Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise.

Autophagy is a conserved pathway where cytoplasmic contents are engulfed by autophagosomes, which then fuse with lysosomes enabling their degradation. Mutations in core autophagy genes cause neurological conditions, and autophagy defects are seen in neurodegenerative diseases such as Parkinson's disease and Huntington's disease. Thus, we have sought to understand the cellular pathway perturbations that autophagy-perturbed cells are vulnerable to by seeking negative genetic interactions such as synthetic lethality in autophagy-null human cells using available data from yeast screens. These revealed that loss of proteasome and nuclear pore complex components cause synergistic viability changes akin to synthetic fitness loss in autophagy-null cells. This can be attributed to the cytoplasm-to-nuclear transport of proteins during autophagy deficiency and subsequent degradation of these erstwhile cytoplasmic proteins by nuclear proteasomes. As both autophagy and cytoplasm-to-nuclear transport are defective in Huntington's disease, such cells are more vulnerable to perturbations of proteostasis due to these synthetic interactions.

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.

Microglial-to-neuronal CCR5 signaling regulates autophagy in neurodegeneration.

In neurodegenerative diseases, microglia switch to an activated state, which results in excessive secretion of pro-inflammatory factors. Our work aims to investigate how this paracrine signaling affects neuronal function. Here, we show that activated microglia mediate non-cell-autonomous inhibition of neuronal autophagy, a degradative pathway critical for the removal of toxic, aggregate-prone proteins accumulating in neurodegenerative diseases. We found that the microglial-derived CCL-3/-4/-5 bind and activate neuronal CCR5, which in turn promotes mTORC1 activation and disrupts autophagy and aggregate-prone protein clearance. CCR5 and its cognate chemokines are upregulated in the brains of pre-manifesting mouse models for Huntington's disease (HD) and tauopathy, suggesting a pathological role of this microglia-neuronal axis in the early phases of these diseases. CCR5 upregulation is self-sustaining, as CCL5-CCR5 autophagy inhibition impairs CCR5 degradation itself. Finally, pharmacological or genetic inhibition of CCR5 rescues mTORC1 hyperactivation and autophagy dysfunction, which ameliorates HD and tau pathologies in mouse models.

Compounds activating VCP D1 ATPase enhance both autophagic and proteasomal neurotoxic protein clearance.

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.

The different autophagy degradation pathways and neurodegeneration.

The term autophagy encompasses different pathways that route cytoplasmic material to lysosomes for degradation and includes macroautophagy, chaperone-mediated autophagy, and microautophagy. Since these pathways are crucial for degradation of aggregate-prone proteins and dysfunctional organelles such as mitochondria, they help to maintain cellular homeostasis. As post-mitotic neurons cannot dilute unwanted protein and organelle accumulation by cell division, the nervous system is particularly dependent on autophagic pathways. This dependence may be a vulnerability as people age and these processes become less effective in the brain. Here, we will review how the different autophagic pathways may protect against neurodegeneration, giving examples of both polygenic and monogenic diseases. We have considered how autophagy may have roles in normal CNS functions and the relationships between these degradative pathways and different types of programmed cell death. Finally, we will provide an overview of recently described strategies for upregulating autophagic pathways for therapeutic purposes.

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

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

Leucine regulates autophagy via acetylation of the mTORC1 component raptor.

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.

Mendelian neurodegenerative disease genes involved in autophagy.

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.

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.

LC3-positive structures are prominent in autophagy-deficient cells.

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.

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

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

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.

Assessing Autophagic Activity and Aggregate Formation of Mutant Huntingtin in Mammalian Cells.

The accumulation of mutant aggregate-prone proteins is a hallmark of the majority of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Autophagy, a cytosolic bulk degradation system, is the major clearance pathway for several aggregate-prone proteins, such as mutant huntingtin. The autophagosome-associated protein LC3-II is a specific marker of autophagic flux within cells, whereas aggregate formation of mutant huntingtin represents a good readout for studying autophagy modulation. Here we describe the method of assessing autophagic flux using LC3-II western blotting and substrate clearance by expressing the N-terminal fragment of huntingtin (htt exon 1) containing an expanded polyglutamine tract in mammalian cells.

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.

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 Gsk3ß. 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.

Postsynaptic assembly: a role for Wnt signaling.

Synapse formation requires the coordinated formation of the presynaptic terminal, containing the machinery for neurotransmitter release, and the postsynaptic side that possesses the machinery for neurotransmitter reception. For coordinated pre- and postsynaptic assembly signals across the synapse are required. Wnt secreted proteins are well-known synaptogenic factors that promote the recruitment of presynaptic components in diverse organisms. However, recent studies demonstrate that Wnts act directly onto the postsynaptic side at both central and peripheral synapses to promote postsynaptic development and synaptic strength. This review focuses on the role of Wnts in postsynaptic development at central synapses and the neuromuscular junction.

Activity-dependent spine morphogenesis: a role for the actin-capping protein Eps8.

Neuronal activity regulates the formation and morphology of dendritic spines through changes in the actin cytoskeleton. However, the molecular mechanisms that regulate this process remain poorly understood. Here we report that Eps8, an actin-capping protein, is required for spine morphogenesis. In rat hippocampal neurons, gain- and loss-of-function studies demonstrate that Eps8 promotes the formation of dendritic spines but inhibits filopodium formation. Loss of function of Eps8 increases actin polymerization and induces fast actin turnover within dendritic spines, as revealed by free-barbed end and FRAP assays, consistent with a role for Eps8 as an actin-capping protein. Interestingly, Eps8 regulates the balance between excitatory synapses on spines and on the dendritic shaft, without affecting the total number of synapses or basal synaptic transmission. Importantly, Eps8 loss of function impairs the structural and functional plasticity of synapses induced by long-term potentiation. These findings demonstrate a novel role for Eps8 in spine formation and in activity-mediated synaptic plasticity.

Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli.

Axon guidance and target-derived signals control axonal behavior by regulating the cytoskeleton through poorly defined mechanisms. In particular, how these signaling molecules regulate the growth and directionality of microtubules is not well understood. Here we examine the effect of Wnts on growth cone remodeling, a process that precedes synapse formation. Time-lapse recordings reveal that Wnt3a rapidly inhibits growth cone translocation while inducing growth cone enlargement. These changes in axonal behavior are associated with changes in the organization of microtubules. Time-lapse imaging of EB3-GFP (green fluorescent protein)-labeled microtubule plus-ends demonstrates that Wnt3a regulates microtubule directionality, resulting in microtubule looping, growth cone pausing, and remodeling. Analyses of Dishevelled-1 (Dvl1) mutant neurons demonstrate that Dvl1 is required for Wnt-mediated microtubule reorganization and axon remodeling. Wnt signaling directly affects the microtubule cytoskeleton by unexpectedly inducing adenomatous polyposis coli (APC) loss from microtubule plus-ends. Consistently, short hairpin RNA knockdown of APC mimics Wnt3a function. Together, our findings define APC as a key Wnt signaling target in the regulation of microtubule growth direction.