Aberrant Protein Aggregation Research Articles

Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein.

To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.

Leucine-rich repeat kinase 2 and lysosomal dyshomeostasis in Parkinson disease.

Over the last two decades, a number of studies have underlined the importance of lysosomal-based degradative pathways in maintaining the homeostasis of post-mitotic cells, and revealed the remarkable contribution of a functional autophagic machinery in the promotion of longevity. In contrast, defects in the clearance of organelles and aberrant protein aggregates have been linked to accelerated neuronal loss and neurological dysfunction. Several neurodegenerative disorders, among which Alzheimer disease (AD), Frontotemporal dementia, and Amyotrophic Lateral Sclerosis to name a few, are associated with alterations of the autophagy and endo-lysosomal pathways. In Parkinson disease (PD), the most prevalent genetic determinant, Leucine-rich repeat kinase 2 (LRRK2), is believed to be involved in the regulation of intracellular vesicle traffic, autophagy and lysosomal function. Here, we review the current understanding of the mechanisms by which LRRK2 regulates lysosomal-based degradative pathways in neuronal and non-neuronal cells and discuss the impact of pathogenic PD mutations in contributing to lysosomal dyshomeostasis.

When few survive to tell the tale: thymus and gonad as auditioning organs: historical overview.

Unlike other organs, the thymus and gonads generate nonuniform cell populations, many members of which perish, and a few survive. While it is recognized that thymic cells are "audited" to optimize an organism's immune repertoire, whether gametogenesis could be orchestrated similarly to favor high-quality gametes is uncertain. Ideally, such quality would be affirmed at early stages before the commitment of extensive parental resources. A case is here made that, along the lines of a previously proposed lymphocyte quality control mechanism, gamete quality can be registered indirectly through detection of incompatibilities between proteins encoded by the grandparental DNA sequences within the parent from which haploid gametes are meiotically derived. This "stress test" is achieved in the same way that thymic screening for potential immunological incompatibilities is achieved-by "promiscuous" expression, under the influence of the AIRE protein, of the products of genes that are not normally specific for that organ. Consistent with this, the Aire gene is expressed in both thymus and gonads, and AIRE deficiency impedes function in both organs. While not excluding the subsequent emergence of hybrid incompatibilities due to the intermixing of genomic sequences from parents (rather than grandparents), many observations, such as the number of proteins that are aberrantly expressed during gametogenesis, can be explained on this basis. Indeed, promiscuous expression could have first evolved in gamete-forming cells where incompatible proteins would be manifest as aberrant protein aggregates that cause apoptosis. This mechanism would later have been co-opted by thymic epithelial cells which display peptides from aggregates to remove potentially autoreactive T cells.

Small Heat Shock Proteins, Big Impact on Protein Aggregation in Neurodegenerative Disease.

Misfolding, aggregation, and aberrant accumulation of proteins are central components in the progression of neurodegenerative disease. Cellular molecular chaperone systems modulate proteostasis, and, therefore, are primed to influence aberrant protein-induced neurotoxicity and disease progression. Molecular chaperones have a wide range of functions from facilitating proper nascent folding and refolding to degradation or sequestration of misfolded substrates. In disease states, molecular chaperones can display protective or aberrant effects, including the promotion and stabilization of toxic protein aggregates. This seems to be dependent on the aggregating protein and discrete chaperone interaction. Small heat shock proteins (sHsps) are a class of molecular chaperones that typically associate early with misfolded proteins. These interactions hold proteins in a reversible state that helps facilitate refolding or degradation by other chaperones and co-factors. These sHsp interactions require dynamic oligomerization state changes in response to diverse cellular triggers and, unlike later steps in the chaperone cascade of events, are ATP-independent. Here, we review evidence for modulation of neurodegenerative disease-relevant protein aggregation by sHsps. This includes data supporting direct physical interactions and potential roles of sHsps in the stewardship of pathological protein aggregates in brain. A greater understanding of the mechanisms of sHsp chaperone activity may help in the development of novel therapeutic strategies to modulate the aggregation of pathological, amyloidogenic proteins. sHsps-targeting strategies including modulators of expression or post-translational modification of endogenous sHsps, small molecules targeted to sHsp domains, and delivery of engineered molecular chaperones, are also discussed.

Highly Dynamic Changes in the Activity and Regulation of Macroautophagy in Hearts Subjected to Increased Proteotoxic Stress

Macroautophagy (referred to as autophagy hereafter) plays an important role in the quality control of cellular proteins and organelles. Transcription Factor EB (TFEB) globally activates the expression of genes in the autophagic-lysosomal pathway (ALP) to replenish lysosomes and ALP machineries. We previously reported that myocardial TFEB signaling was impaired in advanced cardiac proteinopathy; however, myocardial ALP status and TFEB activity at earlier stages of cardiac proteinopathy remain uncharacterized. Here a stable line of CryABR120G transgenic (R120G) and non-transgenic (NTG) littermate mice with cardiomyocyte-restricted overexpression of CryABR120G were used at 1, 3, and 6 months of age. At 1 month when no cardiac phenotypes other than aberrant protein aggregation are discernible, R120G mice displayed a 5-fold increase in myocardial LC3-II flux. Interestingly, the LC3-II flux increase co-existed with increases in mTOR complex 1 (mTORC1) activities as well as cytoplasmic, but not nuclear, TFEB proteins. This increase in cytoplasmic TFEB proteins occurred without any discernible alteration in TFEB activity as reflected by unchanged mRNA levels of representative TFEB target genes (Mcoln1, M6pr, Sqstm1, Vps18, and Uvrag). At 3 months of age when hypertrophy and diastolic malfunction start to develop, the LC3-II flux remained significantly increased but to a lesser degree (2-fold) than at 1 month. The LC3-II flux increase was associated with decreased mTORC1 activities and with increased nuclear TFEB proteins and TFEB activities. At 6 months of age when congestive heart failure is apparent in R120G mice, both LC3-II flux and TFEB activities were severely suppressed, while mTORC1 activity increased. We conclude that changes in both autophagy and TFEB signaling are highly dynamic during the progression of cardiac proteinopathy. Increases in autophagy occur before increases in TFEB activities but both increase in the compensatory stage of cardiac proteinopathy. Once congestive heart failure develops, both autophagy and TFEB signaling become impaired. Our results suggest that TFEB signaling is regulated by both mTORC1-dependent and -independent mechanisms in hearts subjected to increased proteotoxic stress. For therapeutic exploration, it will be important to test the effect of TFEB stimulation at the early, intermediate, and late stages of cardiac proteinopathy.

Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD.

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD.

Spatial control of irreversible protein aggregation.

Liquid cellular compartments form in the cyto- or nucleoplasm and can regulate aberrant protein aggregation. Yet, the mechanisms by which these compartments affect protein aggregation remain unknown. Here, we combine kinetic theory of protein aggregation and liquid-liquid phase separation to study the spatial control of irreversible protein aggregation in the presence of liquid compartments. We find that even for weak interactions aggregates strongly partition into the liquid compartment. Aggregate partitioning is caused by a positive feedback mechanism of aggregate nucleation and growth driven by a flux maintaining the phase equilibrium between the compartment and its surrounding. Our model establishes a link between specific aggregating systems and the physical conditions maximizing aggregate partitioning into the compartment. The underlying mechanism of aggregate partitioning could be used to confine cytotoxic protein aggregates inside droplet-like compartments but may also represent a common mechanism to spatially control irreversible chemical reactions in general.

Differential Effects of Yeast NADH Dehydrogenase (Ndi1) Expression on Mitochondrial Function and Inclusion Formation in a Cell Culture Model of Sporadic Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient’s brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.

Protein Solubility Predictions Using the CamSol Method in the Study of Protein Homeostasis.

One of the major functions of the protein homeostasis system is to maintain proteins in their soluble states, and indeed several human disorders are associated with the aberrant aggregation of proteins. An active involvement of the protein homeostasis system is necessary to avoid aggregation because proteins are expressed at levels close to their solubility limits, hence being poorly soluble. The mechanisms by which the protein homeostasis system acts to control protein aggregation are, however, still not known in much detail. To facilitate systematic investigations of these mechanisms, we describe here the CamSol method of predicting protein solubility, and illustrate its initial applications. We anticipate that with the advent of powerful proteomics and transcriptomic methods, in combination with the use of the CamSol method and related approaches to predict the solubility and other biophysical properties of proteins, it will become possible to increase our understanding of the principles of protein homeostasis related to the maintenance of the proteome in its soluble form.

LRSAM1 E3 ubiquitin ligase: molecular neurobiological perspectives linked with brain diseases.

Cellular protein quality control (PQC) plays a significant role in the maintenance of cellular homeostasis. Failure of PQC mechanism may lead to various neurodegenerative diseases due to accumulation of aberrant proteins. To avoid such fatal neuronal conditions PQC employs autophagy and ubiquitin proteasome system (UPS) to degrade misfolded proteins. Few quality control (QC) E3 ubiquitin ligases interplay an important role to specifically recognize misfolded proteins for their intracellular degradation. Leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1) is a really interesting new gene (RING) class protein that possesses E3 ubiquitin ligase activity with promising applications in PQC. LRSAM1 is also known as RING finger leucine repeat rich (RIFLE) or TSG 101-associated ligase (TAL). LRSAM1 has various cellular functions as it modulates the protein aggregation, endosomal sorting machinery and virus egress from the cells. Thus, this makes LRSAM1 interesting to study not only in protein conformational disorders such as neurodegeneration but also in immunological and other cancerous disorders. Furthermore, LRSAM1 interacts with both cellular protein degradation machineries and hence it can participate in maintenance of overall cellular proteostasis. Still, more research work on the quality control molecular functions of LRSAM1 is needed to comprehend its roles in various protein aggregatory diseases. Earlier findings suggest that in a mouse model of Charcot-Marie-Tooth (CMT) disease, lack of LRSAM1 functions sensitizes peripheral axons to degeneration. It has been observed that in CMT the patients retain dominant and recessive mutations of LRSAM1 gene, which encodes most likely a defective protein. However, still the comprehensive molecular pathomechanism of LRSAM1 in neuronal functions and neurodegenerative diseases is not known. The current article systematically represents the molecular functions, nature and detailed characterization of LRSAM1 E3 ubiquitin ligase. Here, we review emerging molecular mechanisms of LRSAM1 linked with neurobiological functions, with a clear focus on the mechanism of neurodegeneration and also on other diseases. Better understanding of LRSAM1 neurobiological and intracellular functions may contribute to develop promising novel therapeutic approaches, which can also propose new lines of molecular beneficial targets for various neurodegenerative diseases.

Modulation of Amyloid States by Molecular Chaperones.

Aberrant protein aggregation is a defining feature of most neurodegenerative diseases. During pathological aggregation, key proteins transition from their native state to alternative conformations, which are prone to oligomerize into highly ordered fibrillar states. As part of the cellular quality control machinery, molecular chaperones can intervene at many stages of the aggregation process to inhibit or reverse aberrant protein aggregation or counteract the toxicity associated with amyloid species. Although the action of chaperones is considered cytoprotective, essential housekeeping functions can be hijacked for the propagation and spreading of protein aggregates, suggesting the cellular protein quality control system constitutes a double-edged sword in neurodegeneration. Here, we discuss the various mechanisms used by chaperones to influence protein aggregation into amyloid fibrils to understand how the interplay of these activities produces specific cellular outcomes and to define mechanisms that may be targeted by pharmacological agents for the treatment of neurodegenerative conditions.

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles.

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.

PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. We reveal that PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. We find that hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). We further uncover that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, we reveal that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation.

Modeling the Effects of Ligand Binding on the Phase Behavior of Aggregation-Prone Proteins

Many diseases are associated with aberrant protein aggregation. Recently, there has been resurgent interest in co-opting the framework of phase transitions to understand the distinct concentration thresholds separating different phases of aggregation-prone molecules. This analogy, which was purely conceptual, has now been shown to have quantitative significance. Importantly, instead of searching for elusive “toxic conformations”, dealing with the deleterious consequences of protein aggregation and phase separation can be approached via methods that alter or modulate the sequence-specific phase behavior. One route to accomplishing this is by shifting phase boundaries via ligand binding through a phenomenon known as polyphasic linkage. Disease-causing mutations, such as polyglutamine expansions in the N-terminal region of huntingtin, lead to aggregation and phase separation at lower, physiologically relevant concentration thresholds. Ligand binding can shift these phase boundaries based on the phase that is preferentially bound. Here, we show that a ligand can reduce the driving force for protein aggregation in vitro and may help to explain the reduced cellular aggregation and toxicity observed when overexpressed in the context of Huntington's disease. Through a combined biophysical and computational approach, we further show that multi-valency in binding appears to be important for shifting phase boundaries in therapeutically relevant directions. However, in the context of the cell, a protein such as huntingtin has a large interactome. Each of the interaction partners will have a different effect on the intrinsic phase boundaries and therefore, designing a therapeutic approach that leverages the tenets of polyphasic linkage requires a general framework that considers the competing effects within a network of interactions. Using the huntingtin example, we develop a framework for designing modulators of phase behavior via polyphasic linkage that accounts for competing interactions within an interactome.

Atomistic Simulation Tools to Study Protein Self-Aggregation.

Aberrant aggregation of proteins into poorly soluble, toxic structures that accumulate intracellularly or extracellularly leads to a range of disease states including Alzheimer's, Parkinson's, Huntington's, prion diseases, and type II diabetes. Many of the disease-associated amyloidogenic proteins are intrinsically disordered, which makes their experimental investigation challenging due to a limited number of experimental observables to effectively characterize their ensemble of conformations. Molecular dynamics simulations provide dynamic information with atomistic detail, and are increasingly employed to study aggregation processes, offering valuable structural and mechanistic insights. In this chapter, we demonstrate the use of all-atom molecular dynamics simulations to model the self-aggregation of a six-residue amyloidogenic peptide derived from amyloid β, a 39-43 residue-long peptide associated with the pathogenesis of Alzheimer's disease. We provide detailed instructions on how to obtain the initial monomer conformations and build the multichain systems, how to carry out the simulations, and how to analyze the simulation trajectories to investigate the peptide self-aggregation process.

Rethinking to riluzole mechanism of action: the molecular link among protein kinase CK1δ activity, TDP-43 phosphorylation, and amyotrophic lateral sclerosis pharmacological treatment.

Rethinking to riluzole mechanism of action: the molecular link among protein kinase CK1δ activity, TDP-43 phosphorylation, and amyotrophic lateral sclerosis pharmacological treatment.

The regulation of TORC1 pathway by the yeast chaperones Hsp31 is mediated by SFP1 and affects proteasomal activity

The Saccharomyces cerevisiae heat shock proteins Hsp31–34 are members of DJ-1/ThiJ/Pfpl superfamily that includes human DJ-1 (Park7), a protein involved in heritable Parkinsonism. Although, homologs of these proteins can be found in most organisms their functions are unclear. We have carried out a quantitative proteomics analysis of yeast cells devoid of the whole set of Hsp31 family of proteins, as a model of Parkinson Disease (PD), under conditions of glucose availability and starvation. The protein profile indicates a constitutive activation of the enzyme TORC1 that makes the cells more sensitive to stress conditions. TORC1 activation prevents the cells from diauxic shift and entry into the stationary phase inducing cell death. Sfp1 stays at the helm among the several transcription factors governing the cell adaptation to Hsp31–34 deficiency. We show that Sfp1 remains mainly in the nucleus likely releasing TORC1 from inhibition by cytosolic Sfp1. Impairment of glycolysis leads to increased levels of methylglyoxal and accumulation of glycated proteins. We also show an increase in proteasome subunits in the Hsp31–34 mutant, under the control of Rpn4 transcription factor. This increase is abnormally accompanied by a decrease in proteasomal activity which could lead to accumulation of aberrant proteins and contributing to cell death.

Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides Containing Dipeptide Tentacles.

Molecular motifs that could interfere with amyloid fibrillation via non-covalent interactions are vital for aberrant protein aggregation and related human diseases. Mutual aggregation ensues in the presence of these structural motifs, and nucleation on the particle surface leads to inhibition of the fibrillization process. This modular process generates a new generation of inhibitory reagents. Oligomers are the primary toxic species that initiate pathogenic aggregation, leading to toxic β-sheet-rich structures. To inhibit these toxic oligomers, two dipeptide-linked perylenebisimide isomers (PAPAP and APPPA) are developed as selective modulators for insulin fibrillization. Early insulin aggregates are adsorbed onto the modulator surface and stabilize soluble oligomeric aggregates. Fibrillation and inhibition are examined by thioflavin T (ThT) assay in the presence and the absence of both inhibitors PAPAP and APPPA. Conformational modulation using far-UV circular dichroism studies also highlights their role as aggregation inhibitors via reduction of α-helix into β-sheet along with increased random coil contents. Moreover, the inhibitory effects were more pronounced due to the ability of these isomers to undergo varying multiple non-covalent interactions, a performance well beyond all known modulators with respect to selectivity and efficiency, with the more-aggregation-prone derivative due to higher probability of a hydrophobic encounter between the protein and the molecular modulator. These results also lead to the elucidation of an insulin fibril regulating mechanism via selective non-covalent binding and provide fundamental insights into the chemistry of peptide-based probes as tools for developing next-generation therapeutics.

Non-invasive imaging of CSF-mediated brain clearance pathways via assessment of perivascular fluid movement with diffusion tensor MRI.

The glymphatics system describes a CSF-mediated clearance pathway for the removal of potentially harmful molecules, such as amyloid beta, from the brain. As such, its components may represent new therapeutic targets to alleviate aberrant protein accumulation that defines the most prevalent neurodegenerative conditions. Currently, however, the absence of any non-invasive measurement technique prohibits detailed understanding of glymphatic function in the human brain and in turn, it's role in pathology. Here, we present the first non-invasive technique for the assessment of glymphatic inflow by using an ultra-long echo time, low b-value, multi-direction diffusion weighted MRI sequence to assess perivascular fluid movement (which represents a critical component of the glymphatic pathway) in the rat brain. This novel, quantitative and non-invasive approach may represent a valuable biomarker of CSF-mediated brain clearance, working towards the clinical need for reliable and early diagnostic indicators of neurodegenerative conditions such as Alzheimer's disease.

Down-regulating Proteolysis to Enhance Anticancer Activity of Peptide Nanofibers.

Nanofibers of short peptides are emerging as a promising type of agents for inhibiting cancer cells. But the proteolysis of peptides decreases the anticancer efficacy of the peptide nanofibers. Here we show that decreasing the activity of proteasomes enhance the activity of peptide nanofibers for inhibiting cancer cells. Based on the structure of galactin-3, we designed a heptapeptide, which self-assembles to form nanofibers. The nanofibers of the heptapeptide exhibit moderate cytotoxicity to three representative cancer cell lines (HeLa, MCF-7, and HepG2), largely due to the proteolysis of the peptides. Using a clinically approved proteasome inhibitor, bortezomib, to treat the cancer cells significantly decreases the proteolysis of the peptides and enhances the activity of the peptide nanofibers for inhibiting the cancer cells. This work illustrates a promising approach for enhancing the anticancer efficacy of peptide nanofibers by modulating intracellular protein degradation machinery, as well as provides insights for understanding the cytotoxicity of aberrant protein or peptide aggregates in complicated cellular environment.