Molecular and functional architecture of striatal dopamine release sites.

Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.

Membrane interaction and disulphide-bridge formation in the unconventional secretion of Tau.

Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer's disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.

The Cell Biology of Tau Secretion.

The progressive accumulation and spread of misfolded tau protein in the nervous system is the hallmark of tauopathies, progressive neurodegenerative diseases with only symptomatic treatments available. A growing body of evidence suggests that spreading of tau pathology can occur via cell-to-cell transfer involving secretion and internalization of pathological forms of tau protein followed by templated misfolding of normal tau in recipient cells. Several studies have addressed the cell biological mechanisms of tau secretion. It now appears that instead of a single mechanism, cells can secrete tau via three coexisting pathways: (1) translocation through the plasma membrane; (2) membranous organelles-based secretion; and (3) ectosomal shedding. The relative importance of these pathways in the secretion of normal and pathological tau is still elusive, though. Moreover, glial cells contribute to tau propagation, and the involvement of different cell types, as well as different secretion pathways, complicates the understanding of prion-like propagation of tauopathy. One of the important regulators of tau secretion in neuronal activity, but its mechanistic connection to tau secretion remains unclear and may involve all three secretion pathways of tau. This review article summarizes recent advancements in the field of tau secretion with an emphasis on cell biological aspects of the secretion process and discusses the role of neuronal activity and glial cells in the spread of pathological forms of tau.

The interaction of α-synuclein and Tau: A molecular conspiracy in neurodegeneration?

α-synuclein and Tau are proteins prone to pathological misfolding and aggregation that are normally found in the presynaptic and axonal compartments of neurons. Misfolding initiates a homo-oligomerization and aggregation cascade culminating in cerebral accumulation of aggregated α-synuclein and Tau in insoluble protein inclusions in multiple neurodegenerative diseases. Traditionally, α-synuclein-containing Lewy bodies have been associated with Parkinson's disease and Tau-containing neurofibrillary tangles with Alzheimer's disease and various frontotemporal dementia syndromes. However, there is significant overlap and co-occurrence of α-synuclein and Tau pathologies in a spectrum of neurodegenerative diseases. Importantly, α-synuclein and Tau can interact in cells, and their pathological conformations are capable of templating further misfolding and aggregation of each other. They also share a number of protein interactors indicating that network perturbations may contribute to chronic proteotoxic stress and neuronal dysfunction in synucleinopathies and tauopathies, some of which share similarities in both neuropathological and clinical manifestations. In this review, we focus on the protein interactions of these two pathologically important proteins and consider a network biology perspective towards neurodegenerative diseases.

Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation.

The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called 'lipid rafts', in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.

Mechanisms of secretion and spreading of pathological tau protein.

Accumulation of misfolded and aggregated forms of tau protein in the brain is a neuropathological hallmark of tauopathies, such as Alzheimer's disease and frontotemporal lobar degeneration. Tau aggregates have the ability to transfer from one cell to another and to induce templated misfolding and aggregation of healthy tau molecules in previously healthy cells, thereby propagating tau pathology across different brain areas in a prion-like manner. The molecular mechanisms involved in cell-to-cell transfer of tau aggregates are diverse, not mutually exclusive and only partially understood. Intracellular accumulation of misfolded tau induces several mechanisms that aim to reduce the cellular burden of aggregated proteins and also promote secretion of tau aggregates. However, tau may also be released from cells physiologically unrelated to protein aggregation. Tau secretion involves multiple vesicular and non-vesicle-mediated pathways, including secretion directly through the plasma membrane. Consequently, extracellular tau can be found in various forms, both as a free protein and in vesicles, such as exosomes and ectosomes. Once in the extracellular space, tau aggregates can be internalized by neighboring cells, both neurons and glial cells, via endocytic, pinocytic and phagocytic mechanisms. Importantly, accumulating evidence suggests that prion-like propagation of misfolding protein pathology could provide a general mechanism for disease progression in tauopathies and other related neurodegenerative diseases. Here, we review the recent literature on cellular mechanisms involved in cell-to-cell transfer of tau, with a particular focus in tau secretion.

Secretion of Tau via an Unconventional Non-vesicular Mechanism.

Tauopathies are characterized by cerebral accumulation of Tau protein aggregates that appear to spread throughout the brain via a cell-to-cell transmission process that includes secretion and uptake of pathological Tau, followed by templated misfolding of normal Tau in recipient cells. Here, we show that phosphorylated, oligomeric Tau clusters at the plasma membrane in N2A cells and is secreted in vesicle-free form in an unconventional process sensitive to changes in membrane properties, particularly cholesterol and sphingomyelin content. Cell surface heparan sulfate proteoglycans support Tau secretion, possibly by facilitating its release after membrane penetration. Notably, secretion of endogenous Tau from primary cortical neurons is mediated, at least partially, by a similar mechanism. We suggest that Tau is released from cells by an unconventional secretory mechanism that involves its phosphorylation and oligomerization and that membrane interaction may help Tau to acquire properties that allow its escape from cells directly through the plasma membrane.

FRMD4A-cytohesin signaling modulates the cellular release of tau.

One of the defining pathological features of Alzheimer's disease is the intraneuronal accumulation of tau (also known as MAPT) protein. Tau is also secreted from neurons in response to various stimuli and accumulates in the cerebrospinal fluid of Alzheimer's disease patients. Tau pathology might spread from cell to cell through a mechanism involving secretion and uptake. Here, we developed an assay to follow cellular release and uptake of tau dimers. Individual silencing of ten common late-onset Alzheimer's disease risk genes in HEK293T cells expressing the tau reporters suggested that FRMD4A is functionally linked to tau secretion. FRMD4A depletion by using RNA interference (RNAi) reduced and overexpression increased tau secretion. The activity of cytohesins, interactors of FRMD4A and guanine-nucleotide-exchange factors of Arf6, was necessary for FRMD4A-induced tau secretion. Increased Arf6 and cell polarity signaling through Par6 and atypical protein kinase Cζ (aPKCζ) stimulated tau secretion. In mature cortical neurons, FRMD4A RNAi or inhibition of cytohesins strongly upregulated secretion of endogenous tau. These results suggest that FRMD4A, a genetic risk factor for late-onset Alzheimer's disease, regulates tau secretion by activating cytohesin-Arf6 signaling. We conclude that genetic risk factors of Alzheimer's disease might modulate disease progression by altering tau secretion.

LDL cholesterol recycles to the plasma membrane via a Rab8a-Myosin5b-actin-dependent membrane transport route.

Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC- and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a- and Myosin5b (Myo5b)-dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement.

Cytoplasmic oxysterol-binding proteins: sterol sensors or transporters?

Families of oxysterol-binding protein (ORP) homologues are present in eukaryotes from yeast to man. Their hallmark feature is a characteristic ligand binding domain that, for several family members, has been shown to accommodate different oxysterols and/or cholesterol. ORPs of the "long" subtype contain targeting determinants for the endoplasmic reticulum and to other organelle membranes, the most prominent of which are phosphoinositide-binding pleckstrin homology domains, while "short" ORPs comprise a ligand binding domain with little additional sequences. There is increasing evidence that both long and short ORPs can be enriched at membrane contact sites, junctions of the endoplasmic reticulum with other organelles, where they are suggested to execute regulatory or sterol transfer functions. In this review we discuss the current evidence for putative roles of ORPs as sterol sensors or transporters.

Sterol binding by OSBP-related protein 1L regulates late endosome motility and function.

ORP1L is an oxysterol binding homologue that regulates late endosome (LE) positioning. We show that ORP1L binds several oxysterols and cholesterol, and characterize a mutant, ORP1L Δ560-563, defective in oxysterol binding. While wild-type ORP1L clusters LE, ORP1L Δ560-563 induces LE scattering, which is reversed by disruption of the endoplasmic reticulum (ER) targeting FFAT motif, suggesting that it is due to enhanced LE-ER interactions. Endosome motility is reduced upon overexpression of ORP1L. Both wild-type ORP1L and the Δ560-563 mutant induce the recruitment of both dynactin and kinesin-2 on LE. Most of the LE decorated by overexpressed ORP1L fail to accept endocytosed dextran or EGF, and the transfected cells display defective degradation of internalized EGF. ORP1L silencing in macrophage foam cells enhances endosome motility and results in inhibition of [(3)H]cholesterol efflux to apolipoprotein A-I. These data demonstrate that LE motility and functions in both protein and lipid transport are regulated by ORP1L.

Niemann-Pick C1 modulates hepatic triglyceride metabolism and its genetic variation contributes to serum triglyceride levels.

OBJECTIVE: To study how Niemann-Pick disease type C1 (NPC1) influences hepatic triacylglycerol (TG) metabolism and to determine whether this is reflected in circulating lipid levels. METHODS AND RESULTS: In Npc1(-/-) mice, the hepatic cholesterol content is increased but the TG content is decreased. We investigated lipid metabolism in Npc1(-/-) mouse hepatocytes and the association of NPC1 single-nucleotide polymorphisms with circulating TGs in humans. TGs were reduced in Npc1(-/-) mouse serum and hepatocytes. In Npc1(-/-) hepatocytes, the incorporation of [3H]oleic acid and [3H]acetate into TG was decreased, but shunting of oleic acid- or acetate-derived [3H]carbons into cholesterol was increased. Inhibition of cholesterol synthesis normalized TG synthesis, content, and secretion in Npc1(-/-) hepatocytes, suggesting increased hepatic cholesterol neogenesis as a cause for the reduced TG content and secretion. We found a significant association between serum TG levels and 5 common NPC1 single-nucleotide polymorphisms in a cohort of 1053 men, with the lowest P=8.7 x 10(-4) for the single-nucleotide polymorphism rs1429934. The association between the rs1429934 A allele and higher TG levels was replicated in 2 additional cohorts, which included 8041 individuals. CONCLUSIONS: This study provides evidence of the following: (1) in mice, loss of NPC1 function reduces hepatocyte TG content and secretion by increasing the metabolic flux of carbons into cholesterol synthesis; and (2) common variation in NPC1 contributes to serum TG levels in humans.

Rab8 regulates ABCA1 cell surface expression and facilitates cholesterol efflux in primary human macrophages.

OBJECTIVE: ATP-binding cassette transporter A1 (ABCA1) is thought to lipidate apolipoprotein A-I (apoA-I) at the plasma membrane, with endosomal cholesterol contributing as substrate. The mechanisms of ABCA1 surface delivery are not well understood. We have shown that Rab8 regulates endosomal cholesterol removal to apoA-I in human fibroblasts. Here, we investigated whether Rab8 plays a role in ABCA1 plasma membrane expression and cholesterol removal in primary human macrophages. METHODS AND RESULTS: We found that Rab8 was abundantly expressed in human atherosclerotic lesional macrophages and upregulated on lipid loading of macrophages in vitro. Adenoviral overexpression of Rab8 increased ABCA1 protein levels and reduced cholesterol deposition in macrophage foam cells incubated with apoA-I. Depletion of Rab8 decreased the fraction of ABCA1 at the plasma membrane and inhibited the efflux of lipoprotein-derived endosomal cholesterol to apoA-I. In Rab8-depleted cells, ABCA1-GFP localized in beta1 integrin and transferrin receptor containing recycling organelles. CONCLUSIONS: Rab8 reduces foam cell formation by facilitating ABCA1 surface expression and stimulating endosomal cholesterol efflux to apoA-I in primary human macrophages.

BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms.

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol-storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY-cholesterol did not disturb zebrafish development and was targeted to sterol-enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time-lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.

Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells.

The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann-Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.