J Neural Transm DOI 10.1007/s00702-017-1695-x
TRANSLATIONAL NEUROSCIENCES - REVIEW ARTICLE
Alpha-synuclein and iron: two keys unlocking Parkinson’s disease Paul Lingor1,2
•
Eleonora Carboni1,2 • Jan Christoph Koch1
Received: 26 December 2016 / Accepted: 1 February 2017 Ó Springer-Verlag Wien 2017
Abstract Current therapies for Parkinson’s disease (PD) confer symptomatic relief and are particularly efficient in the treatment of motor symptoms in earlier disease stages. However, we are still unable to treat the causes of neurodegeneration by modification of the underlying mechanisms, which is partially due to their insufficient understanding. In this short review, we focus on two pivotal disease mechanisms: alpha-synuclein pathology and dysfunction of iron homeostasis as well as their intricate interaction. Both pathomechanisms have been extensively studied in the past and represent valid targets for disease-modifying pharmacological treatment approaches for PD. We summarize the current attempts to exploit iron chelation and modification of alpha-synuclein pathology as translational therapies in PD and discuss the chances and challenges of prospective disease-modifying approaches. Keywords Disease mechanism Neurodegeneration Translational therapy
Introduction Within the field of neurodegenerative disorders, Parkinson’s disease (PD) is by far leading with the highest number of available therapeutic options. Levodopa plus & Paul Lingor [email protected] 1
Department of Neurology, University Medicine Go¨ttingen, 37075 Go¨ttingen, Germany
2
Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Go¨ttingen, Germany
decarboxylase inhibitors and dopamine agonists dominated the drug therapy regimes of motor symptoms for decades. During the last years, however, we witnessed not only the marketing of numerous novel formulations within these well-established therapeutic classes employing new extended-release kinetics or alternative ways of application, but also the appearance of entirely novel substances, such as opicapone, istradefylline, or safinamide (Oertel and Schulz 2016). In addition, also the repertoire of drugs targeting non-motor symptoms is growing, the latest additions being droxidopa and pimavanserin (Cummings et al. 2014; Hauser et al. 2016). Although the plethora of current treatment options confers significant improvements in the quality of life of PD patients and the life expectancy of PD patients is continuously growing, this cannot hide the fact that all these options are symptomatic, meaning that they do not alter the progressive pathophysiology of the disease. One of the reasons for the lack of disease-modifying treatments is the fact that the pathogenesis of PD is only insufficiently understood and that PD is likely to be more than one disease: not only there are different clinical phenotypes, but also there is also evidence to suggest that PD is histologically diverse (Halliday et al. 2011; Sauerbier et al. 2016). Protein degradation pathways, calcium homeostasis, mitochondrial function, and the innate immune system have all been suggested to contribute to disease mechanisms in PD (De Rosa et al. 2015). In this short review, we will, however, focus on two pivotal mechanisms, which appear instrumental in the development of the disease, and both of which promise to yield approaches for diseasemodifying therapies in the near future: alpha-synuclein pathology and accumulation of iron.
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Alpha-synuclein pathology As early as in 1912, Lewy described inclusion bodies in neurons of PD patients (Lewy 1912), which now bear his name and shortly thereafter, Tretiakoff revealed the degeneration of dopaminergic neurons as major pathoanatomic substrate of the disease (Tre´tiakoff 1919). It took then almost 80 years until the first family with inherited PD, the so-called Contursi-kindred, was described and shown to possess a mutation of the alpha-synuclein (aSyn) gene (Polymeropoulos et al. 1997). Other point mutations as well as multiplications of the aSyn gene have been described thereafter that all cause autosomal dominant forms of PD (Gasser et al. 2011). Shortly thereafter, Spillantini used antibodies against aSyn to demonstrate that it is the major component of the Lewy bodies found in PD (Spillantini et al. 1997), and Braak then used aSyn immunohistochemistry to map the neuroanatomical distribution of Lewy body pathology in over one hundred brains of autopsy cases with and without clinically diagnosed PD (Braak et al. 2003). This allowed demonstrating a stage-dependent distribution of the pathology, which later led up to the spreading hypothesis of aSyn. Correlating with disease severity, Braak described an expanding Lewy body pathology from the lower brainstem to the pons and midbrain, before being found in the basal forebrain and, ultimately, in the neocortex in the most advanced disease stages. In a subgroup of subjects without documented clinical signs of PD, solitary Lewy body pathology was visualized in the olfactory bulb and anterior olfactory nucleus, as well as in the dorsal motor nucleus of the vagus nerve in the medulla oblongata. This suggests that the olfactory bulb and the vagal nerve are the earliest affected brain structures, probably already in a pre-symptomatic stage. This finding gave rise to the ‘‘dual-hit hypothesis’’, since aSyn pathology appears to enter the brain from two different sites (Hawkes et al. 2007; Angot et al. 2010). Moreover, aSyn pathology has been detected outside the central nervous system in individuals with early PD. It was found in neurons of Auerbach’s and Meissner’s plexuses of the enteric nervous system and in autonomic nerves of the submandibular gland (Wakabayashi et al. 1988; Del Tredici et al. 2010). Based on these findings, it is speculated that a yet unknown environmental pathogen might trigger PD pathogenesis by entering the nervous system through the gastrointestinal system (Pan-Montojo et al. 2012). In line with this hypothesis, it was shown in large registry studies that full truncal vagotomy early in life correlates with a significantly decreased risk of developing PD (Svensson et al. 2015). The spreading theory was further fueled by the description of Lewy bodies in transplanted fetal human midbrain neurons, which had been grafted into the striata of patients with advanced PD in transplantation trials in the
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1990s (Li et al. 2008). The fact that young neurons, aged between 11 and 16 years at the time of analysis contained inclusion bodies, which were previously found only in old brains, was highly suggestive of a pathology that could be transmitted to these implanted neurons. The percentage of previously grafted midbrain neurons that display Lewy body pathology increased from 2 to 5% at 11–16 years after transplantation to 11–12% in a recently examined patient at 24 years after transplantation (Li et al. 2016). However, the alternative hypothesis of local environmental factors in the PD brain inducing pathology in susceptible neurons without spread of a pathological protein cannot be ruled out completely by this data. Based on these findings and in an analogy to previously known infectious proteinaceous particles or prions, the question arose whether PD could be a prion-like disorder. In its native form, aSyn is a soluble protein, but it can assemble into a filamentous, beta-sheet-rich conformation that is prone for aggregation. It has been shown that aSyn fibrils can seed aggregation and the molecular properties of these toxic aSyn fibrils have been analyzed in detail (Rodriguez et al. 2015; Tuttle et al. 2016). Moreover, the injection of different strains of aSyn was described to cause specific types of synucleinopathies in rodent models. While aSyn fibrils conferred severe neuronal death, aSyn ribbons resulted in Lewy neurite formation and aSyn pathology in oligodendrocytes partly resembling the pathology of multiple system atrophy (Peelaerts et al. 2015). It still remains to be investigated whether the different forms of synucleinopathies in humans, including PD, dementia with Lewy bodies and multiple system atrophy, are also caused by different strains of aSyn. In vitro experiments showed that aSyn fibrils are taken up by neurons, seed aggregation of expressed protein in the infected neurons, are transported along the axons, and can then be released and taken up again by other adjacent neurons (Guo and Lee 2014). The uptake of aSyn fibrils into neurons is mediated by macropinocytosis requiring heparan sulphate proteoglycans and endocytosis via the transmembrane protein lymphocyteactivation gene 3 (LAG3) (Holmes et al. 2013; Mao et al. 2016). These recent findings offer new possibilities for developing therapeutics against aSyn spread. While the peripheral injection of aggregated aSyn was not shown to cause formation of brain inclusions in wild-type animals up to date, the injection of aSyn aggregates into the substantia nigra of wild-type rodents resulted in spread of aSyn inclusions and functional deficits of the animals (Luk et al. 2012; Masuda-Suzukake et al. 2014; Paumier et al. 2015). Although there is thus much evidence suggesting that toxic forms of aSyn can indeed confer a misfolded conformation to non-aggregated aSyn in animal models, a prion-like spread has not been proven in humans so far and there is so
Alpha-synuclein and iron: two keys unlocking Parkinson’s disease
far no epidemiological evidence that this disease could be transmitted from one patient to the other (Irwin et al. 2013; Beekes et al. 2014; Walsh and Selkoe 2016).
Accumulation of iron Although the discovery of aSyn involvement strongly influenced our understanding of the pathophysiology of PD during the last 20 years, another mechanism was taking the lead as prime candidate in explaining the etiology of PD: already in the beginning of the 20th century iron was shown to accumulate in particularly high amounts in the brain of PD patients (Lhermitte et al. 1924). These initial observations were carried out using histological iron stains, such as Prussian blue, and were later confirmed by more advanced methods, such as X-ray fluorescence and mass spectroscopy (Earle 1968; Dexter et al. 1991). However, iron dys-homeostasis in the brain is not PD specific. Considerable accumulations of iron are also found in the brains of patients with Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, prion disorders, and of course those diseases, which are specifically named for their high iron content: the neurodegeneration with brain iron accumulation syndromes (NBIA). For PD patients, this increased iron content is most consistently documented in the substantia nigra, and the putamen by postmortem histology and more recently also by quantitative MRI imaging using quantitative susceptibility mapping (QSM) (He et al. 2015; Wang et al. 2016). The amount of iron accumulation correlated with disease stage and severity (Martin-Bastida et al. 2017). MRI and QSM were able to detect iron deposits in the brains of PD patients in several areas of the brainstem, indicating a promising application of this technique as a biomarker for Parkinson’s disease (Acosta-Cabronero et al. 2016). Among all the trace elements in the human body, iron is the most abundant (Mills et al. 2010). Iron is required for essential cellular processes, such as the generation of ATP in mitochondria and DNA replication. During neurodevelopment, iron is involved in the formation of myelin and its absence from the diet has been associated with hypomyelination (Todorich et al. 2009). Iron concentration in the brain is physiologically increasing with age and the areas of the brain most abundant in this element are the nucleus accumbens, deep cerebellar nuclei, the red nucleus, and parts of the hippocampus as well as the substantia nigra. In the substantia nigra, one the most important reservoirs of iron is the neuromelanin (Double et al. 2003). Neuromelanin is a dark pigment that is thought to originate from metabolic products of catecholamines (such as dopamine) that get oxidized (Zucca et al. 2015). Neuromelanin content
increases with age causing a physiological increase of iron with age as was shown by synchrotron X-ray fluorescence analysis (Bohic et al. 2008). Due to its ability to get easily reduced and oxidized, iron is an important catalytic co-factor of many enzymes that function through redox reactions, such as cytochrome c oxidase, catalase, and oxygenase. However, this redox ability has a downside, because it can participate in the production of reactive oxygen species through the Fenton and Haber–Weiss reactions, resulting in oxidative stress if not counterbalanced. Therefore, the trafficking of iron inside and outside the brain as well as the neurons is tightly regulated (Zecca et al. 2004). The transferrin receptor allows the uptake of iron from the extracellular space, where it is usually bound to transferrin. In addition, the divalent metal ion transporter 1 (DMT1) contributes to import of iron into the cell, while the export is mediated by ferroportin 1 (Sigel et al. 2006). Iron has been implicated to be involved in the pathogenesis of PD for several reasons. Acting as a catalyst, iron is involved in the production of peroxide and hydroxyl radicals which participate in peroxidation of biological membranes, such as mitochondria, ER, etc. Moreover, the effects of iron are exaggerated by dopamine which is auto-oxidized and this further contributes to the generation of reactive oxygen species (Galvin 2006). Besides iron, the expression levels of several proteins involved in iron metabolism are modulated in PD. For example, ferritin levels were shown to be significantly decreased in the substantia nigra of PD patients, while levels of the DMT1 were upregulated in rodent models of PD (Salazar et al. 2008).
Interaction of alpha-synuclein and iron It is of particular interest that very soon after the discovery of aSyn, the protein was shown to contain binding sites for divalent metals, such as copper, manganese, and iron (Rasia et al. 2005; Binolfi et al. 2006). Binding properties of aSyn also change with its phosphorylation state resulting in a higher affinity of aSyn for the ferrous ion at the c-terminus of the protein (Lu et al. 2011). Phosphorylation of aSyn is a noteworthy modification, because it is thought to be a necessary event during the formation of Lewy bodies (Anderson et al. 2006). Even more surprising was the finding that aSyn contains an iron-responsive element (IRE) in its 50 -untranslated region. In iron-depleted conditions, the iron-responsive particle (IRP) binds to the IRE, thus inhibiting protein synthesis, while increased levels of iron result in an iron binding of the IRP, which consecutively fails to bind to the IRE and thus permits translation and protein synthesis. Remarkably, only a few proteins are regulated by an IRE, including ferritin (Anderson et al.
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2012). Therefore, increased levels of iron could result in an increased translation of aSyn protein. Intriguingly, iron as well as other metals, e.g., aluminum, has also been shown to increase the propensity of aSyn to aggregate (Uversky et al. 2001; Kostka et al. 2008). On the other hand, aSyn in the cell acts as a sponge for divalent metals. Overexpression of aSyn in primary midbrain neurons as well as in PC 12 cells resulted in higher intracellular iron levels, and the same could also be shown for manganese (Ortega et al. 2016; Ducic´ et al. 2015). aSyn has been shown to directly bind iron in both the ferric and ferrous states (Peng et al. 2010), and ferric iron is able to foster its aggregation (Levin et al. 2011). In this context, ROS present in the cells could catalyze the oxidation of iron toward its ferric form resulting in an exacerbation of aSyn aggregation. The pro-aggregative effect of iron on aSyn can be increased by the presence of dopamine (Ostrerova-Golts et al. 2000), and besides aggregation, there is a scientific consensus that these three components can exacerbate oxidative stress in PD (Hare and Double 2016). Intriguingly, upon binding, aSyn is able to reduce ferric iron into ferrous iron in the presence of NADPH (Davies et al. 2011). This could be an interesting physiological feature of this protein, as ferrous iron is the bioavailable form of this metal and it also represents its reduced form leading to a diminished oxidative stress in the cell. There is thus large experimental evidence to suggest that iron and aSyn play major roles in the pathogenesis of PD and that the interaction of both can contribute to a vicious circle, in which increased aSyn levels result in iron accumulation or in which iron can trigger aSyn aggregation. Experimentally, it may not be feasible to unequivocally dissect, which one is the hen and which the egg. In a translational therapeutic approach, however, this may be of secondary importance, since both aSyn and iron represent highly promising therapeutic targets for disease-modifying approaches. The reduction of their levels could be an approachable therapeutic aim.
Therapeutic approaches targeting iron Numerous iron chelators with highly variable chemical and biological properties are currently available. An important prerequisite for clinical iron chelation in neurodegenerative disorders is the ability of the drug to cross the blood brain barrier. In addition, the risk of peripheral iron depletion from plasma should be taken into account. The iron chelator clioquinol was shown to improve Parkinsonism in tau knockout mice and to reduce the amount of insoluble forms of aggregated aSyn in a murine model of genetic PD (Finkelstein et al. 2015). However, due to its toxicity in humans, clioquinol is unlikely to be
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transferred into clinics (Mao and Schimmer 2008). Other iron chelators that have shown promising results in preclinical PD studies include desferrioxamine, deferasirox, epigallocatechin-3-gallate (EGCG), VK28 and a novel multitarget iron chelator, radical scavenger, and MAO-inhibitor M30 (Weinreb et al. 2013). A phase II study of polyphenol, a major ingredient of green tea that is also known to act as an iron chelator, was completed in de novo PD patients, but results have not yet been published (NCT00461942) (Bhullar and Rupasinghe 2013). The iron chelator deferiprone is a particularly interesting substance, since it has already been used routinely for the treatment of thalassaemia for several years. Moreover, it was used in a study to treat Friedreich’s ataxia, a neurodegenerative disorder that is caused by the expansion of a GAA repeat in the first intron of the frataxin gene (Boddaert et al. 2007). This trinucleotide expansion leads to defects in the assembly of mitochondrial iron–sulphur cluster proteins resulting in increased intraneuronal accumulation of labile iron and oxidative stress. Nine patients were treated over 6 months with deferiprone, which resulted in significantly less iron accumulation in the dentate nuclei as visualized by MRI. Moreover, motor skills and signs of neuropathy were improved, especially in the younger patients. No serious side effects were reported in this trial. Deferiprone has shown to translocate iron from cells with iron overload to those with iron deficiency thus keeping the total iron content relatively stable, and this concept was thus termed conservative iron chelation (Cabantchik et al. 2013). Preclinical data have demonstrated that application of deferiprone protected mice from MPTP toxicity. In a translational pilot approach, the FAIRPARK-I study showed that application of deferiprone in 40 patients not only reduced the iron-mediated susceptibility in the substantia nigra measured by MRI, but also resulted in improved UPDRS motor scores after 12 months of treatment (Devos et al. 2014). Based on this data, a diseasemodifying effect was postulated and resulted in the design of a currently ongoing clinical trial (FAIRPARK-II), which will assess the ability of deferiprone to attenuate the progression of PD in a larger patient cohort (NCT02655315). Similarly, another ongoing phase 2 trial in Europe and Canada analyzes the effects of an extended-release tablet formulation of deferiprone on the clinical PD rating score UPDRS part III over a 9-month-treatment period (NCT02728843). Moreover, safety of deferiprone treatment, several biomarkers, and cognition will be assessed in this trial.
Therapeutic approaches targeting alpha-synuclein Alpha-synuclein pathology can be targeted from different sides. One option is the immunological depletion of the protein by active and passive immunization approaches.
Alpha-synuclein and iron: two keys unlocking Parkinson’s disease
Antibodies may act through different mechanisms of action, e.g., by binding and facilitating the uptake of aSyn into phagocytes followed by its degradation, an inhibition or dissociation of intracellular aSyn aggregation, or shielding of aSyn against cleavage (Bergstro¨m et al. 2015). IgG antibodies against aSyn have been shown to penetrate into the CNS and reduce memory and learning deficits in a mouse overexpressing human aSyn under the PDGF promoter (Masliah et al. 2011). C-terminal binding of the antibody may be instrumental for successful targeting, since the C-terminal cleavage of aSyn by calpain has been suggested to contribute to the generation of neurotoxic aggregates (Li et al. 2005; Dufty et al. 2007). This approach has been taken further when a humanized monoclonal antibody specifically binding to the C-terminal domain of alphasynuclein (PRX002, Prothena Corp.) was developed and shown to be safe and well-tolerated in a pilot phase I trial (NCT02095171). A multiple ascending-dose trial was recently reported to dose-dependently reduce levels of aSyn with a good safety profile (NCT02157714). Another example for translated passive immunization approaches is the BIIB054 antibody from Biogen (NCT02459886). In contrast to passive immunization, where specific antibodies are directly applied, active immunization (vaccination) strategies employ peptide fragments to elicit a specific immune response. Whereas passive immunization may require the repetitive application of antibodies, active vaccination may confer more long-lasting immunity (Schneeberger et al. 2015). Although active immunization approaches have been in the focus of neurodegenerative disease research for a long time, the incidence of meningoencephalitis in a vaccination trial in patients with Alzheimer’s disease raised severe safety concerns (Gilman et al. 2005). Recently, however, short peptides mimicking a region of the aSyn molecule, so-called AFFITOPEsÒ, have been shown to elicit a more specific immune answer without activating a T-cell response, which has been made responsible for deleterious autoimmune reactions. In mouse models, overexpressing aSyn under the Thy1and the PDGF-promoter active immunization reduced the level of aSyn and was accompanied by a reduction in oligomers, attenuated nigrostriatal degeneration and behavioral improvements (Mandler et al. 2014). This is, so far, the only active vaccination approach that has advanced to clinical trials with the AFFITOPEÒPD01 (Affiris AG). The company recently announced that their phase I trials with patients receiving four initial vaccinations (AFF008) as well as a boost vaccination (AFF008A) were safe and well tolerated over an observation period of 1 year (Affiris 2016) (NCT01568099 and NCT02216188). Next to immunization approaches, small molecules may tackle aSyn pathology from multiple different angles. One promising approach could be to reduce brain levels of aSyn or increase its degradation. The tyrosine kinase inhibitor
nilotinib, for example, increased the autophagic clearance of aSyn in transgenic mouse models and models of lentiviral aSyn overexpression and was effectively attenuating loss of dopaminergic neurons. Nilotinib is a particularly interesting drug from the translational perspective, since it is approved for the treatment of leukemia and shows sufficient penetration into the brain (Hebron et al. 2013). A small phase I trial with PD and DLB patients suggested possible target engagement and demonstrated that the substance may be safe in this population (Pagan et al. 2016; Wyse et al. 2016) (NCT02281474). Two dosages of nilotinib are currently under investigation in a phase II trial, with safety as primary outcome (NCT02954978). Another already approved drug, the low-molecular weight fatty acid glycerol 4-phenylbutyrate, has been suggested to decrease protein levels of aSyn in mouse brains (Inden et al. 2007). A phase I clinical trial now aims to verify whether glycerol 4-phenylbutyrate is able to increase levels of aSyn in the blood, suggesting that it is cleared from the brain (NCT02046434). Other strategies target the formation of toxic multimeric species of aSyn. For example, the green tea ingredient epigallocatechine-3-gallate (EGCG) was shown to inhibit aSyn aggregation in vitro (Caruana et al. 2011). As mentioned previously, EGCG also seems to act as an iron chelator and antioxidant (Ryan and Hynes 2007). It thus targets several major mechanisms involved in the pathogenesis of PD and, in addition, is freely available as a nutritional supplement, which advocated its evaluation in clinical trials. In a current phase III trial, EGCG is, therefore, tested as a diseasemodifying drug in MSA (NCT02008721) (Levin et al. 2016). The compound NPT200-11, an aSyn-stabilizer, was shown to bind to aSyn and to prevent the formation of toxic oligomers in cell membranes. A phase I trial evaluating safety and tolerability of NPT200-11 in PD patients was already conducted (NCT02606682), but the results are not published yet. The oligomer modulator, anle138b, convincingly demonstrated its ability to attenuate oligomer formation of aSyn and other proteins in multiple animal models (Wagner et al. 2013), arguing for its testing in clinical trials. This drug is of particular interest because it has been proven to increase life span even after the onset of symptoms in a transgenic mouse model (Levin et al. 2014). An inhibition of aSyn aggregation in vitro and in vivo was also demonstrated by the clinically approved rho-kinase inhibitor fasudil (Tatenhorst et al. 2016). Because fasudil has also shown to prolong survival and improve behavior in the SOD1.G93A mouse model of ALS, it will shortly be tested in an international phase II clinical trial with ALS patients, which will be an important step towards its translation for PD.
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Fig. 1 Scheme of the pathological interplay between aSyn and iron (Fe) including therapeutic approaches. aSyn translation is regulated by an iron-responsive element (IRE), which the iron-regulatory protein (IRP) binds to. Intracellularly, aSyn aggregates and forms Lewy bodies, which also contains other ubiquitinated proteins and iron. Iron and other transition metals as well as dopamine foster the aggregation of aSyn. Aggregation of aSyn may also result in the formation of calcium (Ca)-permeable pores. Iron import into the cell is mainly operated by the transferrin receptor (TfR), the divalent
metal transporter 1 (DMT1), and the export by ferroportin 1 (Fpn1). aSyn can act as a ferrireductase reducing Fe(III) into Fe(II). Iron chelation, e.g., by deferiprone, removes iron from the system. In a similar way, antibodies directed against aSyn lower the levels of the protein. Several small molecule compounds, e.g., Anle138b, EGCG and fasudil, attenuate the aggregation propensity of aSyn, whereas EGCG can also act as an iron chelator. Nilotinib lowers aSyn levels by driving autophagic degradation of the protein (for details, see text)
Conclusion
proven successful in animal models and are currently translated into human clinical trials (Fig. 1). The results of these clinical trials are urgently awaited to validate both iron and aSyn as targets for disease modification. However, many parameters in trials for disease modification are derived from educated guesses rather than from firm knowledge, and many questions remain open. How big is the contribution of each of the pathomechanisms? Can we
Accumulation of both iron and aggregated aSyn is pathological hallmarks of PD. Although it is not yet fully understood how either one of them contributes to the pathogenesis of PD, it is well known that aSyn and iron interact and can potentiate their respective toxicity. Several therapeutic approaches targeting aSyn and iron have
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Alpha-synuclein and iron: two keys unlocking Parkinson’s disease
expect that targeting just one mechanism is successful for disease-modification? How long should patients be treated to observe an effect? Can a treatment, even in very early symptomatic disease stages, show sufficient effects, given the fact that pathological alterations precede the onset of motor symptoms by decades? Can one therapeutic approach be successful for all PD patients given the variability of this disease? Although animal models have contributed to the dissection of pathomechanisms and to the selection of promising compounds, only a validation in clinical trials will yield clear answers to these questions. Funding of translational clinical trials is thus more important than ever. In the next years, an earlier diagnosis allowing for treatment of pre-motor patients, the use of surrogate biomarkers to better assess therapeutic response and a selection of more homogenous patient subgroups may contribute to a faster translation of disease-modifying strategies.
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TRANSLATIONAL NEUROSCIENCES - REVIEW ARTICLE
Alpha-synuclein and iron: two keys unlocking Parkinson’s disease Paul Lingor1,2
•
Eleonora Carboni1,2 • Jan Christoph Koch1
Received: 26 December 2016 / Accepted: 1 February 2017 Ó Springer-Verlag Wien 2017
Abstract Current therapies for Parkinson’s disease (PD) confer symptomatic relief and are particularly efficient in the treatment of motor symptoms in earlier disease stages. However, we are still unable to treat the causes of neurodegeneration by modification of the underlying mechanisms, which is partially due to their insufficient understanding. In this short review, we focus on two pivotal disease mechanisms: alpha-synuclein pathology and dysfunction of iron homeostasis as well as their intricate interaction. Both pathomechanisms have been extensively studied in the past and represent valid targets for disease-modifying pharmacological treatment approaches for PD. We summarize the current attempts to exploit iron chelation and modification of alpha-synuclein pathology as translational therapies in PD and discuss the chances and challenges of prospective disease-modifying approaches. Keywords Disease mechanism Neurodegeneration Translational therapy
Introduction Within the field of neurodegenerative disorders, Parkinson’s disease (PD) is by far leading with the highest number of available therapeutic options. Levodopa plus & Paul Lingor [email protected] 1
Department of Neurology, University Medicine Go¨ttingen, 37075 Go¨ttingen, Germany
2
Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Go¨ttingen, Germany
decarboxylase inhibitors and dopamine agonists dominated the drug therapy regimes of motor symptoms for decades. During the last years, however, we witnessed not only the marketing of numerous novel formulations within these well-established therapeutic classes employing new extended-release kinetics or alternative ways of application, but also the appearance of entirely novel substances, such as opicapone, istradefylline, or safinamide (Oertel and Schulz 2016). In addition, also the repertoire of drugs targeting non-motor symptoms is growing, the latest additions being droxidopa and pimavanserin (Cummings et al. 2014; Hauser et al. 2016). Although the plethora of current treatment options confers significant improvements in the quality of life of PD patients and the life expectancy of PD patients is continuously growing, this cannot hide the fact that all these options are symptomatic, meaning that they do not alter the progressive pathophysiology of the disease. One of the reasons for the lack of disease-modifying treatments is the fact that the pathogenesis of PD is only insufficiently understood and that PD is likely to be more than one disease: not only there are different clinical phenotypes, but also there is also evidence to suggest that PD is histologically diverse (Halliday et al. 2011; Sauerbier et al. 2016). Protein degradation pathways, calcium homeostasis, mitochondrial function, and the innate immune system have all been suggested to contribute to disease mechanisms in PD (De Rosa et al. 2015). In this short review, we will, however, focus on two pivotal mechanisms, which appear instrumental in the development of the disease, and both of which promise to yield approaches for diseasemodifying therapies in the near future: alpha-synuclein pathology and accumulation of iron.
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Alpha-synuclein pathology As early as in 1912, Lewy described inclusion bodies in neurons of PD patients (Lewy 1912), which now bear his name and shortly thereafter, Tretiakoff revealed the degeneration of dopaminergic neurons as major pathoanatomic substrate of the disease (Tre´tiakoff 1919). It took then almost 80 years until the first family with inherited PD, the so-called Contursi-kindred, was described and shown to possess a mutation of the alpha-synuclein (aSyn) gene (Polymeropoulos et al. 1997). Other point mutations as well as multiplications of the aSyn gene have been described thereafter that all cause autosomal dominant forms of PD (Gasser et al. 2011). Shortly thereafter, Spillantini used antibodies against aSyn to demonstrate that it is the major component of the Lewy bodies found in PD (Spillantini et al. 1997), and Braak then used aSyn immunohistochemistry to map the neuroanatomical distribution of Lewy body pathology in over one hundred brains of autopsy cases with and without clinically diagnosed PD (Braak et al. 2003). This allowed demonstrating a stage-dependent distribution of the pathology, which later led up to the spreading hypothesis of aSyn. Correlating with disease severity, Braak described an expanding Lewy body pathology from the lower brainstem to the pons and midbrain, before being found in the basal forebrain and, ultimately, in the neocortex in the most advanced disease stages. In a subgroup of subjects without documented clinical signs of PD, solitary Lewy body pathology was visualized in the olfactory bulb and anterior olfactory nucleus, as well as in the dorsal motor nucleus of the vagus nerve in the medulla oblongata. This suggests that the olfactory bulb and the vagal nerve are the earliest affected brain structures, probably already in a pre-symptomatic stage. This finding gave rise to the ‘‘dual-hit hypothesis’’, since aSyn pathology appears to enter the brain from two different sites (Hawkes et al. 2007; Angot et al. 2010). Moreover, aSyn pathology has been detected outside the central nervous system in individuals with early PD. It was found in neurons of Auerbach’s and Meissner’s plexuses of the enteric nervous system and in autonomic nerves of the submandibular gland (Wakabayashi et al. 1988; Del Tredici et al. 2010). Based on these findings, it is speculated that a yet unknown environmental pathogen might trigger PD pathogenesis by entering the nervous system through the gastrointestinal system (Pan-Montojo et al. 2012). In line with this hypothesis, it was shown in large registry studies that full truncal vagotomy early in life correlates with a significantly decreased risk of developing PD (Svensson et al. 2015). The spreading theory was further fueled by the description of Lewy bodies in transplanted fetal human midbrain neurons, which had been grafted into the striata of patients with advanced PD in transplantation trials in the
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1990s (Li et al. 2008). The fact that young neurons, aged between 11 and 16 years at the time of analysis contained inclusion bodies, which were previously found only in old brains, was highly suggestive of a pathology that could be transmitted to these implanted neurons. The percentage of previously grafted midbrain neurons that display Lewy body pathology increased from 2 to 5% at 11–16 years after transplantation to 11–12% in a recently examined patient at 24 years after transplantation (Li et al. 2016). However, the alternative hypothesis of local environmental factors in the PD brain inducing pathology in susceptible neurons without spread of a pathological protein cannot be ruled out completely by this data. Based on these findings and in an analogy to previously known infectious proteinaceous particles or prions, the question arose whether PD could be a prion-like disorder. In its native form, aSyn is a soluble protein, but it can assemble into a filamentous, beta-sheet-rich conformation that is prone for aggregation. It has been shown that aSyn fibrils can seed aggregation and the molecular properties of these toxic aSyn fibrils have been analyzed in detail (Rodriguez et al. 2015; Tuttle et al. 2016). Moreover, the injection of different strains of aSyn was described to cause specific types of synucleinopathies in rodent models. While aSyn fibrils conferred severe neuronal death, aSyn ribbons resulted in Lewy neurite formation and aSyn pathology in oligodendrocytes partly resembling the pathology of multiple system atrophy (Peelaerts et al. 2015). It still remains to be investigated whether the different forms of synucleinopathies in humans, including PD, dementia with Lewy bodies and multiple system atrophy, are also caused by different strains of aSyn. In vitro experiments showed that aSyn fibrils are taken up by neurons, seed aggregation of expressed protein in the infected neurons, are transported along the axons, and can then be released and taken up again by other adjacent neurons (Guo and Lee 2014). The uptake of aSyn fibrils into neurons is mediated by macropinocytosis requiring heparan sulphate proteoglycans and endocytosis via the transmembrane protein lymphocyteactivation gene 3 (LAG3) (Holmes et al. 2013; Mao et al. 2016). These recent findings offer new possibilities for developing therapeutics against aSyn spread. While the peripheral injection of aggregated aSyn was not shown to cause formation of brain inclusions in wild-type animals up to date, the injection of aSyn aggregates into the substantia nigra of wild-type rodents resulted in spread of aSyn inclusions and functional deficits of the animals (Luk et al. 2012; Masuda-Suzukake et al. 2014; Paumier et al. 2015). Although there is thus much evidence suggesting that toxic forms of aSyn can indeed confer a misfolded conformation to non-aggregated aSyn in animal models, a prion-like spread has not been proven in humans so far and there is so
Alpha-synuclein and iron: two keys unlocking Parkinson’s disease
far no epidemiological evidence that this disease could be transmitted from one patient to the other (Irwin et al. 2013; Beekes et al. 2014; Walsh and Selkoe 2016).
Accumulation of iron Although the discovery of aSyn involvement strongly influenced our understanding of the pathophysiology of PD during the last 20 years, another mechanism was taking the lead as prime candidate in explaining the etiology of PD: already in the beginning of the 20th century iron was shown to accumulate in particularly high amounts in the brain of PD patients (Lhermitte et al. 1924). These initial observations were carried out using histological iron stains, such as Prussian blue, and were later confirmed by more advanced methods, such as X-ray fluorescence and mass spectroscopy (Earle 1968; Dexter et al. 1991). However, iron dys-homeostasis in the brain is not PD specific. Considerable accumulations of iron are also found in the brains of patients with Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, prion disorders, and of course those diseases, which are specifically named for their high iron content: the neurodegeneration with brain iron accumulation syndromes (NBIA). For PD patients, this increased iron content is most consistently documented in the substantia nigra, and the putamen by postmortem histology and more recently also by quantitative MRI imaging using quantitative susceptibility mapping (QSM) (He et al. 2015; Wang et al. 2016). The amount of iron accumulation correlated with disease stage and severity (Martin-Bastida et al. 2017). MRI and QSM were able to detect iron deposits in the brains of PD patients in several areas of the brainstem, indicating a promising application of this technique as a biomarker for Parkinson’s disease (Acosta-Cabronero et al. 2016). Among all the trace elements in the human body, iron is the most abundant (Mills et al. 2010). Iron is required for essential cellular processes, such as the generation of ATP in mitochondria and DNA replication. During neurodevelopment, iron is involved in the formation of myelin and its absence from the diet has been associated with hypomyelination (Todorich et al. 2009). Iron concentration in the brain is physiologically increasing with age and the areas of the brain most abundant in this element are the nucleus accumbens, deep cerebellar nuclei, the red nucleus, and parts of the hippocampus as well as the substantia nigra. In the substantia nigra, one the most important reservoirs of iron is the neuromelanin (Double et al. 2003). Neuromelanin is a dark pigment that is thought to originate from metabolic products of catecholamines (such as dopamine) that get oxidized (Zucca et al. 2015). Neuromelanin content
increases with age causing a physiological increase of iron with age as was shown by synchrotron X-ray fluorescence analysis (Bohic et al. 2008). Due to its ability to get easily reduced and oxidized, iron is an important catalytic co-factor of many enzymes that function through redox reactions, such as cytochrome c oxidase, catalase, and oxygenase. However, this redox ability has a downside, because it can participate in the production of reactive oxygen species through the Fenton and Haber–Weiss reactions, resulting in oxidative stress if not counterbalanced. Therefore, the trafficking of iron inside and outside the brain as well as the neurons is tightly regulated (Zecca et al. 2004). The transferrin receptor allows the uptake of iron from the extracellular space, where it is usually bound to transferrin. In addition, the divalent metal ion transporter 1 (DMT1) contributes to import of iron into the cell, while the export is mediated by ferroportin 1 (Sigel et al. 2006). Iron has been implicated to be involved in the pathogenesis of PD for several reasons. Acting as a catalyst, iron is involved in the production of peroxide and hydroxyl radicals which participate in peroxidation of biological membranes, such as mitochondria, ER, etc. Moreover, the effects of iron are exaggerated by dopamine which is auto-oxidized and this further contributes to the generation of reactive oxygen species (Galvin 2006). Besides iron, the expression levels of several proteins involved in iron metabolism are modulated in PD. For example, ferritin levels were shown to be significantly decreased in the substantia nigra of PD patients, while levels of the DMT1 were upregulated in rodent models of PD (Salazar et al. 2008).
Interaction of alpha-synuclein and iron It is of particular interest that very soon after the discovery of aSyn, the protein was shown to contain binding sites for divalent metals, such as copper, manganese, and iron (Rasia et al. 2005; Binolfi et al. 2006). Binding properties of aSyn also change with its phosphorylation state resulting in a higher affinity of aSyn for the ferrous ion at the c-terminus of the protein (Lu et al. 2011). Phosphorylation of aSyn is a noteworthy modification, because it is thought to be a necessary event during the formation of Lewy bodies (Anderson et al. 2006). Even more surprising was the finding that aSyn contains an iron-responsive element (IRE) in its 50 -untranslated region. In iron-depleted conditions, the iron-responsive particle (IRP) binds to the IRE, thus inhibiting protein synthesis, while increased levels of iron result in an iron binding of the IRP, which consecutively fails to bind to the IRE and thus permits translation and protein synthesis. Remarkably, only a few proteins are regulated by an IRE, including ferritin (Anderson et al.
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2012). Therefore, increased levels of iron could result in an increased translation of aSyn protein. Intriguingly, iron as well as other metals, e.g., aluminum, has also been shown to increase the propensity of aSyn to aggregate (Uversky et al. 2001; Kostka et al. 2008). On the other hand, aSyn in the cell acts as a sponge for divalent metals. Overexpression of aSyn in primary midbrain neurons as well as in PC 12 cells resulted in higher intracellular iron levels, and the same could also be shown for manganese (Ortega et al. 2016; Ducic´ et al. 2015). aSyn has been shown to directly bind iron in both the ferric and ferrous states (Peng et al. 2010), and ferric iron is able to foster its aggregation (Levin et al. 2011). In this context, ROS present in the cells could catalyze the oxidation of iron toward its ferric form resulting in an exacerbation of aSyn aggregation. The pro-aggregative effect of iron on aSyn can be increased by the presence of dopamine (Ostrerova-Golts et al. 2000), and besides aggregation, there is a scientific consensus that these three components can exacerbate oxidative stress in PD (Hare and Double 2016). Intriguingly, upon binding, aSyn is able to reduce ferric iron into ferrous iron in the presence of NADPH (Davies et al. 2011). This could be an interesting physiological feature of this protein, as ferrous iron is the bioavailable form of this metal and it also represents its reduced form leading to a diminished oxidative stress in the cell. There is thus large experimental evidence to suggest that iron and aSyn play major roles in the pathogenesis of PD and that the interaction of both can contribute to a vicious circle, in which increased aSyn levels result in iron accumulation or in which iron can trigger aSyn aggregation. Experimentally, it may not be feasible to unequivocally dissect, which one is the hen and which the egg. In a translational therapeutic approach, however, this may be of secondary importance, since both aSyn and iron represent highly promising therapeutic targets for disease-modifying approaches. The reduction of their levels could be an approachable therapeutic aim.
Therapeutic approaches targeting iron Numerous iron chelators with highly variable chemical and biological properties are currently available. An important prerequisite for clinical iron chelation in neurodegenerative disorders is the ability of the drug to cross the blood brain barrier. In addition, the risk of peripheral iron depletion from plasma should be taken into account. The iron chelator clioquinol was shown to improve Parkinsonism in tau knockout mice and to reduce the amount of insoluble forms of aggregated aSyn in a murine model of genetic PD (Finkelstein et al. 2015). However, due to its toxicity in humans, clioquinol is unlikely to be
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transferred into clinics (Mao and Schimmer 2008). Other iron chelators that have shown promising results in preclinical PD studies include desferrioxamine, deferasirox, epigallocatechin-3-gallate (EGCG), VK28 and a novel multitarget iron chelator, radical scavenger, and MAO-inhibitor M30 (Weinreb et al. 2013). A phase II study of polyphenol, a major ingredient of green tea that is also known to act as an iron chelator, was completed in de novo PD patients, but results have not yet been published (NCT00461942) (Bhullar and Rupasinghe 2013). The iron chelator deferiprone is a particularly interesting substance, since it has already been used routinely for the treatment of thalassaemia for several years. Moreover, it was used in a study to treat Friedreich’s ataxia, a neurodegenerative disorder that is caused by the expansion of a GAA repeat in the first intron of the frataxin gene (Boddaert et al. 2007). This trinucleotide expansion leads to defects in the assembly of mitochondrial iron–sulphur cluster proteins resulting in increased intraneuronal accumulation of labile iron and oxidative stress. Nine patients were treated over 6 months with deferiprone, which resulted in significantly less iron accumulation in the dentate nuclei as visualized by MRI. Moreover, motor skills and signs of neuropathy were improved, especially in the younger patients. No serious side effects were reported in this trial. Deferiprone has shown to translocate iron from cells with iron overload to those with iron deficiency thus keeping the total iron content relatively stable, and this concept was thus termed conservative iron chelation (Cabantchik et al. 2013). Preclinical data have demonstrated that application of deferiprone protected mice from MPTP toxicity. In a translational pilot approach, the FAIRPARK-I study showed that application of deferiprone in 40 patients not only reduced the iron-mediated susceptibility in the substantia nigra measured by MRI, but also resulted in improved UPDRS motor scores after 12 months of treatment (Devos et al. 2014). Based on this data, a diseasemodifying effect was postulated and resulted in the design of a currently ongoing clinical trial (FAIRPARK-II), which will assess the ability of deferiprone to attenuate the progression of PD in a larger patient cohort (NCT02655315). Similarly, another ongoing phase 2 trial in Europe and Canada analyzes the effects of an extended-release tablet formulation of deferiprone on the clinical PD rating score UPDRS part III over a 9-month-treatment period (NCT02728843). Moreover, safety of deferiprone treatment, several biomarkers, and cognition will be assessed in this trial.
Therapeutic approaches targeting alpha-synuclein Alpha-synuclein pathology can be targeted from different sides. One option is the immunological depletion of the protein by active and passive immunization approaches.
Alpha-synuclein and iron: two keys unlocking Parkinson’s disease
Antibodies may act through different mechanisms of action, e.g., by binding and facilitating the uptake of aSyn into phagocytes followed by its degradation, an inhibition or dissociation of intracellular aSyn aggregation, or shielding of aSyn against cleavage (Bergstro¨m et al. 2015). IgG antibodies against aSyn have been shown to penetrate into the CNS and reduce memory and learning deficits in a mouse overexpressing human aSyn under the PDGF promoter (Masliah et al. 2011). C-terminal binding of the antibody may be instrumental for successful targeting, since the C-terminal cleavage of aSyn by calpain has been suggested to contribute to the generation of neurotoxic aggregates (Li et al. 2005; Dufty et al. 2007). This approach has been taken further when a humanized monoclonal antibody specifically binding to the C-terminal domain of alphasynuclein (PRX002, Prothena Corp.) was developed and shown to be safe and well-tolerated in a pilot phase I trial (NCT02095171). A multiple ascending-dose trial was recently reported to dose-dependently reduce levels of aSyn with a good safety profile (NCT02157714). Another example for translated passive immunization approaches is the BIIB054 antibody from Biogen (NCT02459886). In contrast to passive immunization, where specific antibodies are directly applied, active immunization (vaccination) strategies employ peptide fragments to elicit a specific immune response. Whereas passive immunization may require the repetitive application of antibodies, active vaccination may confer more long-lasting immunity (Schneeberger et al. 2015). Although active immunization approaches have been in the focus of neurodegenerative disease research for a long time, the incidence of meningoencephalitis in a vaccination trial in patients with Alzheimer’s disease raised severe safety concerns (Gilman et al. 2005). Recently, however, short peptides mimicking a region of the aSyn molecule, so-called AFFITOPEsÒ, have been shown to elicit a more specific immune answer without activating a T-cell response, which has been made responsible for deleterious autoimmune reactions. In mouse models, overexpressing aSyn under the Thy1and the PDGF-promoter active immunization reduced the level of aSyn and was accompanied by a reduction in oligomers, attenuated nigrostriatal degeneration and behavioral improvements (Mandler et al. 2014). This is, so far, the only active vaccination approach that has advanced to clinical trials with the AFFITOPEÒPD01 (Affiris AG). The company recently announced that their phase I trials with patients receiving four initial vaccinations (AFF008) as well as a boost vaccination (AFF008A) were safe and well tolerated over an observation period of 1 year (Affiris 2016) (NCT01568099 and NCT02216188). Next to immunization approaches, small molecules may tackle aSyn pathology from multiple different angles. One promising approach could be to reduce brain levels of aSyn or increase its degradation. The tyrosine kinase inhibitor
nilotinib, for example, increased the autophagic clearance of aSyn in transgenic mouse models and models of lentiviral aSyn overexpression and was effectively attenuating loss of dopaminergic neurons. Nilotinib is a particularly interesting drug from the translational perspective, since it is approved for the treatment of leukemia and shows sufficient penetration into the brain (Hebron et al. 2013). A small phase I trial with PD and DLB patients suggested possible target engagement and demonstrated that the substance may be safe in this population (Pagan et al. 2016; Wyse et al. 2016) (NCT02281474). Two dosages of nilotinib are currently under investigation in a phase II trial, with safety as primary outcome (NCT02954978). Another already approved drug, the low-molecular weight fatty acid glycerol 4-phenylbutyrate, has been suggested to decrease protein levels of aSyn in mouse brains (Inden et al. 2007). A phase I clinical trial now aims to verify whether glycerol 4-phenylbutyrate is able to increase levels of aSyn in the blood, suggesting that it is cleared from the brain (NCT02046434). Other strategies target the formation of toxic multimeric species of aSyn. For example, the green tea ingredient epigallocatechine-3-gallate (EGCG) was shown to inhibit aSyn aggregation in vitro (Caruana et al. 2011). As mentioned previously, EGCG also seems to act as an iron chelator and antioxidant (Ryan and Hynes 2007). It thus targets several major mechanisms involved in the pathogenesis of PD and, in addition, is freely available as a nutritional supplement, which advocated its evaluation in clinical trials. In a current phase III trial, EGCG is, therefore, tested as a diseasemodifying drug in MSA (NCT02008721) (Levin et al. 2016). The compound NPT200-11, an aSyn-stabilizer, was shown to bind to aSyn and to prevent the formation of toxic oligomers in cell membranes. A phase I trial evaluating safety and tolerability of NPT200-11 in PD patients was already conducted (NCT02606682), but the results are not published yet. The oligomer modulator, anle138b, convincingly demonstrated its ability to attenuate oligomer formation of aSyn and other proteins in multiple animal models (Wagner et al. 2013), arguing for its testing in clinical trials. This drug is of particular interest because it has been proven to increase life span even after the onset of symptoms in a transgenic mouse model (Levin et al. 2014). An inhibition of aSyn aggregation in vitro and in vivo was also demonstrated by the clinically approved rho-kinase inhibitor fasudil (Tatenhorst et al. 2016). Because fasudil has also shown to prolong survival and improve behavior in the SOD1.G93A mouse model of ALS, it will shortly be tested in an international phase II clinical trial with ALS patients, which will be an important step towards its translation for PD.
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Fig. 1 Scheme of the pathological interplay between aSyn and iron (Fe) including therapeutic approaches. aSyn translation is regulated by an iron-responsive element (IRE), which the iron-regulatory protein (IRP) binds to. Intracellularly, aSyn aggregates and forms Lewy bodies, which also contains other ubiquitinated proteins and iron. Iron and other transition metals as well as dopamine foster the aggregation of aSyn. Aggregation of aSyn may also result in the formation of calcium (Ca)-permeable pores. Iron import into the cell is mainly operated by the transferrin receptor (TfR), the divalent
metal transporter 1 (DMT1), and the export by ferroportin 1 (Fpn1). aSyn can act as a ferrireductase reducing Fe(III) into Fe(II). Iron chelation, e.g., by deferiprone, removes iron from the system. In a similar way, antibodies directed against aSyn lower the levels of the protein. Several small molecule compounds, e.g., Anle138b, EGCG and fasudil, attenuate the aggregation propensity of aSyn, whereas EGCG can also act as an iron chelator. Nilotinib lowers aSyn levels by driving autophagic degradation of the protein (for details, see text)
Conclusion
proven successful in animal models and are currently translated into human clinical trials (Fig. 1). The results of these clinical trials are urgently awaited to validate both iron and aSyn as targets for disease modification. However, many parameters in trials for disease modification are derived from educated guesses rather than from firm knowledge, and many questions remain open. How big is the contribution of each of the pathomechanisms? Can we
Accumulation of both iron and aggregated aSyn is pathological hallmarks of PD. Although it is not yet fully understood how either one of them contributes to the pathogenesis of PD, it is well known that aSyn and iron interact and can potentiate their respective toxicity. Several therapeutic approaches targeting aSyn and iron have
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Alpha-synuclein and iron: two keys unlocking Parkinson’s disease
expect that targeting just one mechanism is successful for disease-modification? How long should patients be treated to observe an effect? Can a treatment, even in very early symptomatic disease stages, show sufficient effects, given the fact that pathological alterations precede the onset of motor symptoms by decades? Can one therapeutic approach be successful for all PD patients given the variability of this disease? Although animal models have contributed to the dissection of pathomechanisms and to the selection of promising compounds, only a validation in clinical trials will yield clear answers to these questions. Funding of translational clinical trials is thus more important than ever. In the next years, an earlier diagnosis allowing for treatment of pre-motor patients, the use of surrogate biomarkers to better assess therapeutic response and a selection of more homogenous patient subgroups may contribute to a faster translation of disease-modifying strategies.
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