LC3-positive structures are prominent in autophagy-deficient cells

Abstract: Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy

S-acylation of the Wnt receptor Frizzled-5 by zDHHC5 controls its cellular localization and synaptogenic activity in the rodent hippocampus

Proper localization of receptors for synaptic organizing factors is crucial for synapse formation. Wnt proteins promote synapse assembly through Frizzled (Fz) receptors. In hippocampal neurons, the surface and synaptic localization of Fz5 is regulated by neuronal activity, but the mechanisms involved remain poorly understood. Here, we report that all Fz receptors can be post-translationally modified by S-acylation and that Fz5 is S-acylated on three C-terminal cysteines by zDHHC5. S-acylation is essential for Fz5 localization to the cell surface, axons, and presynaptic sites. Notably, S-acylation-deficient Fz5 is internalized faster, affecting its association with signalosome components at the cell surface. S-acylation-deficient Fz5 also fails to activate canonical and divergent canonical Wnt pathways. Fz5 S-acylation levels are regulated by the pattern of neuronal activity. In vivo studies demonstrate that S-acylation-deficient Fz5 expression fails to induce presynaptic assembly. Our studies show that S-acylation of Frizzled receptors is a mechanism controlling their localization and function

Wnt signalling tunes neurotransmitter release by directly targeting Synaptotagmin-1

The functional assembly of the synaptic release machinery is well understood; however, how signalling factors modulate this process remains unknown. Recent studies suggest that Wnts play a role in presynaptic function. To examine the mechanisms involved, we investigated the interaction of release machinery proteins with Dishevelled-1 (Dvl1), a scaffold protein that determines the cellular locale of Wnt action. Here we show that Dvl1 directly interacts with Synaptotagmin-1 (Syt-1) and indirectly with the SNARE proteins SNAP25 and Syntaxin (Stx-1). Importantly, the interaction of Dvl1 with Syt-1, which is regulated by Wnts, modulates neurotransmitter release. Moreover, presynaptic terminals from Wnt signalling-deficient mice exhibit reduced release probability and are unable to sustain high-frequency release. Consistently, the readily releasable pool size and formation of SNARE complexes are reduced. Our studies demonstrate that Wnt signalling tunes neurotransmitter release and identify Syt-1 as a target for modulation by secreted signalling proteins.Fil: Ciani, Lorenza. University College London; Estados UnidosFil: Marzo, Aude. University College London; Estados UnidosFil: Boyle, Kieran. University College London; Estados UnidosFil: Stamatakou, Eleanna. University College London; Estados UnidosFil: Lopes, Douglas M.. University College London; Estados UnidosFil: Anane, Derek. University College London; Estados UnidosFil: McLeod, Faye. University College London; Estados UnidosFil: Rosso, Silvana Beatriz. University College London; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gibb, Alasdair. University College London; Estados UnidosFil: Salinas, Patricia C.. University College London; Estados Unido

A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.

Autophagy involves engulfment of cytoplasmic contents by double-membraned autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This "scission" step is, thus, critical for autophagosome formation, is defective in a human disease, and influences the way we consider how autophagosomes are formed

Author Correction: Mendelian neurodegenerative disease genes involved in autophagy

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Mendelian neurodegenerative disease genes involved in autophagy

Funder: Fondation Roger de Spoelberch (Roger de Spoelberch Foundation); doi: https://doi.org/10.13039/501100008236Funder: Alzheimer's Research UK (ARUK); doi: https://doi.org/10.13039/501100002283Funder: UK Dementia Research InstituteFunder: Takeda Science Foundation; doi: https://doi.org/10.13039/100007449Abstract: The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology by regulating the removal of unwanted cargoes such as protein aggregates and damaged organelles. Over the last five decades, significant progress has been made in understanding the molecular mechanisms that regulate autophagy and its roles in human physiology and diseases. These advances, together with discoveries in human genetics linking autophagy-related gene mutations to specific diseases, provide a better understanding of the mechanisms by which autophagy-dependent pathways can be potentially targeted for treating human diseases. Here, we review mutations that have been identified in genes involved in autophagy and their associations with neurodegenerative diseases

Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration

Erratum: Author Correction: Mendelian neurodegenerative disease genes involved in autophagy.

[This corrects the article DOI: 10.1038/s41421-020-0158-y.]

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling

Leucine regulates autophagy via acetylation of the mTORC1 component raptor

Funder: Alzheimer's Research UK (ARUK); doi: https://doi.org/10.13039/501100002283Funder: CUH | Addenbrooke's Charitable Trust, Cambridge University Hospitals (Addenbrooke's Charitable Trust, Cambridge University Hospitals NHS Foundation Trust); doi: https://doi.org/10.13039/501100002927Abstract: Macroautophagy (“autophagy”) is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism