George K. Tofaris

α-Synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome

α‐Synuclein has been implicated in the pathogenesis of Parkinsons disease based on mutations in familial cases of the disease and its presence in Lewy bodies. Here we show that over‐expression of wild‐type human α‐synuclein is sufficient to induce inclusion formation in SH‐SY5Y cells. In this cellular model, proteasome inhibition leads to an increase of α‐synuclein accumulation in vivo without ubiquitylation. In accordance, we find that in vitro, unmodified α‐synuclein can be directly degraded by the 20S proteasome. These findings suggest an ubiquitin‐independent mechanism of proteasomal degradation for α‐synuclein and other natively unfolded proteins.

Ubiquitination of α-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function

Lewy bodies are intracellular fibrillar inclusions composed of α-synuclein. They constitute the pathological hallmark of Parkinsons disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble α-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic α-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, disease-associated 22–24-kDa α-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22–24-kDa α-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified α-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22–24-kDa α-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated α-synuclein, presumably the result of aberrant proteolysis, is found only in association with α-synuclein aggregates.

Pathological changes in dopaminergic nerve cells of the substantia nigra and olfactory bulb in mice transgenic for truncated human alpha-synuclein(1-120): implications for Lewy body disorders

Dysfunction of the 140 aa protein α-synuclein plays a central role in Lewy body disorders, including Parkinson’s disease, as well as in multiple system atrophy. Here, we show that the expression of truncated human α-synuclein(1–120), driven by the rat tyrosine hydroxylase promoter on a mouse α-synuclein null background, leads to the formation of pathological inclusions in the substantia nigra and olfactory bulb and to a reduction in striatal dopamine levels. At the behavioral level, the transgenic mice showed a progressive reduction in spontaneous locomotion and an increased response to amphetamine. These findings suggest that the C-terminal of α-synuclein is an important regulator of aggregation in vivo and will help to understand the mechanisms underlying the pathogenesis of Lewy body disorders and multiple system atrophy.

SNARE protein redistribution and synaptic failure in a transgenic mouse model of Parkinson’s disease

The pre-synaptic protein alpha-synuclein is the main component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of Parkinsons disease and dementia with Lewy bodies. Mutations in the alpha-synuclein gene cause familial forms of Parkinsons disease and dementia with Lewy bodies. We previously described a transgenic mouse line expressing truncated human alpha-synuclein(1-120) that develops alpha-synuclein aggregates, striatal dopamine deficiency and reduced locomotion, similar to Parkinsons disease. We now show that in the striatum of these mice, as in Parkinsons disease, synaptic accumulation of alpha-synuclein is accompanied by an age-dependent redistribution of the synaptic SNARE proteins SNAP-25, syntaxin-1 and synaptobrevin-2, as well as by an age-dependent reduction in dopamine release. Furthermore, the release of FM1-43 dye from PC12 cells expressing either human full-length alpha-synuclein(1-140) or truncated alpha-synuclein(1-120) was reduced. These findings reveal a novel gain of toxic function of alpha-synuclein at the synapse, which may be an early event in the pathogenesis of Parkinsons disease.

Ubiquitin ligase Nedd4 promotes alpha-synuclein degradation by the endosomal-lysosomal pathway.

α-Synuclein is an abundant brain protein that binds to lipid membranes and is involved in the recycling of presynaptic vesicles. In Parkinson disease, α-synuclein accumulates in intraneuronal inclusions often containing ubiquitin chains. Here we show that the ubiquitin ligase Nedd4, which functions in the endosomal–lysosomal pathway, robustly ubiquitinates α-synuclein, unlike ligases previously implicated in its degradation. Purified Nedd4 recognizes the carboxyl terminus of α-synuclein (residues 120–133) and attaches K63-linked ubiquitin chains. In human cells, Nedd4 overexpression enhances α-synuclein ubiquitination and clearance by a lysosomal process requiring components of the endosomal-sorting complex required for transport. Conversely, Nedd4 down-regulation increases α-synuclein content. In yeast, disruption of the Nedd4 ortholog Rsp5p decreases α-synuclein degradation and enhances inclusion formation and α-synuclein toxicity. In human brains, Nedd4 is present in pigmented neurons and is expressed especially strongly in neurons containing Lewy bodies. Thus, ubiquitination by Nedd4 targets α-synuclein to the endosomal–lysosomal pathway and, by reducing α-synuclein content, may help protect against the pathogenesis of Parkinson disease and other α-synucleinopathies.

Lysosome-dependent pathways as a unifying theme in Parkinson's disease†‡§

Although the pathogenesis of Parkinsons disease (PD) is considered multifactorial, evidence from genetics and cell biology has implicated specific molecular pathways. This article summarizes evidence that suggests that the level of intracellular alpha‐synuclein is critical for the onset of neurodegeneration with Lewy bodies and dependent, to a large extent, on lysosomal degradation. The function of other key proteins that emerged from genetics is discussed: Pink1 and Parkin regulate the degradation of damaged mitochondria by the lysosome (mitophagy). Glucocerebrosidase and ATP13A2 are important components of this degradative organelle. VPS35 and LRRK2 may regulate trafficking within lysosome‐dependent pathways, such as autophagy and endosomal vesicle recycling. Clinically, diffuse alpha‐synucleinopathy or dementia seems to correlate with mutations which interfere with the broader function of lysosomal pathways, whereas a predominantly motor syndrome and nigrostriatal degeneration is associated with specific defects in mitophagy. Based on these studies, it is proposed that a protein network involved in trafficking to, or degradation by, lysosomes could be sufficient to explain the phenotypic spectrum within PD in a unifying biochemical pathway.

ER Stress and Autophagic Perturbations Lead to Elevated Extracellular α-Synuclein in GBA-N370S Parkinson's iPSC-Derived Dopamine Neurons

Summary Heterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinsons disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.

Alpha-synuclein Dysfunction in lewy body diseases

α‐Synuclein belongs to a small group of natively unfolded proteins that can transiently bind to lipid membranes and acquire a partial α‐helical conformation. Its relevance to Parkinsons disease (PD) is based on mutations found in familial cases of the disease and its presence in filaments of Lewy bodies (LB) and Lewy neurites (LN) in sporadic cases where it is packed in a β‐sheet configuration. This structural plasticity of α‐synuclein has raised the possibility that neurodegeneration may be a consequence of abnormal protein folding. The extent to which abnormal folding and aggregation of neuronal proteins is directly toxic to the cell, an inert biochemical marker of an underlying harmful metabolic defect, or a protective reaction remains to be seen. We review the function of α‐synuclein and recent studies that have shed light on the mechanisms by which it aggregates.

Identification of distinct circulating exosomes in Parkinson's disease

Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinsons disease (PD).

The effect of truncated human alpha-synuclein (1-120) on dopaminergic cells in a transgenic mouse model of Parkinson's disease.

α-Synuclein is thought to play an important role in the pathology of Parkinsons disease (PD). Truncated forms of this protein can be found in PD brain extracts, and these species aggregate faster and are more susceptible to oxidative stress than the full-length protein. We investigated the effect of truncated α-synuclein on dopaminergic cells using a transgenic mouse expressing α-synuclein (1–120) driven by the rat tyrosine hydroxylase promoter on a mouse α-synuclein null background. We found a selective reduction in the yield of dopaminergic cells from transgenic embryonic ventral mesencephalic cell cultures. However, in vivo the substantia nigra/ventral tegmentum dopaminergic cell counts were not reduced in transgenics, although these mice are known to have reduced striatal dopamine. When transplanted to the striatum in the unilateral 6-hydroxydopamine-lesioned mouse model of PD, dopaminergic cells derived from transgenic embryonic ventral mesencephala were significantly smaller at 6 weeks, and showed a trend towards being less effective at ameliorating rotational asymmetry than those from control α-synuclein null mice. These results suggest that α-synuclein (1–120) renders dopaminergic cells more susceptible to stress, which may have important implications as to how this truncated protein might contribute to dopaminergic cell death in sporadic PD.