Cellular effects mediated by pathogenic LRRK2: homing in on Rab-mediated processes

Leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease. Mutations in LRRK2 are associated with increased kinase activity that correlates with cytotoxicity, indicating that kinase inhibitors may comprise promising diseasemodifying compounds. However, before embarking on such strategies, detailed knowledge of the cellular deficits mediated by pathogenic LRRK2 in the context of defined and pathologically relevant kinase substrates is essential. LRRK2 has been consistently shown to impair various intracellular vesicular trafficking events, and recent studies have shown that LRRK2 can phosphorylate a subset of proteins that are intricately implicated in those processes. In light of these findings, we here review the link between cellular deficits in intracellular trafficking pathways and the LRRK2-mediated phosphorylation of those newly identified substrates

Mechanisms underlying endolysosomal deficits mediated by the Parkinson’s disease-related kinase LRRK2

Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are a common cause of hereditary Parkinson´s disease (PD), and some variants also confer risk to develop sporadic PD. LRRK2 has been reported to regulate various intracellular vesicular trafficking pathways, including autophagy, retromer-mediated trafficking, synaptic vesicle dynamics and endolysosomal degradative events, although the precise molecular mechanism(s) underlying these alterations remain to be elucidated. Recent studies have revealed that a subset of Rab proteins involved in secretory and endocytic recycling are LRRK2 kinase substrates in vivo. However, the effects of LRRK2-mediated phosphorylation of these substrates on membrane trafficking remain unknown. Here, by studying the well-characterized epidermal growth factor receptor (EGFR) trafficking pathway, we report that expression of active or phospho-deficient, but not phospho-mimetic, RAB8A variants rescues the G2019S LRRK2-mediated effects on endolysosomal membrane trafficking. Similarly, upregulation of the RAB11-Rabin8-RAB8A cascade, which activates RAB8A, also reverted these trafficking deficits. Strikingly, we found that the loss of RAB8A delayed EGFR degradation and decreased RAB7A activity, mimicking the previously described effect of pathogenic LRRK2 on EGFR trafficking. Moreover, expression of pathogenic G2019S LRRK2 or loss of RAB8A resulted in mistargeting of the EGFR into a RAB4-positive endocytic compartment, which was accompanied not only by endocytic degradation defects, but also by a deficit in EGFR recycling to the plasma membrane. Both the accumulation of EGFR in the RAB4-positive compartment and the recycling deficits were rescued upon expression of active RAB7A. Finally, we showed that dominant-negative RAB7A expression resulted in similar defects in EGF degradation, accumulation in a RAB4 compartment, and deficits in EGFR recycling, which were all rescued upon expression of active RAB8A. Taken together, these findings suggest that LRRK2-mediated phosphorylation and subsequent impairment of RAB8A function may form the basis of how pathogenic LRRK2 deregulates endolysosomal transport and endocytic recycling events.Mutaciones en el gen que codifica la quinasa rica en repeticiones de leucina 2 (leucine-rich repeat kinase 2, LRRK2) son una causa común de enfermedad de Parkinson (EP) hereditaria y algunas variantes también aumentan el riesgo de EP esporádica. LRRK2 participa en la regulación de una serie de rutas de tráfico intracelular de membranas, incluyendo autofagia, tráfico mediado por el retrómero, dinámica de las vesículas sinápticas y degradación endolisosomal, si bien no se conocen con exactitud los mecanismos moleculares responsables de estas alteraciones. Estudios recientes han revelado que varias proteínas Rab que participan en las rutas secretora y de reciclaje endocítico son sustratos de la actividad quinasa de LRRK2 in vivo. Sin embargo, los efectos de su fosforilación por LRRK2 sobre las rutas de tráfico intracelular de membranas permanecen desconocidos. En esta tesis, mediante el estudio de la bien caracterizada ruta del receptor del factor de crecimiento epidérmico (epidermal growth factor receptor, EGFR), observamos que la expresión de una versión catalíticamente activa o de una variante fosfodeficiente, pero no así de variantes fosfomiméticas, de RAB8A rescata las alteraciones sobre el tráfico endolisosomal mediadas por G2019S, un mutante patogénico de LRRK2. De forma similar, RAB11 y Rabin8, que forman parte de una cascada de activación de RAB8A, revierten dichas alteraciones. Además, observamos que el silenciamiento de RAB8A retrasa la degradación del EGFR y provoca una disminución de la actividad de RAB7A, al igual que se había descrito anteriormente para LRRK2 patogénica. Tanto la expresión de la variante G2019S como el silenciamiento de RAB8A resultan en la acumulación aberrante del EGFR en un compartimento endocítico positivo para RAB4, lo cual impide su eficiente degradación endolisosomal, así como su reciclaje de vuelta a la membrana plasmática. Tanto la acumulación del EGFR en el compartimento positivo para RAB4 como las alteraciones en su reciclaje son revertidas por una forma activa de RAB7A. Finalmente, mostramos que la expresión de una mutación dominante negativa de RAB7A da como resultado defectos similares en la degradación y en el reciclaje del EGFR, provocando su acumulación en el compartimento positivo para RAB4. En conjunto, nuestros descubrimientos sugieren que la fosforilación y consiguiente inactivación de RAB8A mediada por LRRK2 patogénica podría constituir la base de cómo esta proteína desregula las rutas de tráfico intracelular de membranas.Tesis Univ. Granada

Distinct Roles for RAB10 and RAB29 in Pathogenic LRRK2-Mediated Endolysosomal Trafficking Alterations

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson’s disease, and sequence variations are associated with the sporadic form of the disease. LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling trafficking pathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruit LRRK2 to the Golgi, where it stimulates its kinase activity. Our previous studies revealed that G2019S LRRK2 expression or knockdown of RAB8A deregulate epidermal growth factor receptor (EGFR) trafficking, with a concomitant accumulation of the receptor in a RAB4-positive recycling compartment. Here, we show that the G2019S LRRK2-mediated EGFR deficits are mimicked by knockdown of RAB10 and rescued by expression of active RAB10. By contrast, RAB29 knockdown is without effect, but expression of RAB29 also rescues the pathogenic LRRK2-mediated trafficking deficits independently of Golgi integrity. Our data suggest that G2019S LRRK2 deregulates endolysosomal trafficking by impairing the function of RAB8A and RAB10, while RAB29 positively modulates non-Golgi-related trafficking events impaired by pathogenic LRRK2

Upstream deregulation of calcium signaling in Parkinson’s disease

Parkinson’s disease (PD) is a major health problem affecting millions of people worldwide. Recent studies provide compelling evidence that altered Ca2+ homeostasis may underlie disease pathomechanism and be an inherent feature of all vulnerable neurons. The downstream effects of altered Ca2+ handling in the distinct subcellular organelles for proper cellular function are beginning to be elucidated. Here, we summarize the evidence that vulnerable neurons may be exposed to homeostatic Ca2+ stress which may determine their selective vulnerability, and suggest how abnormal Ca2+ handling in the distinct intracellular compartments may compromise neuronal health in the context of aging, environmental, and genetic stress. Gaining a better understanding of the varied effects of Ca2+ dyshomeostasis may allow novel combinatorial therapeutic strategies to slow PD progression.Spanish Ministry of Economy and Competitiveness (BFU2011-29899), the Junta de Andalucia (grant number CTS 6816), and the Michael J. Fox FoundationPeer reviewe

LRRK2-Related Parkinson’s Disease Due to Altered Endolysosomal Biology With Variable Lewy Body Pathology: A Hypothesis

Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are associated with both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large protein comprised of a GTPase and a kinase domain. All pathogenic variants converge on enhancing LRRK2 kinase substrate phosphorylation, and distinct LRRK2 kinase inhibitors are currently in various stages of clinical trials. Although the precise pathophysiological functions of LRRK2 remain largely unknown, PD-associated mutants have been shown to alter various intracellular vesicular trafficking pathways, especially those related to endolysosomal protein degradation events. In addition, biochemical studies have identified a subset of Rab proteins, small GTPases required for all vesicular trafficking steps, as substrate proteins for the LRRK2 kinase activity in vitro and in vivo. Therefore, it is crucial to evaluate the impact of such phosphorylation on neurodegenerative mechanisms underlying LRRK2-related PD, especially with respect to deregulated Rab-mediated endolysosomal membrane trafficking and protein degradation events. Surprisingly, a significant proportion of PD patients due to LRRK2 mutations display neuronal cell loss in the substantia nigra pars compacta in the absence of any apparent α-synuclein-containing Lewy body neuropathology. These findings suggest that endolysosomal alterations mediated by pathogenic LRRK2 per se are not sufficient to cause α-synuclein aggregation. Here, we will review current knowledge about the link between pathogenic LRRK2, Rab protein phosphorylation and endolysosomal trafficking alterations, and we will propose a testable working model whereby LRRK2-related PD may present with variable LB pathology.This work was supported by the Michael J. Fox Foundation and by intramural support from Rutgers Universit

Distinct Roles for RAB10 and RAB29 in Pathogenic LRRK2-Mediated Endolysosomal Trafficking Alterations

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease, and sequence variations are associated with the sporadic form of the disease. LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling trafficking pathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruit LRRK2 to the Golgi, where it stimulates its kinase activity. Our previous studies revealed that G2019S LRRK2 expression or knockdown of RAB8A deregulate epidermal growth factor receptor (EGFR) trafficking, with a concomitant accumulation of the receptor in a RAB4-positive recycling compartment. Here, we show that the G2019S LRRK2-mediated EGFR deficits are mimicked by knockdown of RAB10 and rescued by expression of active RAB10. By contrast, RAB29 knockdown is without effect, but expression of RAB29 also rescues the pathogenic LRRK2-mediated trafficking deficits independently of Golgi integrity. Our data suggest that G2019S LRRK2 deregulates endolysosomal trafficking by impairing the function of RAB8A and RAB10, while RAB29 positively modulates non-Golgi-related trafficking events impaired by pathogenic LRRK2.This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO)(SAF2017-89402-R) and the Michael J. Fox Foundation (MJFF) (to S.H.). M.R.-L. is enrolled in the PhD programof Biochemistry and Molecular Biology at the University of Granada, and supported by a Fellowship (FPI;BES-2015-075580) from the MINECO

LRRK2 and Parkinson's Disease: From Lack of Structure to Gain of Function

Mutations in LRRK2 comprise the most common cause for familial Parkinson's disease (PD), and variations increase risk for sporadic disease, implicating LRRK2 in the entire disease spectrum. LRRK2 is a large protein harbouring both GTPase and kinase domains which display measurable catalytic activity. Most pathogenic mutations increase the kinase activity, with increased activity being cytotoxic under certain conditions. These findings have spurred great interest in drug development approaches, and various specific LRRK2 kinase inhibitors have been developed. However, LRRK2 is a largely ubiquitously expressed protein, and inhibiting its function in some non-neuronal tissues has raised safety liability issues for kinase inhibitor approaches. Therefore, understanding the cellular and cell type-specific role(s) of LRRK2 has become of paramount importance. This review will highlight current knowledge on the precise biochemical activities of normal and pathogenic LRRK2, and highlight the most common proposed cellular roles so as to gain a better understanding of the cell type-specific effects of LRRK2 modulators.Work in the laboratory is funded by FEDER, the Spanish Ministry of Economy and Competitiveness (SAF2014-58653-R), the Junta de Andalucia (CTS-6816), the BBVA Foundation and the Michael J. Fox Foundation. B.F. was funded by a Juan de la Cierva Fellowship (MINECO; JCI2010-07703).Peer reviewe

Sustained virological response to direct-acting antiviral regimens reduces the risk of hepatocellular carcinoma in HIV/HCV-coinfected patients with cirrhosis

HEPAVIR-Cirrhosis Study Group.[Objectives] To assess the impact of all-oral direct-acting antiviral agent (DAA) regimens on the risk of hepatocellular carcinoma (HCC) in HIV/HCV-coinfected patients with cirrhosis.[Methods] This was a multicentre prospective cohort study recruiting HIV/HCV-coinfected patients with a new diagnosis of compensated cirrhosis. Patients were followed up until HCC, death or the censoring date (March 2017). The primary endpoint was the emergence of HCC. The incidence rate (IR) (95% CI) of HCC in different groups was computed. Time-to-event analyses were performed to identify predictors of HCC emergence.[Results] The study included 495 HIV/HCV-coinfected patients with cirrhosis. After a median (IQR) follow-up of 59 (27–84) months, 22 (4.4%; 95% CI 2.6–6.3) patients developed an HCC. The IR (95% CI) of HCC was 0.93 (0.06–1.42) per 100 person-years (PY). Three hundred and three (61%) patients achieved sustained virological response (SVR) during follow-up, 79 after interferon (IFN)-based regimens and 224 after an all-oral DAA regimen. The IR (95% CI) of HCC after all-oral DAA was 0.35 (0.14–0.85) per 100 PY whereas it was 1.79 (1.11–2.88) per 100 PY in the remaining cohort (P = 0.0005). When only patients with SVR were considered, the IR (95% CI) of HCC after all-oral DAA was 0.32 (0.12–0.86) whereas it was 0 per 100 PY among those with SVR after IFN-based therapies (P = 0.27). Achieving SVR with an all-oral DAA regimen during follow-up was independently associated with a lower risk of HCC emergence (subhazard ratio 0.264; 95% CI 0.070–0.991; P = 0.049).[Conclusions] SVR with all-oral DAA regimens reduces the risk of HCC in HIV/HCV-coinfected patients with compensated cirrhosis.This study was supported by grants from the Consejería de Salud de la Junta de Andalucía (PI-0014/2014), the Servicio Andaluz de Salud (grant number SAS/111239) and the Fondo de Investigaciones Sanitarias ISCIII (grant number PI13/01621 and PI16/01443). J. A. P. is the recipient of an intensification grant from the Instituto de Salud Carlos III (grant number Programa-I3SNS). In addition, this work has been partially funded by the Spanish AIDS Research Network RD16/0025/0010 as part of the Plan Nacional R + D+I and cofinanced by ISCIII Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER).Peer reviewe

Upregulation of the ESCRT pathway and multivesicular bodies accelerates degradation of proteins associated with neurodegeneration

Many neurodegenerative diseases, including Huntington’s disease (HD) and Alzheimer’s disease (AD), occur due to an accumulation of aggregation-prone proteins, which results in neuronal death. Studies in animal and cell models show that reducing the levels of these proteins mitigates disease phenotypes. We previously reported a small molecule, NCT-504, which reduces cellular levels of mutant huntingtin (mHTT) in patient fibroblasts as well as mouse striatal and cortical neurons from an HdhQ111 mutant mouse. Here, we show that NCT-504 has a broader potential, and in addition reduces levels of Tau, a protein associated with Alzheimer’s disease, as well as other tauopathies. We find that in untreated cells, Tau and mHTT are degraded via autophagy. Notably, treatment with NCT-504 diverts these proteins to multivesicular bodies (MVB) and the ESCRT pathway. Specifically, NCT-504 causes a proliferation of endolysosomal organelles including MVB, and an enhanced association of mHTT and Tau with endosomes and MVB. Importantly, depletion of proteins that act late in the ESCRT pathway blocked NCT-504 dependent degradation of Tau. Moreover, NCT-504-mediated degradation of Tau occurred in cells where Atg7 is depleted, which indicates that this pathway is independent of canonical autophagy. Together, these studies reveal that upregulation of traffic through an ESCRT-dependent MVB pathway may provide a therapeutic approach for neurodegenerative diseases. Abbreviations: AD: Alzheimer’s disease CLEAR: Coordinated Lysosomal Expression and Regulation HTT: Huntingtin HD: Huntington’s disease MEF: Mouse embryonic fibroblasts HTT: Mutant Huntingtin MVB: Multivesicular bodies TFEB: Transcription factor E