ß-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits.

Background: Alzheimer's Disease (AD) is the leading cause of dementia globally, affecting around 50 million people and marked by cognitive decline and the accumulation of ß-amyloid plaques and hyperphosphorylated tau. The limited treatment options and numerous failed clinical trials targeting ß-amyloid (Aß) highlight the need for novel approaches. Lowered proteasome activity is a consistent feature in AD, particularly in the hippocampus. Impaired proteasome function in AD is hypothesized to stem from direct inhibition by ß-amyloid or hyperphosphorylated tau, disrupting critical neuronal processes such as memory formation and synaptic plasticity. Objectives: This study tests the hypothesis that AD related deficits are driven in part by impaired proteasome function as a consequence of inhibition by Aß. We evaluated how proteasome function is modulated by Aß and the capacity of two proteasome-activating compounds, TAT1-8,9-TOD and TAT1-DEN to rescue Aß-induced impairment in vitro, as well as survival deficits in cell culture and Aß-induced cognitive deficits in Drosophila and mouse models. Results: Our study demonstrates that oligomeric ß-amyloid binds to the 20S proteasome and impairs its activity and conformational stability. The oligomers also destabilize the 26S proteasome to release the free 20S proteasome. Treatment with proteasome activators TAT1-8,9TOD and TAT1-DEN rescue the 20S proteasome function and reduces cell death caused by Aß42 toxicity in SK-N-SH cells. In Drosophila models overexpressing Aß42, oral administration of proteasome agonists delayed mortality and restored cognitive function. Chronic treatment with TAT1-DEN protected against deficits in working memory caused by Aß42 in mice and in hAPP(J20) mice with established deficits, acute TAT1-DEN treatment significantly improved spatial learning, with treated mice performing comparably to controls. Conclusions: Aß has dual impacts on 20S and 26S proteasome function and stability. Proteasome activation using TAT1-8,9TOD and TAT1-DEN shows promise in mitigating AD-like deficits by protecting against amyloid toxicity and enhancing proteasome function. These findings suggest that targeting proteasome activity could be a viable therapeutic approach for AD, warranting further investigation into the broader impacts of proteasome modulation on AD pathology.

Altered Proteasome Composition in Aging Brains, Genetic Proteasome Augmentation Mitigates Age-Related Cognitive Declines, and Acute Proteasome Agonist Treatment Rescues Age-Related Cognitive Deficits in Mice.

The aging brain experiences a significant decline in proteasome function, The proteasome is critical for many key neuronal functions including neuronal plasticity, and memory formation/retention. Treatment with proteasome inhibitors impairs these processes. Our study reveals a marked reduction in 20S and 26S proteasome activities in aged mice brains driven by reduced functionality of aged proteasome. This is matched by a decline in 20S proteasome but an increase in 26S proteasome. Our data suggests this may be a compensatory response to reduced functionality. By overexpressing the proteasome subunit PSMB5 in the neurons of mice, enhancing proteasome function, we slowed age-related declines in spatial learning and memory as well neuromuscular declines. We then showed acute treatment with a proteasome activator to rescue spatial learning and memory deficits in aged mice. These findings highlight the potential of proteasome augmentation as a therapeutic strategy to mitigate age-related cognitive declines.

Subsyndromes and symptom clusters: Multilevel factor analysis of behavioral and psychological symptoms of dementia with intensive longitudinal data.

INTRODUCTION: Behavioral and psychological symptoms in dementia (BPSD) are dynamic phenomena with a high amount of intraindividual variability. We applied a multilevel framework to identify subsyndromes (between-person factors) that represent clinically relevant profiles of BPSD and identify symptom clusters (within-person factors) that represent contextually driven daily symptom experiences. METHODS: This study used an intensive longitudinal design in which 68 co-residing family caregivers to persons living with dementia were recruited to proxy report on their care recipient's daily symptom experiences of 23 different BPSD for eight consecutive days (n = 443 diaries). A multilevel exploratory/confirmatory factor analysis was used to account for nested data and separate within-person variances from between-level factor estimates. RESULTS: Exploratory factor analysis identified a 4-between 3-within factor structure based on fit statistics and clinical interpretability. DISCUSSION: This study offers major methodological and conceptual advancements for management of BPSD within Alzheimer's disease and related dementias by introducing two related but distinct concepts of subsyndromes and symptom clusters. HIGHLIGHTS: Because behavioral and psychological symptoms of dementia (BPSD) are dynamic temporal phenomenon, this introduces measurement error into aggregate group-level estimates when trying to create subsyndromes. We propose a multilevel analysis to provide a more valid and reliable estimation by separating out variance due to within-person daily fluctuations. Using a multilevel exploratory factor analysis with intensive longitudinal data, we identified distinct and meaningful groups of BPSD. The four factors at the between-person level represented subsyndromes that are based on how BPSD co-occurred among persons with Alzheimer's disease (AD). These subsyndromes are clinically relevant because they share features of established clinical phenomena and may have similar neurobiological etiologies. We also found three within-person factors representing distinct symptom clusters. They are based on how BPSD clustered together on a given day for an individual with AD and related dementias. These clusters may have shared environmental triggers.

Early-adulthood spike in protein translation drives aging via juvenile hormone/germline signaling.

Protein translation (PT) declines with age in invertebrates, rodents, and humans. It has been assumed that elevated PT at young ages is beneficial to health and PT ends up dropping as a passive byproduct of aging. In Drosophila, we show that a transient elevation in PT during early-adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later-life. Blocking the early-life PT elevation robustly improves life-/health-span and prevents age-related protein aggregation, whereas transiently inducing an early-life PT surge in long-lived fly strains abolishes their longevity/proteostasis benefits. The early-life PT elevation triggers proteostatic dysfunction, silences stress responses, and drives age-related functional decline via juvenile hormone-lipid transfer protein axis and germline signaling. Our findings suggest that PT is adaptively suppressed after early-adulthood, alleviating later-life proteostatic burden, slowing down age-related functional decline, and improving lifespan. Our work provides a theoretical framework for understanding how lifetime PT dynamics shape future aging trajectories.

The proteasome: A key modulator of nervous system function, brain aging, and neurodegenerative disease.

The proteasome is a large multi-subunit protease responsible for the degradation and removal of oxidized, misfolded, and polyubiquitinated proteins. The proteasome plays critical roles in nervous system processes. This includes maintenance of cellular homeostasis in neurons. It also includes roles in long-term potentiation via modulation of CREB signaling. The proteasome also possesses roles in promoting dendritic spine growth driven by proteasome localization to the dendritic spines in an NMDA/CaMKIIα dependent manner. Proteasome inhibition experiments in varied organisms has been shown to impact memory, consolidation, recollection and extinction. The proteasome has been further shown to impact circadian rhythm through modulation of a range of 'clock' genes, and glial function. Proteasome function is impaired as a consequence both of aging and neurodegenerative diseases. Many studies have demonstrated an impairment in 26S proteasome function in the brain and other tissues as a consequence of age, driven by a disassembly of 26S proteasome in favor of 20S proteasome. Some studies also show proteasome augmentation to correct age-related deficits. In amyotrophic lateral sclerosis Alzheimer's, Parkinson's and Huntington's disease proteasome function is impaired through distinct mechanisms with impacts on disease susceptibility and progression. Age and neurodegenerative-related deficits in the function of the constitutive proteasome are often also accompanied by an increase in an alternative form of proteasome called the immunoproteasome. This article discusses the critical role of the proteasome in the nervous system. We then describe how proteasome dysfunction contributes to brain aging and neurodegenerative disease.

Protein translation paradox: Implications in translational regulation of aging.

Protein translation is an essential cellular process playing key roles in growth and development. Protein translation declines over the course of age in multiple animal species, including nematodes, fruit flies, mice, rats, and even humans. In all these species, protein translation transiently peaks in early adulthood with a subsequent drop over the course of age. Conversely, lifelong reductions in protein translation have been found to extend lifespan and healthspan in multiple animal models. These findings raise the protein synthesis paradox: age-related declines in protein synthesis should be detrimental, but life-long reductions in protein translation paradoxically slow down aging and prolong lifespan. This article discusses the nature of this paradox and complies an extensive body of work demonstrating protein translation as a modulator of lifespan and healthspan.

Genetic and pharmacologic proteasome augmentation ameliorates Alzheimer's-like pathology in mouse and fly APP overexpression models.

The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer's disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome's increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.

Mitochondrial TrxR2 regulates metabolism and protects from metabolic disease through enhanced TCA and ETC function.

Mitochondrial dysfunction is a key driver of diabetes and other metabolic diseases. Mitochondrial redox state is highly impactful to metabolic function but the mechanism driving this is unclear. We generated a transgenic mouse which overexpressed the redox enzyme Thioredoxin Reductase 2 (TrxR2), the rate limiting enzyme in the mitochondrial thioredoxin system. We found augmentation of TrxR2 to enhance metabolism in mice under a normal diet and to increase resistance to high-fat diet induced metabolic dysfunction by both increasing glucose tolerance and decreasing fat deposition. We show this to be caused by increased mitochondrial function which is driven at least in part by enhancements to the tricarboxylic acid cycle and electron transport chain function. Our findings demonstrate a role for TrxR2 and mitochondrial thioredoxin as metabolic regulators and show a critical role for redox enzymes in controlling functionality of key mitochondrial metabolic systems.

Gut- and oral-dysbiosis differentially impact spinal- and bulbar-onset ALS, predicting ALS severity and potentially determining the location of disease onset.

BACKGROUND: Prior studies on the role of gut-microbiome in Amyotrophic Lateral Sclerosis (ALS) pathogenesis have yielded conflicting results. We hypothesized that gut- and oral-microbiome may differentially impact two clinically-distinct ALS subtypes (spinal-onset ALS (sALS) vs. bulbar-onset ALS (bALS), driving disagreement in the field. METHODS: ALS patients diagnosed within 12 months and their spouses as healthy controls (n = 150 couples) were screened. For eligible sALS and bALS patients (n = 36) and healthy controls (n = 20), 16S rRNA next-generation sequencing was done in fecal and saliva samples after DNA extractions to examine gut- and oral-microbiome differences. Microbial translocation to blood was measured by blood lipopolysaccharide-binding protein (LBP) and 16S rDNA levels. ALS severity was assessed by Revised ALS Functional Rating Scale (ALSFRS-R). RESULTS: sALS patients manifested significant gut-dysbiosis, primarily driven by increased fecal Firmicutes/Bacteroidetes-ratio (F/B-ratio). In contrast, bALS patients displayed significant oral-dysbiosis, primarily driven by decreased oral F/B-ratio. For sALS patients, gut-dysbiosis (a shift in fecal F/B-ratio), but not oral-dysbiosis, was strongly associated with greater microbial translocation to blood (r = 0.8006, P < 0.0001) and more severe symptoms (r = 0.9470, P < 0.0001). In contrast, for bALS patients, oral-dysbiosis (a shift in oral F/B-ratio), but not gut-dysbiosis, was strongly associated with greater microbial translocation to blood (r = 0.9860, P < 0.0001) and greater disease severity (r = 0.9842, P < 0.0001). For both ALS subtypes, greater microbial translocation was associated with more severe symptoms (sALS: r = 0.7924, P < 0.0001; bALS: r = 0.7496, P = 0.0067). Importantly, both sALS and bALS patients displayed comparable oral-motor deficits with associations between oral-dysbiosis and severity of oral-motor deficits in bALS but not sALS. This suggests that oral-dysbiosis is not simply caused by oral/bulbar/respiratory symptoms but represents a pathological driver of bALS. CONCLUSIONS: We found increasing gut-dysbiosis with worsening symptoms in sALS patients and increasing oral-dysbiosis with worsening symptoms in bALS patients. Our findings support distinct microbial mechanisms underlying two ALS subtypes, which have been previously grouped together as a single disease. Our study suggests correcting gut-dysbiosis as a therapeutic strategy for sALS patients and correcting oral-dysbiosis as a therapeutic strategy for bALS patients.

Tumour-reprogrammed stromal BCAT1 fuels branched-chain ketoacid dependency in stromal-rich PDAC tumours.

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-ß-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

New Peptide-Based Pharmacophore Activates 20S Proteasome.

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs' pharmacophore consists of an N-terminal basic pocket-docking "activation anchor" connected via a ß turn inducer to a C-terminal "specificity clamp" that binds on the proteasome α surface. By allosteric effects-including destabilization of the proteasomal gate-the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

Neuronal-specific proteasome augmentation via Prosß5 overexpression extends lifespan and reduces age-related cognitive decline.

Cognitive function declines with age throughout the animal kingdom, and increasing evidence shows that disruption of the proteasome system contributes to this deterioration. The proteasome has important roles in multiple aspects of the nervous system, including synapse function and plasticity, as well as preventing cell death and senescence. Previous studies have shown neuronal proteasome depletion and inhibition can result in neurodegeneration and cognitive deficits, but it is unclear if this pathway is a driver of neurodegeneration and cognitive decline in aging. We report that overexpression of the proteasome ß5 subunit enhances proteasome assembly and function. Significantly, we go on to show that neuronal-specific proteasome augmentation slows age-related declines in measures of learning, memory, and circadian rhythmicity. Surprisingly, neuronal-specific augmentation of proteasome function also produces a robust increase of lifespan in Drosophila melanogaster. Our findings appear specific to the nervous system; ubiquitous proteasome overexpression increases oxidative stress resistance but does not impact lifespan and is detrimental to some healthspan measures. These findings demonstrate a key role of the proteasome system in brain aging.

Cause or casualty: The role of mitochondrial DNA in aging and age-associated disease.

The mitochondrial genome (mtDNA) represents a tiny fraction of the whole genome, comprising just 16.6 kilobases encoding 37 genes involved in oxidative phosphorylation and the mitochondrial translation machinery. Despite its small size, much interest has developed in recent years regarding the role of mtDNA as a determinant of both aging and age-associated diseases. A number of studies have presented compelling evidence for key roles of mtDNA in age-related pathology, although many are correlative rather than demonstrating cause. In this review we will evaluate the evidence supporting and opposing a role for mtDNA in age-associated functional declines and diseases. We provide an overview of mtDNA biology, damage and repair as well as the influence of mitochondrial haplogroups, epigenetics and maternal inheritance in aging and longevity.

Proline- and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity.

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.

Mitochondrial thioredoxin reductase 2 is elevated in long-lived primate as well as rodent species and extends fly mean lifespan.

In a survey of enzymes related to protein oxidation and cellular redox state, we found activity of the redox enzyme thioredoxin reductase (TXNRD) to be elevated in cells from long-lived species of rodents, primates, and birds. Elevated TXNRD activity in long-lived species reflected increases in the mitochondrial form, TXNRD2, rather than the cytosolic forms TXNRD1 and TXNRD3. Analysis of published RNA-Seq data showed elevated TXNRD2 mRNA in multiple organs of longer-lived primates, suggesting that the phenomenon is not limited to skin-derived fibroblasts. Elevation of TXNRD2 activity and protein levels was also noted in liver of three different long-lived mutant mice, and in normal male mice treated with a drug that extends lifespan in males. Overexpression of mitochondrial TXNRD2 in Drosophila melanogaster extended median (but not maximum) lifespan in female flies with a small lifespan extension in males; in contrast, overexpression of the cytosolic form, TXNRD1, did not produce a lifespan extension.

Lifespan of mice and primates correlates with immunoproteasome expression.

There is large variation in lifespan among different species, and there is evidence that modulation of proteasome function may contribute to longevity determination. Comparative biology provides a powerful tool for identifying genes and pathways that control the rate of aging. Here, we evaluated skin-derived fibroblasts and demonstrate that among primate species, longevity correlated with an elevation in proteasomal activity as well as immunoproteasome expression at both the mRNA and protein levels. Immunoproteasome enhancement occurred with a concurrent increase in other elements involved in MHC class I antigen presentation, including ß-2 microglobulin, (TAP1), and TAP2. Fibroblasts from long-lived primates also appeared more responsive to IFN-γ than cells from short-lived primate species, and this increase in IFN-γ responsiveness correlated with elevated expression of the IFN-γ receptor protein IFNGR2. Elevation of immunoproteasome and proteasome activity was also observed in the livers of long-lived Snell dwarf mice and in mice exposed to drugs that have been shown to extend lifespan, including rapamycin, 17-α-estradiol, and nordihydroguaiaretic acid. This work suggests that augmented immunoproteasome function may contribute to lifespan differences in mice and among primate species.

Fibroblasts From Longer-Lived Species of Primates, Rodents, Bats, Carnivores, and Birds Resist Protein Damage.

Species differ greatly in their rates of aging. Among mammalian species life span ranges from 2 to over 60 years. Here, we test the hypothesis that skin-derived fibroblasts from long-lived species of animals differ from those of short-lived animals in their defenses against protein damage. In parallel studies of rodents, nonhuman primates, birds, and species from the Laurasiatheria superorder (bats, carnivores, shrews, and ungulates), we find associations between species longevity and resistance of proteins to oxidative stress after exposure to H(2)O(2) or paraquat. In addition, baseline levels of protein carbonyl appear to be higher in cells from shorter-lived mammals compared with longer-lived mammals. Thus, resistance to protein oxidation is associated with species maximal life span in independent clades of mammals, suggesting that this cellular property may be required for evolution of longevity. Evaluation of the properties of primary fibroblast cell lines can provide insights into the factors that regulate the pace of aging across species of mammals.

A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster.

In mammalian cells, hydrogen peroxide (H(2)O(2))-induced adaptation to oxidative stress is strongly dependent on an Nrf2 transcription factor-mediated increase in the 20S proteasome. Here, we report that both Caenorhabditis elegans nematode worms and Drosophila melanogaster fruit flies are also capable of adapting to oxidative stress with H(2)O(2) pre-treatment. As in mammalian cells, this adaptive response in worms and flies involves an increase in proteolytic activity and increased expression of the 20S proteasome, but not of the 26S proteasome. We also found that the increase in 20S proteasome expression in both worms and flies, as in mammalian cells, is important for the adaptive response, and that it is mediated by the SKN-1 and CNC-C orthologs of the mammalian Nrf2 transcription factor, respectively. These studies demonstrate that stress mechanisms operative in cell culture also apply in disparate intact organisms across a wide biological diversity.

Oxidative stress adaptation with acute, chronic, and repeated stress.

Oxidative stress adaptation, or hormesis, is an important mechanism by which cells and organisms respond to, and cope with, environmental and physiological shifts in the level of oxidative stress. Most studies of oxidative stress adaption have been limited to adaptation induced by acute stress. In contrast, many if not most environmental and physiological stresses are either repeated or chronic. In this study we find that both cultured mammalian cells and the fruit fly Drosophila melanogaster are capable of adapting to chronic or repeated stress by upregulating protective systems, such as their proteasomal proteolytic capacity to remove oxidized proteins. Repeated stress adaptation resulted in significant extension of adaptive responses. Repeated stresses must occur at sufficiently long intervals, however (12-h or more for MEF cells and 7 days or more for flies), for adaptation to be successful, and the levels of both repeated and chronic stress must be lower than is optimal for adaptation to acute stress. Regrettably, regimens of adaptation to both repeated and chronic stress that were successful for short-term survival in Drosophila nevertheless also caused significant reductions in life span for the flies. Thus, although both repeated and chronic stress can be tolerated, they may result in a shorter life.

Degradation of damaged proteins: the main function of the 20S proteasome.

Cellular proteins are exposed to oxidative modification and other forms of damage through oxidative stress and disease, and as a consequence of aging. This oxidative damage results in loss and/or modification of protein function, which in turn compromises cell function and may even cause cell death. Therefore, the removal of damaged proteins is extremely important for the maintenance of normal cell function. The 20S proteasome functions primarily as a system for removal of such damaged proteins. Unlike the 26S proteasome, the 20S proteasome exhibits a high degree of selectivity in degrading the oxidized, or otherwise damaged, forms of cell proteins. The 20S proteasome is broadly distributed throughout the cell and has a range of specific functions in different organelles, which are controlled through a number of proteasome regulators. It is also activated, and its synthesis is induced, under conditions of enhanced oxidative stress, thus permitting greater removal of damaged proteins.