Interplay of Proteostasis Capacity and Protein Aggregation: Implications for Cellular Function and Disease.

Eukaryotic cells are equipped with an intricate proteostasis network (PN), comprising nearly 3,000 components dedicated to preserving proteome integrity and sustaining protein homeostasis. This protective system is particularly important under conditions of external and intrinsic cell stress, where inherently dynamic proteins may unfold and lose functionality. A decline in proteostasis capacity is associated with the aging process, resulting in a reduced folding efficiency of newly synthesized proteins and a deficit in the cellular capacity to degrade misfolded proteins. A critical consequence of PN insufficiency is the accumulation of cytotoxic protein aggregates that underlie various age-related neurodegenerative conditions and other pathologies. By interfering with specific proteostasis components, toxic aggregates place an excessive burden on the PN's ability to maintain proteome integrity. This initiates a feed-forward loop, wherein the generation of misfolded and aggregated proteins ultimately leads to proteostasis collapse and cellular demise.

A Multiscale Closed-Loop Neurotoxicity Model of Alzheimer's Disease Progression Explains Functional Connectivity Alterations.

The accumulation of amyloid-ß (Aß) and hyperphosphorylated-tau (hp-tau) are two classical histopathological biomarkers in Alzheimer's disease (AD). However, their detailed interactions with the electrophysiological changes at the meso- and macroscale are not yet fully understood. We developed a mechanistic multiscale model of AD progression, linking proteinopathy to its effects on neural activity and vice-versa. We integrated a heterodimer model of prion-like protein propagation and a brain network model of Jansen-Rit neural masses derived from human neuroimaging data whose parameters varied due to neurotoxicity. Results showed that changes in inhibition guided the electrophysiological alterations found in AD, and these changes were mainly attributed to Aß effects. Additionally, we found a causal disconnection between cellular hyperactivity and interregional hypersynchrony contrary to previous beliefs. Finally, we demonstrated that early Aß and hp-tau depositions' location determine the spatiotemporal profile of the proteinopathy. The presented model combines the molecular effects of both Aß and hp-tau together with a mechanistic protein propagation model and network effects within a closed-loop model. This holds the potential to enlighten the interplay between AD mechanisms on various scales, aiming to develop and test novel hypotheses on the contribution of different AD-related variables to the disease evolution.

[The new age of neurodegenerative diseases. The basis of the new approaches]. / La nueva era de las enfermedades neurodegenerativas. La base de los nuevos abordajes.

The detection by biomarkers of the pathophysiological and molecular processes involved in misfolding protein diseases making it possible to delineate the natural history of these processes. The great majority of protein misfolding diseases have a prolonged preclinical phase, in which the biological changes are patent. The clinical manifestations (i.e., phenotypes) do not have a univocal correspondence with the underlying pathology, despite the fact that pathological eponyms have been used for the description of the clinical syndromes, which has favored diagnostic inaccuracy. In order to perform an adequate clinical management, we must know the 3 planes that currently define the most common neurodegenerative processes. Diagnostic accuracy will be a prerequisite for new therapies aimed at modifying the course of brain protein misfolding diseases.

TITLE: La nueva era de las enfermedades neurodegenerativas. La base de los nuevos abordajes.La detección por biomarcadores de los procesos fisiopatológicos y moleculares implicados en las enfermedades cerebrales por plegamiento anormal de proteínas está permitiendo delinear la historia natural de estos procesos. La gran mayoría de ellos tiene una fase preclínica prolongada, en la que los cambios biológicos son patentes. Las manifestaciones clínicas (fenotipos) no tienen una correspondencia unívoca con la patología subyacente, a pesar de que se han utilizado los epónimos anatomopatológicos para la descripción de los síndromes clínicos, lo que ha favorecido la imprecisión diagnóstica. Para realizar un adecuado manejo clínico debemos conocer los tres planos que definen actualmente los procesos neurodegenerativos más frecuentes. La precisión diagnóstica será un prerrequisito para las nuevas terapias dirigidas a modificar el curso de las enfermedades por plegamiento proteico cerebrales.

Extracellular protein homeostasis in neurodegenerative diseases.

The protein homeostasis (proteostasis) system encompasses the cellular processes that regulate protein synthesis, folding, concentration, trafficking and degradation. In the case of intracellular proteostasis, the identity and nature of these processes have been extensively studied and are relatively well known. By contrast, the mechanisms of extracellular proteostasis are yet to be fully elucidated, although evidence is accumulating that their age-related progressive impairment might contribute to neuronal death in neurodegenerative diseases. Constitutively secreted extracellular chaperones are emerging as key players in processes that operate to protect neurons and other brain cells by neutralizing the toxicity of extracellular protein aggregates and promoting their safe clearance and disposal. Growing evidence indicates that these extracellular chaperones exert multiple effects to promote cell viability and protect neurons against pathologies arising from the misfolding and aggregation of proteins in the synaptic space and interstitial fluid. In this Review, we outline the current knowledge of the mechanisms of extracellular proteostasis linked to neurodegenerative diseases, and we examine the latest understanding of key molecules and processes that protect the brain from the pathological consequences of extracellular protein aggregation and proteotoxicity. Finally, we contemplate possible therapeutic opportunities for neurodegenerative diseases on the basis of this emerging knowledge.

Current Concepts of Mixed Pathologies in Neurodegenerative Diseases.

Neurodegenerative diseases are a pathologically, clinically and genetically diverse group of disorders without effective disease-modifying therapies. Pathologically, these disorders are characterised by disease-specific protein aggregates in neurons and/or glia and referred to as proteinopathies. Many neurodegenerative diseases show pathological overlap with the same abnormally deposited protein occurring in anatomically distinct regions, which give rise to specific patterns of cognitive and motor clinical phenotypes. Sequential distribution patterns of protein inclusions throughout the brain have been described. Rather than occurring in isolation, it is increasingly recognised that combinations of one or more proteinopathies with or without cerebrovascular disease frequently occur in individuals with neurodegenerative diseases. In addition, complex constellations of ageing-related and incidental pathologies associated with tau, TDP-43, Aß, α-synuclein deposition have been commonly reported in longitudinal ageing studies. This review provides an overview of current classification of neurodegenerative and age-related pathologies and presents the spectrum and complexity of mixed pathologies in community-based, longitudinal ageing studies, in major proteinopathies, and genetic conditions. Mixed pathologies are commonly reported in individuals >65 years with and without cognitive impairment; however, they are increasingly recognised in younger individuals (<65 years). Mixed pathologies are thought to lower the threshold for developing cognitive impairment and dementia. Hereditary neurodegenerative diseases also show a diverse range of mixed pathologies beyond the proteinopathy primarily linked to the genetic abnormality. Cases with mixed pathologies might show a different clinical course, which has prognostic relevance and obvious implications for biomarker and therapy development, and stratifying patients for clinical trials.

Misfolding-induced chronic pancreatitis in CPA1 N256K mutant mice is unaffected by global deletion of Ddit3/Chop.

Genetic mutations in pancreatic digestive enzymes may cause protein misfolding, endoplasmic reticulum (ER) stress and chronic pancreatitis. The CPA1 N256K mouse model carries the human p.N256K carboxypeptidase A1 (CPA1) mutation, a classic example of a pancreatitis-associated misfolding variant. CPA1 N256K mice develop spontaneous, progressive chronic pancreatitis with moderate acinar atrophy, acinar-to-ductal metaplasia, fibrosis, and macrophage infiltration. Upregulation of the ER-stress associated pro-apoptotic transcription factor Ddit3/Chop mRNA was observed in the pancreas of CPA1 N256K mice suggesting that acinar cell death might be mediated through this mechanism. Here, we crossed the CPA1 N256K strain with mice containing a global deletion of the Ddit3/Chop gene (Ddit3-KO mice) and evaluated the effect of DDIT3/CHOP deficiency on the course of chronic pancreatitis. Surprisingly, CPA1 N256K x Ddit3-KO mice developed chronic pancreatitis with a similar time course and features as the CPA1 N256K parent strain. In contrast, Ddit3-KO mice showed no pancreas pathology. The observations indicate that DDIT3/CHOP plays no significant role in the development of misfolding-induced chronic pancreatitis in CPA1 N256K mice and this transcription factor is not a viable target for therapeutic intervention in this disease.

Molecular Mechanisms of Amylin Turnover, Misfolding and Toxicity in the Pancreas.

Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet ß-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of ß-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP-membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed.

Involvement of molecular chaperone in protein-misfolding brain diseases.

Protein misfolding causes aggregation and build-up in a variety of brain diseases. There are numeral molecules that are linked with the protein homeostasis mechanism. Molecular chaperones are one of such molecules that are responsible for protection against protein misfolded and aggregation-induced neurotoxicity. Many studies have explored the participation of molecular chaperones in Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, and Huntington's diseases. In this review, we highlighted the constructive role of molecular chaperones in neurological diseases characterized by protein misfolding and aggregation and their capability to control aberrant protein interactions at an early stage thus successfully suppressing pathogenic cascades. A comprehensive understanding of the protein misfolding associated with brain diseases and the molecular basis of involvement of chaperone against aggregation-induced cellular stress might lead to the progress of new therapeutic intrusion-related to protein misfolding and aggregation.

Emerging fluorescence tools for the study of proteostasis in cells.

Understanding how cells maintain the functional proteome and respond to stress conditions is critical for deciphering molecular pathogenesis and developing treatments for conditions such as neurodegenerative diseases. Efforts towards finer quantification of cellular proteostasis machinery efficiency, phase transitions and local environment changes remain a priority. Herein, we describe recent developments in fluorescence-based strategy and methodology, building on the experimental toolkit, for the study of proteostasis (protein homeostasis) in cells. We hope this review can assist in bridging gaps between a multitude of research disciplines and promote interdisciplinary collaboration to address the crucial topic of proteostasis.

Environmental factors modulating protein conformations and their role in protein aggregation diseases.

The adverse physiological conditions have been long known to impact protein synthesis, folding and functionality. Major physiological factors such as the effect of pH, temperature, salt and pressure are extensively studied for their impact on protein structure and homeostasis. However, in the current scenario, the environmental risk factors (pollutants) have gained impetus in research because of their increasing concentrations in the environment and strong epidemiologic link with protein aggregation disorders. Here, we review the physiological and environmental risk factors for their impact on protein conformational changes, misfolding, aggregation, and associated pathological conditions, especially environmental risk factors associated pathologies.

Carrying Excess Baggage Can Slowdown Life: Protein Clearance Machineries That Go Awry During Aging and the Relevance of Maintaining Them.

Cellular homeostasis is maintained by rapid and systematic cleansing of aberrant and aggregated proteins within cells. Neurodegenerative diseases (NDs) especially Parkinson's and Alzheimer's disease are known to be associated with multiple factors, most important being impaired clearance of aggregates, resulting in the accumulation of specific aggregated protein in the brain. Protein quality control (PQC) of proteostasis network comprises proteolytic machineries and chaperones along with their regulators to ensure precise operation and maintenance of proteostasis. Such regulatory factors coordinate among each other multiple functional aspects related to proteins, including their synthesis, folding, transport, and degradation. During aging due to inevitable endogenous and external stresses, sustaining a proteome balance is a challenging task. Such stresses decline the capacity of the proteostasis network compromising the proteome integrity, affecting the fundamental physiological processes including reproductive fitness of the organism. This review focuses on highlighting proteome-wide changes during aging and the strategies for proteostasis improvements. The possibility of augmenting the proteostasis network either via genetic or pharmacological interventions may be a promising strategy towards delaying age-associated pathological consequences due to proteome disbalance, thus promoting healthy aging and prolonged longevity.

Differential Accumulation of Misfolded Prion Strains in Natural Hosts of Prion Diseases.

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative protein misfolding diseases that invariably cause death. TSEs occur when the endogenous cellular prion protein (PrPC) misfolds to form the pathological prion protein (PrPSc), which templates further conversion of PrPC to PrPSc, accumulates, and initiates a cascade of pathologic processes in cells and tissues. Different strains of prion disease within a species are thought to arise from the differential misfolding of the prion protein and have different clinical phenotypes. Different strains of prion disease may also result in differential accumulation of PrPSc in brain regions and tissues of natural hosts. Here, we review differential accumulation that occurs in the retinal ganglion cells, cerebellar cortex and white matter, and plexuses of the enteric nervous system in cattle with bovine spongiform encephalopathy, sheep and goats with scrapie, cervids with chronic wasting disease, and humans with prion diseases. By characterizing TSEs in their natural host, we can better understand the pathogenesis of different prion strains. This information is valuable in the pursuit of evaluating and discovering potential biomarkers and therapeutics for prion diseases.

Mitochondrial Dysfunction, Protein Misfolding and Neuroinflammation in Parkinson's Disease: Roads to Biomarker Discovery.

Parkinson's Disease (PD) is a highly prevalent neurodegenerative disease among older adults. PD neuropathology is marked by the progressive loss of the dopaminergic neurons of the substantia nigra pars compacta and the widespread accumulation of misfolded intracellular α-synuclein (α-syn). Genetic mutations and post-translational modifications, such as α-syn phosphorylation, have been identified among the multiple factors supporting α-syn accrual during PD. A decline in the clearance capacity of the ubiquitin-proteasome and the autophagy-lysosomal systems, together with mitochondrial dysfunction, have been indicated as major pathophysiological mechanisms of PD neurodegeneration. The accrual of misfolded α-syn aggregates into soluble oligomers, and the generation of insoluble fibrils composing the core of intraneuronal Lewy bodies and Lewy neurites observed during PD neurodegeneration, are ignited by the overproduction of reactive oxygen species (ROS). The ROS activate the α-syn aggregation cascade and, together with the Lewy bodies, promote neurodegeneration. However, the molecular pathways underlying the dynamic evolution of PD remain undeciphered. These gaps in knowledge, together with the clinical heterogeneity of PD, have hampered the identification of the biomarkers that may be used to assist in diagnosis, treatment monitoring, and prognostication. Herein, we illustrate the main pathways involved in PD pathogenesis and discuss their possible exploitation for biomarker discovery.

Bi-aryl Analogues of Salicylic Acids: Design, Synthesis and SAR Study to Ameliorate Endoplasmic Reticulum Stress.

INTRODUCTION: Endoplasmic reticulum (ER) stress condition is characterized as the accumulation of misfolded or unfolded proteins in lumen of ER. This condition has been implicated in various diseases and pathologies including ß-cell apoptosis, Alzheimer's disease and atherosclerosis. We have reported that hydroxynaphthoic acids (HNA), naphthalene analogues of salicylic acid (SA), reduced ER stress. In this study, we explored structural modification to bi-aryl analogues of SA. METHODS: Palladium-catalyzed cross-coupling was applied to synthesize bi-aryl analogues of SA. Anti-ER stress activity was monitored by using our cell-based assay system where ER stress is induced by tunicamycin. To monitor ER stress markers, ER stress was induced physiologically relevant palmitate system. RESULTS: Many analogues decreased ER stress signal induced by tunicamycin. Compounds creating dihedral angle between Ar group and SA moiety generally increased the activity but gave some cytotoxicity to indicate the crucial role of flat conformation of aromatic region. The best compound (16e) showed up to almost 6-fold and 90-fold better activity than 3-HNA and tauro-ursodeoxycholic acid, positive controls, respectively. ER stress markers such as p-PERK and p-JNK were accordingly decreased in Western blotting upon treatment of 16e under palmitate-induced condition. CONCLUSION: Anti-ER stress activity and toxicity profile of bi-aryl analogues of SA could provide a novel platform for potential therapy for protein misfolding diseases.

Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease.

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc-EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc-EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington's disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.

Quality control of mislocalized and orphan proteins.

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively. Quality control systems are in place to handle these aberrant proteins, and to minimize their detrimental impact on cellular functions. Here, we discuss recent findings on quality control mechanisms handling mislocalized and orphan proteins. We highlight common principles involved in their recognition and summarize how accumulation of these aberrant molecules is associated with aging and disease.

Methods to study the structure of misfolded protein states in systemic amyloidosis.

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

Dual amyloid cross-seeding reveals steric zipper-facilitated fibrillization and pathological links between protein misfolding diseases.

Amyloid cross-seeding, as a result of direct interaction and co-aggregation between different disease-causative peptides, is considered as a main mechanism for the spread of the overlapping pathology across different cells and tissues between different protein-misfolding diseases (PMDs). Despite the biomedical significance of amyloid cross-seeding in amyloidogenesis, it remains a great challenge to discover amyloid cross-seeding systems and reveal their cross-seeding structures and mechanisms. Herein, we are the first to report that GNNQQNY - a short fragment from yeast prion protein Sup35 - can cross-seed with both amyloid-ß (Aß, associated with Alzheimer's disease) and human islet amyloid polypeptide (hIAPP, associated with type II diabetes) to form ß-structure-rich assemblies and to accelerate amyloid fibrillization. Dry, steric ß-zippers, formed by the two ß-sheets of different amyloid peptides, provide generally interactive and structural motifs to facilitate amyloid cross-seeding. The presence of different steric ß-zippers in a variety of GNNQQNY-Aß and GNNQQNY-hIAPP assemblies also explains amyloid polymorphism. In addition, alteration of steric zipper formation by single-point mutations of GNNQQNY and interactions of GNNQQNY with different Aß and hIAPP seeds leads to different amyloid cross-seeding efficiencies, further confirming the existence of cross-seeding barriers. This work offers a better structural-based understanding of amyloid cross-seeding mechanisms linked to different PMDs.