Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins.

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Synaptotagmin-11 Inhibits Synaptic Vesicle Endocytosis via Endophilin A1.

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.

Rac1, the actin cytoskeleton and microtubules are key players in clathrin-independent endophilin-A3-mediated endocytosis.

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3, encoded by SH3GL3), which controls the cell surface homeostasis of the tumor marker CD166 (also known as ALCAM). Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here, we investigate the role of actin, Rho GTPases and microtubules, which are major players in CIE processes, in this mechanism. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers, and that its perturbation strongly inhibits the process of CD166 uptake. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms. This article has an associated First Person interview with the first author of the paper.

The CHD Protein Kismet Restricts the Synaptic Localization of Cell Adhesion Molecules at the Drosophila Neuromuscular Junction.

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we show that the Drosophila homolog of the chromatin remodeling enzymes CHD7 and CHD8, Kismet, represses the synaptic levels of several cell adhesion molecules. Neuroligins 1 and 3 and the integrins αPS2 and ßPS are increased at kismet mutant synapses but Kismet only directly regulates transcription of neuroligin 2. Kismet may therefore regulate synaptic CAMs indirectly by activating transcription of gene products that promote intracellular vesicle trafficking including endophilin B (endoB) and/or rab11. Knock down of EndoB in all tissues or neurons increases synaptic FasII while knock down of EndoB in kis mutants does not produce an additive increase in FasII. In contrast, neuronal expression of Rab11, which is deficient in kis mutants, leads to a further increase in synaptic FasII in kis mutants. These data support the hypothesis that Kis influences the synaptic localization of FasII by promoting intracellular vesicle trafficking through the early endosome.

The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis.

The multidomain adaptor protein amphiphysin-1 (Amph1) is an important coordinator of clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid-binding N-BAR (Bin/Amphiphysin/Rvs) domain, central proline-rich (PRD) and clathrin/AP2 (CLAP) domains, and a C-terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain-specific interactions of Amph1 were validated using in vitro GST pull-down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1-endophilin A1 complex is dependent on the phosphorylation status of Amph1-S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation-dependent Amph1-endophilin A1 interaction in efficient SV endocytosis.

Endophilin A2 protects against renal fibrosis by targeting TGF-ß/Smad signaling.

Renal fibrosis underlies all forms of end-stage kidney disease. Endophilin A2 (EndoA2) plays a role in nephrotic syndrome; however, its effect on renal fibrosis remains unknown. Here, we demonstrate that EndoA2 protects against kidney interstitial fibrosis via the transforming growth factor-ß (TGF-ß)/Smad signaling pathway. Mouse kidneys with fibrosis or kidney biopsy specimens from patients with fibrotic nephropathy had lower levels of EndoA2 protein expression than that in kidneys without fibrosis. In vivo overexpression of EndoA2 with the endophilin A2 transgene (EndoA2Tg ) notably prevented renal fibrosis, decreased the protein expression of profibrotic molecules, suppressed tubular injury, and reduced apoptotic tubular cells in the obstructed kidney cortex of mice with unilateral ureteral obstruction (UUO). In vivo and in vitro overexpression of EndoA2 markedly inhibited UUO- or TGF-ß1-induced phosphorylation of Smad2/3 and tubular epithelial cells dedifferentiation. Furthermore, EndoA2 was co-immunoprecipitated with the type II TGF-ß receptor (TßRII), thus inhibiting the binding of the type I TGF-ß receptor (TßRI) to TßRII. These findings indicate that EndoA2 mitigates renal fibrosis, at least partially, via modulating the TGF-ß/Smad signaling. Targeting EndoA2 may be a new potential therapeutic strategy for treatment of renal fibrosis.

Endophilin A2 regulates B-cell endocytosis and is required for germinal center and humoral responses.

Antigen-specific B-cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B-cell endosomal trafficking pathways and their specific roles in B-cell responses have not been systematically investigated. Here, we report high-throughput identification of genes regulating B-cell receptor (BCR)-mediated antigen internalization using genome-wide functional screens. We show that antigen internalization depends both on constitutive, clathrin-mediated endocytosis and on antigen-induced, clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2-mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B-cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2-deficient mice show defects in GC B-cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B-cell clonal expansion and antibody responses.

Endophilin recruitment drives membrane curvature generation through coincidence detection of GPCR loop interactions and negative lipid charge.

Endophilin plays key roles during endocytosis of cellular receptors, including generating membrane curvature to drive internalization. Electrostatic interactions between endophilin's BIN/Amphiphysin/Rvs domain and anionic membrane lipids have been considered the major driving force in curvature generation. However, the SH3 domain of endophilin also interacts with the proline-rich third intracellular loop (TIL) of various G-protein-coupled receptors (GPCRs), and it is unclear whether this interaction has a direct role in generating membrane curvature during endocytosis. To examine this, we designed model membranes with a membrane density of 1400 receptors per µm2 represented by a covalently conjugated TIL region from the ß1-adrenergic receptor. We observed that TIL recruits endophilin to membranes composed of 95 mol% of zwitterionic lipids via the SH3 domain. More importantly, endophilin recruited via TIL tubulates vesicles and gets sorted onto highly curved membrane tubules. These observations indicate that the cellular membrane bending and curvature sensing activities of endophilin can be facilitated through detection of the TIL of activated GPCRs in addition to binding to anionic lipids. Furthermore, we show that TIL electrostatically interacts with membranes composed of anionic lipids. Therefore, anionic lipids can modulate TIL/SH3 domain binding. Overall, our findings imply that an interplay between TIL, charged membrane lipids, BAR domain, and SH3 domain could exist in the biological system and that these components may act in coordination to regulate the internalization of cellular receptors.

Endophilin-A2-dependent tubular endocytosis promotes plasma membrane repair and parasite invasion.

Endocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.

17ß-Estradiol nongenomically induces vasodilation is enhanced by promoting phosphorylation of endophilin A2.

ObjectiveA previous study found that the tyrosine phosphorylation of endophilin A2 (Endo II) was responsible for increase surface expression of MT1-MMP and ECM degradation; however, there is little information about whether Endo II could influence membrane estrogen receptors (mERs) and its functions.Materials and methodsIn the present study, Human umbilical vein endothelial cells (HUVECs) were treated with E2, PPT, DPN, ICI 182780, Endo siRNA or negative control siRNA, and the biological behavior of the treated cells was observed. The mice were randomly divided into AAV-control-shRNA + Ach, AAV-Endo II-shRNA + Ach, AAV-control-shRNA + E2, AAV-Endo II-shRNA + E2 groups and the thoracic aorta were isolated, cut into 2-mm rings, then the wall tension was detected.ResultsWe found that 17ß-Estradiol (E2) enhanced mERα protein level, which was further increased after knocking down Endo II, the mechanism maybe involved in E2-induced tyrosine phosphorylation of Endo II. In addition, we also observed that Endo II blocked the activation of Akt, ERK1/2 and eNOS signaling in HUVECs treated with E2. E2 induced vasodilation was significantly increased by silencing of Endo II expression.ConclusionOur study provided a sound basis to selective modulate Endo II for E2's nongenomic pathway, which can be benefit for cardiovascular system.

BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3.

Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.

The variations of endophilin A2-FoxO3a-autophagy signal in angiotensin II-induced dopaminergic neuron injury mouse model and by biochanin A.

Biochanin A (Bioch A) is a natural plant estrogen, with various biological activities such as anti-apoptosis, anti-oxidation, and suppression of inflammation. In this study, we investigated the protective effects of Bioch A on angiotensin II (AngII) - induced dopaminergic (DA) neuron damage in vivo and on molecular mechanisms. Spontaneous activity and motor ability of mice among groups was detected by open-field test and swim-test. The expression of TH, microtubule-associated proteins light chain 3B II (LC3BII)/LC3BI, beclin-1, P62, forkhead box class O3 (FoxO3), phosphorylated (p) FoxO3a/FoxO3a, FoxO3, and endophilin A2 were determined by Western blot and immunohistochemistry or immunofluorescence staining. Our results showed that AngII treatment significantly increased the behavioral dysfunction of mice and DA neuron damage. Meanwhile, AngII treatment increased the expression of LC3BII/LC3BI, beclin-1, P62, and FoxO3a and decreased the expression of endophilin A2 and p-FoxO3a/FoxO3a, however, Bioch A treatment alleviate these changes. In summary, these results suggest that Bioch A exerts protective effects on AngII-induced mouse model may be related to regulating endophilin A2, FoxO3a, and autophagy-related proteins; however, the specific mechanism is not yet clear and needs further study.

Molecular mechanism of Fast Endophilin-Mediated Endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3ß. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

Knock-Down of HDAC2 in Human Induced Pluripotent Stem Cell Derived Neurons Improves Neuronal Mitochondrial Dynamics, Neuronal Maturation and Reduces Amyloid Beta Peptides.

Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

Endophilin A2 regulates calcium-activated chloride channel activity via selective autophagy-mediated TMEM16A degradation.

TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125-1 µM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.

Modeling endophilin-mediated Aß disposal in glioma cells.

Autophagy dysregulation has emerged in age-related neurological diseases (Ulland et al.; Matheoud et al.; Ashkenazi et al.). Alzheimer Disease (AD), the most common progressive neurodegenerative disorder, is characterized by the accumulation of amyloid-ß (Aß) plaques caused by aberrant Aß metabolism (Qiang et al.; Sevigny et al.; Ittner et al.). Glia constitute the brain immune system and ingest extracellular Aß for degradation via the autophagy-lysosome machinery (Ries and Sastre; Cho et al.). Here, we model the molecular rationale for this clearance process in glioma cells by showing that miR34a inhibits autophagy-mediated disposal of Aß fibrils and identifying two novel direct targets of miR34a, endophilin-3 and cathepsin B (CTSB, a previously reported enzyme for Aß degrading (Sun et al.)). Bioinformatics analyses revealed that endophilin-3 expresses at a significantly lower level in neurodegenerative diseases. Its gain-of-function substantially promotes both uptake and degradation of Aß while small interfering RNA (siRNA)-mediated endophilin-3 knockdown slowed down Aß clearance and blocked autolysosome formation. Mechanistically, gene ontology (GO) analysis of the endophilin-3 interactome identified by mass spectrometry uncovered enriched components involved in actin binding (with the highest score). Importantly, we validated that the actin-binding protein phostensin interacted with endophilin-3. Phostensin knockdown restored endophilin-3-mediated up-regulation of Aß clearance. Thus, our findings indicate that miR34a inhibits Aß clearance by targeting endophilin-3 and CTSB at multiple steps including uptake and autophagy-mediated degradation.

An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2.

BACKGROUND: Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. METHODS: A novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. RESULTS: Our integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. CONCLUSIONS: Our study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.

Crosstalk between presynaptic trafficking and autophagy in Parkinson's disease.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder that profoundly affects one's motor functions. The disease is characterized pathologically by denervation of dopaminergic (DAergic) nigrostriatal terminal and degeneration of DAergic neurons in the substantia nigra par compacta (SNpc); however, the precise molecular mechanism underlying disease pathogenesis remains poorly understood. Animal studies in both toxin-induced and genetic PD models suggest that presynaptic impairments may underlie the early stage of DA depletion and neurodegeneration (reviewed in Schirinzi, T., et al. 2016). Supporting this notion, human genetic studies and genomic analysis have identified an increasing number of PD risk variants that are associated with synaptic vesicle (SV) trafficking, regulation of synaptic function and autophagy/lysosomal system (Chang, D., et al. 2017, reviewed in Trinh, J. & Farrer, M. 2013; Singleton, A.B., et al. 2013). Although the precise mechanism for autophagy regulation in neurons is currently unclear, many studies demonstrate that autophagosomes form at the presynaptic terminal (Maday, S. & Holzbaur, E.L. 2014; Vanhauwaert, R., et al. 2017; reviewed in Yue, Z. 2007). Growing evidence has revealed overlapping genes involved in both SV recycling and autophagy, suggesting that the two membrane trafficking processes are inter-connected. Here we will review emergent evidence linking SV endocytic genes and autophagy genes at the presynaptic terminal. We will discuss their potential relevance to PD pathogenesis.