Relevance of genetic testing in the gene-targeted trial era: the Rostock Parkinson's disease study.

Estimates of the spectrum and frequency of pathogenic variants in Parkinson's disease (PD) in different populations are currently limited and biased. Furthermore, although therapeutic modification of several genetic targets has reached the clinical trial stage, a major obstacle in conducting these trials is that PD patients are largely unaware of their genetic status and, therefore, cannot be recruited. Expanding the number of investigated PD-related genes and including genes related to disorders with overlapping clinical features in large, well-phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene-targeted clinical trials. The Rostock Parkinson's disease (ROPAD) study is an observational clinical study aiming to determine the frequency and spectrum of genetic variants contributing to PD in a large international cohort. We investigated variants in 50 genes with either an established relevance for PD or possible phenotypic overlap in a group of 12 580 PD patients from 16 countries [62.3% male; 92.0% White; 27.0% positive family history (FH+), median age at onset (AAO) 59 years] using a next-generation sequencing panel. Altogether, in 1864 (14.8%) ROPAD participants (58.1% male; 91.0% White, 35.5% FH+, median AAO 55 years), a PD-relevant genetic test (PDGT) was positive based on GBA1 risk variants (10.4%) or pathogenic/likely pathogenic variants in LRRK2 (2.9%), PRKN (0.9%), SNCA (0.2%) or PINK1 (0.1%) or a combination of two genetic findings in two genes (∼0.2%). Of note, the adjusted positive PDGT fraction, i.e. the fraction of positive PDGTs per country weighted by the fraction of the population of the world that they represent, was 14.5%. Positive PDGTs were identified in 19.9% of patients with an AAO ≤ 50 years, in 19.5% of patients with FH+ and in 26.9% with an AAO ≤ 50 years and FH+. In comparison to the idiopathic PD group (6846 patients with benign variants), the positive PDGT group had a significantly lower AAO (4 years, P = 9 × 10-34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 × 10-35). Female patients were 22% more likely to have a positive PDGT (P = 3 × 10-4), and for individuals with FH+ this likelihood was 55% higher (P = 1 × 10-14). About 0.8% of the ROPAD participants had positive genetic testing findings in parkinsonism-, dystonia/dyskinesia- or dementia-related genes. In the emerging era of gene-targeted PD clinical trials, our finding that ∼15% of patients harbour potentially actionable genetic variants offers an important prospect to affected individuals and their families and underlines the need for genetic testing in PD patients. Thus, the insights from the ROPAD study allow for data-driven, differential genetic counselling across the spectrum of different AAOs and family histories and promote a possible policy change in the application of genetic testing as a routine part of patient evaluation and care in PD.

FARS-ADL across Ataxias: Construct Validity, Sensitivity to Change, and Minimal Important Change.

BACKGROUND: Patient-focused outcomes present a central need for trial-readiness across all ataxias. The Activities of Daily Living part of the Friedreich Ataxia Rating Scale (FARS-ADL) captures functional impairment and longitudinal change but is only validated in Friedreich Ataxia. OBJECTIVE: Validation of FARS-ADL regarding disease severity and patient-meaningful impairment, and its sensitivity to change across genetic ataxias. METHODS: Real-world registry data of FARS-ADL in 298 ataxia patients across genotypes were analyzed, including (1) cross-correlation with FARS-stage, Scale for the Assessment and Rating of Ataxia (SARA), Patient-Reported Outcome Measure (PROM)-ataxia, and European Quality of Life 5 Dimensions visual analogue scale (EQ5D-VAS); (2) sensitivity to change within a trial-relevant 1-year median follow-up, anchored in Patient Global Impression of Change (PGI-C); and (3) general linear modeling of factors age, sex, and depression (nine-item Patient Health Questionnaire [PHQ-9]). RESULTS: FARS-ADL correlated with overall disability (rhoFARS-stage = 0.79), clinical disease severity (rhoSARA = 0.80), and patient-reported impairment (rhoPROM-ataxia = 0.69, rhoEQ5D-VAS = -0.37), indicating comprehensive construct validity. Also at item level, and validated within genotype (SCA3, RFC1), FARS-ADL correlated with the corresponding SARA effector domains; and all items correlated to EQ5D-VAS quality of life. FARS-ADL was sensitive to change at a 1-year interval, progressing only in patients with worsening PGI-C. Minimal important change was 1.1. points based on intraindividual variability in patients with stable PGI-C. Depression was captured using FARS-ADL (+0.3 points/PHQ-9 count) and EQ5D-VAS, but not FARS-stage or SARA. CONCLUSION: FARS-ADL reflects both disease severity and patient-meaningful impairment across genetic ataxias, with sensitivity to change in trial-relevant timescales in patients perceiving change. It thus presents a promising patient-focused outcome for upcoming ataxia trials. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Exploring the lived experiences of individuals with Parkinson's disease and their relatives: insights into care provision experiences, disease management support, self-management strategies, and future needs in Germany (qualitative study).

BACKGROUND: Parkinson's disease (PD) significantly impacts the health-related quality of life of affected individuals and their relatives. In order to support the affected individuals and their families in coping with PD, it is essential to offer comprehensive information about their experiences. A comprehensive understanding of their lived experiences with the disease, the healthcare system, applied self-management strategies and their needs is considered crucial for developing a PD support program. Therefore, we aimed to explore the lived experiences and support needs of individuals with PD and their relatives in Germany. METHODS: This non-interventional, qualitative study conducted an explorative status quo and needs assessment. It generated knowledge through semi-structured focus groups and interviews with individuals with PD at various disease stages and their relatives. The interviews were digitally recorded, transcribed verbatim, and analysed using content analysis. RESULTS: Fifty-two individuals with PD and 29 relatives participated in eight focus groups and 13 paired and 13 individual interviews. Four themes with corresponding subthemes emerged: (1) experiences, revealing individuals' experiences around their diagnosis and with disease-specific care provision; (2) management support offers, clarifying who provides support and the type of support offered; (3) self-management, including comprehensibility, meaningfulness and manageability; and (4) future needs, differentiating between deficits and needs. Most participants expressed a sense of abandonment when obtaining self-management strategies and mastering their lives with PD, often referred to as 'life 2.0'. They identified the lack of structured and adequate provision of information, system orientation and social awareness. CONCLUSIONS: In Germany, there is an urgent need for a comprehensive PD care program that addresses the needs of individuals with PD and their relatives from the start of their care trajectory. It could assist individuals in gaining a comprehensive understanding of the disease, obtaining self-management strategies, building a support network, and becoming experts in self-managing their disease. Moreover, it may positively influence their care trajectory and reduce burdens, such as overburdening, fear of progression, and health anxiety. TRIAL REGISTRATION: German Clinical Studies Register ( https://www.drks.de/DRKS00030090 , No. DRKS00030090, Date of registration: 15.12.2022).

[How the implementation of a school for people with Parkinson's disease can succeed-Results of a consensus study and a formative evaluation]. / Wie die Umsetzung einer Schule für Menschen mit Parkinson-Krankheit gelingen kann ­ Ergebnisse eines Konsensusverfahrens und einer formativen Evaluation.

BACKGROUND: As the most rapidly increasing neurodegenerative disease worldwide, Parkinson's disease is highly relevant to society. Successful treatment requires active patient participation. Patient education has been successfully implemented for many chronic diseases, such as diabetes and could also provide people with Parkinson's disease with skills to manage the disease better and to participate in shared decision making. MATERIAL AND METHODS: To prepare the implementation of a concept for patient education for people with Parkinson's disease, a structured consensus study was conducted and a pilot project formatively evaluated. The structured consensus study included experts from all over Germany. It consisted of two online surveys and an online consensus conference. The formative evaluation was conducted as three focus groups. Transcripts were evaluated using content-structuring qualitative content analysis. RESULTS: From the consensus procedure 59 consented statements emerged, mainly regarding the contents of a patient school and a group size of 6-8 persons. Only two statements could not be consented. The formative evaluation detected a tendency towards a positive attitude for a digital training format and a very positive evaluation of the contents. DISCUSSION: Overall, important recommendations for a patient school can be drawn from this study. The following subjects require further investigation: format, inclusion criteria, group composition and inclusion of caregivers.

The heterogeneity of Parkinson's disease.

The heterogeneity of Parkinson's disease (PD), i.e. the various clinical phenotypes, pathological findings, genetic predispositions and probably also the various implicated pathophysiological pathways pose a major challenge for future research projects and therapeutic trail design. We outline several pathophysiological concepts, pathways and mechanisms, including the presumed roles of α-synuclein misfolding and aggregation, Lewy bodies, oxidative stress, iron and melanin, deficient autophagy processes, insulin and incretin signaling, T-cell autoimmunity, the gut-brain axis and the evidence that microbial (viral) agents may induce molecular hallmarks of neurodegeneration. The hypothesis is discussed, whether PD might indeed be triggered by exogenous (infectious) agents in susceptible individuals upon entry via the olfactory bulb (brain first) or the gut (body-first), which would support the idea that disease mechanisms may change over time. The unresolved heterogeneity of PD may have contributed to the failure of past clinical trials, which attempted to slow the course of PD. We thus conclude that PD patients need personalized therapeutic approaches tailored to specific phenomenological and etiologic subtypes of disease.

[Gene Therapy for Huntington Disease]. / Gentherapie der Huntington-Krankheit.

Englisch: Being one of the most common genetic neurodegenerative disease, Huntington's disease has been a model disease - also for gene therapy. Among the various options, the development of antisense oligonucleotides is the most advanced. Further options at the RNA level include micro-RNAs and modulators of RNA processing (splicing), at the DNA level zinc finger proteins. Several products are in clinical trials. These differ in their mode of application and in the extent of systemic availability. Another important difference between therapeutic strategies could be whether all forms of the huntingtin protein are targeted in the same extent, or whether a therapy preferentially targets particular toxic forms such as the exon1 protein. The results of the recently terminated GENERATION HD1 trial were somewhat sobering, most likely due to the side effect-related hydrocephalus. Therefore they represent just one step towards the development of an effective gene therapy against Huntington's disease.

Maturing Autophagosomes are Transported Towards the Cell Periphery.

Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson's disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP "tandem-fluorescence" tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

Adaptive changes in striatal projection neurons explain the long duration response and the emergence of dyskinesias in patients with Parkinson's disease.

Neuronal activity in the brain is tightly regulated. During operation in real time, for instance, feedback and feedforward loops limit excessive excitation. In addition, cell autonomous processes ensure that neurons' average activity is restored to a setpoint in response to chronic perturbations. These processes are summarized as homeostatic plasticity (Turrigiano in Cold Spring Harb Perspect Biol 4:a005736-a005736, 2012). In the basal ganglia, information is mainly transmitted through disinhibition, which already constraints the possible range of neuronal activity. When this tightly adjusted system is challenged by the chronic decline in dopaminergic neurotransmission in Parkinson's disease (PD), homeostatic plasticity aims to compensate for this perturbation. We here summarize recent experimental work from animals demonstrating that striatal projection neurons adapt excitability and morphology in response to chronic dopamine depletion and substitution. We relate these cellular processes to clinical observations in patients with PD that cannot be explained by the classical model of basal ganglia function. These include the long duration response to dopaminergic medication that takes weeks to develop and days to wear off. Moreover, dyskinesias are considered signs of excessive dopaminergic neurotransmission in Parkinson's disease, but they are typically more severe on the body side that is more strongly affected by dopamine depletion. We hypothesize that these clinical observations can be explained by homeostatic plasticity in the basal ganglia, suggesting that plastic changes in response to chronic dopamine depletion and substitution need to be incorporated into models of basal ganglia function. In addition, better understanding the molecular mechanism of homeostatic plasticity might offer new treatment options to avoid motor complications in patients with PD.

GBA-associated PD: chances and obstacles for targeted treatment strategies.

Given the clear role of GBA in the pathogenesis of Parkinson's disease (PD) and its impact on phenotypical characteristics, this review provides an overview of the current knowledge of GBA-associated PD with a special focus on clinical trajectories and the underlying pathological mechanisms. Importantly, differences and characteristics based on mutation severity are recognized, and current as well as potential future treatment options are discussed. These findings will inform future strategies for patient stratification and cohort enrichment as well as suitable outcome measures when designing clinical trials.

Potential of Non-Coding RNA as Biomarkers for Progressive Supranuclear Palsy.

Objective markers for the neurodegenerative disorder progressive supranuclear palsy (PSP) are needed to provide a timely diagnosis with greater certainty. Non-coding RNA (ncRNA), including microRNA, piwi-interacting RNA, and transfer RNA, are good candidate markers in other neurodegenerative diseases, but have not been investigated in PSP. Therefore, as proof of principle, we sought to identify whether they were dysregulated in matched serum and cerebrospinal fluid (CSF) samples of patients with PSP. Small RNA-seq was undertaken on serum and CSF samples from healthy controls (n = 20) and patients with PSP (n = 31) in two cohorts, with reverse transcription-quantitative PCR (RT-qPCR) to confirm their dysregulation. Using RT-qPCR, we found in serum significant down-regulation in hsa-miR-92a-3p, hsa-miR-626, hsa-piR-31068, and tRNA-ValCAC. In CSF, both hsa-let-7a-5p and hsa-piR-31068 showed significant up-regulation, consistent with their changes observed in the RNA-seq results. Interestingly, we saw no correlation in the expression of hsa-piR-31068 within our matched serum and CSF samples, suggesting there is no common dysregulatory mechanism between the two biofluids. While these changes were in a small cohort of samples, we have provided novel evidence that ncRNA in biofluids could be possible diagnostic biomarkers for PSP and further work will help to expand this potential.

[Treatment practice of patients with Parkinson's disease in Saxony : A secondary data-based analysis of utilization in the observation period 2011-2019]. / Versorgungssituation von Parkinson-Patienten in Sachsen : Eine sekundärdatenbasierte Analyse der Inanspruchnahme im Beobachtungszeitraum 2011 bis 2019.

BACKGROUND: The consequences of demographic change are already noticeable in Saxony, the federal state with the highest average age in Germany and predominantly rural areas. In order to improve medical care for patients with Parkinson's disease (PwP), a status quo analysis of current care practice is required. OBJECTIVE: To what extent does the utilization of medical services by PwP differ a) between urban and rural areas in Saxony and b) between PwP with and without neurologist contact in the observation period from 2011 to 2019? MATERIAL AND METHODS: The cohort study was based on extensive routine data for Saxony from the health insurance company AOK PLUS from 2010 to 2019. A cohort of 15,744 PwP (n = 67,448 patient-years) was compared to a matched cohort (n = 674,480 patient-years; criteria: year of birth, gender, year of insurance, place of residence: urban/rural) without an ICD-10 coding of a movement disorder. RESULTS: Overall, there was a steady increase in the number of PwP in the dynamic cohort from 2011 (n = 6829) to 2019 (n = 8254). Urban-rural differences included a smaller proportion of patients being seen by a neurologist in rural areas. The PwP had a 3.5 to 4­fold higher risk of dying compared to those in the comparison cohort. Changes in drug therapy for Parkinson's disease (i.e., increases in COMT and MAO inhibitors) and in remedy delivery (i.e., increases in occupational therapy and speech therapy) over the observation period were primarily seen in PwP who were seen by a neurologist. DISCUSSION: The study identified increased morbidity and mortality in PwP who are suitable targets for innovative care concepts. The increasing number of patients and the described differences document the need for this. At the same time, changes in prescription practice show that innovative forms of treatment are being used by neurologists in outpatient care.

Improved in vivo PET imaging of the adenosine A2A receptor in the brain using [18F]FLUDA, a deuterated radiotracer with high metabolic stability.

PURPOSE: The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. METHODS: [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. RESULTS: [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72-180 GBq/µmol. Autoradiography proved A2A receptor-specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 µg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. CONCLUSIONS: The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application.

Emotional well-being and pain could be a greater determinant of quality of life compared to motor severity in cervical dystonia.

Non-motor symptoms (NMS) occur in patients with cervical dystonia (CD) but with variable frequencies and impact on health-related quality of life (HRQoL). To define non-motor and motor profiles and their respective impact on HRQoL in CD patients using the newly validated Dystonia Non-Motor Symptoms Questionnaire (DNMSQuest). In an observational prospective multicentre case-control study, we enrolled 61 patients with CD and 61 age- and sex-matched healthy controls (HC) comparing demographic data, motor and non-motor symptoms and HRQoL measurements. 95% CD patients reported at least one NMS. Mean total NMS score was significantly higher in CD patients (5.62 ± 3.33) than in HC (1.74 ± 1.52; p < 0.001). Pain, insomnia and stigma were the most prevalent NMS and HRQoL was significantly impaired in CD patients compared to HC. There was strong correlation of NMS burden with HRQoL (CDQ-24: r = 0.72, EQ-5D: r = - 0.59; p < 0.001) in CD patients. Regression analysis between HRQoL and NMS suggested that emotional well-being (standardized beta = - 0.352) and pain (standardized beta = - 0.291) had a major impact on HRQoL while, in contrast motor severity had no significant impact in this model. Most NMS with the exception of pain, stigma and ADL did not correlate with motor severity. NMS are highly prevalent in CD patients and occur independent of age, sex, disease duration, duration of botulinum neurotoxin therapy and socio-economic status. Specific NMS such as emotional well-being and pain have a major impact on HRQoL and are more relevant than motor severity.

The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta.

BACKGROUND: ADP-ribosylation is a ubiquitous post-translational modification that involves both mono- and poly-ADP-ribosylation. ARTD10, also known as PARP10, mediates mono-ADP-ribosylation (MARylation) of substrate proteins. A previous screen identified protein kinase C delta (PKCδ) as a potential ARTD10 substrate, among several other kinases. The voltage-gated K+ channel Kv1.1 constitutes one of the dominant Kv channels in neurons of the central nervous system and the inactivation properties of Kv1.1 are modulated by PKC. In this study, we addressed the role of ARTD10-PKCδ as a regulator of Kv1.1. RESULTS: We found that ARTD10 inhibited PKCδ, which increased Kv1.1 current amplitude and the proportion of the inactivating current component in HeLa cells, indicating that ARTD10 regulates Kv1.1 in living cells. An inhibitor of ARTD10, OUL35, significantly decreased peak amplitude together with the proportion of the inactivating current component of Kv1.1-containing channels in primary hippocampal neurons, demonstrating that the ARTD10-PKCδ signaling cascade regulates native Kv1.1. Moreover, we show that the pharmacological blockade of ARTD10 increases excitability of hippocampal neurons. CONCLUSIONS: Our results, for the first time, suggest that MARylation by ARTD10 controls neuronal excitability.

Advancement of PD Is Reflected by White Matter Changes in Olfactory Areas: A Pilot Study.

Loss of sense of smell is a well-known non-motor symptom of Parkinson's disease (PD). Here, we present insight into the association between PD advancement and equivalents of smell loss in olfactory-eloquent brain areas, such as the posterior cortex and orbitofrontal cortex. Twelve PD patients in different Hoehn and Yahr stages and 12 healthy normosmic individuals were examined with diffusion tensor imaging. Tract-based spatial statistics were used to analyze microstructural changes in white matter adjacent to the bilateral posterior and orbitofrontal cortex. Axial diffusivity, mean diffusivity, and radial diffusivity were significantly higher in olfactory ROIs in advanced PD patients. The results of this preliminary study indicate that PD advancement is associated with progressive neurodegeneration in olfactory-related brain areas.

[Huntington's disease and Sydenham's chorea]. / Huntington-Krankheit und Chorea minor.

Huntington's disease was an important example for discussing the problem of predictive genetic testing. Like other movement disorders, it includes non-motor symptoms and a prodromal phase. As a rapidly progressive monogenetic disease, it is an important model disease for the study of neurodegenerative pathomechanisms and one of the first movement disorders for which causal therapies seem to be reachable.

[Structure and efferences of the substantia nigra pars compacta in Parkinson's disease]. / Struktur und Efferenzen der Substantia nigra pars compacta beim idiopathischen Parkinson-Syndrom.

There is consensus that the neuropathological characteristic of Parkinson's disease (PD) is the neuronal cell loss of the substantia nigra pars compacta (SNc) in connection with a Lewy pathology. The transsynaptic spread of Lewy pathology is considered essential in PD pathogenesis. Therefore, the knowledge of pre-existing neuroanatomical connections of the SNc is essential. We describe recent animal experiments on the afferent and efferent projections of the SNc and discuss the evidence for and against the sequential transsynaptic spread of Lewy pathology in the pathogenesis of PD.

Fingolimod (FTY720) is not protective in the subacute MPTP mouse model of Parkinson's disease and does not lead to a sustainable increase of brain-derived neurotrophic factor.

Parkinson's disease (PD) is characterized by the loss of midbrain dopaminergic neurons and aggregates of α-synuclein termed Lewy bodies. Fingolimod (FTY720) is an agonist of sphingosine-1 phosphate receptors and an approved oral treatment for multiple sclerosis. Fingolimod elevates brain-derived neurotrophic factor (BDNF), an important neurotrophic factor for dopaminergic neurons. BDNF and fingolimod are beneficial in several animal models of PD. In order to validate the therapeutic potential of fingolimod for the treatment of PD, we tested its effect in the subacute MPTP mouse model of PD. MPTP or vehicle was applied i.p. in doses of 30 mg/kg MPTP on five consecutive days. In order to recapitulate the combination of dopamine loss and α-synuclein aggregates found in PD, MPTP was first administered in Thy1-A30P-α-synuclein transgenic mice. Fingolimod was administered i.p. at a dose of 0.1 mg/kg every second day. Nigrostriatal degeneration was assayed by stereologically counting the number of dopaminergic neurons in the substantia nigra pars compacta, by analysing the concentration of catecholamines and the density of dopaminergic fibres in the striatum. MPTP administration produced a robust nigrostriatal degeneration, comparable to previous studies. Unexpectedly, we found no difference between mice with and without fingolimod treatment, neither at baseline, nor at 14 or 90 days after MPTP. Also, we found no effect of fingolimod in the subacute MPTP mouse model when we used wildtype mice instead of α-synuclein transgenic mice, and no effect with an increased dose of 1 mg/kg fingolimod administered every day. In order to explain these findings, we analysed BDNF regulation by fingolimod. We did find an increase of BDNF protein after a single injection of fingolimod 0.1 or 1.0 mg/kg, but not after multiple injections, indicating that the BDNF response to fingolimod is unsustainable over time. Taken together we did not observe a neuroprotective effect of fingolimod in the subacute MPTP mouse model of PD. We discuss possible explanations for this discrepancy with previous findings and conclude fingolimod might be beneficial for the nonmotor symptoms of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* and *Open Data* because it provided all relevant information to reproduce the study in the manuscript and because it made the data publicly available. The data can be accessed at https://osf.io/6xgfn/. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that over-expression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson's disease, we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP-decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 over-expression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7-induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7-induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α-synuclein and could be beneficial in patients with Parkinson's disease. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Faim2 contributes to neuroprotection by erythropoietin in transient brain ischemia.

Delayed cell death in the penumbra region of acute ischemic stroke occurs through apoptotic mechanisms, making it amenable to therapeutic interventions. Fas/CD95 mediates apoptotic cell death in response to external stimuli. In mature neurons, Fas/CD95 signaling is modulated by Fas-apoptotic inhibitory molecule 2 (Faim2), which reduces cell death in animal models of stroke, meningitis, and Parkinson disease. Erythropoietin (EPO) has been studied as a therapeutic strategy in ischemic stroke. Erythropoietin stimulates the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway, which regulates Faim2 expression. Therefore, up-regulation of Faim2 may contribute to neuroprotection by EPO. Male Faim2-deficient mice (Faim2-/- ) and wild-type littermates (WT) were subjected to 30 min of middle cerebral artery occlusion (MCAo) followed by 72 h of reperfusion. EPO was applied before (30 min) and after (24 and 48 h) MCAo. In WT mice application of EPO at a low dose (5000 U/kg) significantly reduced stroke volume, whereas treatment with high dose (90 000 U/kg) did not. In Faim2-/- animals administration of low-dose EPO did not result in a significant reduction in stroke volume. Faim2 expression as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) increased after low-dose EPO but not with high dose. An extensive phenotyping including analysis of cerebral vessel architecture did not reveal confounding differences between the genotypes. In human post-mortem brain Faim2 displayed a differential expression in areas of penumbral ischemia. Faim2 up-regulation may contribute to the neuroprotective effects of low-dose erythropoietin in transient brain ischemia. The dose-dependency may explain mixed effects of erythropoietin observed in clinical stroke trials.