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

Funder: Alzheimer’s Research UKFunder: Michael J. Fox Foundation for Parkinson’s ResearchFunder: Medical Research CouncilFunder: Deutsche ForschungsgemeinschaftHuntington'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

Crosstalk Between Chaperone-Mediated Protein Disaggregation and Proteolytic Pathways in Aging and Disease

A functional protein quality control machinery is crucial to maintain cellular and organismal physiology. Perturbation in the protein homeostasis network can lead to the formation of misfolded and aggregated proteins that are a hallmark of protein conformational disorders and aging. Protein aggregation is counteracted by the action of chaperones that can resolubilize aggregated proteins. An alternative protein aggregation clearance strategy is the elimination by proteolysis employing the ubiquitin proteasome system (UPS) or autophagy. Little is known how these three protein aggregate clearance strategies are regulated and coordinated in an organism with the progression of aging or upon expression of disease-associated proteins. To unravel the crosstalk between the protein aggregate clearance options, we investigated how autophagy and the UPS respond to perturbations in protein disaggregation capacity. We found that autophagy is induced as a potential compensatory mechanism, whereas the UPS exhibits reduced capacity upon depletion of disaggregating chaperones in C. elegans and HEK293 cells. The expression of amyloid proteins A beta(3-42) and Q(40) result in an impairment of autophagy as well as the UPS within the same and even across tissues. Our data indicate a tight coordination between the different nodes of the proteostasis network (PN) with the progression of aging and upon imbalances of the capacity of each clearance mechanism.Peer reviewe

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 (A beta)-induced detrimental effects in different C. elegans AD models and it reduces A beta-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 A beta toxicity. Our data support the power of C. elegans as an in vivo model to investigate therapeutic options for AD

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

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects.By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spa¨ tzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases

Mutant huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

27 p.-7 fig.Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.We acknowledge support from the Deutsche Forschungsgemeinschaft (DFG)(PR1527/5-1 to A.P., RTG 2155 ProMoAge to H.O. and L.A.M.K., SFB167 B07 to J.P., RU2795: “Synapses under stress”: PR-1527/6-1 to A.P. and AN-1440/4-1 to R.A.), the Berlin Institute of Health (BIH) (to S.D., J.P., R.K., and A.P.),the Bundesministerium für Bildung und Forschung (BMBF) (AZ. 031L0211 and 01GM2002A to A.P. and 01EE2303B to J.P.), the Medical Faculty of Heinrich Heine University (FoKo grant to A.P. and S.C.), the European Commission’s Horizon Europe Program (SIMPATHIC #101080249 to A.P.),the National Science Centre, Poland (NCN grant No. 20 16/22/M/NZ2/00548 and 2017/27/B/NZ1/02401 to P.L.), the UK Dementia Research Institute programme grant (to J.P.), the Instituto de Salud Carlos III (ISCIII) grant PI20-00057 (to C.U.), the Berlin School of Integrative Oncology through the GSSP program of the German Academy of Exchange Service (DAAD) and the Joachim Herz Foundation through the Add-on Fellowship program (to T.M.P.), and the Studienstiftung des deutschen Volkes (to Se.Li.). We acknowledge the Center for Advanced Imaging (CAi) at Heinrich Heine University Düsseldorf for providing access to the Perki- nElmer Operetta CLS (DFG grant number INST 208/760-1 FUGG) and Olympus FV3000 microscope.Peer reviewe

Mutant huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington’s disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD

Regulation des AAA+ Proteins ClpC durch Adaptorproteine

0\. Title, Content, Summary 1\. Summary 6 2\. Introduction 10 3\. Results and Discussion 30 3.1. A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis 30 3.2. Adaptor protein controlled oligomerization activates the AAA+ protein ClpC 36 3.3. Cyanobacterial ClpC/HSP100 protein displays intrinsic chaperone activity 42 3.4. The tyrosine kinase McsB is a regulated adaptor protein for ClpCP 44 4\. Outlook and Discussion 53 5\. Literature 61 7\. Abbreviations 71 8\. Acknowledgements 72 PublicationsThe main objective of my PhD thesis was the analysis of the functional relationship of the HSP100/Clp protein ClpC with its adaptor proteins. ClpC is not only involved in the removal of misfolded and aggregated proteins, but also controls, through regulated proteolysis, key steps of several developmental processes in the Gram-positive bacterium Bacillus subtilis. ClpC differs from other members of the HSP100 family in that it requires an adaptor protein for virtually all its activities. A variety of molecular biological, biochemical and biophysical methods were employed to address the mechanistic and functional interplay of ClpC and its adaptor protein network. (1) It could be demonstrated that the activation of ClpC is based on the adaptor mediated assembly of the ClpC hexamer and that the oligomerized complex constitutes the functional and substrate binding species of this chaperone complex. (2) It could be shown that the formation of the whole proteolytic complex, ClpCP, depends on the preceding adaptor mediated assembly of the ClpC oligomer. Once assembled, hexameric ClpC facilitates the oligomerization and thereby activation of the otherwise monomeric proteolytic component, ClpP. Thus, ClpCP mediated proteolysis appears to be regulated in a hierarchic mode governed by an adaptor protein. (3) A functional characterization of a ClpC homolog from a photobiontic organism revealed that the adaptor dependent activation is not a common feature among the ClpC homologs. Thus, ClpC of B. subtilis exhibits a unique characteristic even within the ClpC family. Besides the activation of ClpC, adaptor proteins fulfill also a substrate recognition role enabling the specific degradation of a huge variety of substrate proteins by ClpCP. The substrate spectrum of ClpCP includes key regulators such as ComK, ComS, SpoIIAB, CtsR and MurAA. The wide range of ClpC substrates argues for a substantial number of ClpC adaptors to allow a specifically controlled degradation. The cognate adaptor protein for one of these regulatory proteins, CtsR, could be identified and thus (4), adding McsB to the adaptor protein network of ClpC. McsB could be characterized as tyrosine kinase, exhibiting adaptor properties only in its kinase-on state. The kinase activity is crucial for the regulation of the class III heat shock genes in B. subtilis. (i) McsB phosphorylates CtsR, which diminishes the DNA-binding ability of CtsR and marks it for degradation and (ii) only phosphorylated McsB is enabled to subsequently target CtsR for proteolysis by ClpCP. The kinase activity of McsB is tightly controlled, as it is induced by its activator McsA and inhibited by the partner ATPase ClpC and the cognate phosphatase YwlE. Thus, McsB can be considered as a regulated adaptor protein. (5) Although McsB exhibits no similarity to the two established ClpC adaptor proteins, MecA and YpbH, all three use the same binding sites on ClpC. The interaction of adaptor proteins with ClpC might therefore not occur simultaneously. This assumption was hold up by the finding that McsB-P could successfully compete with MecA. Finally, a model of the class III heat shock regulation is proposed, integrating the novel aspects of the specific adaptor protein for CtsR, McsB, the phospho- relay between McsB, CtsR and YwlE and the interplay of ClpC with additional adaptor proteins. The competition of the different adaptor proteins for ClpC binding might probably constitute a central part of the control of regulated proteolysis in general.Das Ziel meiner Dissertation war die Aufklärung des funktionellen Zusammenhangs zwischen dem HSP100/Clp Protein ClpC und seiner Adaptorproteine. ClpC nimmt eine duale Funktion wahr. Zum einen ist ClpC durch die unspezifische Proteolyse missgefalteter und aggregierter Proteine ein Bestandteil der Proteinqualitätskontrolle, darüber hinaus, ist ClpC aber auch in den gezielten Abbau spezifischer Regulatorproteine involviert. Da diese Regulatorproteine Schlüsselpositionen in Signaltransduktionswegen einnehmen, ist ClpC in eine Vielzahl von Entwicklungs-, Differenzierungs- und Adaptionsprozessen involviert. ClpC bedarf bereits für basale Funktionen einem Adaptorprotein und in dieser Abhängigkeit unterscheidet sich ClpC von von homologen Clp ATPasen. In dieser Arbeit wurde eine Vielzahl von molekularbiologischen, biochemischen und biophysikalischen Methoden genutzt, um den funktionellen Mechanismus zwischen ClpC und seiner Adaptorproteine aufzuklären. (1) Es konnte gezeigt werden, dass der zu Grunde liegende Mechanismus der ClpC Aktivierung in der Adaptor vermittelten ClpC Oligomerisierung liegt. Der assemblierte Komplex ist zur Substratinteraktion befähigt und stellt somit den aktiven ATPase-Adaptor-Komplex dar. (2) Dieser vermittelt darüber hinaus aber auch die weitere Assemblierung zum funktionalen proteolytischen ClpCP Komplex. Die proteolytische Untereinheit, ClpP, liegt in B. subtilis als Monomer vor und bedarf für seine Assemblierung in ein aktives Tetradekamer einem oligomerisiertem ATPase-Hexamer. ClpCP-abhängige Proteolyse ist daher einer hierarchischen Kontrolle unterlegen: zunächst wird durch ein Adaptorprotein ClpC in ein aktives Hexamer überführt, welches sodann ClpP rekrutieren und assemblieren kann. (3) Die Charakterisierung eines cyanobakteriellen ClpC-Homologs ergab, das dieses für seine Basisaktivität keinen Adaptor benötigt. Die Abhängigkeit zwischen ClpC und Adaptor ist daher nicht innerhalb der ClpC-Familie konserviert und scheint somit ein spezifisches Merkmal des B. subtilis ClpC zu sein. (4) Neben ihrer Aktivierungsfunktion, vermitteln Adaptorproteine auch die Substraterkennung für ClpC. Das bisherige ClpC Substraspektrum umfasst die regulatorischen Proteine ComK, ComS, SpoIIAB, CtsR und MurAA. Die Vielzahl der Substrate lässt eine entsprechend große Anzahl an Adaptoren vermuten. Der spezifische Adaptor für CtsR, dem Regulator der Klasse III Hitzeschockgene war bisher unbekannt und konnte in dieser Arbeit in McsB identifiziert werden. McsB ist damit neben MecA and seinem Paralog, YpbH, das dritte Adaptorprotein für ClpC. Die Besonderheit von McsB liegt in seiner zusätzlichen Aktivität als Tyrosinkinase und dass diese direkt mit der Adaptorfunktion gekopelt ist. Der Kinase kommt eine zweifache Bedeutung zu (i) McsB phosphoryliert CtsR, welches die CtsR- DNA-Interaktion aufhebt und (ii) nur im phosphorylierten Zustand ist McsB befähigt, CtsR an ClpCP zur anschließenden Proteolyse zu zuführen. Die Kinaseaktivität unterliegt einer strengen Kontrolle. Die Kinaseaktivität von McsB wird durch sein Aktivatorprotein McsA induziert, durch ClpC inhibiert und McsB durch seine spezifische Phosphatase YwlE dephosphoryliert. Aufgrund der Kopplung zwischen Kinase- und Adaptoraktivität kann McsB als regulierter Adaptor angesehen werden. (5) Obwohl McsB keinerlei Ähnlichkeit zu MecA und YpbH aufweist, interagiert es mit den gleichen ClpC-Domänen. Dies wiederum legt die Vermutung nahe, dass sich die simultane Interaktion zweier verschiedener Adaptorproteine mit ClpC ausschließt. Die Annahme einer potentiellen Kompetition konnte experimentell erhärtet werden. In seiner phosphorlyierten Form konnte McsB die Adaptorfunktion von MecA unterdrücken. Abschließend konnte ein Modell zur Regulation der CtsR Aktivität aufgestellt werden, das die neuen Erkenntnisse z.B. die Identifizierung des spezifischen Adaptors für CtsR, McsB, den Phosphattransfer zwischen McsB, CtsR und YwlE und der Interaktion von ClpC mit seinen weiteren Adaptorproteinen, berücksichtigt. Die Konkurrenz verschiedener Adaptorproteine könnte generell ein wesentlicher Bestandteil der Kontrolle der regulierten Proteolyse sein

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