Accumulation Of Toxic Protein Aggregates Research Articles

Exploiting sweet relief for preeclampsia by targeting autophagy-lysosomal machinery and proteinopathy.

The etiology of preeclampsia (PE), a severe complication of pregnancy with several clinical manifestations and a high incidence of maternal and fetal morbidity and mortality, remains unclear. This issue is a major hurdle for effective treatment strategies. We recently demonstrated that PE exhibits an Alzheimer-like etiology of impaired autophagy and proteinopathy in the placenta. Targeting of these pathological pathways may be a novel therapeutic strategy for PE. Stimulation of autophagy with the natural disaccharide trehalose and its lacto analog lactotrehalose in hypoxia-exposed primary human trophoblasts restored autophagy, inhibited the accumulation of toxic protein aggregates, and restored the ultrastructural features of autophagosomes and autolysosomes. Importantly, trehalose and lactotrehalose inhibited the onset of PE-like features in a humanized mouse model by normalizing autophagy and inhibiting protein aggregation in the placenta. These disaccharides restored the autophagy-lysosomal biogenesis machinery by increasing nuclear translocation of the master transcriptional regulator TFEB. RNA-seq analysis of the placentas of mice with PE indicated the normalization of the PE-associated transcriptome profile in response to trehalose and lactotrehalose. In summary, our results provide a novel molecular rationale for impaired autophagy and proteinopathy in patients with PE and identify treatment with trehalose and its lacto analog as promising therapeutic options for this severe pregnancy complication.

Implications of organophosphate pesticides on brain cells and their contribution towardprogression of Alzheimer's disease.

The most widespread neurodegenerative disorder, Alzheimer's disease (AD) is marked by severe behavioral abnormalities, cognitive and functional impairments. It is inextricably linked with the deposition of amyloid β (Aβ) plaques and tau protein in the brain. Loss of white matter, neurons, synapses, and reactive microgliosis are also frequently observed in patients of AD. Although the causative mechanisms behind the neuropathological alterations in AD are not fully understood, they are likely influenced by hereditary and environmental factors. The etiology and pathogenesis of AD are significantly influenced by the cells of the central nervous system, namely, glial cells and neurons, which are directly engaged in the transmission of electrical signals and the processing of information. Emerging evidence suggests that exposure to organophosphate pesticides (OPPs) can trigger inflammatory responses in glial cells, leading to various cascades of events that contribute to neuroinflammation, neuronal damage, and ultimately, AD pathogenesis. Furthermore, there are striking similarities between the biomarkers associated with AD and OPPs, including neuroinflammation, oxidative stress, dysregulation of microRNA, and accumulation of toxic protein aggregates, such as amyloid β. These shared markers suggest a potential mechanistic link between OPP exposure and AD pathology. In this review, we attempt to address the role of OPPs on altered cell physiology of the brain cells leading to neuroinflammation, mitochondrial dysfunction, and oxidative stress linked with AD pathogenesis.

Abstract C091: Harnessing proteostatic vulnerabilities in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) harbors mutations in KRAS in greater than 90% of cases and is characterized by a dense, fibrotic stroma that fosters the formation of a hypoxic, nutrient-poor tumor microenvironment, rendering tumor cells susceptible to proteotoxic stress. In order to prevent endoplasmic reticulum (ER) stress-induced cell death caused by accumulation of toxic protein aggregates, tumor cells must adapt proteostatic mechanisms including activation of the unfolded protein response (UPR) pathway and degradation of misfolded or aggregated proteins through proteasome-dependent or -independent mechanisms, such as ER-specific autophagy (ERphagy). Despite evidence suggesting that tumor cells may experience proteotoxic stress, little is known about the mechanisms pancreatic cancer cells employ to maintain proteostasis. We found that pancreatic cancer cells exhibit KRas-dependent constitutive UPR activation at the expense of ERphagy, suggesting that tumor cells preferentially employ UPR signaling to maintain proteostasis. Accordingly, mutant KRas pancreatic cancer cells have little to no aggregated proteins at baseline as a consequence of UPR activation. Furthermore, upon pharmacological enhancement of ER stress by 26S proteasome inhibition with bortezomib, ERphagy is engaged as a protective mechanism against UPR hyperactivation and apoptosis. In order to target protective ERphagy to promote apoptosis, we suppressed ERphagy activation through siRNA-mediated knockdown of ERphagy receptors under conditions of bortezomib-induced ER stress and found that depletion of CCPG1 but not other ERphagy receptors augmented the growth-suppressive effects of bortezomib due to greater UPR activation and accumulation of aggregated proteins. Overall, our findings implicate context-dependent reliance on both UPR signaling and ERphagy in pancreatic cancer cell survival and identify a targetable cell-intrinsic vulnerability that can leveraged for enhancing the cytotoxic effects of bortezomib, which alone is clinically ineffective in treating PDAC. Citation Format: Sandra Vogt, Dafna Bar-Sagi. Harnessing proteostatic vulnerabilities in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C091.

Abstract P2019: Diastolic Dysfunction In Alzheimer's Disease Is Associated With aβ- Amyloid Aggregate Formation And Mitochondrial Dysfunction

Background: Alzheimer's disease (AD) is a neurodegenerative disease with memory impairment caused by an accumulation of toxic protein aggregates (Aβ- amyloid plaques and tau-derived neurofibrillary tangles). Several studies reported cardiovascular abnormalities with diastolic dysfunction in AD patients; however, the direct mechanism of cardiac dysfunction in AD patients remains unexplored. Therefore, we investigated the pathophysiological mechanism of cardiac dysfunction in the mice model of AD. Hypothesis: Two most commonly used mice models of AD was thoroughly evaluated for cardiac dysfunction, histopathology, and biochemical changes over aging. Methods and Result: To determine the mechanism of AD-associated cardiac dysfunction, we used two different AD mice models: double transgenic mice expressing a chimeric mouse/human amyloid precursor protein and a mutant human presenilin 1 (APP/PS1 Tg), and transgenic mice that is homozygous for a Psen1 mutation and the co-injected APPSwe and tauP301L transgenes (3xTg-AD). Cardiac function measurement using non-invasive echocardiography in aged Ntg and AD mice models showed no difference in systolic cardiac functional parameters. However, diastolic function assessment using Doppler echocardiographic measurements showed ventricular diastolic dysfunction with reduced diastolic filling in APP/PS1 Tg and 3xTg-AD mice. Western blot analysis showed Aβ- amyloid protein expression in APP/PS1 Tg hearts. Immunohistochemical and immunofluorescence staining of the APP/PS1 Tg heart sections further confirmed the Aβ- amyloid protein expression and Aβ- amyloid aggregate formation in the cardiomyocytes. Further evaluation of APP/PS1 Tg hearts demonstrated increased collagen deposition (Picro-Sirius red staining) and increased cardiomyocyte cross-sectional area. Interestingly, APP/PS1 Tg hearts also displayed mitochondrial dysfunction with decreased mitochondrial bioenergetics and significantly reduced mitochondrial function regulatory parameters. Conclusion: Overall, we found diastolic dysfunction development with Aβ- amyloid aggregate formation, fibrotic remodeling, increased cardiomyocyte cross-sectional area, and mitochondrial dysfunction in the AD mice hearts.

Ubiquitin-mediated degradation at the Golgi apparatus.

The Golgi apparatus is an essential organelle of the secretory pathway in eukaryotic cells. It processes secretory and transmembrane proteins and orchestrates their transport to other endomembrane compartments or the plasma membrane. The Golgi apparatus thereby shapes the cell surface, controlling cell polarity, cell-cell communication, and immune signaling. The cytosolic face of the Golgi hosts and regulates signaling cascades, impacting most notably the DNA damage response and mitosis. These essential functions strongly depend on Golgi protein homeostasis and Golgi integrity. Golgi fragmentation and consequent malfunction is associated with neurodegenerative diseases and certain cancer types. Recent studies provide first insight into the critical role of ubiquitin signaling in maintaining Golgi integrity and in Golgi protein quality control. Similar to well described pathways at the endoplasmic reticulum, ubiquitin-dependent degradation of non-native proteins prevents the accumulation of toxic protein aggregates at the Golgi. Moreover, ubiquitination regulates Golgi structural rearrangements in response to cellular stress. Advances in elucidating ubiquitination and degradation events at the Golgi are starting to paint a picture of the molecular machinery underlying Golgi (protein) homeostasis.

Distinct Signaling Pathways for Autophagy-Driven Cell Death and Survival in Adult Hippocampal Neural Stem Cells.

Autophagy is a cellular catabolic process that degrades and recycles cellular materials. Autophagy is considered to be beneficial to the cell and organism by preventing the accumulation of toxic protein aggregates, removing damaged organelles, and providing bioenergetic substrates that are necessary for survival. However, autophagy can also cause cell death depending on cellular contexts. Yet, little is known about the signaling pathways that differentially regulate the opposite outcomes of autophagy. We have previously reported that insulin withdrawal (IW) or corticosterone (CORT) induces autophagic cell death (ACD) in adult hippocampal neural stem (HCN) cells. On the other hand, metabolic stresses caused by 2-deoxy-D-glucose (2DG) and glucose-low (GL) induce autophagy without death in HCN cells. Rather, we found that 2DG-induced autophagy was cytoprotective. By comparing IW and CORT conditions with 2DG treatment, we revealed that ERK and JNK are involved with 2DG-induced protective autophagy, whereas GSK-3β regulates death-inducing autophagy. These data suggest that cell death and survival-promoting autophagy undergo differential regulation with distinct signaling pathways in HCN cells.

Novel therapeutic approaches to target neurodegeneration.

Ageing is the main risk factor common to most primary neurodegenerative disorders. Indeed, age-related brain alterations have been long considered to predispose to neurodegeneration. Although protein misfolding and the accumulation of toxic protein aggregates have been considered as causative events in neurodegeneration, several other biological pathways affected by brain ageing also contribute to pathogenesis. Here, we discuss the evidence showing the involvement of the mechanisms controlling neuronal structure, gene expression, autophagy, cell metabolism and neuroinflammation in the onset and progression of neurodegenerative disorders. Furthermore, we review the therapeutic strategies currently under development or as future approaches designed to normalize these pathways, which may then increase brain resilience to cope with toxic protein species. In addition to therapies targeting the insoluble protein aggregates specifically associated with each neurodegenerative disorder, these novel pharmacological approaches may be part of combined therapies designed to rescue brain function.

Multiparameter phenotypic screening for endogenous TFEB and TFE3 translocation identifies novel chemical series modulating lysosome function

ABSTRACT The accumulation of toxic protein aggregates in multiple neurodegenerative diseases is associated with defects in the macroautophagy/autophagy-lysosome pathway. The amelioration of disease phenotypes across multiple models of neurodegeneration can be achieved through modulating the master regulator of lysosome function, TFEB (transcription factor EB). Using a novel multi-parameter high-throughput screen for cytoplasmic:nuclear translocation of endogenous TFEB and the related transcription factor TFE3, we screened the Published Kinase Inhibitor Set 2 (PKIS2) library as proof of principle and to identify kinase regulators of TFEB and TFE3. Given that TFEB and TFE3 are responsive to cellular stress we have established assays for cellular toxicity and lysosomal function, critical to ensuring the identification of hit compounds with only positive effects on lysosome activity. In addition to AKT inhibitors which regulate TFEB localization, we identified a series of quinazoline-derivative compounds that induced TFEB and TFE3 translocation. A novel series of structurally-related analogs was developed, and several compounds induced TFEB and TFE3 translocation at higher potency than previously screened compounds. KINOMEscan and cell-based KiNativ kinase profiling revealed high binding for the PRKD (protein kinase D) family of kinases, suggesting good selectivity for these compounds. We describe and utilize a cellular target-validation platform using CRISPRi knockdown and orthogonal PRKD inhibitors to demonstrate that the activity of these compounds is independent of PRKD inhibition. The more potent analogs induced subsequent upregulation of the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy, demonstrating their potential to alleviate neurodegeneration-relevant phenotypes.Abbreviations: AD: Alzheimer disease; AK: adenylate kinase; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; HD: Huntington disease; PD: Parkinson disease; PKIS2: Published Kinase Inhibitor Set 2; PRKD: protein kinase D; TFEB: transcription factor EB.

Autophagy Dysfunction in ALS: from Transport to Protein Degradation

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motor neurons (MNs). Since the identification of the first ALS mutation in 1993, more than 40 genes have been associated with the disorder. The most frequent genetic causes of ALS are represented by mutated genes whose products challenge proteostasis, becoming unable to properly fold and consequently aggregating into inclusions that impose proteotoxic stress on affected cells. In this context, increasing evidence supports the central role played by autophagy dysfunctions in the pathogenesis of ALS. Indeed, in early stages of disease, high levels of proteins involved in autophagy are present in ALS MNs; but at the same time, with neurodegeneration progression, autophagy-mediated degradation decreases, often as a result of the accumulation of toxic protein aggregates in affected cells. Autophagy is a complex multistep pathway that has a central role in maintaining cellular homeostasis. Several proteins are involved in its tight regulation, and importantly a relevant fraction of ALS-related genes encodes products that directly take part in autophagy, further underlining the relevance of this key protein degradation system in disease onset and progression. In this review, we report the most relevant findings concerning ALS genes whose products are involved in the several steps of the autophagic pathway, from phagophore formation to autophagosome maturation and transport and finally to substrate degradation.

The Effect of Increasing Levels of Hyperthermia on Autophagy in Peripheral Blood Mononuclear Cells from Young Adults

Exposure to an elevated state of hyperthermia is associated with heat‐induced cytotoxicity, thereby resulting in impaired cellular function. Cellular dysfunction during heat stress leads to the initiation of systemic inflammation and apoptotic cell death, which if left unabated, can cause tissue damage and death. To counter heat‐induced cellular injury, cells employ the vital mechanism of autophagy which acts to prevent the accumulation of toxic protein aggregates and dysfunctional organelles to promote cellular survival. In our recent ex vivomodel, we demonstrated that the process of autophagy is activated during exposure to increased temperatures (37‐41°C) in peripheral blood mononuclear cells (PBMCs) from young adults which acts to suppress heat‐induced apoptotic cell death. Despite this, the dose‐dependent autophagic response to heat stress has yet to be evaluated in an in vivo human model and thus the relationship between elevations in core body temperature (Tcore) and autophagy have yet to be established in humans. Therefore, we sought to extend upon our previous work by assessing if in vivorises in Tcore are associated with greater autophagic activation in healthy young adults. We evaluated the hypothesis that in parallel to elevations in Tcore(indexed by esophageal temperature), greater autophagic initiation would occur in a dose‐dependent manner in young adults during immersion in mild to very warm water. To assess this hypothesis, on separate days 6 young adults (mean [SD] 22 [3] years; 3 women) were immersed (up to the clavicle) in water set at a temperature to clamp Tcore at either thermoneutral (TN, 37°C), 38°C, or 39°C for 60‐min. Autophagy was characterized in PBMCs prior to and immediately post‐water immersion, as well as following 3‐h of seated recovery. Western blot was used to assess autophagy proteins microtubule associated protein 1 light chain 3 beta (LC3)‐II and sequesterome‐1/p62 (p62). All proteins were normalized to β‐actin and reported as fold change relative to baseline. Data were compared via a two‐way repeated measures ANOVA with Fisher’s LSD (α=0.05). No changes in either LC3‐II or p62 were observed during TN, indicating no time‐dependent changes in autophagy (all p≥0.28). Immediately following the 38°C clamp, LC3‐II increased (1.56 [0.32]; p=0.02) and p62 decreased (0.52 [0.30]; p=0.049) from baseline, with both proteins returning to basal levels within 3‐h (both, p≥0.22). Similarly, LC3‐II increased (1.90 [0.70]; p=0.04) and p62 decreased (0.58 [0.21]; p=0.03) immediately after clamping Tcore at 39°C, however LC3‐II remained elevated above baseline at 3‐h (2.54 [1.08]; p=0.03) while p62 did not differ from pre‐immersion levels at the end of recovery (p=0.57). Given LC3‐II accumulation and p62 degradation indicate elevated autophagy, our preliminary findings indicate greater autophagy during increasing levels of hyperthermia in healthy young adults. Further, we showed increased reliance on autophagy to restore cellular homeostasis after exposure to an elevated state of hyperthermia of 39°C, although the mechanism of activation is unclear.

Chaperone-Mediated Autophagy in Neurodegenerative Diseases and Acute Neurological Insults in the Central Nervous System.

Autophagy is an important function that mediates the degradation of intracellular proteins and organelles. Chaperone-mediated autophagy (CMA) degrades selected proteins and has a crucial role in cellular proteostasis under various physiological and pathological conditions. CMA dysfunction leads to the accumulation of toxic protein aggregates in the central nervous system (CNS) and is involved in the pathogenic process of neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. Previous studies have suggested that the activation of CMA to degrade aberrant proteins can provide a neuroprotective effect in the CNS. Recent studies have shown that CMA activity is upregulated in damaged neural tissue following acute neurological insults, such as cerebral infarction, traumatic brain injury, and spinal cord injury. It has been also suggested that various protein degradation mechanisms are important for removing toxic aberrant proteins associated with secondary damage after acute neurological insults in the CNS. Therefore, enhancing the CMA pathway may induce neuroprotective effects not only in neurogenerative diseases but also in acute neurological insults. We herein review current knowledge concerning the biological mechanisms involved in CMA and highlight the role of CMA in neurodegenerative diseases and acute neurological insults. We also discuss the possibility of developing CMA-targeted therapeutic strategies for effective treatments.

Therapeutic Potential of Vital Transcription Factors in Alzheimer's and Parkinson's Disease With Particular Emphasis on Transcription Factor EB Mediated Autophagy.

Autophagy is an important cellular self-digestion and recycling pathway that helps in maintaining cellular homeostasis. Dysregulation at various steps of the autophagic and endolysosomal pathway has been reported in several neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington disease (HD) and is cited as a critically important feature for central nervous system (CNS) proteostasis. Recently, another molecular target, namely transcription factor EB (TFEB) has been explored globally to treat neurodegenerative disorders. This TFEB, is a key regulator of autophagy and lysosomal biogenesis pathway. Multiple research studies suggested therapeutic potential by targeting TFEB to treat human diseases involving autophagy-lysosomal dysfunction, especially neurodegenerative disorders. A common observation involving all neurodegenerative disorders is their poor efficacy in clearing and recycle toxic aggregated proteins and damaged cellular organelles due to impairment in the autophagy pathway. This dysfunction in autophagy characterized by the accumulation of toxic protein aggregates leads to a progressive loss in structural integrity/functionality of neurons and may even result in neuronal death. In recent years TFEB, a key regulator of autophagy and lysosomal biogenesis, has received considerable attention. It has emerged as a potential therapeutic target in numerous neurodegenerative disorders like AD and PD. In various neurobiology studies involving animal models, TFEB has been found to ameliorate neurotoxicity and rescue neurodegeneration. Since TFEB is a master transcriptional regulator of autophagy and lysosomal biogenesis pathway and plays a crucial role in defining autophagy activation. Studies have been done to understand the mechanisms for TFEB dysfunction, which may yield insights into how TFEB might be targeted and used for the therapeutic strategy to develop a treatment process with extensive application to neurodegenerative disorders. In this review, we explore the role of different transcription factor-based targeted therapy by some natural compounds for AD and PD with special emphasis on TFEB.

Proteostasis and Proteotoxicity in the Network Medicine Era

Neurodegenerative proteinopathies are complex diseases that share some pathogenetic processes. One of these is the failure of the proteostasis network (PN), which includes all components involved in the synthesis, folding, and degradation of proteins, thus leading to the aberrant accumulation of toxic protein aggregates in neurons. The single components that belong to the three main modules of the PN are highly interconnected and can be considered as part of a single giant network. Several pharmacological strategies have been proposed to ameliorate neurodegeneration by targeting PN components. Nevertheless, effective disease-modifying therapies are still lacking. In this review article, after a general description of the PN and its failure in proteinopathies, we will focus on the available pharmacological tools to target proteostasis. In this context, we will discuss the main advantages of systems-based pharmacology in contrast to the classical targeted approach, by focusing on network pharmacology as a strategy to innovate rational drug design.

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V.

The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.

Interferon-β-induced miR-1 alleviates toxic protein accumulation by controlling autophagy.

Appropriate regulation of autophagy is crucial for clearing toxic proteins from cells. Defective autophagy results in accumulation of toxic protein aggregates that detrimentally affect cellular function and organismal survival. Here, we report that the microRNA miR-1 regulates the autophagy pathway through conserved targeting of the orthologous Tre-2/Bub2/CDC16 (TBC) Rab GTPase-activating proteins TBC-7 and TBC1D15 in Caenorhabditis elegans and mammalian cells, respectively. Loss of miR-1 causes TBC-7/TBC1D15 overexpression, leading to a block on autophagy. Further, we found that the cytokine interferon-β (IFN-β) can induce miR-1 expression in mammalian cells, reducing TBC1D15 levels, and safeguarding against proteotoxic challenges. Therefore, this work provides a potential therapeutic strategy for protein aggregation disorders.

Age-related neurodegenerative diseases.

Converging evidence indicates the dysregulation of unique cytosolic compartments called stress granules (SGs) might facilitate the accumulation of toxic protein aggregates that underlie many age-related neurodegenerative pathologies (ANPs). SG dynamics are particularly susceptible to the cellular conditions that are commonly induced by aging, including the elevation in reactive oxygen species and increased concentration of aggregate-prone proteins. In turn, the persistent formation of these compartments is hypothesized to serve as a seed for subsequent protein aggregation. Notably, the protein quality control (PQC) machinery responsible for inhibiting persistent SGs (e.g., Hsc70-BAG3) can become compromised with age, suggesting that the modulation of such PQC mechanisms could reliably inhibit pathological processes of ANPs. As exemplified in the context of accelerated aging syndromes (i.e., Hutchinson-Gilford progeria), PQC enhancement is emerging as a potential therapeutic strategy, indicating similar techniques might be applied to ANPs. Collectively, these recent findings advance our understanding of how the processes that might facilitate protein aggregation are particularly susceptible to aging conditions, and present investigators with an opportunity to develop novel targets for ANPs.

Abstract 849: Drp1-dependent Altered Mitochondrial Dynamics Contribute to Protein Aggregation and Mitochondrial Dysfunction in R120G αB-crystallin-induced Proteotoxicity

Introduction: αB-crystallin (CryAB) is a chaperone protein that plays a pivotal role in the maintenance of the structural and mechanical integrity of the sarcomere. Mutation in αB-crystallin (CryAB R120G) causes sarcomere disorganization leading to protein aggregation. Accumulation of toxic protein aggregates causes cardiac contractile dysfunction and perturbed mitochondrial spatial organization. However, the role of mitochondria in the pathogenesis of protein aggregation-induced cardiomyopathies remain obscure. Objective: In this study, we investigated the role of mitochondrial dynamics and function in the development of R120G αB-crystallin induced cardiac proteotoxicity. Methods and Results: CryAB R120G Tg hearts showed accumulation of toxic protein aggregates resulting in disruption of the sarcomere structure, development of cardiomyocyte hypertrophy, and contractile dysfunction. Hearts obtained from CryAB R120G Tg mice showed increased expression and localization of Drp1 on the mitochondrial membrane. Alteration in the expression of mitochondrial dynamics regulatory proteins was observed at two months age when these mice exhibited normal cardiac morphometry and systolic function. Adenoviral-mediated expression of CryAB R120G in cardiomyocytes also showed increased mitochondrial fission, expression dependent inhibition of mitochondrial respiration and activation of cellular toxicity. Inhibition mitochondrial fission by Drp1 partial knockdown in CryABR120G expressing cardiomyocytes significantly decreased protein aggregation and improved mitochondrial respiration. Conclusion: Drp1-dependent excessive mitochondrial fission results in defects in mitochondrial respiration and accumulation of protein aggregates in CryABR120G expressing cardiomyocytes. Therefore, our study shows that maladaptive aberrant mitochondrial fission causes CryABR120G -induced mitochondrial dysfunction and cardiac proteotoxicity-associated cellular dysfunction.

Cinnamic acid activates PPARα to stimulate Lysosomal biogenesis and lower Amyloid plaque pathology in an Alzheimer's disease mouse model

The response of the lysosomes, the waste clearance machinery of the cell, to different environmental stimuli is coordinated by a gene network with a master regulator Transcription factor EB (TFEB) at the core. Disruption of multiple facets of the lysosomal and autophagic network has been linked to various neurodegenerative and lysosomal storage disorders, making TFEB an attractive therapeutic target to rescue or augment lysosomal function under pathological scenario. In this study, we demonstrate that cinnamic acid, a naturally occurring plant-based product, induces lysosomal biogenesis in mouse primary brain cells via upregulation of TFEB. We delineate that cinnamic acid activates the nuclear hormone receptor PPARα to transcriptionally upregulate TFEB and stimulate lysosomal biogenesis. Moreover, using in-silico and biochemical approaches we established that cinnamic acid serves as a potent ligand for peroxisome proliferator-activated receptor α (PPARα). Finally, cinnamic acid treatment in male and female 5× Familial Alzheimer's disease (5XFAD) mice remarkably reduced cerebral amyloid-beta plaque burden and improved memory via PPARα. Therefore, stimulation of lysosomal biogenesis by cinnamic acid may have therapeutic implications for treatment of Alzheimer's disease and other lysosomal disorders originating from accumulation of toxic protein aggregates.

Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils’ detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose.The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM).It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases.We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.

Autophagy in Age-Associated Neurodegeneration.

The elimination of abnormal and dysfunctional cellular constituents is an essential prerequisite for nerve cells to maintain their homeostasis and proper function. This is mainly achieved through autophagy, a process that eliminates abnormal and dysfunctional cellular components, including misfolded proteins and damaged organelles. Several studies suggest that age-related decline of autophagy impedes neuronal homeostasis and, subsequently, leads to the progression of neurodegenerative disorders due to the accumulation of toxic protein aggregates in neurons. Here, we discuss the involvement of autophagy perturbation in neurodegeneration and present evidence indicating that upregulation of autophagy holds potential for the development of therapeutic interventions towards confronting neurodegenerative diseases in humans.