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.

Vinexin contributes to autophagic decline in brain ageing across species.

Autophagic decline is considered a hallmark of ageing. The activity of this intracytoplasmic degradation pathway decreases with age in many tissues and autophagy induction ameliorates ageing in many organisms, including mice. Autophagy is a critical protective pathway in neurons and ageing is the primary risk factor for common neurodegenerative diseases. Here, we describe that autophagosome biogenesis declines with age in mouse brains and that this correlates with increased expression of the SORBS3 gene (encoding vinexin) in older mouse and human brain tissue. We characterise vinexin as a negative regulator of autophagy. SORBS3 knockdown increases F-actin structures, which compete with YAP/TAZ for binding to their negative regulators, angiomotins, in the cytosol. This promotes YAP/TAZ translocation into the nucleus, thereby increasing YAP/TAZ transcriptional activity and autophagy. Our data therefore suggest brain autophagy decreases with age in mammals and that this is likely, in part, mediated by increasing levels of vinexin.

Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro.

The regulation of lipid kinases has remained elusive given the difficulties of assessing changes in lipid levels. Here, we describe the isolation of protein and lipid kinases to determine the regulation of lipid kinases in vitro. This can be followed by analysis of effects of regulators on lipid kinase-mediated changes in phospholipids without the use of radioactivity, with a specific focus on PI(5)P generation by the enzyme PIKfyve. For complete details on the use and execution of this protocol, please refer to Karabiyik et al. (2021).

AMPK-activated ULK1 phosphorylates PIKFYVE to drive formation of PtdIns5P-containing autophagosomes during glucose starvation.

The induction of macroautophagy/autophagy upon glucose deprivation can occur independently of the PIK3C3/VPS34 complex. Recently, we described a non-canonical signaling pathway involving the kinases AMPK, ULK1 and PIKFYVE that are induced during glucose starvation, leading to the formation of PtdIns5P-containing autophagosomes, resulting in increased autophagy flux and clearance of autophagy substrates. In this cascade, the activation of AMPK leads to ULK1 phosphorylation. ULK1 then phosphorylates PIKFYVE at S1548, leading to its activation and increased PtdIns5P formation, which enables the recruitment of machinery required for autophagosome biogenesis.

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner.

Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.

Lysosome positioning and mTOR activity in Lowe syndrome.

Lowe syndrome is a rare, developmental disorder caused by mutations in the phosphatase, OCRL. A study in this issue of EMBO Reports shows that OCRL is required for microtubule nucleation and that mutations in this protein lead to an inability to activate mTORC1 signaling and consequent cell proliferation in the presence of nutrients. These defects are the result of impaired microtubule-dependent lysosomal trafficking to the cell periphery and are independent of OCRL phosphatase activity.

mTORC2 Assembly Is Regulated by USP9X-Mediated Deubiquitination of RICTOR.

The mechanistic target of rapamycin complex 2 (mTORC2) controls cell metabolism and survival in response to environmental inputs. Dysregulation of mTORC2 signaling has been linked to diverse human diseases, including cancer and metabolic disorders, highlighting the importance of a tightly controlled mTORC2. While mTORC2 assembly is a critical determinant of its activity, the factors regulating this event are not well understood, and it is unclear whether this process is regulated by growth factors. Here, we present data, from human cell lines and mice, describing a mechanism by which growth factors regulate ubiquitin-specific protease 9X (USP9X) deubiquitinase to stimulate mTORC2 assembly and activity. USP9X removes Lys63-linked ubiquitin from RICTOR to promote its interaction with mTOR, thereby facilitating mTORC2 signaling. As mTORC2 is central for cellular homeostasis, understanding the mechanisms regulating mTORC2 activation toward its downstream targets is vital for our understanding of physiological processes and for developing new therapeutic strategies in pathology.

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.

Neuronal Rho GTPase Rac1 elimination confers neuroprotection in a mouse model of permanent ischemic stroke.

The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our results show that pMCAO significantly increased total Rac1 levels in wild type mice, mainly through rising nuclear Rac1, while a reduction in Rac1 activation was observed. Such changes preceded cell death induced by excitotoxic stress. Pharmacological inhibition of Rac1 in primary neuronal cortical cells prevented the increase in oxidative stress induced after overactivation of glutamate receptors. However, this was not sufficient to prevent the associated neuronal cell death. In contrast, RNAi-mediated knock down of Rac1 in primary cortical neurons prevented cell death elicited by glutamate excitotoxicity and decreased the activity of NADPH oxidase. To test whether in vivo down regulation of neuronal Rac1 was neuroprotective after pMCAO, we used tamoxifen-inducible neuron-specific conditional Rac1-knockout mice. We observed a significant 50% decrease in brain infarct volume of knockout mice and a concomitant increase in HIF-1α expression compared to littermate control mice, demonstrating that ablation of Rac1 in neurons is neuroprotective. Transmission electron microscopy performed in the ischemic brain showed that lysosomes in the infarct of Rac1- knockout mice were preserved at similar levels to those of non-infarcted tissue, while littermate mice displayed a decrease in the number of lysosomes, further corroborating the notion that Rac1 ablation in neurons is neuroprotective. Our results demonstrate that Rac1 plays important roles in the ischemic pathological cascade and that modulation of its levels is of therapeutic interest.

Autophagy impairment in Parkinson's disease.

Parkinson's disease (PD) is a debilitating movement disorder typically associated with the accumulation of intracytoplasmic aggregate prone protein deposits. Over recent years, increasing evidence has led to the suggestion that the mutations underlying certain forms of PD impair autophagy. Autophagy is a degradative pathway that delivers cytoplasmic content to lysosomes for degradation and represents a major route for degradation of aggregated cellular proteins and dysfunctional organelles. Autophagy up-regulation is a promising therapeutic strategy that is being explored for its potential to protect cells against the toxicity of aggregate-prone proteins in neurodegenerative diseases. Here, we describe how the mutations in different subtypes of PD can affect different stages of autophagy.

Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities.

Autophagy is a conserved pathway that delivers cytoplasmic contents to the lysosome for degradation. Here we consider its roles in neuronal health and disease. We review evidence from mouse knockout studies demonstrating the normal functions of autophagy as a protective factor against neurodegeneration associated with intracytoplasmic aggregate-prone protein accumulation as well as other roles, including in neuronal stem cell differentiation. We then describe how autophagy may be affected in a range of neurodegenerative diseases. Finally, we describe how autophagy upregulation may be a therapeutic strategy in a wide range of neurodegenerative conditions and consider possible pathways and druggable targets that may be suitable for this objective.