Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (α-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced α-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of α-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on α-Syn pathology in vivo in a transgenic mouse model overexpressing human α-Syn bearing the A53T mutation (α-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in α-Syn(A53T) mice as determined by Catwalk(TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of α-Syn pathology in the midbrain of α-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with α-Syn and attenuates α-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies.

LRRK2 Promotes Tau Accumulation, Aggregation and Release.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are known as the most frequent cause of familial Parkinson's disease (PD), but are also present in sporadic cases. The G2019S-LRRK2 mutation is located in the kinase domain of the protein, and has consistently been reported to promote a gain of kinase function. Several proteins have been reported as LRRK2 substrates and/or interactors, suggesting possible pathways involved in neurodegeneration in PD. Hyperphosphorylated Tau protein accumulates in neurofibrillary tangles, a typical pathological hallmark in Alzheimer's disease and frontotemporal dementia. In addition, it is also frequently found in the brains of PD patients. Although LRRK2 is a kinase, it appears that a putative interaction with Tau is phosphorylation-independent. However, the underlying mechanisms and the cellular consequences of this interaction are still unclear. In this study, we demonstrate an interaction between LRRK2 and Tau and that LRRK2 promotes the accumulation of non-monomeric and high-molecular weight (HMW) Tau species independent of its kinase activity. Interestingly, we found that LRRK2 increases Tau secretion, possibly as a consequence of an impairment of Tau proteasomal degradation. Our data highlight a mechanism through which LRRK2 regulates intracellular Tau levels, contributing to the progression of the pathology caused by the LRRK2-mediated proteasome impairment. In total, our findings suggest that the interplay between LRRK2 and proteasome activity might constitute a valid target for therapeutic intervention in PD.

Dimerization propensities of Synucleins are not predictive for Synuclein aggregation.

Aggregation and fibril formation of human alpha-Synuclein (αS) are neuropathological hallmarks of Parkinson's disease and other synucleinopathies. The molecular mechanisms of αS aggregation and fibrillogenesis are largely unknown. Several studies suggested a sequence of events from αS dimerization via oligomerization and pre-fibrillar aggregation to αS fibril formation. In contrast to αS, little evidence suggests that γS can form protein aggregates in the brain, and for ßS its neurotoxic properties and aggregation propensities are controversially discussed. These apparent differences in aggregation behavior prompted us to investigate the first step in Synuclein aggregation, i.e. the formation of dimers or oligomers, by Bimolecular Fluorescence Complementation in cells. This assay showed some Synuclein-specific limitations, questioning its performance on a single cell level. Nevertheless, we unequivocally demonstrate that all Synucleins can interact with each other in a very similar way. Given the divergent aggregation properties of the three Synucleins this suggests that formation of dimers is not predictive for the aggregation of αS, ßS or γS in the aged or diseased brain.

Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

Sumoylation of p35 modulates p35/cyclin-dependent kinase (Cdk) 5 complex activity.

Cyclin-dependent kinase (Cdk) 5 is critical for central nervous system development and neuron-specific functions including neurite outgrowth as well as synaptic function and plasticity. Cdk5 activity requires association with one of the two regulatory subunits, called p35 and p39. p35 redistribution as well as misregulation of Cdk5 activity is followed by cell death in several models of neurodegeneration. Posttranslational protein modification by small ubiquitin-related modifier (SUMO) proteins (sumoylation) has emerged as key regulator of protein targeting and protein/protein interaction. Under cell-free in vitro conditions, we found p35 covalently modified by SUMO1. Using both biochemical and FRET-/FLIM-based approaches, we demonstrated that SUMO2 is robustly conjugated to p35 in cells and identified the two major SUMO acceptor lysines in p35, K246 and K290. Furthermore, different degrees of oxidative stress resulted in differential p35 sumoylation, linking oxidative stress that is encountered in neurodegenerative diseases to the altered activity of Cdk5. Functionally, sumoylation of p35 increased the activity of the p35/Cdk5 complex. We thus identified a novel neuronal SUMO target and show that sumoylation is a likely candidate mechanism for the rapid modulation of p35/Cdk5 activity in physiological situations as well as in disease.

Protein phosphorylation in neurodegeneration: friend or foe?

Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's 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.

SUMO and Parkinson's disease.

Parkinson's disease (PD) is one of the most common degenerative disorders of the central nervous system that produces motor and non-motor symptoms. The majority of cases are idiopathic and characterized by the presence of Lewy bodies containing fibrillar α-synuclein. Small ubiquitin-related modifier (SUMO) immunoreactivity was observed among others in cases with PD. Key disease-associated proteins are SUMO-modified, linking this posttranslational modification to neurodegeneration. SUMOylation and SUMO-mediated mechanisms have been intensively studied in recent years, revealing nuclear and extranuclear functions for SUMO in a variety of cellular processes, including the regulation of transcriptional activity, modulation of signal transduction pathways, and response to cellular stress. This points to a role for SUMO more than just an antagonist to ubiquitin and proteasomal degradation. The identification of risk and age-at-onset gene loci was a breakthrough in PD and promoted the understanding of molecular mechanisms in the pathology. PD has been increasingly linked with mitochondrial dysfunction and impaired mitochondrial quality control. Interestingly, SUMO is involved in many of these processes and up-regulated in response to cellular stress, further emphasizing the importance of SUMOylation in physiology and disease.

The potential for beta-structure in the repeat domain of tau protein determines aggregation, synaptic decay, neuronal loss, and coassembly with endogenous Tau in inducible mouse models of tauopathy.

We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimer's disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation deltaK280 (Tau(RD)/deltaK280), or the deltaK280 plus two proline mutations in the hexapeptide motifs (Tau(RD)/deltaK280/I277P/I308P). The deltaK280 mutation accelerates aggregation ("proaggregation mutant"), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models ("antiaggregation mutant"). The inducible transgene expression was driven by the forebrain-specific CaMKIIalpha (calcium/calmodulin-dependent protein kinase IIalpha) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2-3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant Tau(RD) coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation Tau(RD) is switched off, soluble and aggregated exogenous Tau(RD) disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.

The beta-propensity of Tau determines aggregation and synaptic loss in inducible mouse models of tauopathy.

Neurofibrillary lesions are characteristic for a group of human diseases, named tauopathies, which are characterized by prominent intracellular accumulations of abnormal filaments formed by the microtubule-associated protein Tau. The tauopathies are accompanied by abnormal changes in Tau protein, including pathological conformation, somatodendritic mislocalization, hyperphosphorylation, and aggregation, whose interdependence is not well understood. To address these issues we have created transgenic mouse lines in which different variants of full-length Tau are expressed in a regulatable fashion, allowing one to switch the expression on and off at defined time points. The Tau variants differ by small mutations in the hexapeptide motifs that control the ability of Tau to adopt a beta-structure conformation and hence to aggregate. The "pro-aggregation" mutant DeltaK280, derived from one of the mutations observed in frontotemporal dementias, aggregates avidly in vitro, whereas the "anti-aggregation" mutant DeltaK280/PP cannot aggregate because of two beta-breaking prolines. In the transgenic mice, the pro-aggregation Tau induces a pathological conformation and pre-tangle aggregation, even at low expression levels, the anti-aggregation mutant does not. This illustrates that abnormal aggregation is primarily controlled by the molecular structure of Tau in vitro and in the organism. Both variants of Tau become mislocalized and hyperphosphorylated independently of aggregation, suggesting that localization and phosphorylation are mainly a consequence of increased concentration. These pathological changes are reversible when the expression of Tau is switched off. The pro-aggregation Tau causes a strong reduction in spine synapses.