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-SynA53T mice). Fasudil treatment improved motor and cognitive functions in α-SynA53T mice as determined by CatwalkTM 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-SynA53T 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.

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.

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.

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 βS 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, βS or γS in the aged or diseased brain.

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.

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.

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.

Accumulations of tau intracellular deposits are characteristic for tauopathies and Alzheimer's disease. In order to understand the toxicity of tau aggregation and the reversibility of the pathologic processes we generated inducible transgenic mouse lines, which express either the 4-repeat tau domain with the FTDP-17 mutation Delta-K280 (TauRD/DeltaK280 – “pro-aggregation mutant”) or the 4-repeat tau domain with Delta-K280 deletion and two proline mutations in the hexapeptide motifs (TauRD/DeltaK280/I277P/I308 – “anti-aggregation mutant”). The DeltaK280 mutation accelerates the aggregation of tau, but the inserted proline residues inhibit the tau aggregation in vitro and in cell models. Inducible transgene expression in mice was driven by a forebrain-specific CaMKII promoter in a Tet-Off system and can be suppressed by doxycycline. The pro-aggregation mutant showed aggregated tau in sarkosyl insoluble fractions and Gallyas silver stained neurofibrillary tangles from 3 months onwards, even though the level of the human tau protein was lower than endogenous mouse tau. Tau preparations from pro-aggregation mutant mice revealed PHFs by electron microscopy. Consistent with the tau pathology the neuronal loss was age-dependent and visible in the dentate gyrus as early as 5 months. The immunohistochemisty results showed phosphorylated tau at S262 missorted into the somatodendritic compartment of cortical and hippocampal neurons. The anti-aggregation mutant with a similar expression level as the pro-aggregation mutant did not show aggregated tau or neuronal loss, but missorting into the somatodendritic compartment. The level of synaptophysin, a presynaptic marker and the number of spine-synapses were reduced in the stratum radiatum of the pro-aggregation mutant, but not of the anti-aggregation mutant. Six weeks of switching off the tau transgene in the pro-aggregation mutant lead to ∼90% reduction in the level of soluble human tau protein, to complete reversal of the pathological somatodendritic localization and of phosphorylation in the repeat domain. Remarkably, the aggregation was only partly reversed. The remaining aggregates did not contain the exogenous human tau, but the endogenous mouse tau. The results argue that a toxic species of tau, once introduced into neurons, can “poison” endogenous tau and propagate its aggregation for extended time periods. Supported by MPG and DFG.

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.

In order to understand the toxicity of tau aggregation we monitored the overexpression and aggregation of tau in transgenic mouse models. We have generated several inducible transgenic mouse lines which express different tau constructs. Here we describe two lines which express the 4–repeat tau domain with the FTDP17– mutation DelK280 (K18DelK280, ‘pro–aggregation mutant’), and the 4–repeat tau domain with DelK280 mutation plus two proline mutations in the hexapeptide motifs (K18DelK280/I277P/I308P, ‘anti–aggregation mutant’). The DelK280 mutant is known to accelerate the aggregation of tau (Barghorn et al., Biochemistry 2000). Previous studies have demonstrated that the two Ile>Pro mutations inserted into 4R tau domains inhibit tau aggregation in vitro and in cell models (von Bergen et al., PNAS 2000; Khlistunova et al., JBC 2005). Inducible transgene expression in mice was driven by the forebrain–specific CaMKII promoter in a Tet–Off system and can be suppressed by doxycycline. Biochemical studies of the pro–aggregation mutant show the presence of soluble human tau protein as well as aggregated tau from 3 months onwards. The double proline mutant shows a similar pattern of expression, but does not form aggregated tau. By immunohistochemistry, the pro–aggregation mutant shows relocalization of tau from the axonal to the somatodendritic compartment in a phosphorylated form (pS262/pS256 recognized by antibody 12E8, in the KXGS motifs that are targets of the kinase MARK). Gallyas silver staining confirms the presence of aggregated tau in the limbic system starting as early as 3 months of tau expression. Consistent with the tau pathology, there is a noticeable neuronal loss in the dentate gyrus of aged mice. The anti–aggregation transgenic mice show similar tau expression but less phosphorylated tau at pS262/pS256 by immunohistochemistry, and no aggregated tau by Gallyas staining, and no neuronal loss. Further behavioural and LTP studies are underway in order to evaluate the pathological functions of tau constructs in the double transgenic mouse models.

Immunohistological studies of inducible transgenic mice expressing the tau mutant K18delK280 (one of the FTDP17 mutations) have shown the translocation of tau from the axonal to the somatodendritic compartment of hippocampal CA1 neurons. The mice express the 4 repeats of the human tau (K18) with a delK280 mutation under the control of the doxycyclin–dependent regulation system (tet–off system), driven by the CaMKII promoter in forebrain structures like hippocampus and cortex. To determine whether pathological tau in the somatodendritic compartment is associated with morphological neuronal changes, CA1 neurons were analyzed by “DiOlistic”–labeling – a technique for labeling neurons with fluorescent dyes. To prepare tissue slices, hippocampi from perfused transgenic mice and same aged control littermates were removed and sagittally sliced into 400 μm thick sections using a tissue chopper. For preparing the DiI–coated gold particles, 0.75 mg gold particles (1.6 μm, BIORAD) were spread on a slide and 20 μl DiI solution (Invitrogen, 1 mg DiI solved into 100 μl methylene chloride) were applied. The hippocampal slices were postfixed in 4% paraformaldehyde and sprayed with the lipophilic DiI–coated gold particles, using a diolistic system developed for gene transfections (Particle Delivery System, BIORAD; ∼ 1100 psi). Labeled CA1 neurons of the pyramidal layer were imaged using a confocal microscope (Zeiss LSM510). To investigate the pathological function of tau in the stained CA1 neurons, the thickness of their apical dendrites was measured. The results show that the apical dendrites of transgenic animals are thicker than in the same age control littermates. Detailed analysis of dendritic arbor and dendritic spines is underway.

In order to explore the physiological function of tau in the brain and to conceive its pathological mechanisms in tauopathies we generated inducible transgenic mice carrying the longest human tau isoform with the FTDP–17 mutation delK280. We used the tetracycline–responsive transactivator (Mayford et al., Science 1996) under the control of the forebrain–specific CaMKII–promoter to achieve a regional and temporal control of the transgene expression. The human tau mutant is expressed with high levels in the hippocampus and the cortex. Most of the protein is still soluble in younger animals, but aggregates and accumulates in sarcosyl–insoluble fractions of older animals as shown by biochemical analysis. Immunohistochemical staining of brain sections indicates a pathological somatodendritic distribution of the human tau mutant as well as changes in conformation and phosphorylation. Most prominent are a conformational change seen with the antibody MC1 and the pathological phosphorylation at the KXGS motifs (targets of kinase MARK, antibody 12E8) starting to develop in 3–month–old transgenic animals. Further abnormalities in phosphorylation are later identified at the Ser/Thr–Pro sites pT231/pS235 (AT180), pS46, pS199 and pT231. Switching off the tau gene in the mice for 6 weeks at different ages between 4 and 9 months reduces the amount of tau protein to nearly control levels. Likewise, the pathological conformation of tau as seen by the MC1 antibody, the phosphorylation at KXGS motifs and other Ser/Thr–Pro motifs are reversible. Functional analysis of the hippocampal region reveals a significant decrease in the number of spine–synapses in animals carrying the human tau with delK280 mutation and we find the long–term potentiation (LTP) reduced. In parallel, the mouse line with the expression of the anti–aggregating human tau mutant delK280/PP was analyzed. This tau mutant cannot aggregate in vitro. In the mouse brain it shows comparable expression and phosphorylation to the delK280 mutant but no pathological changes in conformation (MC1), aggregation (sarkosyl), number of synapses or synaptic function (LTP). Comparing the pro– and antiaggregation, tau mouse models clearly demonstrate that tau aggregation is a prerequisite of toxicity or is limited to tau toxicity.