The interrelationship of proteasome impairment and oligomeric intermediates in neurodegeneration

Various neurodegenerative diseases are characterized by the accumulation of amyloidogenic proteins such as tau, α‐synuclein, and amyloid‐β. Prior to the formation of these stable aggregates, intermediate species of the respective proteins—oligomers—appear. Recently acquired data have shown that oligomers may be the most toxic and pathologically significant to neurodegenerative diseases such as Alzheimer's and Parkinson's. The covalent modification of these oligomers may be critically important for biological processes in disease. Ubiquitin and small ubiquitin‐like modifiers are the commonly used tags for degradation. While the modification of large amyloid aggregates by ubiquitination is well established, very little is known about the role ubiquitin may play in oligomer processing and the importance of the more recently discovered sumoylation. Many proteins involved in neurodegeneration have been found to be sumoylated, notably tau protein in brains afflicted with Alzheimer's. This evidence suggests that while the cell may not have difficulty recognizing dangerous proteins, in brains afflicted with neurodegenerative disease, the proteasome may be unable to properly digest the tagged proteins. This would allow toxic aggregates to develop, leading to even more proteasome impairment in a snowball effect that could explain the exponential progression in most neurodegenerative diseases. A better understanding of the covalent modifications of oligomers could have a huge impact on the development of therapeutics for neurodegenerative diseases. This review will focus on the proteolysis of tau and other amyloidogenic proteins induced by covalent modification, and recent findings suggesting a relationship between tau oligomers and sumoylation.     

Myricetin slows liquid–liquid phase separation of Tau and activates ATG5-dependent autophagy to suppress Tau toxicity

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer''s disease. A current therapeutic avenue for treating Alzheimer''s disease is aimed at inhibiting Tau accumulation with small molecules such as natural flavonoids. Liquid–liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregates can then be degraded by autophagy. However, it is unclear whether natural flavonoids modulate the formation of phase-separated Tau droplets or promote autophagy and Tau clearance. Here, using confocal microscopy and fluorescence recovery after photobleaching assays, we report that a natural antioxidant flavonoid compound myricetin slows LLPS of full-length human Tau, shifting the equilibrium phase boundary to a higher protein concentration. This natural flavonoid also significantly inhibits pathological phosphorylation and abnormal aggregation of Tau in neuronal cells and blocks mitochondrial damage and apoptosis induced by Tau aggregation. Importantly, using coimmunoprecipitation and Western blotting, we show that treatment of cells with myricetin stabilizes the interaction between Tau and autophagy-related protein 5 (ATG5) to promote clearance of phosphorylated Tau to indirectly limit its aggregation. Consistently, this natural flavonoid inhibits mTOR pathway, activates ATG5-dependent Tau autophagy, and almost completely suppresses Tau toxicity in neuronal cells. Collectively, these results demonstrate how LLPS and abnormal aggregation of Tau are inhibited by natural flavonoids, bridging the gap between Tau LLPS and aggregation in neuronal cells, and also establish that myricetin could act as an ATG5-dependent autophagic activator to ameliorate the pathogenesis of Alzheimer''s disease.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

The Parkinson's disease protein LRRK2 impairs proteasome substrate clearance without affecting proteasome catalytic activity

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common known cause of Parkinson''s disease (PD). The clinical features of LRRK2 PD are indistinguishable from idiopathic PD, with accumulation of α-synuclein and/or tau and/or ubiquitin in intraneuronal aggregates. This suggests that LRRK2 is a key to understanding the aetiology of the disorder. Although loss-of-function does not appear to be the mechanism causing PD in LRRK2 patients, it is not clear how this protein mediates toxicity. In this study, we report that LRRK2 overexpression in cells and in vivo impairs the activity of the ubiquitin-proteasome pathway, and that this accounts for the accumulation of diverse substrates with LRRK2 overexpression. We show that this is not mediated by large LRRK2 aggregates or sequestration of ubiquitin to the aggregates. Importantly, such abnormalities are not seen with overexpression of the related protein LRRK1. Our data suggest that LRRK2 inhibits the clearance of proteasome substrates upstream of proteasome catalytic activity, favouring the accumulation of proteins and aggregate formation. Thus, we provide a molecular link between LRRK2, the most common known cause of PD, and its previously described phenotype of protein accumulation.     

An apparent core/shell architecture of polyQ aggregates in the aging Caenorhabditis elegans neuron

Huntington''s disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein which results in its abnormal aggregation in the nervous system. Huntingtin aggregates are linked to toxicity and neuronal dysfunction, but a comprehensive understanding of the aggregation mechanism in vivo remains elusive. Here, we examine the morphology of polyQ aggregates in Caenorhabditis elegans mechanosensory neurons as a function of age using confocal and fluorescence lifetime imaging microscopy. We find that aggregates in young worms are mostly spherical with homogenous intensity, but as the worm ages aggregates become substantially more heterogeneous. Most prominently, in older worms we observe an apparent core/shell morphology of polyQ assemblies with decreased intensity in the center. The fluorescence lifetime of polyQ is uniform across the aggregate indicating that the dimmed intensity in the assembly center is most likely not due to quenching or changes in local environment, but rather to displacement of fluorescent polyQ from the central region. This apparent core/shell architecture of polyQ aggregates in aging C. elegans neurons contributes to the diverse landscape of polyQ aggregation states implicated in Huntington''s disease.     

Extracellular vesicles,the emerging mirrors of brain physiopathology

Extracellular vesicles are secreted by a wide variety of cells, and their primary functions include intercellular communication, immune responses, human reproduction, and synaptic plasticity. Their molecular cargo reflects the physiological processes that their cells of origin are undergoing. Thus, many studies have suggested that extracellular vesicles could be a promising biomarker tool for many diseases, mainly due to their biological relevance and easy accessibility to a broad range of body fluids. Moreover, since their biological composition leads them to cross the blood-brain barrier bidirectionally, growing evidence points to extracellular vesicles as emerging mirrors of brain diseases processes. In this regard, this review explores the biogenesis and biological functions of extracellular vesicles, their role in different physiological and pathological processes, their potential in clinical practice, and the recent outstanding studies about the role of exosomes in major human brain diseases, such as Alzheimer''s disease (AD), Parkinson''s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or brain tumors.     

Protein misfolding in the late‐onset neurodegenerative diseases: Common themes and the unique case of amyotrophic lateral sclerosis

Enormous strides have been made in the last 100 years to extend human life expectancy and to combat the major infectious diseases. Today, the major challenges for medical science are age‐related diseases, including cancer, heart disease, lung disease, renal disease, and late‐onset neurodegenerative disease. Of these, only the neurodegenerative diseases represent a class of disease so poorly understood that no general strategies for prevention or treatment exist. These diseases, which include Alzheimer's disease, Parkinson's disease, Huntington's disease, the transmissible spongiform encephalopathies, and amyotrophic lateral sclerosis (ALS), are generally fatal and incurable. The first section of this review summarizes the diversity and common features of the late‐onset neurodegenerative diseases, with a particular focus on protein misfolding and aggregation—a recurring theme in the molecular pathology. The second section focuses on the particular case of ALS, a late‐onset neurodegenerative disease characterized by the death of central nervous system motor neurons, leading to paralysis and patient death. Of the 10% of ALS cases that show familial inheritance (familial ALS), the largest subset is caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). The unusual kinetic stability of SOD1 has provided a unique opportunity for detailed structural characterization of conformational states potentially involved in SOD1‐associated ALS. This review discusses past studies exploring the stability, folding, and misfolding behavior of SOD1, as well as the therapeutic possibilities of using detailed knowledge of misfolding pathways to target the molecular mechanisms underlying ALS and other neurodegenerative diseases. Proteins 2013; 81:1285–1303. © 2013 Wiley Periodicals, Inc.     

NAD/NADP及其介导氧化应激在神经退行性疾病中的作用

神经退行性疾病如阿尔茨海默病、帕金森病、亨廷顿病等疾病的发生与氧化应激紧密相关。NAD和NADP是维持氧化系统和抗氧化系统平衡的两个关键物质。NAD和NADP的生物合成和降解有多种途径,参与其生物途径的物质如NAMPT、NADK、PARP1、SIRT1、CD38等,均报道在神经退行性疾病发挥一定的作用。因此,本文分别从NAD和NADP的合成和降解途径中的一些关键物质出发,结合氧化应激总结并探讨它们在神经退行性疾病的作用,以期为临床治疗神经退行性疾病提供新思路。     

Abnormal cortical lysosomal β-hexosaminidase and β-galactosidase activity at post-synaptic sites during Alzheimer's disease progression

A critical role of endosomal–lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimer's disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimer's disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of β-hexosaminidase and β-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimer's disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that β-hexosaminidase and β-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimer's disease. The early increase of lipid microdomain-associated β-hexosaminidase and β-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimer's disease.     

Dissecting the copper bioinorganic chemistry of the functional and pathological roles of the prion protein: Relevance in Alzheimer's disease and cancer

The cellular prion protein (PrP^C) is a metal-binding biomolecule that can interact with different protein partners involved in pivotal physiological processes, such as neurogenesis and neuronal plasticity. Recent studies profile copper and PrP^C as important players in the pathological mechanisms of Alzheimer's disease and cancer. Although the copper-PrP^C interaction has been characterized extensively, the role of the metal ion in the physiological and pathological roles of PrP^C has been barely explored. In this article, we discuss how copper binding and proteolytic processing may impact the ability of PrP^C to recruit protein partners for its functional roles. The importance to dissect the role of copper-PrP^C interactions in health and disease is also underscored.     

Expression Pattern of Synucleins (Non-Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α-Synuclein) During Murine Brain Development

Abstract: The non-Aβ component of Alzheimer's disease amyloid precursor protein (NACP) is predominantly a neuron-specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimer's disease amyloid and a mutation in the NACP gene is associated with familial Parkinson's disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.     

Molecular docking analysis of hyperphosphorylated tau protein with compounds derived from Bacopa monnieri and Withania somnifera

Tau protein, the major player in Alzheimer’s disease forms neurofibrillary tangles in elderly people. Bramhi (Baccopa Monniera) is often used as an ayurvedic treatment for Alzheimer''s disease. Therefore it is of interest to study the interaction of compounds derived from Baccopa with the Tau protein involved in tangle formation. We show that compounds such as bacopaside II, bacopaside XII, and nicotine showed optimal binding features with the R2 repeat domain of hyperphosphorylated tau protein for further consideration in the context of Alzheimer''s disease (AD).     

Modelling neurodegenerative diseases with 3D brain organoids

Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post‐mortem brain tissue, non‐human primate tissue, or in vitro two‐dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three‐dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.     

Comparison of force fields for Alzheimer's A : A case study for intrinsically disordered proteins

Intrinsically disordered proteins are essential for biological processes such as cell signalling, but are also associated to devastating diseases including Alzheimer's disease, Parkinson's disease or type II diabetes. Because of their lack of a stable three‐dimensional structure, molecular dynamics simulations are often used to obtain atomistic details that cannot be observed experimentally. The applicability of molecular dynamics simulations depends on the accuracy of the force field chosen to represent the underlying free energy surface of the system. Here, we use replica exchange molecular dynamics simulations to test five modern force fields, OPLS, AMBER99SB, AMBER99SB*ILDN, AMBER99SBILDN‐NMR and CHARMM22*, in their ability to model Aβ₄₂, an intrinsically disordered peptide associated with Alzheimer's disease, and compare our results to nuclear magnetic resonance (NMR) experimental data. We observe that all force fields except AMBER99SBILDN‐NMR successfully reproduce local NMR observables, with CHARMM22* being slightly better than the other force fields.     

Aggresomes——错误折叠蛋白的一种普遍细胞反应

通常,细胞中的错误折叠蛋白质会被蛋白酶体降解。但是在一定的病理和生理条件下,一些错误折叠蛋白质几乎不被降解,反而可以形成蛋白质聚集体。研究表明,许多疾病,如神经退行性疾病的发病机理与错误折叠蛋白质在细胞内的聚集体沉积有关。这些蛋白质聚集体可以通过微管上动力蛋白依赖的逆行性运输形式传送、聚集,最终形成aggresomes。早新的报道还指出,蛋白质聚集体能直接损伤泛素—蛋白酶体系统的功能,从而引起细胞的调控紊乱和细胞死亡。     

Immunoglobulin G transport increases in an in vitro blood–brain barrier model with amyloid-β and with neuroinflammatory cytokines

Immunotherapies are a promising strategy for the treatment of neurological diseases such as Alzheimer's disease (AD), however, transport of antibodies to the brain is severely restricted by the blood–brain barrier (BBB). Furthermore, molecular transport at the BBB is altered in disease, which may affect the mechanism and quantity of therapeutic antibody transport. To better understand the transport of immunotherapies at the BBB in disease, an in vitro BBB model derived from human induced pluripotent stem cells (iPSCs) was used to investigate the endocytic uptake route of immunoglobulin G (IgG). In this model, uptake of fluorescently labeled IgGs is a saturable process. Inhibition of clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis demonstrated that macropinocytosis is a major transport route for IgGs at the BBB. IgG uptake and transport were increased after the addition of stimuli to mimic AD (Aβ_1–40 and Aβ_1–42) and neuroinflammation (tumor necrosis factor-α and interleukin-6). Lastly, caveolar endocytosis increased in the AD model, which may be responsible for the increase in IgG uptake in disease. This study presents an iPSC-derived BBB model that responds to disease stimuli with physiologically relevant changes to molecular transport and can be used to understand fundamental questions about transport mechanisms of immunotherapies in health and neurodegenerative disease.     

When amyloids become prions

The conformational diseases, linked to protein aggregation into amyloid conformations, range from non-infectious neurodegenerative disorders, such as Alzheimer''s disease (AD), to highly infectious ones, such as human transmissible spongiform encephalopathies (TSEs). They are commonly known as prion diseases. However, since all amyloids could be considered prions (from those involved in cell-to-cell transmission to those responsible for real neuronal invasion), it is necessary to find an underlying cause of the different capacity to infect that each of the proteins prone to form amyloids has. As proposed here, both the intrinsic cytotoxicity and the number of nuclei of aggregation per cell could be key factors in this transmission capacity of each amyloid.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Novel compounds for the modulation of mTOR and autophagy to treat neurodegenerative diseases

Most neurodegenerative diseases show a disruption of autophagic function and display abnormal accumulation of toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. The phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway, which promotes cellular survival, is the main modulator upstream of mTOR, and alterations in this pathway are common in neurodegenerative diseases, e.g. Alzheimer’s disease (AD) and Parkinson’s disease (PD). In the present work we revised mammalian target of rapamycin complex 1 (mTORC1) pathway and mTORC2 as a complementary an important element in mTORC1 signaling. In addition, we revised the extracellular signal regulated kinase (ERK) pathway, which has become relevant in the regulation of the autophagic process and cellular survival through mTORC2 signaling. Finally, we summarize novel compounds that promote autophagy and neuronal protection in the last five years.