Upregulated pexophagy limits the capacity of selective autophagy.

Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson's Disease and Huntington's Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.

The G51D SNCA mutation generates a slowly progressive α-synuclein strain in early-onset Parkinson's disease.

Unique strains of α-synuclein aggregates have been postulated to underlie the spectrum of clinical and pathological presentations seen across the synucleinopathies. Whereas multiple system atrophy (MSA) is associated with a predominance of oligodendroglial α-synuclein inclusions, α-synuclein aggregates in Parkinson's disease (PD) preferentially accumulate in neurons. The G51D mutation in the SNCA gene encoding α-synuclein causes an aggressive, early-onset form of PD that exhibits clinical and neuropathological traits reminiscent of both PD and MSA. To assess the strain characteristics of G51D PD α-synuclein aggregates, we performed propagation studies in M83 transgenic mice by intracerebrally inoculating patient brain extracts. The properties of the induced α-synuclein aggregates in the brains of injected mice were examined using immunohistochemistry, a conformational stability assay, and by performing α-synuclein seed amplification assays. Unlike MSA-injected mice, which developed a progressive motor phenotype, G51D PD-inoculated animals remained free of overt neurological illness for up to 18 months post-inoculation. However, a subclinical synucleinopathy was present in G51D PD-inoculated mice, characterized by the accumulation of α-synuclein aggregates in restricted regions of the brain. The induced α-synuclein aggregates in G51D PD-injected mice exhibited distinct properties in a seed amplification assay and were much more stable than those present in mice injected with MSA extract, which mirrored the differences observed between human MSA and G51D PD brain samples. These results suggest that the G51D SNCA mutation specifies the formation of a slowly propagating α-synuclein strain that more closely resembles α-synuclein aggregates associated with PD than MSA.

α-Synuclein Conformational Strains as Drivers of Phenotypic Heterogeneity in Neurodegenerative Diseases.

The synucleinopathies, which include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are a class of human neurodegenerative disorders unified by the presence of α-synuclein aggregates in the brain. Considerable clinical and pathological heterogeneity exists within and among the individual synucleinopathies. A potential explanation for this variability is the existence of distinct conformational strains of α-synuclein aggregates that cause different disease manifestations. Like prion strains, α-synuclein strains can be delineated based on their structural architecture, with structural differences among α-synuclein aggregates leading to unique biochemical attributes and neuropathological properties in humans and animal models. Bolstered by recent high-resolution structural data from patient brain-derived material, it has now been firmly established that there are conformational differences among α-synuclein aggregates from different human synucleinopathies. Moreover, recombinant α-synuclein can be polymerized into several structurally distinct aggregates that exhibit unique pathological properties. In this review, we outline the evidence supporting the existence of α-synuclein strains and highlight how they can act as drivers of phenotypic heterogeneity in the human synucleinopathies.

Alpha-synuclein seeding shows a wide heterogeneity in multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a neurodegenerative condition characterized by variable combinations of parkinsonism, autonomic failure, cerebellar ataxia and pyramidal features. Although the distribution of synucleinopathy correlates with the predominant clinical features, the burden of pathology does not fully explain observed differences in clinical presentation and rate of disease progression. We hypothesized that the clinical heterogeneity in MSA is a consequence of variability in the seeding activity of α-synuclein both between different patients and between different brain regions. METHODS: The reliable detection of α-synuclein seeding activity derived from MSA using cell-free amplification assays remains challenging. Therefore, we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with fully characterized brain homogenates from one MSA and one PD patient. We then validated the two conditions that conferred the optimal ability to discriminate between PD- and MSA-derived samples in a larger cohort of 40 neuropathologically confirmed cases, including 15 MSA. Finally, in a subset of brains, we conducted the first multi-region analysis of seeding behaviour in MSA. RESULTS: Using our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of α-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived α-synuclein. Using this novel approach, we were able to sub-categorize the 15 MSA brains into 3 groups: high, intermediate and low seeders. To further demonstrate heterogeneity in α-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of α-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders. CONCLUSIONS: We have identified unexpected differences in seed-competent α-synuclein across a cohort of neuropathologically comparable MSA brains. Furthermore, our work has revealed a substantial heterogeneity in seeding activity, driven by the PBS-soluble α-synuclein, between different brain regions of a given individual that goes beyond immunohistochemical observations. Our observations pave the way for future subclassification of MSA, which exceeds conventional clinical and neuropathological phenotyping and considers the structural and biochemical heterogeneity of α-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA.

α-Synuclein strains target distinct brain regions and cell types.

The clinical and pathological differences between synucleinopathies such as Parkinson's disease and multiple system atrophy have been postulated to stem from unique strains of α-synuclein aggregates, akin to what occurs in prion diseases. Here we demonstrate that inoculation of transgenic mice with different strains of recombinant or brain-derived α-synuclein aggregates produces clinically and pathologically distinct diseases. Strain-specific differences were observed in the signs of neurological illness, time to disease onset, morphology of cerebral α-synuclein deposits and the conformational properties of the induced aggregates. Moreover, different strains targeted distinct cellular populations and cell types within the brain, recapitulating the selective targeting observed among human synucleinopathies. Strain-specific clinical, pathological and biochemical differences were faithfully maintained after serial passaging, which implies that α-synuclein propagates via prion-like conformational templating. Thus, pathogenic α-synuclein exhibits key hallmarks of prion strains, which provides evidence that disease heterogeneity among the synucleinopathies is caused by distinct α-synuclein strains.

Application of CRISPR genetic screens to investigate neurological diseases.

The adoption of CRISPR-Cas9 technology for functional genetic screens has been a transformative advance. Due to its modular nature, this technology can be customized to address a myriad of questions. To date, pooled, genome-scale studies have uncovered genes responsible for survival, proliferation, drug resistance, viral susceptibility, and many other functions. The technology has even been applied to the functional interrogation of the non-coding genome. However, applications of this technology to neurological diseases remain scarce. This shortfall motivated the assembly of a review that will hopefully help researchers moving in this direction find their footing. The emphasis here will be on design considerations and concepts underlying this methodology. We will highlight groundbreaking studies in the CRISPR-Cas9 functional genetics field and discuss strengths and limitations of this technology for neurological disease applications. Finally, we will provide practical guidance on navigating the many choices that need to be made when implementing a CRISPR-Cas9 functional genetic screen for the study of neurological diseases.

Brain-Derived Neurotrophic Factor Elevates Activating Transcription Factor 4 (ATF4) in Neurons and Promotes ATF4-Dependent Induction of Sesn2.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.