (PD). We previously created a yeast model of α-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to α-syn expression. We also uncovered a core group of proteins with diverse activities related to α-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of α-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress α-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of α-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced α-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of α-syn foci, re-established ER-to-Golgi trafficking and ameliorated α-syn-mediated damage to mitochondria. They also corrected the toxicity of α-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of α-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by targeted over-expression of alpha-synuclein in mouse substantia nigra

Several transgenic mouse lines with altered α‐synuclein expression have been developed that show a variety of Parkinson’s disease‐like symptoms without specific loss of dopaminergic neurons. Targeted over‐expression of human α‐synuclein using viral‐vector mediated gene delivery into the substantia nigra of rats and non‐human primates leads to dopaminergic cell loss and the formation of α‐synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno‐associated virus (AAV) to over‐express wild type human α‐synuclein in the substantia nigra of mice. We hypothesized that this over‐expression would recapitulate pathological hallmarks of Parkinson’s disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing α‐synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress‐related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over‐expression of α‐synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson’s disease pathogenesis can be further elucidated.

The sour side of neurodegenerative disorders: the effects of protein glycation.

Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins, either intra‐ or extracellularly in the nervous system. Although familial mutations play an important role in protein misfolding and aggregation, the majority of cases of neurodegenerative diseases are sporadic, suggesting that other factors must contribute to the onset and progression of these disorders. Post‐translational modifications are known to influence protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play important roles in modifying proteins, forming advanced glycation end‐products (AGEs) in a non‐enzymatic process named glycation. Several proteins linked to neurodegenerative diseases, such as amyloid β, tau, prions and transthyretin, were found to be glycated in patients, and this is thought to be associated with increased protein stability through the formation of crosslinks that stabilize protein aggregates. Moreover, glycation may be responsible, via the receptor for AGE (RAGE), for an increase in oxidative stress and inflammation through the formation of reactive oxygen species and the induction of NF‐κB. Therefore, it is essential to unravel the molecular mechanisms underlying protein glycation to understand their role in neurodegeneration. Here, we reviewed the role of protein glycation in the major neurodegenerative disorders and highlight the potential value of protein glycation as a biomarker or target for therapeutic intervention. Copyright

Extracellular Alpha-Synuclein Oligomers Modulate Synaptic Transmission and Impair LTP Via NMDA-Receptor Activation

Parkinsons disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

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.