Parkinson's disease can be caused by mutations in the α‐synuclein gene and is characterized by aggregates of α‐synuclein protein. Aggregates are degraded by the autophago‐lysosomal pathway. Since ...Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over‐expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α‐synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP‐Rab7‐decorated vesicles contain α‐synuclein. Rab7 over‐expression reduced the percentage of cells with α‐synuclein particles and the amount of α‐synuclein protein. Time‐lapse microscopy confirmed that particles frequently disappeared with Rab7 over‐expression. Clearance of α‐synuclein is explained by the increased occurrence of acidified α‐synuclein vesicles with Rab7 over‐expression, presumably representing autolysosomes. Rab7 over‐expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T‐α‐synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome‐like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α‐synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate‐limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease.
Cells over‐expressing aggregation‐prone A53T alpha‐synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time‐lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over‐expression, indicating that availability of Rab7 is rate‐limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.
Cells over‐expressing aggregation‐prone A53T alpha‐synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time‐lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over‐expression, indicating that availability of Rab7 is rate‐limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.
Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative diseases. Our aim was to study the conversion to manifest ataxia among apparently healthy carriers of mutations associated with ...the most common SCAs (SCA1, SCA2, SCA3, and SCA6), and the sensitivity of clinical and functional measures to detect change in these individuals.
In this prospective, longitudinal, observational cohort study, based at 14 referral centres in seven European countries, we enrolled children or siblings of patients with SCA1, SCA2, SCA3, or SCA6. Eligible individuals were those without ataxia, defined by a score on the Scale for the Assessment and Rating of Ataxia (SARA) of less than 3; participants had to be aged 18–50 years for children or siblings of patients with SCA1, SCA2, or SCA3, and 35–70 years for children or siblings of patients with SCA6. Study visits took place at recruitment and after 2, 4, and 6 years (plus or minus 3 months). We did genetic testing to identify mutation carriers, with results concealed to the participant and clinical investigator. We assessed patients with clinical scales, questionnaires of patient-reported outcome measures, a rating of the examiner's confidence of presence of ataxia, and performance-based coordination tests. Conversion to ataxia was defined by an SARA score of 3 or higher. We analysed the association of factors at baseline with conversion to ataxia and the evolution of outcome parameters on temporal scales (time from inclusion and time to predicted age at ataxia onset) in the context of mutation status and conversion status. This study is registered with ClinicalTrials.gov, NCT01037777.
Between Sept 13, 2008, and Oct 28, 2015, 302 participants were enrolled. We analysed data for 252 participants with at least one follow-up visit. 83 (33%) participants were from families affected by SCA1, 99 (39%) by SCA2, 46 (18%) by SCA3, and 24 (10%) by SCA6. In participants who carried SCA mutations, 26 (52%) of 50 SCA1 carriers, 22 (59%) of 37 SCA2 carriers, 11 (42%) of 26 SCA3 carriers, and two (13%) of 15 SCA6 carriers converted to ataxia. One (3%) of 33 SCA1 non-carriers and one (2%) of 62 SCA2 non-carriers converted to ataxia. Owing to the small number of people who met our criteria for ataxia, subsequent analyses could not be done in carriers of the SCA6 mutation. Baseline factors associated with conversion were age (hazard ratio 1·13 95% CI 1·03–1·24; p=0·011), CAG repeat length (1·25 1·11–1·41; p=0·0002), and ataxia confidence rating (1·72 1·23–2·41; p=0·0015) for SCA1; age (1·08 1·02–1·14; p=0·0077) and CAG repeat length (1·65 1·27–2·13; p=0·0001) for SCA2; and age (1·27 1·09–1·50; p=0·0031), confidence rating (2·60 1·23–5·47; p=0·012), and double vision (14·83 2·15–102·44; p=0·0063) for SCA3. From the time of inclusion, the SARA scores of SCA1, SCA2, and SCA3 mutation carriers increased, whereas they remained stable in non-carriers. On a timescale defined by the predicted time of ataxia onset, SARA progression in SCA1, SCA2, and SCA3 mutation carriers was non-linear, with marginal progression before ataxia and increasing progression after ataxia onset.
Our study provides quantitative data on the conversion of non-ataxic SCA1, SCA2, and SCA3 mutation carriers to manifest ataxia. Our data could prove useful for the design of preventive trials aimed at delaying the onset of ataxia by aiding sample size calculations and stratification of study participants.
European Research Area Network for Research Programmes on Rare Diseases, Polish Ministry of Science and Higher Education, Italian Ministry of Health, European Community's Seventh Framework Programme.
TDP‐43 is an RNA/DNA‐binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP‐43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. ...Besides aggregation of TDP‐43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP‐43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP‐43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH‐SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl‐tubulin. HDAC6 levels were restored by re‐expression of TDP‐43, dependent on RNA binding and the C‐terminal protein interaction domains. Moreover, TDP‐43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP‐43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6‐dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ‐expanded ataxin‐3 were found in TDP‐43 silenced cells. In conclusion, loss of functional TDP‐43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.
Objectives
We aimed to objectify and compare persisting self‐reported symptoms in initially hospitalized and non‐hospitalized patients after infection with severe acute respiratory syndrome ...coronavirus 2 (SARS‐CoV‐2) by applying clinical standardized measures.
Methods
We conducted a cross‐sectional study of adult patients with confirmed SARS‐CoV‐2 infection including medical history, neurological examination, blood markers, neuropsychological testing, patient‐reported outcome measures (PROMs), and brain magnetic resonance imaging (MRI).
Results
Fifty patients with persisting symptoms for at least 4 weeks were included and classified by initial hospitalization status. Median time from SARS‐CoV‐2 detection to investigation was 29.3 weeks (range 3.3–57.9). Although individual cognitive performance was generally within the normative range in both groups, mostly mild deficits were found in attention, executive functions, and memory. Hospitalized patients performed worse in global cognition, logical reasoning, and processes of verbal memory. In both groups, fatigue severity was associated with reduced performance in attention and psychomotor speed tasks (rs = −0.40, p < 0.05) and reduced quality of life (EQ5D, rs = 0.57, p < 0.001) and with more persisting symptoms (median 3 vs. 6, p < 0.01). PROMs identified fatigue, reduced sleep quality, and increased anxiety and depression in both groups but more pronounced in non‐hospitalized patients. Brain MRI revealed microbleeds exclusively in hospitalized patients (n = 5).
Interpretation
Regardless of initial COVID‐19 severity, an individuals' mental and physical health can be severely impaired in the long‐term limitedly objectified by clinical standard diagnostic with abnormalities primarily found in hospitalized patients. This needs to be considered when planning rehabilitation therapies and should give rise to new biomarker research.
Many homodimeric enzymes tune their functions by exploiting either negative or positive cooperativity between subunits. In the SARS-CoV-2 Main protease (Mpro) homodimer, the latter has been suggested ...by symmetry in most of the 500 reported protease/ligand complex structures solved by macromolecular crystallography (MX). Here we apply the latter to both covalent and noncovalent ligands in complex with Mpro. Strikingly, our experiments show that the occupation of both active sites of the dimer originates from an excess of ligands. Indeed, cocrystals obtained using a 1:1 ligand/protomer stoichiometry lead to single occupation only. The empty binding site exhibits a catalytically inactive geometry in solution, as suggested by molecular dynamics simulations. Thus, Mpro operates through negative cooperativity with the asymmetric activity of the catalytic sites. This allows it to function with a wide range of substrate concentrations, making it resistant to saturation and potentially difficult to shut down, all properties advantageous for the virus’ adaptability and resistance.
β-Secretase plays a critical role in β-amyloid formation and thus provides a therapeutic target for Alzheimer's disease. Inhibitor design has usually focused on active-site binding, neglecting the ...subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a β-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active β-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting β-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.
Background and purpose
Ehlers–Danlos syndromes are hereditary disorders of connective tissue that are characterized by joint hypermobility, skin hyperextensibility and tissue fragility. The most ...common subtype is the hypermobile type. In addition to symptoms of small fibre neuropathy (SFN) due to damage to the small peripheral nerve fibres, with degeneration of the distal nerve endings, autonomic disorders such as postural tachycardia syndrome (PoTS) are frequently reported features in patients with hypermobile Ehlers–Danlos syndrome (hEDS). To date, the underlying pathophysiological mechanisms are still not completely understood.
Study Purpose
To better understand pathophysiological mechanisms of small fiber neuropathy and autonomic neuropathy in hypermobile Ehlers‐Danlos Syndromes.
Methods
We prospectively investigated 31 patients with hEDS compared to 31 healthy controls by using skin biopsy, quantitative sensory testing, tilt‐table testing, the painDetect, Small Fibre Neuropathy Screening List and the COMPASS‐31 (Composite Autonomic Symptom Score 31) questionnaire.
Results
Nineteen (61%) patients with hEDS were diagnosed with SFN, and 10 (32%) fulfilled the criteria for PoTS. Patients with hEDS had significantly higher heart rates than controls. According to quantitative sensory testing, these patients had generalized thermal and tactile hypesthesia. Skin biopsy revealed significantly reduced intraepithelial nerve fibre density proximally (thigh) and distally (lower leg) in patients compared to controls. This was consistent with various complaints of pain and sensory disturbances in both the proximal and distal body regions.
Conclusion
These results confirm histologically proven SFN as a common feature in patients with hEDS, revealing a generalized distribution of nerve fibre loss. Regarding the frequently reported autonomic and neuropathic dysfunctions, the findings support SFN as an important, but not the only, underlying pathomechanism.
The gene mapt codes for the microtubule-associated protein Tau. The R406W amino acid substitution in Tau is associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) ...characterized by Tau-positive filamentous inclusions. These filamentous Tau inclusions are present in a group of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To gain more insights into the pathomechanism of tauopathies, we performed an RNAi-based large-scale screen in Drosophila melanogaster to identify genetic modifiers of TauR406W-induced toxicity. A collection of RNAi lines, putatively silencing more than 7000 genes, was screened for the ability to modify TauR406W-induced toxicity in vivo. This collection covered more than 50% of all protein coding fly genes and more than 90% of all fly genes known to have a human ortholog. Hereby, we identified 62 genes that, when silenced by RNAi, modified Tau-induced toxicity specifically. Among these 62 modifiers were three subunits of the Dynein/Dynactin complex. Analysis on segmental nerves of fly larvae showed that pan neural TauR406W expression and concomitant silencing of Dynein/Dynactin complex members synergistically caused strong pathological changes within the axonal compartment, but only minor changes at synapses. At the larval stage, these alterations did not cause locomotion deficits, but became evident in adult flies. Our data suggest that Tau-induced detrimental effects most likely originate from axonal rather than synaptic dysfunction and that impaired retrograde transport intensifies detrimental effects of Tau in axons. In conclusion, our findings contribute to the elucidation of disease mechanisms in tauopathies like FTDP-17 or AD.
Far-infrared images and photometry are presented for 201 Luminous and Ultraluminous Infrared Galaxies LIRGs: log (L sub(IR)/L sub(middot in circle)) = 11.00-11.99, ULIRGs: log (L sub(IR)/L sub(middot ...in circle)) = 12.00-12.99, in the Great Observatories All-Sky LIRG Survey (GOALS), based on observations with the Herschel Space Observatory Photodetector Array Camera and Spectrometer (PACS) and the Spectral and Photometric Imaging Receiver (SPIRE) instruments. The image atlas displays each GOALS target in the three PACS bands (70, 100, and 160 mu m) and the three SPIRE bands (250, 350, and 500 mu m), optimized to reveal structures at both high and low surface brightness levels, with images scaled to simplify comparison of structures in the same physical areas of ~100 x 100 kpc super(2). Flux densities of companion galaxies in merging systems are provided where possible, depending on their angular separation and the spatial resolution in each passband, along with integrated system fluxes (sum of components). This data set constitutes the imaging and photometric component of the GOALS Herschel OT1 observing program, and is complementary to atlases presented for the Hubble Space Telescope, Spitzer Space Telescope, and Chandra X-ray Observatory. Collectively, these data will enable a wide range of detailed studies of active galactic nucleus and starburst activity within the most luminous infrared galaxies in the local universe.
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