Marion Delenclos

Alpha-synuclein and tau: teammates in neurodegeneration?

The accumulation of α-synuclein aggregates is the hallmark of Parkinson’s disease, and more generally of synucleinopathies. The accumulation of tau aggregates however is classically found in the brains of patients with dementia, and this type of neuropathological feature specifically defines the tauopathies. Nevertheless, in numerous cases α-synuclein positive inclusions are also described in tauopathies and vice versa, suggesting a co-existence or crosstalk of these proteinopathies. Interestingly, α-synuclein and tau share striking common characteristics suggesting that they may work in concord. Tau and α-synuclein are both partially unfolded proteins that can form toxic oligomers and abnormal intracellular aggregates under pathological conditions. Furthermore, mutations in either are responsible for severe dominant familial neurodegeneration. Moreover, tau and α-synuclein appear to promote the fibrillization and solubility of each other in vitro and in vivo. This suggests that interactions between tau and α-synuclein form a deleterious feed-forward loop essential for the development and spreading of neurodegeneration. Here, we review the recent literature with respect to elucidating the possible links between α-synuclein and tau.

Biomarkers in Parkinson's disease: Advances and strategies

Parkinsons disease (PD) is a neurodegenerative disorder characterized by progressive motor disturbances and affects more than 1% of the worldwide population. Despite considerable progress in understanding PD pathophysiology, including genetic and biochemical causes, diagnostic approaches lack accuracy and interventions are restricted to symptomatic treatments. PD is a complex syndrome with different clinical subtypes and a wide variability in disorder course. In order to deliver better clinical management of PD patients and discovery of novel therapies, there is an urgent need to find sensitive, specific, and reliable biomarkers. The development of biomarkers will not only help the scientific community to identify populations at risk, but also facilitate clinical diagnosis. Furthermore, these tools could monitor progression, which could ultimately deliver personalized therapeutic strategies. The field of biomarker discovery in PD has attracted significant attention and there have been numerous contributions in recent years. Although none of the parameters have been validated for clinical practice, some candidates hold promise. This review summarizes recent advances in the development of PD biomarkers and discusses new strategies for their utilization.

Transmission of Soluble and Insoluble α-Synuclein to Mice.

Abstract The neurodegenerative synucleinopathies, which include Parkinson disease, multiple-system atrophy, and Lewy body disease, are characterized by the presence of abundant neuronal inclusions called Lewy bodies and Lewy neurites. These disorders remain incurable, and a greater understanding of the pathologic processes is needed for effective treatment strategies to be developed. Recent data suggest that pathogenic misfolding of the presynaptic protein, &agr;-synuclein (&agr;-syn), and subsequent aggregation and accumulation are fundamental to the disease process. It is hypothesized that the misfolded isoform is able to induce misfolding of normal endogenous &agr;-syn, much like what occurs in the prion diseases. Recent work highlighting the seeding effect of pathogenic &agr;-syn has largely focused on the detergent-insoluble species of the protein. In this study, we performed intracerebral inoculations of the sarkosyl-insoluble or sarkosyl-soluble fractions of human Lewy body disease brain homogenate and show that both fractions induce CNS pathology in mice at 4 months after injection. Disease-associated deposits accumulated both near and distal to the site of the injection, suggesting a cell-to-cell spread via recruitment of &agr;-syn. These results provide further insight into the prion-like mechanisms of &agr;-syn and suggest that disease-associated &agr;-syn is not homogeneous within a single patient but might exist in both soluble and insoluble isoforms.

Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein.

Misfolding and aggregation of alpha-synuclein (αsyn) resulting in cytotoxicity is a hallmark of Parkinsons disease (PD) and related synucleinopathies. The recent body of evidence indicates that αsyn can be released from neuronal cells by nonconventional exocytosis involving extracellular vesicles (EVs) such as exosomes. The transfer of αsyn between cells has been proposed to be an important mechanism of disease propagation in PD. To date, exosome trafficking mechanisms, including release and cell-cell transmission, have not been fully described. To gain insight into the mechanisms involved, exosomes were purified from conditioned media of stable cells secreting αsyn oligomers. A novel bimolecular protein complementation assay was used to detect exosomes containing αsyn oligomers. Recipient cells were treated with exosomes containing αsyn oligomers or “free” non-exosome-associated αsyn oligomers and internalization was monitored. We demonstrate that cell-derived exosome-associated αsyn oligomers can be efficiently internalized by recipient cells. Interestingly exosome-free αsyn oligomers isolated from conditioned medium were not internalized but remained bound to the extracellular surface. To investigate the endocytic pathway(s) required for the exosome uptake different pharmacological inhibitors of caveolin-dependent, clathrin-dependent, and macropinocytosis pathways were utilized. Surprisingly, none of these pathways appear to play a significant role in the internalization of exosome-associated αsyn oligomers. Finally, the role of heparin sulfate proteoglycans (HSPGs) in exosome-associated αsyn internalization was investigated using genetic approach. Despite previous studies showing HSPGs can modulate internalization of fibrillar αsyn, genetic manipulations did not attenuate internalization of exosome-associated αsyn oligomers in our hands, suggesting that exosome-associated αsyn is internalized via an alternative endocytic pathway(s) that has yet to be elucidated.

A Rapid, Semi-Quantitative Assay to Screen for Modulators of Alpha-Synuclein Oligomerization Ex vivo.

Alpha synuclein (αsyn) aggregates are associated with the pathogenesis of Parkinsons disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here, we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at 1 week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain.

Untangling a Role for Tau in Synucleinopathies

In this issue of Biological Psychiatry, Sengupta et al. (1) present novel evidence supporting a role for tau protein in the pathogenesis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Synucleinopathies are a group of neurodegenerative disorders characterized by the abnormal deposition of α-synuclein in filamentous intracellular inclusions called Lewy bodies. The most well-known synucleinopathy is PD, but other neurodegenerative diseases, including DLB and multiple system atrophy, also manifest abnormal α-synuclein deposition as a major pathologic feature. Neurofibrillary tangles composed of hyperphosphorylated tau protein, the major pathologic hallmark of tauopathies (e.g., Alzheimer’s disease), have also been observed in brain specimens from patients with DLB and PD. Using Braak staging of neurofibrillary tangle pathology, clinicopathologic studies identified 18% and 75% of PD and DLB cases, respectively, as Braak stage IV or greater (2). Conversely, Lewy bodies are observed in 60% of patients with Alzheimer’s disease (3). Although synucleinopathies and tauopathies are historically considered distinct disease entities, evidence now suggests that common mechanisms and interplay of α-synuclein and tau may determine susceptibility to developing disease (4). Additional data supporting a connection between α-synuclein and tau comes from genetic studies that link the MAPT gene encoding tau with an increased risk of PD (5). Although the co-occurrence of αsynuclein and tau pathologies has been widely reported, the functional consequences of the primary deposited protein on secondary pathology has been poorly investigated to date, and the common mechanisms remain to be elucidated Figure 1. At the molecular level, α-synuclein and tau were first shown to interact physically in the late 1990s by Jensen et al. (6) with a binding inhibitory concentration of 50% of 50 pM. Although this observation has been confirmed in later studies using cellular models and brain tissue analyses (4), the relevance of an α-synuclein-tau interaction has still not been revealed. A possible physiologic function, such as modulation of cytoskeleton dynamics, has been proposed, but coexpression of tau and α-synuclein has proved deleterious in vitro and in vivo, suggesting that the interaction potentially exacerbates a pathologic process (4). One of the first proposed pathologic mechanisms for an α-synuclein-tau interaction was the αsynuclein-induced recruitment of kinases to potentiate abnormal tau phosphorylation (6). Later, using recombinant proteins, Waxman and Giasson (7) showed that tau aggregation could be specifically induced by α-synuclein and vice versa. Nübling et al. (8) were able to detect in vitro co-oligomerization of tau and α-synuclein using tagged recombinant proteins and fluorescent intensity distribution analysis. Sengupta et al., in an analogous study using human brain tissues and oligomerspecific antibodies, present strong evidence for a detrimental α-synuclein-tau relationship via a co-oligomerization process.

Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway.

The sirtuins are highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent enzymes that play a broad role in cellular metabolism and aging. Mitochondrial sirtuin 3 (SIRT3) is downregulated in aging and age-associated diseases such as cancer and neuro-degeneration and plays a major role in maintaining mitochondrial function and preventing oxidative stress. Mitochondria dysfunction is central to the pathogenesis of Parkinson disease with mutations in mitochondrial-associated proteins such as PINK1 and parkin causing familial Parkinson disease. Here, we demonstrate that the presence of alpha-synuclein (αsyn) oligomers in mitochondria induce a corresponding decrease in mitochondrial SIRT3 activity and decreased mitochondrial biogenesis. We show that SIRT3 downregulation in the presence of αsyn accumulation is accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK) and cAMP-response element binding protein (CREB), as well as increased phosphorylation of dynamin-related protein 1 (DRP1) and decreased levels of optic atrophy 1 (OPA1), which is indicative of impaired mitochondrial dynamics. Treatment with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) restores SIRT3 expression and activity and improves mitochondrial function by decreasing αsyn oligomer formation. The accumulation of αsyn oligomers in mitochondria corresponds with SIRT3 down-regulation not only in an experimental cellular model, but also in vivo in a rodent model of Parkinson disease, and importantly, in human post mortem brains with neuropathologically confirmed Lewy body disease (LBD). Taken together our findings suggest that pharmacologically increasing SIRT3 levels will counteract αsyn-induced mitochondrial dysfunction by normalizing mitochondrial bioenergetics. These data support a protective role for SIRT3 in Parkinson disease-associated pathways and reveals significant mechanistic insight into the interplay of SIRT3 and αsyn.

A CRISPR/Cas9 based strategy to manipulate the Alzheimer\'s amyloid pathway

The gradual accumulation of amyloid-β (Aβ) is a neuropathologic hallmark of Alzheimer’s disease (AD); playing a key role in disease progression. Aβ is generated by the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases, with BACE-1 (β-site APP cleaving enzyme-1) cleavage as the rate limiting step 1–3. CRISPR/Cas9 guided gene-editing is emerging as a promising tool to edit pathogenic mutations and hinder disease progression 4,5,6 However, few studies have applied this technology to neurologic diseases 7–9. Besides technical caveats such as low editing efficiency in brains and limited in vivo validation 7, the canonical approach of ‘mutation-correction’ would only be applicable to the small fraction of neurodegenerative cases that are inherited (i.e. < 10% of AD, Parkinson’s, ALS); with a new strategy needed for every gene. Moreover, feasibility of CRISPR/Cas9 as a therapeutic possibility in sporadic AD has not been explored. Here we introduce a strategy to edit endogenous APP at the extreme C-terminus and reciprocally manipulate the amyloid pathway – attenuating β-cleavage and Aβ, while up-regulating neuroprotective a-cleavage. APP N-terminus, as well as compensatory APP homologues remain intact, and key physiologic parameters remain unaffected. Robust APP-editing is seen in cell lines, cultured neurons, human embryonic stem cells/iPSC-neurons, and mouse brains. Our strategy works by limiting the physical association of APP and BACE-1, and we also delineate the mechanism that abrogates APP/BACE-1 interaction in this setting. Our work offers an innovative ‘cut and silence’ gene-editing strategy that could be a new therapeutic paradigm for AD.

A CRISPR/CAS9 BASED STRATEGY TO ATTENUATE THE β-AMYLOID PATHWAY

C-terminus

Lewy body dementia

Lewy body dementia (LBD) is a term used to encompass both Parkinson’s disease dementia and dementia with Lewy body disorders. They are the second most common type of dementia after Alzheimer’s disease but are yet often misdiagnosed. Indeed, LBD appears to fall somewhere in the middle of a disease spectrum ranging from Alzheimer’s to Parkinson’s disease. LBD is characterized by manifestations of neuropsychiatric symptoms with motor impairment, and both disorders can be distinguished by the temporal manifestations of motor symptoms. The pathological hallmark of LBD is the presence of Lewy bodies with aggregated alpha-synuclein being a main component. To date, no cures are available for LBD. Consequently identifying and dissecting disease-modifying pathway for LBD is a priority for the discovery of neuroprotective therapeutics. Herein, we review promising targets and new innovative approaches. In this regard, strategies targeting the clearance of alpha-synuclein and prevention of its propagation will be discussed.