The endolysosomal system in conventional and unconventional protein secretion.

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

Mechanisms of autophagy-lysosome dysfunction in neurodegenerative diseases.

Autophagy is a lysosome-based degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and aggregate-prone proteins. Their postmitotic nature and extremely polarized morphologies make neurons particularly vulnerable to disruptions caused by autophagy-lysosomal defects, especially as the brain ages. Consequently, mutations in genes regulating autophagy and lysosomal functions cause a wide range of neurodegenerative diseases. Here, we review the role of autophagy and lysosomes in neurodegenerative diseases such as Alzheimer disease, Parkinson disease and frontotemporal dementia. We also consider the strong impact of cellular ageing on lysosomes and autophagy as a tipping point for the late-age emergence of related neurodegenerative disorders. Many of these diseases have primary defects in autophagy, for example affecting autophagosome formation, and in lysosomal functions, especially pH regulation and calcium homeostasis. We have aimed to provide an integrative framework for understanding the central importance of autophagic-lysosomal function in neuronal health and disease.

Trial to assess the tolerability of using felodipine to upregulate autophagy as a treatment of Huntington's disease (FELL-HD): a phase II, single-centre, open-label, dose-finding trial protocol.

INTRODUCTION: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that presents with a progressive movement disorder along with cognitive and psychiatric problems. It is caused by a Cytosine-adenin-guanine (CAG) expansion in exon 1 of the huntingtin gene which codes for mutant huntingtin (mHTT) that over time accumulates in cells, causing dysfunction and then death through new toxic gain-of-function mechanisms. Autophagy has been shown to be critical for the degradation of diverse intracytoplasmic aggregate-prone proteins that cause neurodegenerative disease, including mHTT. From a screen of a library enriched in approved drugs, felodipine was selected as the most suitable candidate showing strong autophagy-inducing effects in preclinical models of HD. We are, therefore, conducting a trial to assess the safety and tolerability of felodipine in people with early HD. METHODS AND ANALYSIS: FELL-HD is a phase II, single-centre, open-label, dose-finding trial in people with early HD. 18 participants with early clinical features of the disease will be treated with felodipine for 58 weeks, with a further 4-week follow-up. The primary outcome measure is the number of adverse events attributable to felodipine. Exploratory outcomes include additional measures of motor and cognitive function, non-motor symptoms and quality of life scales, as well as peripheral and central disease biomarkers assessed through brain MRI. Analysis of blood and cerebrospinal fluid will also be performed through an associated sample study, FELL HD-s. ETHICS AND DISSEMINATION: The study was approved by the London-Brent Research Ethics Committee (reference 22/LO/0387) and has been accepted by the Medicines and Healthcare products Regulatory Agency for clinical trials authorisation (reference CTA 12854/0256/001-0001). A lay summary of the results of the trial will be uploaded to our research group website which is publicly accessible. A webinar or in-person open day, to present results of the trial to participants and our wider cohort of patients who attend our centre, will be held once the trial is completed. The results of the trial will also be published in scientific journals and presented at national and international conferences. TRIAL REGISTRATION NUMBERS: EudraCT-2021-000897-27, ISRCTN56240656.

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.

PSMC5 insufficiency and P320R mutation impair proteasome function.

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.

p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol.

The AAA+-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis.

ß-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain iron accumulation. Mutations in the WD repeat domain 45 (WDR45) gene encoding WIPI4 lead to loss of function in BPAN but the cellular mechanisms of how these trigger pathology are unclear. The prevailing view in the literature is that BPAN is simply the consequence of autophagy deficiency given that WIPI4 functions in this degradation pathway. However, our data indicate that WIPI4 depletion causes ferroptosis-a type of cell death induced by lipid peroxidation-via an autophagy-independent mechanism, as demonstrated both in cell culture and in zebrafish. WIPI4 depletion increases ATG2A localization at endoplasmic reticulum-mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria. This results in increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation, thus enabling ferroptosis. This mechanism has minimal overlap with classical ferroptosis stimuli but provides insights into the causes of neurodegeneration in BPAN and may provide clues for therapeutic strategies.

Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly.

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.

p300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.

Meeting Summary of The NYO3 5th NO-Age/AD Meeting and the 1st Norway-UK Joint Meeting on Aging and Dementia: Recent Progress on the Mechanisms and Interventional Strategies.

Unhealthy aging poses a global challenge with profound healthcare and socioeconomic implications. Slowing down the aging process offers a promising approach to reduce the burden of a number of age-related diseases, such as dementia, and promoting healthy longevity in the old population. In response to the challenge of the aging population and with a view to the future, Norway and the United Kingdom are fostering collaborations, supported by a "Money Follows Cooperation agreement" between the 2 nations. The inaugural Norway-UK joint meeting on aging and dementia gathered leading experts on aging and dementia from the 2 nations to share their latest discoveries in related fields. Since aging is an international challenge, and to foster collaborations, we also invited leading scholars from 11 additional countries to join this event. This report provides a summary of the conference, highlighting recent progress on molecular aging mechanisms, genetic risk factors, DNA damage and repair, mitophagy, autophagy, as well as progress on a series of clinical trials (eg, using NAD+ precursors). The meeting facilitated dialogue among policymakers, administrative leaders, researchers, and clinical experts, aiming to promote international research collaborations and to translate findings into clinical applications and interventions to advance healthy aging.

The autophagy of stress granules.

Our understanding of stress granule (SG) biology has deepened considerably in recent years, and with this, increased understanding of links has been made between SGs and numerous neurodegenerative diseases. One of the proposed mechanisms by which SGs and any associated protein aggregates may become pathological is based upon defects in their autophagic clearance, and so the precise processes governing the degradation of SGs are important to understand. Mutations and disease-associated variants implicated in amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease and frontotemporal lobar dementia compromise autophagy, whilst autophagy-inhibiting drugs or knockdown of essential autophagy proteins result in the persistence of SGs. In this review, we will consider the current knowledge regarding the autophagy of SG.

Mammalian phagophores with finger-like shapes emerge from recycling endosomes.

Autophagosomes are double-membraned vesicles that engulf cytoplasmic contents, which are ultimately degraded after autophagosome-lysosome fusion. The prevailing view, largely inferred from EM-based studies, was that mammalian autophagosomes evolved from disc-shaped precursors that invaginated and then were closed at the single opening. Many site(s) of origin of these precursors have been proposed. Using superresolution structured illumination microscopy and electron microscopy, we find that mammalian autophagosomes derive from finger-like outgrowths from the recycling endosome. These "fingers" survey a large cell volume and then close into a "fist" and the openings are sealed in an ESCRT-dependent fashion, while the precursors are still attached to the recycling endosome. We call this transient recycling endosome-attached, closed, autophagic structure an "autophago-dome". DNM2-dependent scission of the autophago-dome from the recycling endosomes liberates free autophagosomes from this compartment. These data reveal unexpected morphologies of autophagosome precursors and raise new questions about the control of this process.

Mammalian autophagosomes form from finger-like phagophores.

The sequence of morphological intermediates that leads to mammalian autophagosome formation and closure is a crucial yet poorly understood issue. Previous studies have shown that yeast autophagosomes evolve from cup-shaped phagophores with only one closure point, and mammalian studies have inferred that mammalian phagophores also have single openings. Our superresolution microscopy studies in different human cell lines in conditions of basal and nutrient-deprivation-induced autophagy identified autophagosome precursors with multifocal origins that evolved into unexpected finger-like phagophores with multiple openings before becoming more spherical structures. Compatible phagophore structures were observed with whole-mount and conventional electron microscopy. This sequence of events was visualized using advanced SIM2 superresolution live microscopy. The finger-shaped phagophore apertures remained open when ESCRT function was compromised. The efficient closure of autophagic structures is important for their release from the recycling endosome. This has important implications for understanding how autophagosomes form and capture various cargoes.

The post-translational regulation of transcription factor EB (TFEB) in health and disease.

Transcription factor EB (TFEB) is a basic helix-loop-helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro-autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA-binding capacity, all of which are regulated at the post-translational level. Pathological states are characterised by a specific set of stressors, which elicit post-translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post-translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post-translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state.

Microglial cytokines poison neuronal autophagy via CCR5, a druggable target.

In the prodromal phase of neurodegenerative diseases, microglia switch to an activated state resulting in increased secretion of pro-inflammatory factors. We reported that C - C chemokine ligand 3 (CCL3), C - C chemokine ligand 4 (CCL4) and C - C chemokine ligand 5 (CCL5) contained in the secretome of activated microglia inhibit neuronal autophagy via a non-cell autonomous mechanism. These chemokines bind and activate neuronal C - C chemokine receptor type 5 (CCR5), which, in turn, promotes phosphoinositide 3-kinase (PI3K) - protein kinase B (PKB, or AKT) - mammalian target of rapamycin complex 1 (mTORC1) pathway activation, which inhibits autophagy, thus causing the accumulation of aggregate-prone proteins in the cytoplasm of neurons. The levels of CCR5 and its chemokine ligands are increased in the brains of pre-manifesting Huntington disease (HD) and tauopathy mouse models. CCR5 accumulation might be due to a self-amplifying mechanism, since CCR5 is a substrate of autophagy and CCL5-CCR5-mediated autophagy inhibition impairs CCR5 degradation. Furthermore, pharmacological, or genetic inhibition of CCR5 rescues mTORC1-autophagy dysfunction and improves neurodegeneration in HD and tauopathy mouse models, suggesting that CCR5 hyperactivation is a pathogenic signal driving the progression of these diseases.

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.

SMER28 binding to VCP/p97 enhances both autophagic and proteasomal neurotoxic protein clearance.

The ability to maintain a functional proteome by clearing damaged or misfolded proteins is critical for cell survival, and aggregate-prone proteins accumulate in many neurodegenerative diseases, such as Huntington, Alzheimer, and Parkinson diseases. The removal of such proteins is mainly mediated by the ubiquitin-proteasome system and autophagy, and the activity of these systems declines in disease or with age. We recently found that targeting VCP/p97 with compounds like SMER28 enhances macroautophagy/autophagy flux mediated by the increased activity of the PtdIns3K complex I. Additionally, we found that SMER28 binding to VCP stimulates aggregate-prone protein clearance via the ubiquitin-proteasome system. This concurrent action of SMER28 on both degradation pathways resulted in the selective decrease in disease-causing proteins but not their wild-type counterparts. These results reveal a promising mode of VCP activation to counteract the toxicity caused by aggregate-prone proteins.

An inducible expression system for the manipulation of autophagic flux in vivo.

Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo.Abbreviations: ANOVA: analysis of variance; Atg: autophagy related; Bcl2l11/Bim: BCL2 like 11; d.p.f.: days post-fertilization; Cryaa: crystallin, alpha a: DMSO: dimethyl sulfoxide; Elavl3: ELAV like neuron-specific RNA binding protein 3; ER: estrogen receptor; ERT2: modified ligand-binding domain of human ESR1/estrogen receptor α; Gal4: galactose-responsive transcription factor 4; GFP: green fluorescent protein; h.p.f.: hours post-fertilization; HSP: heat-shock protein; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; SD: standard deviation; SEM: standard error of the mean; UAS: upstream activating sequence; Ubb: ubiquitin b.

Increased SORBS3 expression in brain ageing contributes to autophagic decline via YAP1-WWTR1/TAZ signaling.

Impaired autophagosome formation and reduced flux through the macroautophagy/autophagy pathway occurs outside the brain as part of normal aging in various species. We recently identified autophagic decline in mouse brain tissue dependent on aging. This sits alongside significantly increased expression of the Sorbs3/SORBS3/vinexin (sorbin and SH3 domain containing 3) gene in older mouse and human brains. We found that SORBS3 negatively regulates autophagy in several cell lines, including mouse primary neurons. SORBS3 depletion increases F-actin structures, which compete with YAP1-WWTR1/TAZ to bind AMOT (angiomotin) proteins in the cytosol. Unbound YAP1-WWTR1/TAZ is free to move into the nucleus and upregulate YAP1-WWTR1/TAZ target gene expression. This upregulates autophagosome formation, in part through increased expression of myosin- and actin-related genes. Moreover, we have shown these YAP1-WWTR1/TAZ target genes are downregulated in older mouse and human brains. Taken together, our findings suggest that increased SORBS3 expression contributes to autophagic decline in normal brain aging across species.