Molecular Mechanisms of Cellular Senescence in Neurodegenerative Diseases.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by several pathological features, including selective neuronal loss, aggregation of specific proteins, and chronic inflammation. Aging is the most critical risk factor of these disorders. However, the mechanism by which aging contributes to the pathogenesis of neurodegenerative diseases is not clearly understood. Cellular senescence is a cell state or fate in response to stimuli. It is typically associated with a series of changes in cellular phenotypes such as abnormal cellular metabolism and proteostasis, reactive oxygen species (ROS) production, and increased secretion of certain molecules via senescence-associated secretory phenotype (SASP). In this review, we discuss how cellular senescence contributes to brain aging and neurodegenerative diseases, and the relationship between protein aggregation and cellular senescence. Finally, we discuss the potential of senescence modifiers and senolytics in the treatment of neurodegenerative diseases.

Inflammation promotes synucleinopathy propagation.

The clinical progression of neurodegenerative diseases correlates with the spread of proteinopathy in the brain. The current understanding of the mechanism of proteinopathy spread is far from complete. Here, we propose that inflammation is fundamental to proteinopathy spread. A sequence variant of α-synuclein (V40G) was much less capable of fibril formation than wild-type α-synuclein (WT-syn) and, when mixed with WT-syn, interfered with its fibrillation. However, when V40G was injected intracerebrally into mice, it induced aggregate spreading even more effectively than WT-syn. Aggregate spreading was preceded by sustained microgliosis and inflammatory responses, which were more robust with V40G than with WT-syn. Oral administration of an anti-inflammatory agent suppressed aggregate spreading, inflammation, and behavioral deficits in mice. Furthermore, exposure of cells to inflammatory cytokines increased the cell-to-cell propagation of α-synuclein. These results suggest that the inflammatory microenvironment is the major driver of the spread of synucleinopathy in the brain.

Senescence and impaired DNA damage responses in alpha-synucleinopathy models.

α-Synuclein is a crucial element in the pathogenesis of Parkinson's disease (PD) and related neurological diseases. Although numerous studies have presented potential mechanisms underlying its pathogenesis, the understanding of α-synuclein-mediated neurodegeneration remains far from complete. Here, we show that overexpression of α-synuclein leads to impaired DNA repair and cellular senescence. Transcriptome analysis showed that α-synuclein overexpression led to cellular senescence with activation of the p53 pathway and DNA damage responses (DDRs). Chromatin immunoprecipitation analyses using p53 and γH2AX, chromosomal markers of DNA damage, revealed that these proteins bind to promoters and regulate the expression of DDR and cellular senescence genes. Cellular marker analyses confirmed cellular senescence and the accumulation of DNA double-strand breaks. The non-homologous end joining (NHEJ) DNA repair pathway was activated in α-synuclein-overexpressing cells. However, the expression of MRE11, a key component of the DSB repair system, was reduced, suggesting that the repair pathway induction was incomplete. Neuropathological examination of α-synuclein transgenic mice showed increased levels of phospho-α-synuclein and DNA double-strand breaks, as well as markers of cellular senescence, at an early, presymptomatic stage. These results suggest that the accumulation of DNA double-strand breaks (DSBs) and cellular senescence are intermediaries of α-synuclein-induced pathogenesis in PD.

Alpha-Synuclein Inclusion Formation in Human Oligodendrocytes.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by presence of α-synuclein-positive inclusions in the cytoplasm of oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are considered an integral part of the pathogenesis of MSA, leading to demyelination and neuronal demise. What is most puzzling in the research fields of GCIs is the origin of α-synuclein aggregates in GCIs, since adult oligodendrocytes do not express high levels of α-synuclein. The most recent leading hypothesis is that GCIs form via transfer and accumulation of α-synuclein from neurons to oligodendrocytes. However, studies regarding this subject are limited due to the absence of proper human cell models, to demonstrate the entry and accumulation of neuronal α-synuclein in human oligodendrocytes. Here, we generated mature human oligodendrocytes that can take up neuronderived α-synuclein and form GCI-like inclusions. Mature human oligodendrocytes are derived from neural stem cells via "oligosphere" formation and then into oligodendrocytes, treating the cells with the proper differentiation factors at each step. In the final cell preparations, oligodendrocytes consist of the majority population, while some astrocytes and unidentified stem cell-like cells were present as well. When these cells were exposed to α-synuclein proteins secreted from neuron-like human neuroblastoma cells, oligodendrocytes developed perinuclear inclusion bodies with α-synuclein immunoreactivity, resembling GCIs, while the stem cell-like cells showed α-synuclein-positive, scattered puncta in the cytoplasm. In conclusion, we have established a human oligodendrocyte model for the study of GCI formation, and the characterization and use of this model might pave the way for understanding the pathogenesis of MSA.

Models of multiple system atrophy.

Multiple system atrophy (MSA) is a neurodegenerative disease with diverse clinical manifestations, including parkinsonism, cerebellar syndrome, and autonomic failure. Pathologically, MSA is characterized by glial cytoplasmic inclusions in oligodendrocytes, which contain fibrillary forms of α-synuclein. MSA is categorized as one of the α-synucleinopathy, and α-synuclein aggregation is thought to be the culprit of the disease pathogenesis. Studies on MSA pathogenesis are scarce relative to studies on the pathogenesis of other synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies. However, recent developments in cellular and animal models of MSA, especially α-synuclein transgenic models, have driven advancements in research on this disease. Here, we review the currently available models of MSA, which include toxicant-induced animal models, α-synuclein-overexpressing cellular models, and mouse models that express α-synuclein specifically in oligodendrocytes through cell type-specific promoters. We will also discuss the results of studies in recently developed transmission mouse models, into which MSA brain extracts were intracerebrally injected. By reviewing the findings obtained from these model systems, we will discuss what we have learned about the disease and describe the strengths and limitations of the models, thereby ultimately providing direction for the design of better models and future research.

Arylsulfatase A, a genetic modifier of Parkinson's disease, is an α-synuclein chaperone.

Mutations in lysosomal genes increase the risk of neurodegenerative diseases, as is the case for Parkinson's disease. Here, we found that pathogenic and protective mutations in arylsulfatase A (ARSA), a gene responsible for metachromatic leukodystrophy, a lysosomal storage disorder, are linked to Parkinson's disease. Plasma ARSA protein levels were changed in Parkinson's disease patients. ARSA deficiency caused increases in α-synuclein aggregation and secretion, and increases in α-synuclein propagation in cells and nematodes. Despite being a lysosomal protein, ARSA directly interacts with α-synuclein in the cytosol. The interaction was more extensive with protective ARSA variant and less with pathogenic ARSA variant than wild-type. ARSA inhibited the in vitro fibrillation of α-synuclein in a dose-dependent manner. Ectopic expression of ARSA reversed the α-synuclein phenotypes in both cell and fly models of synucleinopathy, the effects correlating with the extent of the physical interaction between these molecules. Collectively, these results suggest that ARSA is a genetic modifier of Parkinson's disease pathogenesis, acting as a molecular chaperone for α-synuclein.

LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation.

Propagation of α-synuclein aggregates has been suggested as a contributing factor in Parkinson's disease (PD) progression. However, the molecular mechanisms underlying α-synuclein aggregation are not fully understood. Here, we demonstrate in cell culture, nematode, and rodent models of PD that leucine-rich repeat kinase 2 (LRRK2), a PD-linked kinase, modulates α-synuclein propagation in a kinase activity-dependent manner. The PD-linked G2019S mutation in LRRK2, which increases kinase activity, enhances propagation efficiency. Furthermore, we show that the role of LRRK2 in α-synuclein propagation is mediated by RAB35 phosphorylation. Constitutive activation of RAB35 overrides the reduced α-synuclein propagation phenotype in lrk-1 mutant C. elegans. Finally, in a mouse model of synucleinopathy, administration of an LRRK2 kinase inhibitor reduced α-synuclein aggregation via enhanced interaction of α-synuclein with the lysosomal degradation pathway. These results suggest that LRRK2-mediated RAB35 phosphorylation is a potential therapeutic target for modifying disease progression.

Immunotherapy targeting toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating α-synuclein transmission and neuroinflammation.

BACKGROUND: Synucleinopathies of the aging population are an heterogeneous group of neurological disorders that includes Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and are characterized by the progressive accumulation of α-synuclein in neuronal and glial cells. Toll-like receptor 2 (TLR2), a pattern recognition immune receptor, has been implicated in the pathogenesis of synucleinopathies because TLR2 is elevated in the brains of patients with PD and TLR2 is a mediator of the neurotoxic and pro-inflammatory effects of extracellular α-synuclein aggregates. Therefore, blocking TLR2 might alleviate α-synuclein pathological and functional effects. For this purpose, herein, we targeted TLR2 using a functional inhibitory antibody (anti-TLR2). METHODS: Two different human α-synuclein overexpressing transgenic mice were used in this study. α-synuclein low expresser mouse (α-syn-tg, under the PDGFß promoter, D line) was stereotaxically injected with TLR2 overexpressing lentivirus to demonstrate that increment of TLR2 expression triggers neurotoxicity and neuroinflammation. α-synuclein high expresser mouse (α-Syn-tg; under mThy1 promoter, Line 61) was administrated with anti-TLR2 to examine that functional inhibition of TLR2 ameliorates neuropathology and behavioral defect in the synucleinopathy animal model. In vitro α-synuclein transmission live cell monitoring system was used to evaluate the role of TLR2 in α-synuclein cell-to-cell transmission. RESULTS: We demonstrated that administration of anti-TLR2 alleviated α-synuclein accumulation in neuronal and astroglial cells, neuroinflammation, neurodegeneration, and behavioral deficits in an α-synuclein tg mouse model of PD/DLB. Moreover, in vitro studies with neuronal and astroglial cells showed that the neuroprotective effects of anti-TLR2 antibody were mediated by blocking the neuron-to-neuron and neuron-to-astrocyte α-synuclein transmission which otherwise promotes NFκB dependent pro-inflammatory responses. CONCLUSION: This study proposes TLR2 immunotherapy as a novel therapeutic strategy for synucleinopathies of the aging population.

Mechanism of neuroprotection by trehalose: controversy surrounding autophagy induction.

Trehalose is a non-reducing disaccharide with two glucose molecules linked through an α, α-1,1-glucosidic bond. Trehalose has received attention for the past few decades for its role in neuroprotection especially in animal models of various neurodegenerative diseases, such as Parkinson and Huntington diseases. The mechanism underlying the neuroprotective effects of trehalose remains elusive. The prevailing hypothesis is that trehalose protects neurons by inducing autophagy, thereby clearing protein aggregates. Some of the animal studies showed activation of autophagy and reduced protein aggregates after trehalose administration in neurodegenerative disease models, seemingly supporting the autophagy induction hypothesis. However, results from cell studies have been less certain; although many studies claim that trehalose induces autophagy and reduces protein aggregates, the studies have their weaknesses, failing to provide sufficient evidence for the autophagy induction theory. Furthermore, a recent study with a thorough examination of autophagy flux showed that trehalose interfered with the flux from autophagosome to autolysosome, raising controversy on the direct effects of trehalose on autophagy. This review summarizes the fundamental properties of trehalose and the studies on its effects on neurodegenerative diseases. We also discuss the controversy related to the autophagy induction theory and seek to explain how trehalose works in neuroprotection.

Is trehalose an autophagic inducer? Unraveling the roles of non-reducing disaccharides on autophagic flux and alpha-synuclein aggregation.

Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

Amplification of distinct α-synuclein fibril conformers through protein misfolding cyclic amplification.

Amyloid fibril formation has been implicated in the pathogenesis of neurodegenerative diseases. Fibrillation generates numerous conformers. Presumably, the conformers may possess specific biological properties, thus providing a biochemical framework for strains of prions. However, the precise relationship between various fibril conformers and their pathogenic functions has not been determined because of limited accessibility to adequate amounts of fibrils from tissue samples. α-Synuclein is one such protein, and it has been implicated in Parkinson disease. Using a technique known as protein misfolding cyclic amplification, originally developed for amplifying prions, we established a procedure through which the amplification of α-synuclein fibrils is possible. With a trace amount of seeds, we succeeded in amplifying α-synuclein fibrils. The replication of the seeds was faithful in terms of conformation even after multiple rounds of cyclic amplification. Moreover, two transgenic mouse strains each representing a distinct synucleinopathy were used to investigate different conformers by using this technique. The amplified α-synuclein fibrils derived from the tissue extracts of these two strains led to the production of two different fibril conformers with distinct proteinase K digestion profiles. Together, our results demonstrated that a trace amount of α-synuclein fibrils in tissue extracts could be amplified with their conformations conserved. This procedure should be useful in amplifying α-synuclein fibrils from the brains and body fluids of patients afflicted with synucleinopathies and may serve as a potential diagnostic tool for Parkinson disease and other synucleinopathies.

Cell-to-cell Transmission of Polyglutamine Aggregates in C. elegans.

Huntington disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive dysfunction caused by expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin (HTT). In patients, the number of glutamine residues in polyQ tracts are over 35, and it is correlated with age of onset, severity, and disease progression. Expansion of polyQ increases the propensity for HTT protein aggregation, process known to be implicated in neurodegeneration. These pathological aggregates can be transmitted from neuron to another neuron, and this process may explain the pathological spreading of polyQ aggregates. Here, we developed an in vivo model for studying transmission of polyQ aggregates in a highly quantitative manner in real time. HTT exon 1 with expanded polyQ was fused with either N-terminal or C-terminal fragments of Venus fluorescence protein and expressed in pharyngeal muscles and associated neurons, respectively, of C. elegans. Transmission of polyQ proteins was detected using bimolecular fluorescence complementation (BiFC). Mutant polyQ (Q97) was transmitted much more efficiently than wild type polyQ (Q25) and forms numerous inclusion bodies as well. The transmission of Q97 was gradually increased with aging of animal. The animals with polyQ transmission exhibited degenerative phenotypes, such as nerve degeneration, impaired pharyngeal pumping behavior, and reduced life span. The C. elegans model presented here would be a useful in vivo model system for the study of polyQ aggregate propagation and might be applied to the screening of genetic and chemical modifiers of the propagation.

Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.

Aging is the major risk factor for neurodegenerative diseases that are also associated with impaired proteostasis, resulting in abnormal accumulation of protein aggregates. However, the role of aging in development and progression of disease remains elusive. Here, we used Caenorhabditis elegans models to show that aging-promoting genetic variations accelerated the rate of cell-to-cell transmission of SNCA/α-synuclein aggregates, hallmarks of Parkinson disease, and the progression of disease phenotypes, such as nerve degeneration, behavioral deficits, and reduced life span. Genetic and pharmacological anti-aging manipulations slowed the spread of aggregates and the associated phenotypes. Lysosomal degradation was significantly impaired in aging models, while anti-aging treatments reduced the impairment. Transgenic expression of hlh-30p::hlh-30, the master controller of lysosomal biogenesis, alleviated intercellular transmission of aggregates in the aging model. Our results demonstrate that the rate of aging closely correlates with the rate of aggregate propagation and that general anti-aging treatments can slow aggregate propagation and associated disease progression by restoring lysosomal function.

Exposure to bacterial endotoxin generates a distinct strain of α-synuclein fibril.

A single amyloidogenic protein is implicated in multiple neurological diseases and capable of generating a number of aggregate "strains" with distinct structures. Among the amyloidogenic proteins, α-synuclein generates multiple patterns of proteinopathies in a group of diseases, such as Parkinson disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, the link between specific conformations and distinct pathologies, the key concept of the strain hypothesis, remains elusive. Here we show that in the presence of bacterial endotoxin, lipopolysaccharide (LPS), α-synuclein generated a self-renewable, structurally distinct fibril strain that consistently induced specific patterns of synucleinopathies in mice. These results suggest that amyloid fibrils with self-renewable structures cause distinct types of proteinopathies despite the identical primary structure and that exposure to exogenous pathogens may contribute to the diversity of synucleinopathies.

Non-cell-autonomous Neurotoxicity of α-synuclein Through Microglial Toll-like Receptor 2.

Synucleinopathies are a collection of neurological diseases that are characterized by deposition of α-synuclein aggregates in neurons and glia. These diseases include Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. Although it has been increasingly clear that α-synuclein is implicated in the pathogenesis of PD and other synucleinopathies, the precise mechanism underlying the disease process remains to be unraveled. The past studies on how α-synuclein exerts pathogenic actions have focused on its direct, cell-autonomous neurotoxic effects. However, recent findings suggested that there might be indirect, non-cell-autonomous pathways, perhaps through the changes in glial cells, for the pathogenic actions of this protein. Here, we present evidence that α-synuclein can cause neurodegeneration through a non-cell-autonomous manner. We show that α-synuclein can be secreted from neurons and induces inflammatory responses in microglia, which in turn secreted neurotoxic agents into the media causing neurodegeneration. The neurotoxic response of microglia was mediated by activation of toll-like receptor 2 (TLR2), a receptor for neuron-derived α-synuclein. This work suggests that TLR2 is the key molecule that mediates non-cell-autonomous neurotoxic effects of α-synuclein, hence a candidate for the therapeutic target.

Cell Models to Study Cell-to-Cell Transmission of α-Synuclein.

The cell-to-cell transmission of protein aggregates has been implicated in the progression of pathological phenotypes in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In recent years, several experimental model systems have been developed to study the mechanisms of cell-to-cell transmission. Herein, we describe cell culture models with which cell-to-cell transmission of α-synuclein can be quantitatively analyzed. The principle underlying these models could be applied to developing model systems for transmission of other protein aggregates, such as tau and TDP-43.

Antagonizing Neuronal Toll-like Receptor 2 Prevents Synucleinopathy by Activating Autophagy.

Impaired autophagy has been implicated in many neurodegenerative diseases, such as Parkinson's disease (PD), and might be responsible for deposition of aggregated proteins in neurons. However, little is known about how neuronal autophagy and clearance of aggregated proteins are regulated. Here, we show a role for Toll-like receptor 2 (TLR2), a pathogen-recognizing receptor in innate immunity, in regulation of neuronal autophagy and clearance of α-synuclein, a protein aggregated in synucleinopathies, including in PD. Activation of TLR2 resulted in the accumulation of α-synuclein aggregates in neurons as a result of inhibition of autophagic activity through regulation of the AKT/mTOR pathway. In contrast, inactivation of TLR2 resulted in autophagy activation and increased clearance of neuronal α-synuclein, and hence reduced neurodegeneration, in transgenic mice and in in vitro models. These results uncover roles of TLR2 in regulating neuronal autophagy and suggest that the TLR2 pathway may be targeted for autophagy activation strategies in treating neurodegenerative disorders.

Loss of glucocerebrosidase 1 activity causes lysosomal dysfunction and α-synuclein aggregation.

Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes ß-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When α-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of α-synuclein aggregates.