The accumulation of damaged mitochondria causes the death of dopaminergic neurons. The Parkin-mediated mitophagy pathway functions to remove these mitochondria from cells. Targeting this pathway ...represents a therapeutic strategy for several neurodegenerative diseases, most notably Parkinson's disease. We describe a discovery pipeline to identify small molecules that increase Parkin recruitment to damaged mitochondria and ensuing mitophagy. We show that ROCK inhibitors promote the activity of this pathway by increasing the recruitment of HK2, a positive regulator of Parkin, to mitochondria. This leads to the increased targeting of mitochondria to lysosomes and removal of damaged mitochondria from cells. Furthermore, ROCK inhibitors demonstrate neuroprotective effects in flies subjected to paraquat, a parkinsonian toxin that induces mitochondrial damage. Importantly, parkin and rok are required for these effects, revealing a signaling axis which controls Parkin-mediated mitophagy that may be exploited for the development of Parkinson's disease therapeutics.
The regulation of organelle abundance is critical for cell function and survival; however, the mechanisms responsible are not fully understood. In this study, we characterize a role of the ...deubiquitinating enzyme USP30 in peroxisome maintenance. Peroxisomes are highly dynamic, changing in abundance in response to metabolic stress. In our recent study identifying the role of USP30 in mitophagy, we observed USP30 to be localized to punctate structures resembling peroxisomes. We report here that USP30, best known as a mitophagy regulator, is also necessary for regulating pexophagy, the selective autophagic degradation of peroxisomes. We find that overexpressing USP30 prevents pexophagy during amino acid starvation, and its depletion results in pexophagy induction under basal conditions. We demonstrate that USP30 prevents pexophagy by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2. Finally, we show that USP30 can rescue the peroxisome loss observed in some disease-causing peroxisome mutations, pointing to a potential therapeutic target.
The process of autophagy is an essential cellular mechanism, required to maintain general cell health through the removal of dysfunctional organelles, such as the ER, peroxisomes and mitochondria, as ...well as protein aggregates, and bacteria. Autophagy is an extremely dynamic process, and tools are constantly being developed to study the various steps of this process. This protocol details a method to study the end steps of autophagy-lysosomal fusion and the formation of the autolysosome. Many techniques have been used to study the various steps of the autophagy process. Here we describe the RedGreen-assay (RG-assay), an immunofluorescence-based technique used to visualize the targeting of substrates to the autolysosome in live cells. This technique takes advantage of the low lysosomal pH and over-expression of a tandem GFP-mCherry tagged protein targeted to an organelle of interest. While in the neutral cytosol or autophagosome, both GFP and RFP will fluoresce. However, within the autolysosome, the GFP signal is quenched due to the low pH environment and the RFP emission signal will predominate. This technique is readily quantifiable and amenable to high throughput experiments. Additionally, by tagging the GFP-RFP tandem fluorescent protein with organelle specific targeting sequences, it can be used to measure a wide range of substrates of autophagy.
In our recent publication, we describe a mechanism by which peroxisomes are protected from degradation by autophagy under basal conditions. Taking a page from mitophagy, peroxisomes also recruit the ...mitochondria deubiquitinating enzyme USP30 to counter the action of PEX2, the peroxisomal E3 ubiquitin ligase to regulate pexophagy.
Peroxisomes are rapidly degraded during amino acid and oxygen deprivation by a type of selective autophagy called pexophagy. However, how damaged peroxisomes are detected and removed from the cell is ...poorly understood. Recent studies suggest that the peroxisomal matrix protein import machinery may serve double duty as a quality control machinery, where they are directly involved in activating pexophagy. Here, we explored whether any matrix import factors are required to prevent pexophagy, such that their loss designates peroxisomes for degradation. Using gene editing and quantitative fluorescence microscopy on culture cells and a zebrafish model system, we found that PEX13, a component of the peroxisomal matrix import system, is required to prevent the degradation of otherwise healthy peroxisomes. The loss of PEX13 caused an accumulation of ubiquitinated PEX5 on peroxisomes and an increase in peroxisome-dependent reactive oxygen species that coalesce to induce pexophagy. We also found that PEX13 protein level is downregulated to aid in the induction of pexophagy during amino acid starvation. Together, our study points to PEX13 as a novel pexophagy regulator that is modulated to maintain peroxisome homeostasis.
Abbreviations: AAA ATPases: ATPases associated with diverse cellular activities; ABCD3: ATP binding cassette subfamily D member; 3ACOX1: acyl-CoA oxidase; 1ACTA1: actin alpha 1, skeletal muscle; ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; CAT: catalase; CQ: chloroquine; Dpf: days post fertilization: FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H
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O
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hydrogen peroxide; HA - human influenza hemagglutinin; HBSS: Hanks' Balanced Salt Solution; HCQ; hydroxychloroquine; KANL: lysine alanine asparagine leucine; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; MYC: MYC proto-oncogene, bHLH transcription factor; MZ: maternal and zygotic; NAC: N-acetyl cysteine; NBR1 - NBR1 autophagy cargo receptor; PBD: peroxisome biogenesis disorder; PBS: phosphate-buffered saline; PEX: peroxisomal biogenesis factor; PTS1: peroxisome targeting sequence 1; RFP: red fluorescent protein; ROS: reactive oxygen speciess; iRNA: short interfering RNA; SKL: serine lysine leucine; SLC25A17/PMP34: solute carrier family 25 member 17; Ub: ubiquitin; USP30: ubiquitin specific peptidase 30.
Although adolescent flexible flatfoot deformity (FFD) is common, little is known regarding the effect of weight on associated symptomatology. This study uses pedobarography and patient-reported ...outcome measures (PROs) to determine if overweight adolescents with FFD have more severe alterations in dynamic plantar pressures than normal body mass index percentiles (wnBMI) with FFD and if such alterations correlate with pain and activity.
A retrospective review of patients aged 10 to 18 years with nonsyndromic symptomatic FFD was performed. Overweight (BMI percentile ≥ 85%) patients were compared with wnBMI patients with regard to dynamic plantar pressure measures and PRO scores. Pedobarographic data were subdivided into regions: medial/lateral hindfoot and midfoot, and first, second, and third to fifth metatarsals. Plantar pressure variables were normalized to account for differences in foot size, body weight, and walking speed. Contact area (CA%), maximum force by body weight (MF%), and contact time as a percentage of the rollover process (CT%) were calculated. Two foot-specific PROs were assessed, including the Foot and Ankle Outcome Score and the Oxford Ankle Foot Measure for Children.
Of the 48 adolescents studied, 27 (56%) were overweight and 21 (44%) were wnBMI. After normalization of the data, overweight patients had significantly greater medial midfoot MF%, whereas CT% was increased across the medial and lateral midfoot and hindfoot regions. Correlations showed positive trends: as BMI percentile increases, so will CA and MF in the medial midfoot, as well as CT in the medial and lateral midfoot and hindfoot. Significant differences were seen between groups, with the overweight group reporting lower sports and recreation subscores than the wnBMI group. No significant differences were seen in the pain and disability subscores.
Although overweight adolescents with FFD exhibit greater forces and more time spent during the rollover process in the medial midfoot than normal-weight patients, they did not report worse pain or disability associated with their flat foot deformity.
Therapeutic level 3.
Cheng (1986) trained male rats to search for food in a rectangular arena that also contained distinctive visual patterns. He found that the rats used mainly the geometric framework of the box itself ...to find the food and claimed that geometrical information is processed in a specialized module, which is independent of feature information. The aim of the present set of experiments was to check if the previous results with male rats and an appetitive task could be extended to an aversive task while using both male and female rats and three-dimensional landmarks. In Experiments 1 and 2, rats were trained in a rectangular-shaped pool to find a hidden platform that had a location defined in terms of two sources of information-landmarks of different salience (less salient in Experiment 1, more salient in Experiment 2) outside the pool and a particular corner of the pool. The results showed that both males and females used mainly the particular corner of the pool, supporting Cheng's suggestion. In addition, in the two experiments, what the rats learned with respect to the landmarks was negligible. Experiment 3 used a more difficult triangular pool in addition to the rectangular pool, in the absence of landmarks. The results revealed sex differences in the triangular pool but not in the easier rectangular pool. These results suggest that task difficulty is a factor when it comes to finding sex differences in rats in spatial tasks.
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Background: Belamaf, a BCMA-targeted antibody drug conjugate, has previously demonstrated clinical activity and a manageable safety profile, supporting its use in combination with ...standard-of-care (SoC) therapies in RRMM. DREAMM-7 (NCT04246047) is a global, randomized, open-label, phase III head-to-head trial evaluating the efficacy and safety of BVd triplet vs SoC triplet, DVd, in patients (pts) with RRMM with ≥1 prior line of therapy (LOT). Methods: Pts with ≥1 prior LOT were randomized (1:1) to BVd: B 2.5 mg/kg IV Q3W + V 1.3 mg/m
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(D1, 4, 8, 11 of 21-day cycles (C); up to 8 C) + d 20 mg (D1, 2, 4, 5, 8, 9, 11, 12; up to 8 C) or DVd: D 16 mg/kg (21-day C), C1-3, Q1W, C4-8, Q3W, and Q4W from C 9 on); V and d schedules were the same. The primary endpoint was independent review committee-assessed progression free survival (PFS). Secondary endpoints available include overall survival (OS), duration of response (DOR), overall response rate (ORR). Results: 494 pts were randomized (BVd n = 243, DVd n = 251). With a median (range) follow-up of 28.2 mo (0.10-40 mo), the median PFS (mPFS) in the BVd arm was 36.6 mo (95% CI, 28.4 mo-NR) vs 13.4 mo (11.1-17.5 mo) with DVd; hazard ratio (HR) 0.41 (95% CI, 0.31-0.53; p < 0.00001). OS data was 29% mature; median OS was not reached in either arm; HR, 0.57 (95% CI, 0.40-0.80; nominal p < 0.0005). ORR was 82.7% (95% CI, 77.4-87.3%) with BVd and 71.3% (65.3-76.8%) with DVd. Median DOR (mDOR) was 35.6 mo (95% CI, 30.5 mo-NR) for BVd vs 17.8 mo (13.8-23.6 mo) for DVd. All pts in both arms experienced ≥1 AE (Table). Grade 3/4 treatment-related AEs (TRAE) were reported in 90% of pts in the BVd arm and 67% with DVd. Serious AEs (SAE) were reported in 50% of pts in BVd vs 37% in DVd arm. Ocular AEs were more frequent on BVd vs DVd (79% vs 29%) and were manageable. Conclusions: The DREAMM-7 head-to-head study of BVd vs DVd demonstrated statistically significant PFS benefit of BVd with a mPFS improvement of 23 mo in pts with RRMM with ≥1 prior LOT. A strong and clinically meaningful OS benefit (nominal p < 0.0005) was observed. Additionally, BVd led to greater depth of response and doubling of mDOR vs DVd and had a manageable safety profile. These results support BVd as a potential new SoC in this setting. Clinical trial information: NCT04246047 . Table: see text
Functional enrichment analysis is a cornerstone in bioinformatics as it makes possible to identify functional information by using a gene list as source. Different tools are available to compare gene ...ontology (GO) terms, based on a directed acyclic graph structure or content-based algorithms which are time-consuming and require a priori information of GO terms. Nevertheless, quantitative procedures to compare GO terms among gene lists and species are not available. Here we present a computational procedure, implemented in R, to infer functional information derived from comparative strategies. GOCompare provides a framework for functional comparative genomics starting from comparable lists from GO terms. The program uses functional enrichment analysis (FEA) results and implement graph theory to identify statistically relevant GO terms for both, GO categories and analyzed species. Thus, GOCompare allows finding new functional information complementing current FEA approaches and extending their use to a comparative perspective. To test our approach GO terms were obtained for a list of aluminum tolerance-associated genes in Oryza sativa subsp. japonica and their orthologues in Arabidopsis thaliana. GOCompare was able to detect functional similarities for reactive oxygen species and ion binding capabilities which are common in plants as molecular mechanisms to tolerate aluminum toxicity. Consequently, the R package exhibited a good performance when implemented in complex datasets, allowing to establish hypothesis that might explain a biological process from a functional perspective, and narrowing down the possible landscapes to design wet lab experiments.
•Methods to analyze functional enrichment results for several gene lists are scarce.•Reactive oxygen species and ion-binding processes are conserved mechanisms associated with aluminum stress tolerance.•GOCompare are tools that generate statistical knowledge about experiments or contrasted biological conditions.•Graph theory explores results of functional enrichment analysis with advantages over traditional forms of interpretation.
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