Silencing of the Pink1 gene expression by conditional RNAi does not induce dopaminergic neuron death in mice.

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson\u27s disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson\u27s disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson\u27s disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo

Разработка бизнес-модели стартапа системы совместного использования велосипедов

В работе приведены теоретические основы построения бизнес-моделей и оценки их эффективности. Исследована сфера систем совместного использования велосипедов, выявлены проблемы и перспективы. На основе проведенной работы была проведена разработка и тестирование бизнес-модели стартап-проекта системы совместного использования велосипедов. В результате была описана концепция данного проекта, практическая значимость которой заключается в запуске проекта системы совместного использования велосипедов в формате стартапа. Методы исследования включают в себя: конкурентный анализ, анализ научной и бизнес литературы, изучение актуальной практики, сравнение, а также customer development, инвестиционный анализ.The theoretical foundations of building business models and assessing their effectiveness are presented in the dissertation. The bicycle sharing systems has been investigated, problems and prospects have been identified. Based on the work carried out, the development and testing of a business model of a startup project for a bicycle sharing system was carried out. As a result, the concept of this project was described, the practical significance of which lies in the launch of a project for a bicycle sharing system in a startup format. Research methods include: competitive analysis, analysis of scientific and business literature, study of current practice, comparison, as well as customer development, investment analysis

Loss of Neuroligin3 specifically downregulates retinal GABAAα2 receptors without abolishing direction selectivity.

The postsynaptic adhesion proteins Neuroligins (NLs) are essential for proper synapse function, and their alterations are associated with a variety of neurodevelopmental disorders. It is increasingly clear that each NL isoform occupies specific subsets of synapses and is able to regulate the function of discrete networks. Studies of NL2 and NL4 in the retina in particular have contributed towards uncovering their role in inhibitory synapse function. In this study we show that NL3 is also predominantly expressed at inhibitory postsynapses in the retinal inner plexiform layer (IPL), where it colocalizes with both GABAA- and glycinergic receptor clusters in a 3:2 ratio. In the NL3 deletion-mutant (knockout or KO) mouse, we uncovered a dramatic reduction of the number of GABAAα2-subunit containing GABAA receptor clusters at the IPL. Retinal activity was thereafter assessed in KO and wild-type (WT) littermates by multi-electrode-array recordings of the output cells of retina, the retinal ganglion cells (RGCs). RGCs in the NL3 KO showed reduced spontaneous activity and an altered response to white noise stimulation. Moreover, upon application of light flashes, the proportion of cells firing at light offset (OFF RGCs) was significantly lower in the NL3 KO compared to WT littermates, whereas the relative number of cells firing at light onset (ON RGCs) increased. Interestingly, although GABAAα2-bearing receptors have been related to direction-selective circuits of the retina, features of direction selective-retinal ganglion cells recorded remained unperturbed in the NL3 KO. Together our data underscore the importance of NL3 for the integrity of specific GABAAergic retinal circuits and identifies NL3 as an important regulator of retinal activity

Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future

Exploration potenzieller Barrieren für die Akzeptanz eines interdisziplinären sektorenübergreifenden Versorgungsnetzwerkes für Patient*innen mit Morbus Parkinson

Hintergrund Mit dem ParkinsonNetzwerk Ostsachsen (PANOS) soll ein intersektorales, pfadbasiertes und plattformunterstütztes Versorgungskonzept etabliert werden, um trotz steigender Behandlungszahlen eine flächendeckende Parkinson-Versorgung mit adäquaten Therapien zu unterstützen. Fragestellung Welche Barrieren könnten die Akzeptanz und eine erfolgreiche Verstetigung des PANOS-Behandlungspfades gefährden? Methode Implementierungsbarrieren wurden über eine selektive Literaturrecherche identifiziert und in einer Onlinebefragung von 36 projektassoziierten Neurolog*innen und Hausärzt*innen priorisiert. Die Auswertung der Ergebnisse erfolgte anonymisiert und deskriptiv. Ergebnisse Dreizehn mögliche Implementierungsbarrieren wurden identifiziert. Es nahmen 11 Neurolog*innen und 7 Hausärzt*innen an der Onlineumfrage teil. Die befragten Neurolog*innen sahen in Doppeldokumentationen sowie in unzureichender Kommunikation und Kooperation zwischen den Leistungserbringenden die größten Hindernisse für eine Akzeptanz von PANOS. Hausärzt*innen beurteilten u. a. die restriktiven Verordnungs- und Budgetgrenzen und den möglicherweise zu hohen Zeitaufwand für Netzwerkprozesse als hinderlich. Diskussion Doppeldokumentationen von Patienten- und Behandlungsdaten sind zeitintensiv und fehleranfällig. Die Akzeptanz kann durch adäquate finanzielle Kompensation der Leistungserbringenden erhöht werden. Das hausärztliche Verordnungsverhalten könnte durch die Verwendung interventionsbezogener Abrechnungsziffern positiv beeinflusst werden. Die Ergebnisse zeigen u. a. einen Bedarf an integrativen technischen Systemlösungen und sektorenübergreifenden Dokumentationsstrukturen, um den Mehraufwand für Leistungserbringende zu reduzieren. Schlussfolgerung Eine Vorabanalyse der Einflussfaktoren von PANOS sowie die Sensibilisierung aller mitwirkenden Akteure für potenzielle Barrieren sind entscheidend für die Akzeptanz des Versorgungsnetzwerkes. Gezielte Maßnahmen zur Reduzierung und Vermeidung identifizierter Barrieren können die anwenderseitige Akzeptanz erhöhen und die Behandlungsergebnisse optimieren.Introduction The ParkinsonNetwork Eastern Saxony (PANOS) aims to establish an intersectoral, path-based and platform-supported care concept in order to support comprehensive care with adequate therapies despite the increasing number of patients to be treated. Objective Which barriers may limit the acceptance and successful implementation of PANOS? Methods Implementation barriers were identified through a selective literature review and prioritized in an online survey of 36 project-associated neurologists and general practitioners. The results were analyzed anonymously and descriptively. Results Thirteen potential implementation barriers were identified. Eleven neurologists and seven general practitioners participated in the online survey. The surveyed neurologists assessed double documentation and inadequate communication and cooperation between the service providers as the biggest obstacles to the acceptance of PANOS. General practitioners rated the restrictions for prescription and budget and the potentially high time expenditure required for network activities as barriers. Discussion Double documentation of patient and treatment data is time consuming and prone to errors. Adequate financial compensation could increase service providers’ willingness to participate in such measures. In addition, the prescribing behavior of general practitioners may be influenced positively by the use of intervention-related accounting numbers. The results indicate a need for integrative technical system solutions and intersectoral documentation structures in order to reduce the additional effort for service providers. Conclusion Analyzing the influencing factors of the PANOS network, and raising the awareness of all participating service providers to potential barriers, are decisive measures for the acceptance of the care network. Targeted measures to reduce and avoid identified barriers can increase user acceptance and optimize treatment results

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC

The Mitochondrial Chaperone Protein TRAP1 Mitigates α-Synuclein Toxicity

Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein–induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein–expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein–induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein–induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein

Relevance of genetic testing in the gene-targeted trial era: the Rostock Parkinson\u27s disease study

\ua9 The Author(s) 2024. 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 7 10−34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 7 10−35). Female patients were 22% more likely to have a positive PDGT (P = 3 7 10−4), and for individuals with FH+ this likelihood was 55% higher (P = 1 7 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

Autosomal Recessive Cerebellar Ataxias in Europe: Frequency, Onset, and Severity in 677 Patients

© 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.This work was supported by the European Union's Horizon 2020 research and innovation program as part of the innovation project EVIDENCE-RND under the EJP RD COFUND-EJP (825575 to M.S.), as part of Solve-RD (779257 to J.B., M.S., and B.P.v.d.W.), by the DFG under the frame of EJP-RD network PROSPAX (441409627 to M.S. and B.P.v.d.W.), and by the Clinician Scientist program “PRECISE.net” funded by the Else Kröner-Fresenius-Stiftung (to A.T.). The study was further funded by the Federal Ministry of Education and Research, Germany, and through the TreatHSP network (01GM1905 to L.S.). B.P.v.d.W. receives additional research support from ZonMW, NWO, Hersenstichting, Brugling fonds, Gossweiler Foundation, and Radboud university medical center. L.S., T.K., G.Z., B.P.v.d.W., and M.S. are members of the European Reference Network for Rare Neurological Diseases—Project ID 739510. A.T. receives funding from the University of Tübingen, medical faculty, for the Clinician Scientist Program Grant 439-0-0. P.K. receives funding from University of Szeged (Hetényi Géza: 5S330 A202) and Ministry of Innovation and Technology of Hungary, National Research, Development and Innovation Fund (TKP2021-EGA). J.B. was supported by a Senior Clinical Researcher mandate of the Research Fund—Flanders (FWO) under grant agreement number 1805021N and is a member of the μNEURO Research Centre of Excellence of the University of Antwerp. F.M.S. was supported by the Italian Ministry of Health (the EJP-RD network PROSPAX; Ricerca Finalizzata RF-2016-02361610; RF-2019-12370417; Ricerca Corrente, RC 5x1000). Several authors of this publication are members of the European Reference Network for Rare Neuromuscular Diseases (ERN EURO-NMD) and of the European Reference Network for Rare Neurological Diseases (ERN-RND). Open Access funding enabled and organized by Projekt DEAL.Peer reviewe