Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.

Autophagy gene expression in skeletal muscle of older individuals is associated with physical performance, muscle volume and mitochondrial function in the study of muscle, mobility and aging (SOMMA).

Autophagy is essential for proteostasis, energetic balance, and cell defense and is a key pathway in aging. Identifying associations between autophagy gene expression patterns in skeletal muscle and physical performance outcomes would further our knowledge of mechanisms related with proteostasis and healthy aging. Muscle biopsies were obtained from participants in the Study of Muscle, Mobility, and Aging (SOMMA). For 575 participants, RNA was sequenced and expression of 281 genes related to autophagy regulation, mitophagy, and mTOR/upstream pathways was determined. Associations between gene expression and outcomes including mitochondrial respiration in muscle fiber bundles (MAX OXPHOS), physical performance (VO2 peak, 400 m walking speed, and leg power), and thigh muscle volume, were determined using negative binomial regression models. For autophagy, key transcriptional regulators including TFE3 and NFKB-related genes (RELA, RELB, and NFKB1) were negatively associated with outcomes. On the contrary, regulators of oxidative metabolism that also promote overall autophagy, mitophagy, and pexophagy (PPARGC1A, PPARA, and EPAS1) were positively associated with multiple outcomes. In line with this, several mitophagy, fusion, and fission-related genes (NIPSNAP2, DNM1L, and OPA1) were also positively associated with outcomes. For mTOR pathway and related genes, expression of WDR59 and WDR24, both subunits of GATOR2 complex (an indirect inhibitor of mTORC1), and PRKAG3, which is a regulatory subunit of AMPK, were negatively correlated with multiple outcomes. Our study identifies autophagy and selective autophagy such as mitophagy gene expression patterns in human skeletal muscle related to physical performance, muscle volume, and mitochondrial function in older persons which may lead to target identification to preserve mobility and independence.

Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.

NGLY1 mutations cause protein aggregation in human neurons.

Biallelic mutations in the gene that encodes the enzyme N-glycanase 1 (NGLY1) cause a rare disease with multi-symptomatic features including developmental delay, intellectual disability, neuropathy, and seizures. NGLY1's activity in human neural cells is currently not well understood. To understand how NGLY1 gene loss leads to the specific phenotypes of NGLY1 deficiency, we employed direct conversion of NGLY1 patient-derived induced pluripotent stem cells (iPSCs) to functional cortical neurons. Transcriptomic, proteomic, and functional studies of iPSC-derived neurons lacking NGLY1 function revealed several major cellular processes that were altered, including protein aggregate-clearing functionality, mitochondrial homeostasis, and synaptic dysfunctions. These phenotypes were rescued by introduction of a functional NGLY1 gene and were observed in iPSC-derived mature neurons but not astrocytes. Finally, laser capture microscopy followed by mass spectrometry provided detailed characterization of the composition of protein aggregates specific to NGLY1-deficient neurons. Future studies will harness this knowledge for therapeutic development.

Autophagy gene expression in skeletal muscle of older individuals is associated with physical performance, muscle volume and mitochondrial function in the Study of Muscle, Mobility and Aging (SOMMA).

Autophagy is an essential component of proteostasis and a key pathway in aging. Identifying associations between autophagy gene expression patterns in skeletal muscle and physical performance outcomes would further our knowledge of mechanisms related with proteostasis and healthy aging. Muscle biopsies were obtained from participants in the Study of Muscle, Mobility and Aging (SOMMA). For 575 participants, RNA was sequenced and expression of 281 genes related to autophagy regulation, mitophagy and mTOR/upstream pathways were determined. Associations between gene expression and outcomes including mitochondrial respiration in muscle fiber bundles (MAX OXPHOS), physical performance (VO2 peak, 400m walking speed, and leg power), and thigh muscle volume were determined using negative binomial regression models. For autophagy, key transcriptional regulators including TFE3 and NFKB-related genes (RELA, RELB, NFKB1) were negatively associated with outcomes. On the contrary, regulators of oxidative metabolism that also promote overall autophagy, mitophagy and pexophagy (PPARGC1A, PPARA, EPAS1) were positively associated with multiple outcomes. In line with this, several mitophagy, fusion and fission related genes (NIPSNAP2, DNM1L, OPA1) were also positively associated with outcomes. For mTOR pathway and related genes, expression of WDR59 and WDR24, both subunits of GATOR2 complex (an indirect inhibitor of mTORC1) and PRKAG3, which is a regulatory subunit of AMPK, were negatively correlated with multiple outcomes. Our study identifies autophagy and selective autophagy such as mitophagy gene expression patterns in human skeletal muscle related to physical performance, muscle volume and mitochondrial function in older persons which may lead to target identification to preserve mobility and independence.

A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion.

Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.

Chaperone-mediated autophagy regulates adipocyte differentiation.

Adipogenesis is a tightly orchestrated multistep process wherein preadipocytes differentiate into adipocytes. The most studied aspect of adipogenesis is its transcriptional regulation through timely expression and silencing of a vast number of genes. However, whether turnover of key regulatory proteins per se controls adipogenesis remains largely understudied. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation that, in response to diverse cues, remodels the proteome for regulatory purposes. We report here the activation of CMA during adipocyte differentiation and show that CMA regulates adipogenesis at different steps through timely degradation of key regulatory signaling proteins and transcription factors that dictate proliferation, energetic adaptation, and signaling changes required for adipogenesis.

The lysosomal proteome of senescent cells contributes to the senescence secretome.

Senescent cells accumulate in tissues over time, favoring the onset and progression of multiple age-related diseases. Senescent cells present a remarkable increase in lysosomal mass and elevated autophagic activity. Here, we report that two main autophagic pathways macroautophagy (MA) and chaperone-mediated autophagy (CMA) are constitutively upregulated in senescent cells. Proteomic analyses of the subpopulations of lysosomes preferentially engaged in each of these types of autophagy revealed profound quantitative and qualitative changes in senescent cells, affecting both lysosomal resident proteins and cargo proteins delivered to lysosomes for degradation. These studies have led us to identify resident lysosomal proteins that are highly augmented in senescent cells and can be used as novel markers of senescence, such as arylsulfatase ARSA. The abundant secretome of senescent cells, known as SASP, is considered their main pathological mediator; however, little is known about the mechanisms of SASP secretion. Some secretory cells, including melanocytes, use the small GTPase RAB27A to perform lysosomal secretion. We found that this process is exacerbated in the case of senescent melanoma cells, as revealed by the exposure of lysosomal membrane integral proteins LAMP1 and LAMP2 in their plasma membrane. Interestingly, a subset of SASP components, including cytokines CCL2, CCL3, CXCL12, cathepsin CTSD, or the protease inhibitor SERPINE1, are secreted in a RAB27A-dependent manner in senescent melanoma cells. Finally, proteins previously identified as plasma biomarkers of aging are highly enriched in the lysosomes of senescent cells, including CTSD. We conclude that the lysosomal proteome of senescent cells is profoundly reconfigured, and that some senescent cells can be highly active in lysosomal exocytosis.

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst-mediated protein secretion.

Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age-related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub-proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age-related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein-loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst-RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst-RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst-RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.

Targeting retinoic acid receptor alpha-corepressor interaction activates chaperone-mediated autophagy and protects against retinal degeneration.

Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration.

Chaperone-mediated autophagy protects against atherosclerosis.

Atherosclerosis, the leading cause of cardiovascular death, is driven by hyperlipidemia, inflammation and aggravated by aging. As chaperone-mediated autophagy (CMA), a selective type of lysosomal degradation for intracellular proteins, diminishes with age and is inhibited by lipid excess, we studied if the decline in CMA could contribute to atherosclerosis pathogenesis. We found that CMA declines in human and murine vasculature with disease progression. Inhibition and reactivation of CMA using transgenic mouse models establishes a protective effect of CMA against atherogenesis. CMA upregulation ameliorates both systemic metabolic parameters, and vascular cell function. Our work suggests CMA reactivation could be a viable therapeutic strategy to prevent and reduce cardiovascular disease.

Methamphetamine Dysregulates Macrophage Functions and Autophagy to Mediate HIV Neuropathogenesis.

HIV-neurocognitive impairment (HIV-NCI) can be a debilitating condition for people with HIV (PWH), despite the success of antiretroviral therapy (ART). Substance use disorder is often a comorbidity with HIV infection. The use of methamphetamine (meth) increases systemic inflammation and CNS damage in PWH. Meth may also increase neuropathogenesis through the functional dysregulation of cells that harbor HIV. Perivascular macrophages are long-lived reservoirs for HIV in the CNS. The impaired clearance of extracellular debris and increased release of reactive oxygen species (ROS) by HIV-infected macrophages cause neurotoxicity. Macroautophagy is a vital intracellular pathway that can regulate, in part, these deleterious processes. We found in HIV-infected primary human macrophages that meth inhibits phagocytosis of aggregated amyloid-ß, increases total ROS, and dysregulates autophagic processes. Treatment with widely prescribed ART drugs had minimal effects, although there may be an improvement in phagocytosis when co-administered with meth. Pharmacologically inhibited lysosomal degradation, but not induction of autophagy, further increased ROS in response to meth. Using mass spectrometry, we identified the differentially expressed proteins in meth-treated, HIV-infected macrophages that participate in phagocytosis, mitochondrial function, redox metabolism, and autophagy. Significantly altered proteins may be novel targets for interventional strategies that restore functional homeostasis in HIV-infected macrophages to improve neurocognition in people with HIV-NCI using meth.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Microglial NF-κB drives tau spreading and toxicity in a mouse model of tauopathy.

Activation of microglia is a prominent pathological feature in tauopathies, including Alzheimer's disease. How microglia activation contributes to tau toxicity remains largely unknown. Here we show that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, activated by tau, drives microglial-mediated tau propagation and toxicity. Constitutive activation of microglial NF-κB exacerbated, while inactivation diminished, tau seeding and spreading in young PS19 mice. Inhibition of NF-κB activation enhanced the retention while reduced the release of internalized pathogenic tau fibrils from primary microglia and rescued microglial autophagy deficits. Inhibition of microglial NF-κB in aged PS19 mice rescued tau-mediated learning and memory deficits, restored overall transcriptomic changes while increasing neuronal tau inclusions. Single cell RNA-seq revealed that tau-associated disease states in microglia were diminished by NF-κB inactivation and further transformed by constitutive NF-κB activation. Our study establishes a role for microglial NF-κB signaling in mediating tau spreading and toxicity in tauopathy.

Misfolded GBA/ß-glucocerebrosidase impairs ER-quality control by chaperone-mediated autophagy in Parkinson disease.

Inhibition of chaperone-mediated autophagy (CMA), a selective type of lysosomal degradation for intracellular proteins, may contribute to pathogenesis in neurodegenerative diseases including Parkinson disease (PD). Pathogenic variants of PD-related proteins that reside in the cytosol, including SNCA/alpha-synuclein, LRRK2 (leucine rich repeat kinase 2), UCHL1 (ubiquitin Cterminal hydrolase 1) and VPS35 (VPS35 retromer complex component), exert inhibitory effects on CMA. Decreased CMA activity has also been reported in sporadic PD patients, consistent with an association between CMA inhibition and PD. We have now reported the first example of CMA dysfunction caused by a non-cytosolic PD-related protein, GBA/ß-glucocerebrosidase, the most common genetic risk factor for PD, which uncovers a new role for CMA in endoplasmic reticulum (ER) quality control.

Mutant glucocerebrosidase impairs α-synuclein degradation by blockade of chaperone-mediated autophagy.

The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes ß-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.

Circadian remodeling of the proteome by chaperone-mediated autophagy.

The circadian clock drives daily cycles of physiology and behavioral outputs to keep organisms in tune with the environment. Cyclic oscillations in levels of the clock proteins maintain circadian rhythmicity. In our recent work, we have discovered the interdependence of the circadian clock and chaperone-mediated autophagy (CMA), a selective form of lysosomal protein degradation. Central and peripheral degradation of core clock proteins by CMA (selective chronophagy) modulates circadian rhythm. Loss of CMA in vivo disrupts physiological circadian cycling, resembling defects observed in aging, a condition with reduced CMA. Conversely, the circadian clock temporally regulates CMA activity in a tissue-specific manner, contributing to remodeling of a distinct subproteome at different circadian times. This timely remodeling cannot be sustained when CMA fails, despite rerouting of some CMA substrates to other degradation pathways.

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.