Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade in vitro and in vivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.

Polyamines in Parkinson's Disease: Balancing Between Neurotoxicity and Neuroprotection.

The polyamines putrescine, spermidine, and spermine are abundant polycations of vital importance in mammalian cells. Their cellular levels are tightly regulated by degradation and synthesis, as well as by uptake and export. Here, we discuss the delicate balance between the neuroprotective and neurotoxic effects of polyamines in the context of Parkinson's disease (PD). Polyamine levels decline with aging and are altered in patients with PD, whereas recent mechanistic studies on ATP13A2 (PARK9) demonstrated a driving role of a disturbed polyamine homeostasis in PD. Polyamines affect pathways in PD pathogenesis, such as α-synuclein aggregation, and influence PD-related processes like autophagy, heavy metal toxicity, oxidative stress, neuroinflammation, and lysosomal/mitochondrial dysfunction. We formulate outstanding research questions regarding the role of polyamines in PD, their potential as PD biomarkers, and possible therapeutic strategies for PD targeting polyamine homeostasis.

TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P.

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.

Copper-independent lysosomal localisation of the Wilson disease protein ATP7B.

In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.

HERV-K (HML-2) Envelope Protein Induces Mitochondrial Depolarization and Neurotoxicity via Endolysosome Iron Dyshomeostasis.

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe2+) generates reactive oxygen species (ROS). Endolysosome stores of Fe2+ are central to iron trafficking and endolysosome deacidification releases Fe2+ into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe2+ released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe2+ concentrations, (4) increased cytosolic and mitochondrial Fe2+ and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe2+ and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe2+ efflux into the cytoplasm.

Endolysosomal Disorders Affecting the Proximal Tubule of the Kidney: New Mechanistic Insights and Therapeutics.

Epithelial cells that line the proximal tubule of the kidney rely on an intertwined ecosystem of vesicular membrane trafficking pathways to ensure the reabsorption of essential nutrients. To function effectively and to achieve homeostasis, these specialized cells require the sorting and recycling of a wide array of cell surface proteins within the endolysosomal network, including signaling receptors, nutrient transporters, ion channels, and polarity markers. The dysregulation of the endolysosomal system can lead to a generalized proximal tubule dysfunction, ultimately causing severe metabolic complications and kidney disease.In this chapter, we highlight the biological functions of the genes that code endolysosomal proteins from the perspective of understanding - and potentially reversing - the pathophysiology of endolysosomal disorders affecting the proximal tubule of the kidney. These insights might ultimately lead to potential treatments for currently intractable diseases and transform our ability to regulate kidney homeostasis and health.

Impaired cathepsin D in retinal pigment epithelium cells mediates Stargardt disease pathogenesis.

Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.

Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD.

A GGGGCC (G4 C2 ) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. Here, we performed live imaging of Drosophila motor neurons expressing expanded G4 C2 repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G4 C2 repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G4 C2 repeats. Thus, our data indicate that expanded G4 C2 repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD.

HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

Endolysosomal cholesterol export: More than just NPC1.

NPC1 plays a central role in cholesterol egress from endolysosomes, a critical step for maintaining intracellular cholesterol homeostasis. Despite recent advances in the field, the full repertoire of molecules and pathways involved in this process remains unknown. Emerging evidence suggests the existence of NPC1-independent, alternative routes. These may involve vesicular and non-vesicular mechanisms, as well as release of extracellular vesicles. Understanding the underlying molecular mechanisms that bypass NPC1 function could have important implications for the development of therapies for lysosomal storage disorders. Here we discuss how cholesterol may be exported from lysosomes in which NPC1 function is impaired.

NAADP-Dependent TPC Current.

Two-pore channels, TPC1 and TPC2, are Ca2+- and Na+-permeable cation channels expressed on the membranes of endosomes and lysosomes in nearly all mammalian cells. These channels have been implicated in Ca2+ signaling initiated from the endolysosomes, vesicular trafficking, cellular metabolism, macropinocytosis, and viral infection. Although TPCs have been shown to mediate Ca2+ release from acidic organelles in response to NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca2+ mobilizing messenger, questions remain whether NAADP is a direct ligand of these channels. In whole-endolysosomal patch clamp recordings, it has been difficult to detect NAADP-evoked currents in vacuoles that expressed TPC1 or TPC2, while PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) activated a highly Na+-selective current under the same experimental configuration. In this chapter, we summarize recent progress in this area and provide our observations on NAADP-elicited TPC2 currents from enlarged endolysosomes as well as their possible relationships with the currents evoked by PI(3,5)P2. We propose that TPCs are channels dually regulated by PI(3,5)P2 and NAADP in an interdependent manner and the two endogenous ligands may have both distinguished and cooperative roles.

Lysosomal size matters.

Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra- and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.

Retromer retrieves the Wilson disease protein ATP7B from endolysosomes in a copper-dependent manner.

The Wilson disease protein, ATP7B maintains copper (herein referring to the Cu+ ion) homeostasis in the liver. ATP7B traffics from trans-Golgi network to endolysosomes to export excess copper. Regulation of ATP7B trafficking to and from endolysosomes is not well understood. We investigated the fate of ATP7B after copper export. At high copper levels, ATP7B traffics primarily to acidic, active hydrolase (cathepsin-B)-positive endolysosomes and, upon subsequent copper chelation, returns to the trans-Golgi network (TGN). At high copper, ATP7B colocalizes with endolysosomal markers and with a core member of retromer complex, VPS35. Knocking down VPS35 did not abrogate the copper export function of ATP7B or its copper-responsive anterograde trafficking to vesicles; rather upon subsequent copper chelation, ATP7B failed to relocalize to the TGN, which was rescued by overexpressing wild-type VPS35. Overexpressing mutants of the retromer complex-associated proteins Rab7A and COMMD1 yielded a similar non-recycling phenotype of ATP7B. At high copper, VPS35 and ATP7B are juxtaposed on the same endolysosome and form a large complex that is stabilized by in vivo photoamino acid labeling and UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of copper transporter ATP7B in a manner that is dependent upon intracellular copper.

Skewed endosomal RNA responses from TLR7 to TLR3 in RNase T2-deficient macrophages.

RNase T2, a ubiquitously expressed RNase, degrades RNAs in the endosomal compartments. RNA sensors, double-stranded RNA (dsRNA)-sensing Toll-like receptor 3 (TLR3) and single-stranded RNA (ssRNA)-sensing TLR7, are localized in the endosomal compartment in mouse macrophages. We here studied the role of RNase T2 in TLR3 and TLR7 responses in macrophages. Macrophages expressed RNase T2 and a member of the RNase A family RNase 4. RNase T2 was also expressed in plasmacytoid and conventional dendritic cells. Treatment with dsRNAs or type I interferon (IFN) up-regulated expression of RNase T2 but not RNase 4. RNase T2-deficiency in macrophages up-regulated TLR3 responses but impaired TLR7 responses. Mechanistically, RNase T2 degraded both dsRNAs and ssRNAs in vitro, and its mutants showed a positive correlation between RNA degradation and the rescue of altered TLR3 and TLR7 responses. H122A and C188R RNase T2 mutations, not H69A and E118V mutations, impaired both RNA degradation and the rescue of altered TLR3 and TLR7 responses. RNase T2 in bone marrow-derived macrophages was broadly distributed from early endosomes to lysosomes, and colocalized with the internalized TLR3 ligand poly(I:C). These results suggest that RNase T2-dependent RNA degradation in endosomes/lysosomes negatively and positively regulates TLR3 and TLR7 responses, respectively, in macrophages.

Human SLC15A4 is crucial for TLR-mediated type I interferon production and mitochondrial integrity.

Solute carrier family 15 member 4 (SLC15A4) is an endolysosome-resident amino acid transporter that regulates innate immune responses, and is genetically associated with inflammatory diseases such as systemic lupus erythematosus (SLE) and colitis. SLC15A4-deficient mice showed the amelioration of symptoms of these model diseases, and thus SLC15A4 is a promising therapeutic target of SLE and colitis. For developing a SLC15A4-based therapeutic strategy, understanding human SLC15A4's properties is essential. Here, we characterized human SLC15A4 and demonstrated that human SLC15A4 possessed pH- and temperature-dependent activity for the transportation of dipeptides or tripeptides. Human SLC15A4 localized in LAMP1+ compartments and constitutively associated with Raptor and LAMTORs. We also investigated SLC15A4's role in inflammatory responses using the human plasmacytoid dendritic cell line, CAL-1. Knock down (KD) of the SLC15A4 gene in CAL-1 (SLC15A4-KD CAL-1) impaired Toll-like receptor (TLR) 7/8 or TLR9-triggered type I interferon (IFN-I) production and mTORC1 activity, indicating that human SLC15A4 is critical for TLR7/8/9-mediated inflammatory signaling. We also examined SLC15A4's role in the autophagy response since SLC15A4 loss caused the decrease of mTORC1 activity, which greatly influences autophagy. We found that SLC15A4 was not required for autophagy induction, but was critical for autophagy sustainability. Notably, SLC15A4-KD CAL-1 severely decreased mitochondrial membrane potential in starvation conditions. Our findings revealed that SLC15A4 plays a key role in mitochondrial integrity in human cells, which might benefit immune cells in fulfilling their functions in an inflammatory milieu.

Lysosomal amino acid transporters as key players in inflammatory diseases.

Controlling inflammation can alleviate immune-mediated, lifestyle-related and neurodegenerative diseases. The endolysosome system plays critical roles in inflammatory responses. Endolysosomes function as signal transduction hubs to convert various environmental danger signals into gene expression, enabling metabolic adaptation of immune cells and efficient orchestration of inflammation. Solute carrier family 15 member A3 (SLC15A3) and member A4 (SLC15A4) are endolysosome-resident amino acid transporters that are preferentially expressed in immune cells. These transporters play essential roles in signal transduction through endolysosomes, and the loss of either transporter can alleviate multiple inflammatory diseases because of perturbed endolysosome-dependent signaling events, including inflammatory and metabolic signaling. Here, we summarize the findings leading to a proof-of-concept for anti-inflammatory strategies based on targeting SLC15 transporters.

Multiphasic changes in smooth muscle Ca2+ transporters during the progression of coronary atherosclerosis.

Ischemic heart disease due to macrovascular atherosclerosis and microvascular dysfunction is the major cause of death worldwide and the unabated increase in metabolic syndrome is a major reason why this will continue. Intracellular free Ca2+ ([Ca2+]i) regulates a variety of cellular functions including contraction, proliferation, migration, and transcription. It follows that studies of vascular Ca2+ regulation in reductionist models and translational animal models are vital to understanding vascular health and disease. Swine with metabolic syndrome (MetS) develop the full range of coronary atherosclerosis from mild to severe disease. Intravascular imaging enables quantitative measurement of atherosclerosis in vivo, so viable coronary smooth muscle (CSM) cells can be dispersed from the arteries to enable Ca2+ transport studies in native cells. Transition of CSM from the contractile phenotype in the healthy swine to the proliferative phenotype in mild atherosclerosis was associated with increases in SERCA activity, sarcoplasmic reticulum Ca2+, and voltage-gated Ca2+ channel function. In vitro organ culture confirmed that SERCA activation induces CSM proliferation. Transition from the proliferative to a more osteogenic phenotype was associated with decreases in all three Ca2+ transporters. Overall, there was a biphasic change in Ca2+ transporters over the progression of atherosclerosis in the swine model and this was confirmed in CSM from failing explanted hearts of humans. A major determinant of endolysosome content in human CSM is the severity of atherosclerosis. In swine CSM endolysosome Ca2+ release occurred through the TPC2 channel. We propose a multiphasic change in Ca2+ transporters over the progression of coronary atherosclerosis.

Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications.

More than just sugars: Conserved oligomeric Golgi complex deficiency causes glycosylation-independent cellular defects.

The conserved oligomeric Golgi (COG) complex controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multisystemic diseases known as COG-congenital disorders of glycosylation (COG-CDG). To gain better understanding of COG-CDGs, we compared COG knockout cells with cells deficient to 2 key enzymes, Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase and uridine diphosphate-glucose 4-epimerase (GALE), which contribute to proper N- and O-glycosylation. While all knockout cells share similar defects in glycosylation, these defects only account for a small fraction of observed COG knockout phenotypes. Glycosylation deficiencies were not associated with the fragmented Golgi, abnormal endolysosomes, defective sorting and secretion or delayed retrograde trafficking, indicating that these phenotypes are probably not due to hypoglycosylation, but to other specific interactions or roles of the COG complex. Importantly, these COG deficiency specific phenotypes were also apparent in COG7-CDG patient fibroblasts, proving the human disease relevance of our CRISPR knockout findings. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis in eukaryotic cells, and for better understanding human diseases associated with COG/Golgi impairment.

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.