Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Correction: Proteome-wide analysis of chaperone-mediated autophagy targeting motifs.

[This corrects the article DOI: 10.1371/journal.pbio.3000301.].

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.

Tau seeds from Alzheimer's disease brains trigger tau spread in macaques while oligomeric-Aß mediates pathology maturation.

INTRODUCTION: The "prion-like" features of Alzheimer's disease (AD) tauopathy and its relationship with amyloid-ß (Aß) have never been experimentally studied in primates phylogenetically close to humans. METHODS: We injected 17 macaques in the entorhinal cortex with nanograms of seeding-competent tau aggregates purified from AD brains or control extracts from aged-matched healthy brains, with or without intracerebroventricular co-injections of oligomeric-Aß. RESULTS: Pathological tau injection increased cerebrospinal fluid (CSF) p-tau181 concentration after 18 months. Tau pathology spreads from the entorhinal cortex to the hippocampal trisynaptic loop and the cingulate cortex, resuming the experimental progression of Braak stage I to IV. Many AD-related molecular networks were impacted by tau seeds injections regardless of Aß injections in proteomic analyses. However, we found mature neurofibrillary tangles, increased CSF total-tau concentration, and pre- and postsynaptic degeneration only in Aß co-injected macaques. DISCUSSION: Oligomeric-Aß mediates the maturation of tau pathology and its neuronal toxicity in macaques but not its initial spreading. HIGHLIGHTS: This study supports the "prion-like" properties of misfolded tau extracted from AD brains. This study empirically validates the Braak staging in an anthropomorphic brain. This study highlights the role of oligomeric Aß in driving the maturation and toxicity of tau pathology. This work establishes a novel animal model of early sporadic AD that is closer to the human pathology.

Brain injections of glial cytoplasmic inclusions induce a multiple system atrophy-like pathology.

Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson's disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development.

Proteome-wide analysis of chaperone-mediated autophagy targeting motifs.

Chaperone-mediated autophagy (CMA) contributes to the lysosomal degradation of a selective subset of proteins. Selectivity lies in the chaperone heat shock cognate 71 kDa protein (HSC70) recognizing a pentapeptide motif (KFERQ-like motif) in the protein sequence essential for subsequent targeting and degradation of CMA substrates in lysosomes. Interest in CMA is growing due to its recently identified regulatory roles in metabolism, differentiation, cell cycle, and its malfunctioning in aging and conditions such as cancer, neurodegeneration, or diabetes. Identification of the subset of the proteome amenable to CMA degradation could further expand our understanding of the pathophysiological relevance of this form of autophagy. To that effect, we have performed an in silico screen for KFERQ-like motifs across proteomes of several species. We have found that KFERQ-like motifs are more frequently located in solvent-exposed regions of proteins, and that the position of acidic and hydrophobic residues in the motif plays the most important role in motif construction. Cross-species comparison of proteomes revealed higher motif conservation in CMA-proficient species. The tools developed in this work have also allowed us to analyze the enrichment of motif-containing proteins in biological processes on an unprecedented scale and discover a previously unknown association between the type and combination of KFERQ-like motifs in proteins and their participation in specific biological processes. To facilitate further analysis by the scientific community, we have developed a free web-based resource (KFERQ finder) for direct identification of KFERQ-like motifs in any protein sequence. This resource will contribute to accelerating understanding of the physiological relevance of CMA.

Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates.

In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson's disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson's disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson's disease.

Rare variants in the neuronal ceroid lipofuscinosis gene MFSD8 are candidate risk factors for frontotemporal dementia.

Pathogenic variation in MAPT, GRN, and C9ORF72 accounts for at most only half of frontotemporal lobar degeneration (FTLD) cases with a family history of neurological disease. This suggests additional variants and genes that remain to be identified as risk factors for FTLD. We conducted a case-control genetic association study comparing pathologically diagnosed FTLD patients (n = 94) to cognitively normal older adults (n = 3541), and found suggestive evidence that gene-wide aggregate rare variant burden in MFSD8 is associated with FTLD risk. Because homozygous mutations in MFSD8 cause neuronal ceroid lipofuscinosis (NCL), similar to homozygous mutations in GRN, we assessed rare variants in MFSD8 for relevance to FTLD through experimental follow-up studies. Using post-mortem tissue from middle frontal gyrus of patients with FTLD and controls, we identified increased MFSD8 protein levels in MFSD8 rare variant carriers relative to non-variant carrier patients with sporadic FTLD and healthy controls. We also observed an increase in lysosomal and autophagy-related proteins in MFSD8 rare variant carrier and sporadic FTLD patients relative to controls. Immunohistochemical analysis revealed that MFSD8 was expressed in neurons and astrocytes across subjects, without clear evidence of abnormal localization in patients. Finally, in vitro studies identified marked disruption of lysosomal function in cells from MFSD8 rare variant carriers, and identified one rare variant that significantly increased the cell surface levels of MFSD8. Considering the growing evidence for altered autophagy in the pathogenesis of neurodegenerative disorders, our findings support a role of NCL genes in FTLD risk and suggest that MFSD8-associated lysosomal dysfunction may contribute to FTLD pathology.

In vitro α-synuclein neurotoxicity and spreading among neurons and astrocytes using Lewy body extracts from Parkinson disease brains.

Synucleinopathies are a group of diseases characterized by the presence of intracellular protein aggregates containing α-synuclein (α-syn). While α-syn aggregates have been shown to induce multimodal cellular dysfunctions, uptake and transport mechanisms remain unclear. Using high-content imaging on cortical neurons and astrocytes, we here define the kinetics of neuronal and astrocytic abnormalities induced by human-derived α-syn aggregates grounding the use of such system to identify and test putative therapeutic compounds. We then aimed at characterizing uptake and transport mechanisms using primary cultures of cortical neurons and astrocytes either in single well or in microfluidic chambers allowing connection between cells and cell-types. We report that astrocytes take up α-syn-aggregates far more efficiently than neurons through an endocytic event. We also highlight that active α-syn transport occurs between cells and any cell-types. Of special interest regarding the disease, we also show that uptake and spreading of α-syn from astrocytes to neurons can lead to neuronal death. Altogether, we here show that patients-derived α-synuclein aggregates, which are taken up by neurons and astrocytes, induce a differential endogenous response in the two cell types including a peculiar astrocytic toxic gain-of-function that leads to neuronal death.

Targeting α-synuclein: Therapeutic options.

The discovery of the central role of α-synuclein (αSyn) in the pathogenesis of Parkinson's disease (PD) has powered, in the last decade, the emergence of novel relevant models of this condition based on viral vector-mediated expression of the disease-causing protein or inoculation of toxic species of αSyn. Although the development of these powerful tools and models has provided considerable insights into the mechanisms underlying neurodegeneration in PD, it has also been translated into the expansion of the landscape of preclinical therapeutic strategies. Much attention is now brought to the proteotoxic mechanisms induced by αSyn and how to block them using strategies inspired by intrinsic cellular pathways such as the enhancement of cellular clearance by the lysosomal-autophagic system, through proteasome-mediated degradation or through immunization. The important effort undertaken by several laboratories and consortia to tackle these issues and identify novel targets warrants great promise for the discovery not only of neuroprotective approaches but also of restorative strategies for PD and other synucleinopathies. In this viewpoint, we summarize the latest advances in this new area of PD research and will discuss promising approaches and ongoing challenges. © 2016 International Parkinson and Movement Disorder Society.

Early prenatal exposure to MPTP does not affect nigrostrial neurons in macaque monkey.

The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that induces parkinsonism in both human and primate, has prompted the search for environmental toxins potentially responsible for idiopathic Parkinson's disease (PD). The present study reports the ultimate effects of MPTP intoxication of a female macaque monkey, which unraveled to be pregnant after parkinsonism had developed, upon its fetus. Detailed examination of the offpsring nigrostriatal pathway showed that tyrosine hydroxylase immunoreactivity in caudate-putamen nuclei and substantia nigra compacta (SNc) was not different from an age-matched control. Biochemical analysis of the tissue content of dopaminergic markers further suggested modification of metabolism in the MPTP-exposed monkey. These data suggest that early prenatal intoxication does not destroy nigrostriatal neurons, most likely because dopamine neurons had not developed yet when exposed to MPTP.

D1 dopamine receptor stimulation impairs striatal proteasome activity in Parkinsonism through 26S proteasome disassembly.

Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization.

Experimental modelling of -synuclein aggregation and spreading in synucleinopathies.

During the past two decades, a myriad of studies have suggested a central pathogenic role for a-synuclein in Parkinson's disease. Recent studies have unravelled self-aggregation and prion-like spreading properties for a-synuclein. Of particular importance was the seminal observation of Lewy body-like structures in grafted fetal dopaminergic neurons of patients with Parkinson's disease. This conceptual breakthrough generated the " host-to-the-graft " hypothesis orprion-like hypothesis. Nowadays, mechanisms underlying these new properties appear as putative disease-modifying targets. As the lack of valid animal models for Parkinson's disease is considered as a roadblock toward therapeutic intervention, the use of the newly developed models based on the prion-like properties of a-synuclein should allow future target validation.

Abnormal structure-specific peptide transmission and processing in a primate model of Parkinson's disease and l-DOPA-induced dyskinesia.

A role for enhanced peptidergic transmission, either opioidergic or not, has been proposed for the generation of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) on the basis of in situ hybridization studies showing that striatal peptidergic precursor expression consistently correlates with LID severity. Few studies, however, have focused on the actual peptides derived from these precursors. We used mass-spectrometry to study peptide profiles in the putamen and globus pallidus (internalis and externalis) collected from 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine treated macaque monkeys, acutely or chronically treated with l-DOPA. We identified that parkinsonian and dyskinetic states are associated with an abnormal production of proenkephalin-, prodynorphin- and protachykinin-1-derived peptides in both segments of the globus pallidus. Moreover, we report that peptidergic processing is dopamine-state dependent and highly structure-specific, possibly explaining the failure of previous clinical trials attempting to rectify abnormal peptidergic transmission.

From Association to Intervention: The Alzheimer's Disease-Associated Processes and Targets (ADAPT) Ontology.

BACKGROUND: Many putative causes and risk factors have been associated with outcomes in Alzheimer's disease (AD) but all attempts at disease-modifying treatment have failed to be clinically significant. Efforts to address this "association-intervention" mismatch have tended to focus on the novel design of interventions. OBJECTIVE: Here, we instead deal with the notion of association in depth. We introduce the concept of disease-associated process (DAP) as a flexible concept that can unite different areas of study of AD from genetics to epidemiology to identify disease-modifying targets. METHODS: We sort DAPs using three properties: specificity for AD, frequency in patients, and pathogenic intensity for dementia before using a literature review to apply these properties in three ways. Firstly, we describe and visualize known DAPs. Secondly, we exemplify qualitative specificity analysis with the DAPs of tau protein pathology and autophagy to reveal their differential implication in AD. Finally, we use DAP properties to define the terms "risk factor," "cause," and "biomarker." RESULTS: We show how DAPs fit into our collaborative disease ontology, the Alzheimer's Disease-Associated Processes and Targets (ADAPT) ontology. We argue that our theoretical system can serve as a democratic research forum, offering a more biologically adequate view of dementia than reductionist models. CONCLUSION: The ADAPT ontology is a tool that could help to ground debates around priority setting using objective criteria for the identifying of targets in AD. Further efforts are needed to address issues of how biomedical research into AD is prioritized and funded.

mTORC2-NDRG1-CDC42 axis couples fasting to mitochondrial fission.

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

Cortical Lewy body injections induce long-distance pathogenic alterations in the non-human primate brain.

Aggregation of α-synuclein (α-syn) is the cornerstone of neurodegenerative diseases termed synucleinopathies, which include Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA). These synucleinopathies are characterized by the deposit of aggregated α-syn in intracellular inclusions observable in neurons and glial cells. In PD and DLB, these aggregates, predominantly located in neurons, are called Lewy Bodies (LBs). These LBs are one of the pathological hallmarks of PD and DLB, alongside dopaminergic neuron loss in the substantia nigra. Previous studies have demonstrated the ability of PD patient-derived LB fractions to induce nigrostriatal neurodegeneration and α-syn pathology when injected into the striatum or the enteric nervous system of non-human primates. Here, we report the pathological consequences of injecting these LB fractions into the cortex of non-human primates. To this end, we inoculated mesencephalic PD patient-derived LB fractions into the prefrontal cortex of baboon monkeys terminated one year later. Extensive analyses were performed to evaluate pathological markers known to be affected in LB pathologies. We first assessed the hypothesized presence of phosphorylated α-syn at S129 (pSyn) in the prefrontal cortices. Second, we quantified the neuronal, microglial, and astrocytic cell survival in the same cortices. Third, we characterized these cortical LB injections' putative impact on the integrity of the nigrostriatal system. Overall, we observed pSyn accumulation around the injection site in the dorsal prefrontal cortex, in connected cortical regions, and further towards the striatum, suggesting α-syn pathological propagation. The pathology was also accompanied by neuronal loss in these prefrontal cortical regions and the caudate nucleus, without, however, loss of nigral dopamine neurons. In conclusion, this pilot study provides novel data demonstrating the toxicity of patient-derived extracts, their potential to propagate from the cortex to the striatum in non-human primates, and a possible primate model of DLB.

An Argument for Simple Tests of Treatment of Alzheimer's Disease.

Two potential disease-modifying approaches for dementia are being vigorously tested: the early targeting of the neuropathology of Alzheimer's disease (AD) and multi-domain lifestyle interventions to promote resilience to neuropathology. We apply the "web of information" model of clinical translation to both approaches to argue firstly that tests of treatments aiming to achieve clinically meaningful outcomes should remain simple, and secondly, that building clinically-meaningful treatments should be kept separate from public health policy which means promoting wide-reaching action against risk factors now with available information.

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.

Chaperone-mediated autophagy: a gatekeeper of neuronal proteostasis.

Different types of autophagy co-exist in all mammalian cells, however, the specific contribution of each of these autophagic pathways to the maintenance of cellular proteostasis and cellular function remains unknown. In this work, we have investigated the consequences of failure of chaperone-mediated autophagy (CMA) in neurons and compared the impact, on the neuronal proteome, of CMA loss to that of macroautophagy loss. We found that these autophagic pathways are non-redundant and that CMA is the main one responsible for maintenance of the metastable proteome (the one at risk of aggregation). We demonstrate that loss of CMA, as the one that occurs in aging, has a synergistic effect with the proteotoxicity associated with neurodegenerative conditions such as Alzheimer disease (AD) and, conversely, that, pharmacological enhancement of CMA is effective in improving both behavior and pathology in two different AD mouse models.