Discovery of Therapeutic Approaches for Polyglutamine Diseases: A Summary of Recent Efforts

Polyglutamine (PolyQ) diseases are a group of neurodegenerative disorders caused by the expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the coding region of specific genes. This leads to the production of pathogenic proteins containing critically expanded tracts of glutamines. Although polyQ diseases are individually rare, the fact that these nine diseases are irreversibly progressive over 10 to 30 years, severely impairing and ultimately fatal, usually implicating the full-time patient support by a caregiver for long time periods, makes their economic and social impact quite significant. This has led several researchers worldwide to investigate the pathogenic mechanism(s) and therapeutic strategies for polyQ diseases. Although research in the field has grown notably in the last decades, we are still far from having an effective treatment to offer patients, and the decision of which compounds should be translated to the clinics may be very challenging. In this review, we provide a comprehensive and critical overview of the most recent drug discovery efforts in the field of polyQ diseases, including the most relevant findings emerging from two different types of approaches-hypothesis-based candidate molecule testing and hypothesis-free unbiased drug screenings. We hereby summarize and reflect on the preclinical studies as well as all the clinical trials performed to date, aiming to provide a useful framework for increasingly successful future drug discovery and development efforts.Project ON.2 SR&TD Integrated Program (NORTE-07-0124-FEDER-000021), co-funded by North Portugal Regional Operational Program (ON.2-O Novo Norte), under the National Strategic Reference Framework, through the European Regional Development Fund (ERDF) and also supported by Fundação para a Ciência e Tecnologia through the project POCI-01-0145-FEDER-016818 (PTDC/NEU-NMC/3648/2014)info:eu-repo/semantics/publishedVersio

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Sox11 Reduces Caspase-6 Cleavage and Activity.

The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP) family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS) all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11

Phosphorylation of mutant huntingtin at serine 116 modulates neuronal toxicity.

Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites

Sox11 reduces caspase-3 and -7 activity but not caspase-8 or -9 activity.

<p>(A and C) Top panels show caspase-7 and caspase-3 expression levels with and without sox11 co-expressed. Sox11 co-expression with caspase-3 appears to affect its levels more than caspase-7. The second panel shows the large subunit fragments cleaved from caspase-7 and caspase-3. Both caspase-3 and 7 self- cleavage are reduced. The bottom two panels show sox11 was successfully co-expressed and actin levels indicate protein loading was equal. (B and D) Caspase-3/7 substrate luminescent based assay showed sox11 to reduce both caspase-3 and 7 activities to almost control levels. One-way anova, Sidak’s multiple comparsion test, ****p<0.0001 (E and G) Sox11 co-expression with either caspase-8 or 9 does not affect their steady state levels or proteolysis. (F and H) Caspase-8 and 9 activity assays showed no significant effect of sox11 (n = 3, one-way anova, Sidak’s multiple comparsion test).</p

Sox11 is protective against apoptotic insult and other sox proteins can also reduce caspase-6 activity.

<p>(A) Mouse cortical primary neurons were transfected with either sox11 or sox11 ΔNLS. 24 hours post-transfection, neurons were either treated with toxic agents or subjected to serum starvation for a further 24 hours. Neurons were fixed and stained and assessed for cell death via nuclear condensation (n = 8, one-way anova, Sidak’s multiple comparison test ****p<0.0001, **p<0.01). (B) Caspase-6 activity was measured using a luciferase-based substrate assay from cells transfected with indicated plasmids. Sox11 reduced caspase-6 activity to control levels. Sox4 and sox7 partially reduced caspase-6 activation (n = 2, one-way anova, Sidak’s multiple comparison test ****p<0.0001, **p<0.01).</p

Sox11 prevents caspase-6 autocleavage.

<p>(A) Protein lysates were harvested and separated on a 4–12% bis-tris gel. Top panel shows a western blot of caspase-6 cleavage, the slower migrating band represents the uncleaved caspase-6 zymogen (note caspase-6 runs slighter higher than caspase-6 C163A because of an N-terminal flag tag) and the lower band represents caspase-6 following removal of its prodomain (lane 2). Cleavage of caspase-6 into caspase-6 Δprodomain is diminishedd in the presence of sox11 (lane 6). The lower panel is a western blot probed with an antibody capable of detecting the caspase-6 large subunit. Generation of the caspase-6 large subunit was suppressed in the presence of sox11 (compare lanes 2 and 4 with lanes 6 and 8, respectively). (B) Negative control experiments were performed using two other proteins identified in the yeast-two hybrid screen, COP1 and FEZ1. Neither FEZ1 nor COP1 affected caspase-6 self-cleavage. (C and D) Caspase-6 protein levels quantified using densitometry showed sox11 to significantly decrease caspase-6 protein levels by 30% but caspase-6 activity is completely blocked in the presence of sox11 (n = 8, unpaired t-test, **** p<0.0001).</p

Nuclear localization of sox11 contributes to the loss of caspase-6 activity.

<p>(A i-ix) Immunofluorescent images of caspase-6 and sox11, as well as caspase-6 and sox11 deletion mutants captured using a confocal microscope. Several sox11 mutants lose their nuclear localization i.e. (v), (vii), and (ix). Neither sox11 nor sox11 mutants altered the distribution of caspase-6 within the cell. (B) Microscopy images taken to compare the cellular distribution of sox11 and sox11 with the deletion of a putative NLS. Sox11 ΔNLS loses its nuclear localization and becomes more cytoplasmic (lower panel). (C) Western blot comparing caspase-6 proteolysis in the presence of sox11 versus sox11 ΔNLS. Sox11 ΔNLS is no longer capable of completely blocking caspase-6 cleavage (compare lanes 5 and 6). (D) Quantification of caspase-6 FL (full length, zymogen) levels vs caspase-6 post prodomain cleavage with and without sox11 ΔNLS from (C). No significant change was seen in caspase-6 full length levels in the presence of sox11 ΔNLS. However, caspase-6 prodomain levels were increased 56% in the presence of sox11 ΔNLS.</p

Sox11 reduces caspase-6 activity to control levels.

<p>(A) Lysates from HEK293FT cells were collected and assayed for caspase-6 activity via a luciferase based substrate assay system. NB caspase-6 Δpro refers to caspase-6 Δprodomain. The western blot shows caspase-6 activity is decreased in the presence of sox11. Caspase-6 activity was reduced to control levels by sox11. Statistical analysis showed this reduction was very significant (n = 3, one-way anova, **** p<0001). (B) Negative control experiments were performed using two other proteins, FEZ1 and DISC1. Neither FEZ1 nor DISC1 caused a significant decrease in caspase-6 activity but a one-way anova, followed by a Sidak’s multiple comparsion test showed DISC1 caused a slight increase in caspase-6 activity (n = 3, **** p<0001, *<0.05) (C) RNA was extracted from HEK293FT cells transfected for 24hours with indicated plasmids. Reverse transcription and PCR amplification using caspase-6 primers showed no significant change in mRNA levels between single and co-transfected samples (compare lanes 2, 3, and 4 with 6, 7, and 8, respectively). GAPDH levels were equal across all samples tested (n = 3). (D) A luciferase based reporter assay was used to assay sox11 transcriptional activity in the presence of caspase-6. Sox11 transactivation potential was not significantly affected by caspase-6 or caspase-6 C163A.</p