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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
- Chaudhuri, Kallol Ray, et al.
(författare)
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A Proposal for a Comprehensive Grading of Parkinson's Disease Severity Combining Motor and Non-Motor Assessments: Meeting an Unmet Need
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:2
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Tidskriftsartikel (refereegranskat)abstract
- Background: Non-motor symptoms are present in Parkinson's disease (PD) and a key determinant of quality of life. The Non-motor Symptoms Scale (NMSS) is a validated scale that allows quantifying frequency and severity (burden) of NMS. We report a proposal for using NMSS scores to determine levels of NMS burden (NMSB) and to complete PD patient classification. Methods: This was an observational, cross-sectional international study of 935 consecutive patients. Using a distribution of NMSS scores by quartiles, a classification based on levels from 0 (no NMSB at all) to 4 (very severe NMSB) was obtained and its relation with Hoehn and Yahr (HY) staging, motor and health-related quality of life scales was analyzed. Concordance between NMSB levels and grouping based on clinician's global impression of severity, using categorical regression, was determined. Disability and HRQoL predictors were identified by multiple regression models. Results: The distribution of motor and QoL scales scores by HY and NMSB levels was significantly discriminative. The difference in the classification of cases for both methods, HY and NMSB, was significant (gamma = 0.45; ASE = 0.032). Concordance between NMSB and global severity-based levels from categorical regression was 91.8%, (kappa(w) = 0.97). NMS score was predictor of disability and QoL. Conclusions: Current clinical practice does not address a need for inclusion of non-motor scores in routine assessment of PD in spite of the overwhelming influence of NMS on disability and quality of life. Our data overcome the problems of "pure motor assessment'' and we propose a combined approach with addition of NMSB levels to standard motor assessments.
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| 3. |
- Mullin, Stephen, et al.
(författare)
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Ambroxol for the Treatment of Patients With Parkinson Disease With and Without Glucocerebrosidase Gene Mutations: A Nonrandomized, Noncontrolled Trial.
- 2020
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Ingår i: JAMA neurology. - : American Medical Association (AMA). - 2168-6157 .- 2168-6149. ; 77:4, s. 427-34
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Tidskriftsartikel (refereegranskat)abstract
- Mutations of the glucocerebrosidase gene, GBA1 (OMIM 606463), are the most important risk factor for Parkinson disease (PD). In vitro and in vivo studies have reported that ambroxol increases β-glucocerebrosidase (GCase) enzyme activity and reduces α-synuclein levels. These observations support a potential role for ambroxol therapy in modifying a relevant pathogenetic pathway in PD.To assess safety, tolerability, cerebrospinal fluid (CSF) penetration, and target engagement of ambroxol therapy with GCase in patients with PD with and without GBA1 mutations.An escalating dose of oral ambroxol to 1.26 g per day.This single-center open-label noncontrolled clinical trial was conducted between January 11, 2017, and April 25, 2018, at the Leonard Wolfson Experimental Neuroscience Centre, a dedicated clinical research facility and part of the University College London Queen Square Institute of Neurology in London, United Kingdom. Participants were recruited from established databases at the Royal Free London Hospital and National Hospital for Neurology and Neurosurgery in London. Twenty-four patients with moderate PD were evaluated for eligibility, and 23 entered the study. Of those, 18 patients completed the study; 1 patient was excluded (failed lumbar puncture), and 4 patients withdrew (predominantly lumbar puncture-related complications). All data analyses were performed from November 1 to December 14, 2018.Primary outcomes at 186 days were the detection of ambroxol in the CSF and a change in CSF GCase activity.Of the 18 participants (15 men [83.3%]; mean [SD] age, 60.2 [9.7] years) who completed the study, 17 (8 with GBA1 mutations and 9 without GBA1 mutations) were included in the primary analysis. Between days 0 and 186, a 156-ng/mL increase in the level of ambroxol in CSF (lower 95% confidence limit, 129 ng/mL; P < .001) was observed. The CSF GCase activity decreased by 19% (0.059 nmol/mL per hour; 95% CI, -0.115 to -0.002; P = .04). The ambroxol therapy was well tolerated, with no serious adverse events. An increase of 50 pg/mL (13%) in the CSF α-synuclein concentration (95% CI, 14-87; P = .01) and an increase of 88 ng/mol (35%) in the CSF GCase protein levels (95% CI, 40-137; P = .002) were observed. Mean (SD) scores on part 3 of the Movement Disorders Society Unified Parkinson Disease Rating Scale decreased (ie, improved) by 6.8 (7.1) points (95% CI, -10.4 to -3.1; P = .001). These changes were observed in patients with and without GBA1 mutations.The study results suggest that ambroxol therapy was safe and well tolerated; CSF penetration and target engagement of ambroxol were achieved, and CSF α-synuclein levels were increased. Placebo-controlled clinical trials are needed to examine whether ambroxol therapy is associated with changes in the natural progression of PD.ClinicalTrials.gov identifier: NCT02941822; EudraCT identifier: 2015-002571-24.
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| 4. |
- Piston, Dominik, et al.
(författare)
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DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis
- 2017
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Ingår i: Human Molecular Genetics. - : Oxford University Press. - 0964-6906 .- 1460-2083. ; 26:20, s. 4028-4041
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Tidskriftsartikel (refereegranskat)abstract
- DJ-1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinson's disease (PD), but growing evidence suggests involvement of DJ-1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH-SY5Y neuroblastoma cell lines that DJ-1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ-1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ-1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ-1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ-1 complexes. In the KD model, the absence of DJ-1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re-expression of DJ-1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7-related PD. Notably, oxidised DJ-1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.
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| 5. |
- Wray, Selina, et al.
(författare)
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Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:8
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Tidskriftsartikel (refereegranskat)abstract
- Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.
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