A signaling lipid drives synapse formation.

Phosphatidylinositol 3,5-bisphosphate enables transport of proteins to synaptic sites.

Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

Trafficking of cell-surface proteins from endosomes to the plasma membrane is a key mechanism to regulate synaptic function. In non-neuronal cells, proteins recycle to the plasma membrane either via the SNX27-Retromer-WASH pathway or via the recently discovered SNX17-Retriever-CCC-WASH pathway. While SNX27 is responsible for the recycling of key neuronal receptors, the roles of SNX17 in neurons are less understood. Here, using cultured hippocampal neurons, we demonstrate that the SNX17 pathway regulates synaptic function and plasticity. Disruption of this pathway results in a loss of excitatory synapses and prevents structural plasticity during chemical long-term potentiation (cLTP). cLTP drives SNX17 recruitment to synapses, where its roles are in part mediated by regulating the surface expression of ß1-integrin. SNX17 recruitment relies on NMDAR activation, CaMKII signaling, and requires binding to the Retriever and PI(3)P. Together, these findings provide molecular insights into the regulation of SNX17 at synapses and define key roles for SNX17 in synaptic maintenance and in regulating enduring forms of synaptic plasticity.

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.

Iron-induced cytotoxicity mediated by endolysosomal TRPML1 channels is reverted by TFEB.

Increased brain iron content has been consistently reported in sporadic Parkinson's disease (PD) patients, and an increase in cytosolic free iron is known to cause oxidative stress and cell death. However, whether iron also accumulates in susceptible brain areas in humans or in mouse models of familial PD remains unknown. In addition, whilst the lysosome functions as a critical intracellular iron storage organelle, little is known about the mechanisms underlying lysosomal iron release and how this process is influenced by lysosome biogenesis and/or lysosomal exocytosis. Here, we report an increase in brain iron content also in PD patients due to the common G2019S-LRRK2 mutation as compared to healthy age-matched controls, whilst differences in iron content are not observed in G2019S-LRRK2 knockin as compared to control mice. Chemically triggering iron overload in cultured cells causes cytotoxicity via the endolysosomal release of iron which is mediated by TRPML1. TFEB expression reverts the iron overload-associated cytotoxicity by causing lysosomal exocytosis, which is dependent on a TRPML1-mediated increase in cytosolic calcium levels. Therefore, approaches aimed at increasing TFEB levels, or pharmacological TRPML1 activation in conjunction with iron chelation may prove beneficial against cell death associated with iron overload conditions such as those associated with PD.

Roles of PIKfyve in multiple cellular pathways.

Phosphoinositide signaling lipids are crucial for eukaryotes and regulate many aspects of cell function. These signaling molecules are difficult to study because they are extremely low abundance. Here, we focus on two of the lowest abundance phosphoinositides, PI(3,5)P2 and PI(5)P, which play critical roles in cellular homeostasis, membrane trafficking and transcription. Their levels are tightly regulated by a protein complex that includes PIKfyve, Fig4 and Vac14. Importantly, mutations in this complex that decrease PI(3,5)P2 and PI(5)P are linked to human diseases, especially those of the nervous system. Paradoxically, PIKfyve inhibitors which decrease PI(3,5)P2 and PI(5)P, are currently being tested for some neurodegenerative diseases, as well as other diverse diseases including some cancers, and as a treatment for SARS-CoV2 infection. A more comprehensive picture of the pathways that are regulated by PIKfyve will be critical to understand the roles of PI(3,5)P2 and PI(5)P in normal human physiology and in disease.

Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate retriever-mediated recycling on endosomes.

Cell surface receptors control how cells respond to their environment. Many cell surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH, and CCC complexes. Here, using mammalian cells, we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces phosphatidylinositol 3-phosphate (PI3P), which is the substrate for PIKfyve to generate PI3,5P2. We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH, and CCC complexes on endosomes. Importantly, PIKfyve inhibition results in displacement of Retriever and CCC from endosomes. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration.

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.

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.

The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A.

Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are a common cause of hereditary Parkinson's disease. LRRK2 regulates various intracellular vesicular trafficking pathways, including endolysosomal degradative events such as epidermal growth factor receptor (EGFR) degradation. 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, using an array of immunofluorescence and pulldown assays, we report that expression of active or phosphodeficient RAB8A variants rescues the G2019S LRRK2-mediated effects on endolysosomal membrane trafficking. Similarly, up-regulation of the RAB11-Rabin8-RAB8A cascade, which activates RAB8A, also reverted these trafficking deficits. Loss of RAB8A mimicked the effects of G2019S LRRK2 on endolysosomal trafficking and decreased RAB7A activity. Expression of pathogenic G2019S LRRK2 or loss of RAB8A interfered with EGFR degradation by causing its accumulation in a RAB4-positive endocytic compartment, which was accompanied by a deficit in EGFR recycling and was rescued upon expression of active RAB7A. Dominant-negative RAB7A expression resulted in similar deficits 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, by impairing RAB8A function, pathogenic G2019S LRRK2 deregulates endolysosomal transport and endocytic recycling events.

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 disease-modifying 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.

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.

Two-Pore Channels and Parkinson's Disease: Where's the Link?

Two-pore channels are endolysosomal Ca2+ release channels involved in proper trafficking to and from those organelles. They are the likely targets for the Ca2+-mobilizing messenger NAADP, and are further regulated by a variety of mechanisms including phosphoinositide levels and Rab proteins. As discussed here, recent studies highlight a role for these channels in the pathomechanism(s) underlying Parkinson's disease, with important implications for possible alternative treatment strategies.

Targeting the Autophagy/Lysosomal Degradation Pathway in Parkinson's Disease.

Autophagy is a cellular quality control mechanism crucial for neuronal homeostasis. Defects in autophagy are critically associated with mechanisms underlying Parkinson's disease (PD), a common and debilitating neurodegenerative disorder. Autophagic dysfunction in PD can occur at several stages of the autophagy/lysosomal degradative machinery, contributing to the formation of intracellular protein aggregates and eventual neuronal cell death. Therefore, autophagy inducers may comprise a promising new therapeutic approach to combat neurodegeneration in PD. Several currently available FDA-approved drugs have been shown to enhance autophagy, which may allow for their repurposing for use in novel clinical conditions including PD. This review summarizes our current knowledge of deficits in the autophagy/lysosomal degradation pathways associated with PD, and highlight current approaches which target this pathway as possible means towards novel therapeutic strategies.

Alterations in late endocytic trafficking related to the pathobiology of LRRK2-linked Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene comprise the most common cause of familial Parkinson's disease (PD), and variants increase the risk for sporadic PD. LRRK2 displays kinase and GTPase activity, and altered catalytic activity correlates with neurotoxicity, making LRRK2 a promising therapeutic target. Despite the importance of LRRK2 for disease pathogenesis, its normal cellular function, and the mechanism(s) by which pathogenic mutations cause neurodegeneration remain unclear. LRRK2 seems to regulate a variety of intracellular vesicular trafficking events to and from the late endosome in a manner dependent on various Rab proteins. At least some of those events are further regulated by LRRK2 in a manner dependent on two-pore channels (TPCs). TPCs are ionic channels localized to distinct endosomal structures and can cause localized calcium release from those acidic stores, with downstream effects on vesicular trafficking. Here, we review current knowledge about the link between LRRK2, TPC- and Rab-mediated vesicular trafficking to and from the late endosome, highlighting a possible cross-talk between endolysosomal calcium stores and Rab proteins underlying pathomechanism(s) in LRRK2-related PD.

LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in an Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pulldown assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients when compared with healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway.

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 Ca(2) (+) homeostasis may underlie disease pathomechanism and be an inherent feature of all vulnerable neurons. The downstream effects of altered Ca(2) (+) 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 Ca(2) (+) stress which may determine their selective vulnerability, and suggest how abnormal Ca(2) (+) 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 Ca(2) (+) dyshomeostasis may allow novel combinatorial therapeutic strategies to slow PD progression.