RAB8, RAB10 and RILPL1 contribute to both LRRK2 kinase-mediated centrosomal cohesion and ciliogenesis deficits

Mutations in the LRRK2 kinase are the most common cause of familial Parkinson's disease, and variants increase risk for the sporadic form of the disease. LRRK2 phosphorylates multiple RAB GTPases including RAB8A and RAB10. Phosphorylated RAB10 is recruited to centrosome-localized RILPL1, which may interfere with ciliogenesis in a disease-relevant context. Our previous studies indicate that the centrosomal accumulation of phosphorylated RAB8A causes centrosomal cohesion deficits in dividing cells, including in peripheral patient-derived cells. Here, we show that both RAB8 and RAB10 contribute to the centrosomal cohesion deficits. Pathogenic LRRK2 causes the centrosomal accumulation not only of phosho-RAB8 but also of phospho-RAB10, and the effects on centrosomal cohesion are dependent on RAB8, RAB10 and RILPL1. Conversely, the pathogenic LRRK2-mediated ciliogenesis defects correlate with the centrosomal accumulation of both phospho-RAB8 and phospho-RAB10. LRRK2-mediated centrosomal cohesion and ciliogenesis alterations are observed in patient-derived peripheral cells, as well as in primary astrocytes from mutant LRRK2 mice, and are reverted upon LRRK2 kinase inhibition. These data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are distinct cellular readouts of the same underlying phospho-RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD

ATP13A2 deficiency disrupts lysosomal polyamine export

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome—a parkinsonism with dementia1—and early-onset Parkinson’s disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson’s disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system

PAK6 Phosphorylates 14-3-3 gamma to Regulate Steady State Phosphorylation of LRRK2

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson’s disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain

Reduced expression of BAX is associated with poor prognosis in patients with epithelial ovarian cancer: a multifactorial analysis of TP53, p21, BAX and BCL-2

Traditional clinicopathological features do not predict which patients will develop chemotherapy resistance. The TP53 gene is frequently altered in ovarian cancer but its prognostic implications are controversial. Little is known on the impact of TP53-downstream genes on prognosis. Using molecular and immunohistochemical analyses we examined TP53 and its downstream genes p21 BAX and BCL-2 in ovarian tumour tissues and have evaluated the results in relation to clinico-pathological parameters, clinical outcome and response to platinum-based chemotherapy. Associations of tested factors and patient and tumour characteristics were studied by Spearman rank correlation and Pearsons χ2 test. The Cox proportional hazard model was used for univariate and multivariate analysis. The associations of tested factors with response was tested using logistic regression analysis. TP53 mutation, p21 and BCL-2 expression were not associated with increased rates of progression and death. Expression of TP53 was associated with a shorter overall survival only (relative hazard rate [RHR] 2.01 P = 0.03). Interestingly, when combining TP53 mutation and expression data, this resulted in an increased association with overall survival (P = 0.008). BAX expression was found to be associated with both progression-free (RHR 0.44 P = 0.05) and overall survival (RHR 0.42 P = 0.03). Those patients who simultaneously expressed BAX and BCL-2 had a longer progression-free and overall survival compared to patients whose tumours did not express BCL-2 (P = 0.05 and 0.015 respectively). No relations were observed between tested factors and response to platinum-based chemotherapy. We conclude that BAX expression may represent a prognostic indicator for patients with ovarian cancer and that the combined evaluation of BAX and BCL-2 may provide additional prognostic significance.   http://www.bjcancer.com © 2001 Cancer Research Campaig

High performance burst-mode upstream transmission for next generation PONs (invited paper)

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LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease (PD), with growing importance also for Crohn’s disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology

TP53 status and taxane-platinum versus platinum-based therapy in ovarian cancer patients: A non-randomized retrospective study

<p>Abstract</p> <p>Background</p> <p>Taxane-platinum therapy (TP) has replaced platinum-based therapy (PC or PAC, DNA damaging chemotherapy) in the postoperative treatment of ovarian cancer patients; however, it is not always effective. TP53 protein plays a differential role in response to DNA-damaging agents and taxanes. We sought to define profiles of patients who benefit the most from TP and also of those who can be treated with PC.</p> <p>Methods</p> <p>We compared the effectiveness of PC/PAC (n = 253) and TP (n = 199) with respect to tumor TP53 accumulation in ovarian cancer patients with FIGO stage IIB-IV disease; this was a non-randomized retrospective study. Immunohistochemical analysis was performed on 452 archival tumors; univariate and multivariate analysis by the Cox's and logistic regression models was performed in all patients and in subgroups with [TP53(+)] and without TP53 accumulation [TP53(-)].</p> <p>Results</p> <p>The advantage of taxane-platinum therapy over platinum-based therapy was seen in the TP53(+), and not in the TP53(-) group. In the TP53(+) group taxane-platinum therapy enhanced the probability of complete remission (p = .018), platinum sensitivity (p = .014), platinum highly sensitive response (p = .038) and longer survival (OS, p = .008). Poor tumor differentiation diminished the advantage from taxane-platinum therapy in the TP53(+) group. In the TP53(-) group PC/PAC was at least equally efficient as taxane-platinum therapy and it enhanced the chance of platinum highly sensitive response (p = .010). However, in the TP53(-) group taxane-platinum therapy possibly diminished the risk of death in patients over 53 yrs (p = .077). Among factors that positively interacted with taxane-platinum therapy in some analyses were endometrioid and clear cell type, FIGO III stage, bulky residual tumor, more advanced age of patient and moderate tumor differentiation.</p> <p>Conclusion</p> <p>Our results suggest that taxane-platinum therapy is particularly justified in patients with TP53(+) tumors or older than 53 years. In the group of patients ≤53 yrs and with TP53(-) tumors platinum-based therapy is possibly equally efficient. We provide hints for planning randomized trials to verify these observations.</p

Lack of EGF receptor contributes to drug sensitivity of human germline cells

Germline mutations have been associated with generation of various types of tumour. In this study, we investigated genetic alteration of germline tumours that affect the drug sensitivity of cells. Although all germline tumour cells we tested were hypersensitive to DNA-damaging drugs, no significant alteration was observed in their DNA repair activity or the expression of DNA repair proteins. In contrast, germline tumours expressed very low level of epidermal growth factor receptor (EGFR) compared to drug-resistant ovarian cancer cells. An immunohistochemical analysis indicated that most of the primary germline tumours we tested expressed very low level of EGFR. In accordance with this, overexpression of EGFR in germline tumour cells showed an increase in drug resistance, suggesting that a lack of EGFR, at least in part, contributes to the drug sensitivity of germline tumours