Dissecting aggregation and seeding dynamics of α-Syn polymorphs using the phasor approach to FLIM.

Synucleinopathies are a heterogenous group of neurodegenerative diseases characterized by the progressive accumulation of pathological α-synuclein (α-Syn). The importance of structural polymorphism of α-Syn assemblies for distinct synucleinopathies and their progression is increasingly recognized. However, the underlying mechanisms are poorly understood. Here we use fluorescence lifetime imaging microscopy (FLIM) to investigate seeded aggregation of α-Syn in a biosensor cell line. We show that conformationally distinct α-Syn polymorphs exhibit characteristic fluorescence lifetimes. FLIM further revealed that α-Syn polymorphs were differentially processed by cellular clearance pathways, yielding fibrillar species with increased seeding capacity. Thus, FLIM is not only a powerful tool to distinguish different amyloid structures, but also to monitor the dynamic process of amyloid remodeling by the cellular environment. Our data suggest that the accumulation of highly seeding competent degradation products for particular polymorphs may account for accelerated disease progression in some patients.

Molecular Chaperones: A Double-Edged Sword in Neurodegenerative Diseases.

Aberrant accumulation of misfolded proteins into amyloid deposits is a hallmark in many age-related neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Pathological inclusions and the associated toxicity appear to spread through the nervous system in a characteristic pattern during the disease. This has been attributed to a prion-like behavior of amyloid-type aggregates, which involves self-replication of the pathological conformation, intercellular transfer, and the subsequent seeding of native forms of the same protein in the neighboring cell. Molecular chaperones play a major role in maintaining cellular proteostasis by assisting the (re)-folding of cellular proteins to ensure their function or by promoting the degradation of terminally misfolded proteins to prevent damage. With increasing age, however, the capacity of this proteostasis network tends to decrease, which enables the manifestation of neurodegenerative diseases. Recently, there has been a plethora of studies investigating how and when chaperones interact with disease-related proteins, which have advanced our understanding of the role of chaperones in protein misfolding diseases. This review article focuses on the steps of prion-like propagation from initial misfolding and self-templated replication to intercellular spreading and discusses the influence that chaperones have on these various steps, highlighting both the positive and adverse consequences chaperone action can have. Understanding how chaperones alleviate and aggravate disease progression is vital for the development of therapeutic strategies to combat these debilitating diseases.

C. elegans Models to Study the Propagation of Prions and Prion-Like Proteins.

A hallmark common to many age-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), is that patients develop proteinaceous deposits in their central nervous system (CNS). The progressive spreading of these inclusions from initially affected sites to interconnected brain areas is reminiscent of the behavior of bona fide prions in transmissible spongiform encephalopathies (TSEs), hence the term prion-like proteins has been coined. Despite intensive research, the exact mechanisms that facilitate the spreading of protein aggregation between cells, and the associated loss of neurons, remain poorly understood. As population demographics in many countries continue to shift to higher life expectancy, the incidence of neurodegenerative diseases is also rising. This represents a major challenge for healthcare systems and patients' families, since patients require extensive support over several years and there is still no therapy to cure or stop these diseases. The model organism Caenorhabditis elegans offers unique opportunities to accelerate research and drug development due to its genetic amenability, its transparency, and the high degree of conservation of molecular pathways. Here, we will review how recent studies that utilize this soil dwelling nematode have proceeded to investigate the propagation and intercellular transmission of prions and prion-like proteins and discuss their relevance by comparing their findings to observations in other model systems and patients.

DNA Methyltransferase 1 (DNMT1) Function Is Implicated in the Age-Related Loss of Cortical Interneurons.

Increased life expectancy in modern society comes at the cost of age-associated disabilities and diseases. Aged brains not only show reduced excitability and plasticity, but also a decline in inhibition. Age-associated defects in inhibitory circuits likely contribute to cognitive decline and age-related disorders. Molecular mechanisms that exert epigenetic control of gene expression contribute to age-associated neuronal impairments. Both DNA methylation, mediated by DNA methyltransferases (DNMTs), and histone modifications maintain neuronal function throughout lifespan. Here we provide evidence that DNMT1 function is implicated in the age-related loss of cortical inhibitory interneurons. Dnmt1 deletion in parvalbumin-positive interneurons attenuates their age-related decline in the cerebral cortex. Moreover, conditional Dnmt1-deficient mice show improved somatomotor performance and reduced aging-associated transcriptional changes. A decline in the proteostasis network, responsible for the proper degradation and removal of defective proteins, is implicated in age- and disease-related neurodegeneration. Our data suggest that DNMT1 acts indirectly on interneuron survival in aged mice by modulating the proteostasis network during life-time.

The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species.

The accumulation and prion-like propagation of α-synuclein and other amyloidogenic proteins are associated with devastating neurodegenerative diseases. Metazoan heat shock protein HSP70 and its co-chaperones DNAJB1 and HSP110 constitute a disaggregation machinery that is able to disassemble α-synuclein fibrils in vitro, but its physiological effects on α-synuclein toxicity are unknown. Here, we depleted Caenorhabditis elegans HSP-110 and monitored the consequences on α-synuclein-related pathological phenotypes such as misfolding, intercellular spreading, and toxicity in C. elegans in vivo models. Depletion of HSP-110 impaired HSP70 disaggregation activity, prevented resolubilization of amorphous aggregates, and compromised the overall cellular folding capacity. At the same time, HSP-110 depletion reduced α-synuclein foci formation, cell-to-cell transmission, and toxicity. These data demonstrate that the HSP70 disaggregation activity constitutes a double-edged sword, as it is essential for maintaining cellular proteostasis but also involved in the generation of toxic amyloid-type protein species.

DNA Methylation-Mediated Modulation of Endocytosis as Potential Mechanism for Synaptic Function Regulation in Murine Inhibitory Cortical Interneurons.

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.