mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation.

While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q48 The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Caenorhabditis elegans Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q97 aggregation and represents a target of future therapeutic avenues for HD.

J-domain proteins interaction with neurodegenerative disease-related proteins.

HSP70 chaperones, J-domain proteins (JDPs) and nucleotide exchange factors (NEF) form functional networks that have the ability to prevent and reverse the aggregation of proteins associated with neurodegenerative diseases. JDPs can interact with specific substrate proteins, hold them in a refolding-competent conformation and target them to specific HSP70 chaperones for remodeling. Thereby, JDPs select specific substrates and constitute an attractive target for pharmacological intervention of neurodegenerative diseases. This, under the condition that the exact mechanism of JDPs interaction with specific substrates is unveiled. In this review, we provide an overview of the structural and functional variety of JDPs that interact with neurodegenerative disease-associated proteins and we highlight those studies that identified specific residues, domains or regions of JDPs that are crucial for substrate binding.

The noncanonical small heat shock protein HSP-17 from Caenorhabditis elegans is a selective protein aggregase.

Small heat shock proteins (sHsps) are conserved, ubiquitous members of the proteostasis network. Canonically, they act as "holdases" and buffer unfolded or misfolded proteins against aggregation in an ATP-independent manner. Whereas bacteria and yeast each have only two sHsps in their genomes, this number is higher in metazoan genomes, suggesting a spatiotemporal and functional specialization in higher eukaryotes. Here, using recombinantly expressed and purified proteins, static light-scattering analysis, and disaggregation assays, we report that the noncanonical sHsp HSP-17 of Caenorhabditis elegans facilitates aggregation of model substrates, such as malate dehydrogenase (MDH), and inhibits disaggregation of luciferase in vitro Experiments with fluorescently tagged HSP-17 under the control of its endogenous promoter revealed that HSP-17 is expressed in the digestive and excretory organs, where its overexpression promotes the aggregation of polyQ proteins and of the endogenous kinase KIN-19. Systemic depletion of hsp-17 shortens C. elegans lifespan and severely reduces fecundity and survival upon prolonged heat stress. HSP-17 is an abundant protein exhibiting opposing chaperone activities on different substrates, indicating that it is a selective protein aggregase with physiological roles in development, digestion, and osmoregulation.

Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation.

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy.

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments.

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub-cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter- and intra-molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle-specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Protein quality control: from molecular mechanisms to therapeutic intervention-EMBO workshop, May 21-26 2023, Srebreno, Croatia.

Protein quality control pathways ensure a functional proteome and rely on a complex proteostasis network (PN) that is composed of molecular chaperones and proteases. Failures in the PN can lead to a broad spectrum of diseases, including neurodegenerative disorders like Alzheimer's, Parkinson's, and a range of motor neuron diseases. The EMBO workshop "Protein quality control: from molecular mechanisms to therapeutic intervention" covered all aspects of protein quality control from underlying molecular mechanisms of chaperones and proteases to stress signaling pathways and medical implications. This report summarizes the workshop and highlights selected presentations.

Lack of L-type amino acid transporter 2 in murine thyroid tissue induces autophagy.

Proteolytic cleavage of thyroglobulin (Tg) for thyroid hormone (TH) liberation is followed by TH release from thyroid follicles into the circulation, enabled by TH transporters. The existence of a functional link between Tg-processing cathepsin proteases and TH transporters has been shown to be independent of the hypothalamus-pituitary-thyroid axis. Thus, lack of cathepsin K, combined with genetic defects in the TH transporters Mct8 and Mct10, that is the Ctsk-/-/Mct8-/y/Mct10-/- genotype, results in persistent Tg proteolysis due to autophagy induction. Because amino acid transport by L-type amino acid transporter 2 (Lat2) has been described to regulate autophagy, we asked whether Lat2 availability is affected in Ctsk-/-/Mct8-/y/Mct10-/- thyroid glands. Our data revealed that while mRNA amounts and subcellular localization of Lat2 remained unaltered in thyroid tissue of Ctsk-/-/Mct8-/y/Mct10-/- mice in comparison to WT controls, the Lat2 protein amounts were significantly reduced. These data suggest a direct link between Lat2 function and autophagy induction in Ctsk-/-/Mct8-/y/Mct10-/- mice. Indeed, thyroid tissue of Lat2-/- mice showed enhanced endo-lysosomal cathepsin activities, increased autophagosome formation, and enhanced autophagic flux. Collectively, these results suggest a mechanistic link between insufficient Lat2 protein function and autophagy induction in the thyroid gland of male mice.

Abl depletion via autophagy mediates the beneficial effects of quercetin against Alzheimer pathology across species.

Alzheimer's disease is the most common age-associated neurodegenerative disorder and the most frequent form of dementia in our society. Aging is a complex biological process concurrently shaped by genetic, dietary and environmental factors and natural compounds are emerging for their beneficial effects against age-related disorders. Besides their antioxidant activity often described in simple model organisms, the molecular mechanisms underlying the beneficial effects of different dietary compounds remain however largely unknown. In the present study, we exploit the nematode Caenorhabditis elegans as a widely established model for aging studies, to test the effects of different natural compounds in vivo and focused on mechanistic aspects of one of them, quercetin, using complementary systems and assays. We show that quercetin has evolutionarily conserved beneficial effects against Alzheimer's disease (AD) pathology: it prevents Amyloid beta (Aß)-induced detrimental effects in different C. elegans AD models and it reduces Aß-secretion in mammalian cells. Mechanistically, we found that the beneficial effects of quercetin are mediated by autophagy-dependent reduced expression of Abl tyrosine kinase. In turn, autophagy is required upon Abl suppression to mediate quercetin's protective effects against Aß toxicity. Our data support the power of C. elegans as an in vivo model to investigate therapeutic options for AD.

An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.

Huntington's disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

Novel amyloid-beta pathology C. elegans model reveals distinct neurons as seeds of pathogenicity.

Protein misfolding and aggregation are hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). In AD, the accumulation and aggregation of tau and the amyloid-beta peptide Aß1-42 precedes the onset of AD symptoms. Modelling the aggregation of Aß is technically very challenging in vivo due to its size of only 42 aa. Here, we employed sub-stoichiometric labelling of Aß1-42 in C. elegans to enable tracking of the peptide in vivo, combined with the "native" aggregation of unlabeled Aß1-42. Expression of Aß1-42 leads to severe physiological defects, neuronal dysfunction and neurodegeneration. Moreover, we can demonstrate spreading of neuronal Aß to other tissues. Fluorescence lifetime imaging microscopy enabled a quantification of the formation of amyloid fibrils with ageing and revealed a heterogenic yet specific pattern of aggregation. Notably, we found that Aß aggregation starts in a subset of neurons of the anterior head ganglion, the six IL2 neurons. We further demonstrate that cell-specific, RNAi-mediated depletion of Aß in these IL2 neurons systemically delays Aß aggregation and pathology.

In-depth profiling of the LiaR response of Bacillus subtilis.

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2.

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is key to understanding not only its life cycle, but also to uncover imbalances in the proteostasis network. This method shows how to track the degradation of the disease-causing protein huntingtin. Two versions of huntingtin fused to Dendra2 are expressed in the C. elegans nervous system: a physiological version or one with an expanded and pathogenic stretch of glutamines. Dendra2 is a photoconvertible fluorescent protein; upon a short ultraviolet (UV) irradiation pulse, Dendra2 switches its excitation/emission spectra from green to red. Similar to a pulse-chase experiment, the turnover of the converted red-Dendra2 can be monitored and quantified, regardless of the interference from newly synthesized green-Dendra2. Using confocal-based microscopy and due to the optical transparency of C. elegans, it is possible to monitor and quantify the degradation of huntingtin-Dendra2 in a living, aging organism. Neuronal huntingtin-Dendra2 is partially degraded soon after conversion and cleared further over time. The systems controlling degradation are deficient in the presence of mutant huntingtin and are further impaired with aging. Neuronal subtypes within the same nervous system exhibit different turnover capacities for huntingtin-Dendra2. Overall, monitoring any protein of interest fused to Dendra2 can provide important information not only on its degradation and the players of the proteostasis network involved, but also on its location, trafficking, and transport.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington's, Parkinson's, or Alzheimer's disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Reducing INS-IGF1 signaling protects against non-cell autonomous vesicle rupture caused by SNCA spreading.

Aging is associated with a gradual decline of cellular proteostasis, giving rise to devastating protein misfolding diseases, such as Alzheimer disease (AD) or Parkinson disease (PD). These diseases often exhibit a complex pathology involving non-cell autonomous proteotoxic effects, which are still poorly understood. Using Caenorhabditis elegans we investigated how local protein misfolding is affecting neighboring cells and tissues showing that misfolded PD-associated SNCA/α-synuclein is accumulating in highly dynamic endo-lysosomal vesicles. Irrespective of whether being expressed in muscle cells or dopaminergic neurons, accumulated proteins were transmitted into the hypodermis with increasing age, indicating that epithelial cells might play a role in remote degradation when the local endo-lysosomal degradation capacity is overloaded. Cell biological and genetic approaches revealed that inter-tissue dissemination of SNCA was regulated by endo- and exocytosis (neuron/muscle to hypodermis) and basement membrane remodeling (muscle to hypodermis). Transferred SNCA conformers were, however, inefficiently cleared and induced endo-lysosomal membrane permeabilization. Remarkably, reducing INS (insulin)-IGF1 (insulin-like growth factor 1) signaling provided protection by maintaining endo-lysosomal integrity. This study suggests that the degradation of lysosomal substrates is coordinated across different tissues in metazoan organisms. Because the chronic dissemination of poorly degradable disease proteins into neighboring tissues exerts a non-cell autonomous toxicity, this implies that restoring endo-lysosomal function not only in cells with pathological inclusions, but also in apparently unaffected cell types might help to halt disease progression.Abbreviations: AD: Alzheimer disease; BM: basement membrane; BWM: body wall muscle; CEP: cephalic sensilla; CLEM: correlative light and electron microscopy; CTNS-1: cystinosin (lysosomal protein) homolog; DA: dopaminergic; DAF-2: abnormal dauer formation; ECM: extracellular matrix; FLIM: fluorescence lifetime imaging microscopy; fps: frames per second; GFP: green fluorescent protein; HPF: high pressure freezing; IGF1: insulin-like growth factor 1; INS: insulin; KD: knockdown; LMP: lysosomal membrane permeabilization; MVB: multivesicular body; NOC: nocodazole; PD: Parkinson disease; RFP: red fluorescent protein; RNAi: RNA interference; sfGFP: superfolder GFP; SNCA: synuclein alpha; TEM: transmission electron microscopy; TNTs: tunneling nanotubes; TCSPC: time correlated single photon counting; YFP: yellow fluorescent protein.

Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains.

Interactome maps are valuable resources to elucidate protein function and disease mechanisms. Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. This network reveals interconnectivity across diseases and links many known ND-causing proteins, such as α-synuclein, TDP-43, and ATXN1, to a host of proteins previously unrelated to NDs. It facilitates the identification of interacting proteins that significantly influence mutant TDP-43 and HTT toxicity in transgenic flies, as well as of ARF-GEP100 that controls misfolding and aggregation of multiple ND-causing proteins in experimental model systems. Furthermore, it enables the prediction of ND-specific subnetworks and the identification of proteins, such as ATXN1 and MKL1, that are abnormally aggregated in postmortem brains of Alzheimer's disease patients, suggesting widespread protein aggregation in NDs.