Preclinical evaluation of the novel [18F]CHDI-650 PET ligand for non-invasive quantification of mutant huntingtin aggregates in Huntington's disease.

PURPOSE: Positron emission tomography (PET) imaging of mutant huntingtin (mHTT) aggregates is a potential tool to monitor disease progression as well as the efficacy of candidate therapeutic interventions for Huntington's disease (HD). To date, the focus has been mainly on the investigation of 11C radioligands; however, favourable 18F radiotracers will facilitate future clinical translation. This work aimed at characterising the novel [18F]CHDI-650 PET radiotracer using a combination of in vivo and in vitro approaches in a mouse model of HD. METHODS: After characterising [18F]CHDI-650 using in vitro autoradiography, we assessed in vivo plasma and brain radiotracer stability as well as kinetics through dynamic PET imaging in the heterozygous (HET) zQ175DN mouse model of HD and wild-type (WT) littermates at 9 months of age. Additionally, we performed a head-to-head comparison study at 3 months with the previously published [11C]CHDI-180R radioligand. RESULTS: Plasma and brain radiometabolite profiles indicated a suitable metabolic profile for in vivo imaging of [18F]CHDI-650. Both in vitro autoradiography and in vivo [18F]CHDI-650 PET imaging at 9 months of age demonstrated a significant genotype effect (p < 0.0001) despite the poor test-retest reliability. [18F]CHDI-650 PET imaging at 3 months of age displayed higher differentiation between genotypes when compared to [11C]CHDI-180R. CONCLUSION: Overall, [18F]CHDI-650 allows for discrimination between HET and WT zQ175DN mice at 9 and 3 months of age. [18F]CHDI-650 represents the first suitable 18F radioligand to image mHTT aggregates in mice and its clinical evaluation is underway.

Genetic Variants Associated with Suicide Risk in the Mexican Population: A Systematic Literature Review.

Suicide is defined as the action of harming oneself with the intention of dying. It is estimated that worldwide, one person dies by suicide every 40 s, making it a major health problem. Studies in families have suggested that suicide has a genetic component, so the search for genetic variants associated with suicidal behavior could be useful as potential biomarkers to identify people at risk of suicide. In Mexico, some studies of gene variants related to neurotransmission and other important pathways have been carried out and potential association of variants located in the following genes has been suggested: SLC6A4, SAT-1, TPH-2, ANKK1, GSHR, SCARA50, RGS10, STK33, COMT, and FKBP5. This systematic review shows the genetic studies conducted on the Mexican population. This article contributes by compiling the existing information on genetic variants and genes associated with suicidal behavior, in the future could be used as potential biomarkers to identify people at risk of suicide.

Design and Characterization of pMyc/pMax Peptide-Coupled Gold Nanosystems for Targeting Myc in Prostate Cancer Cell Lines.

Myc and Max are essential proteins in the development of prostate cancer. They act by dimerizing and binding to E-box sequences. Disrupting the Myc:Max heterodimer interaction or its binding to E-box sequences to interrupt gene transcription represent promising strategies for treating cancer. We designed novel pMyc and pMax peptides from reference sequences, and we evaluated their ability to bind specifically to E-box sequences using an electrophoretic mobility shift assay (EMSA). Then, we assembled nanosystems (NSs) by coupling pMyc and pMax peptides to AuNPs, and determined peptide conjugation using UV-Vis spectroscopy. After that, we characterized the NS to obtain the nanoparticle's size, hydrodynamic diameter, and zeta potential. Finally, we evaluated hemocompatibility and cytotoxic effects in three different prostate adenocarcinoma cell lines (LNCaP, PC-3, and DU145) and a non-cancerous cell line (Vero CCL-81). EMSA results suggests peptide-nucleic acid interactions between the pMyc:pMax dimer and the E-box. The hemolysis test showed little hemolytic activity for the NS at the concentrations (5, 0.5, and 0.05 ng/µL) we evaluated. Cell viability assays showed NS cytotoxicity. Overall, results suggest that the NS with pMyc and pMax peptides might be suitable for further research regarding Myc-driven prostate adenocarcinomas.

Green Synthesis of Mesquite-Gum-Stabilized Gold Nanoparticles for Biomedical Applications: Physicochemical Properties and Biocompatibility Assessment.

Using cytotoxic reducing and stabilizing agents in the synthesis of gold nanoparticles (AuNPs) limits their use in biomedical applications. One strategy to overcome this problem is using "green" synthesis methodologies using polysaccharides. In the present study, we propose a green methodology for synthetizing AuNPs with mesquite gum (MG) as a reducing agent and steric stabilizer in Gold(III) chloride trihydrate aqueous solutions to obtain biocompatible nanoparticles that can be used for biomedical applications. Through this method, AuNPs can be produced without using elevated temperatures or pressures. For synthetizing gold nanoparticles coated with mesquite gum (AuNPs@MG), Gold(III) chloride trihydrate was used as a precursor, and mesquite gum was used as a stabilizing and reducing agent. The AuNPs obtained were characterized using UV-Vis spectroscopy, dynamic light scattering, transmission electron microscopy, scanning transmission electron microscopy, and FT-IR spectroscopy. The stability in biological media (phosphate buffer solution), cytotoxicity (MTT assay, hematoxylin, and eosin staining), and hemocompatibility (Hemolysis assay) were measured at different concentrations and exposure times. The results showed the successful synthesis of AuNPs@MG with sizes ranging from 3 to 30 nm and a zeta potential of -31 mV. The AuNPs@MG showed good colloidal stability in PBS (pH 7.4) for up to 24 h. Finally, cytotoxicity assays showed no changes in cell metabolism or cell morphology. These results suggest that these gold nanoparticles have potential biomedical applications because of their low cytotoxicity and hemotoxicity and improved stability at a physiological pH.

In Vivo Cerebral Imaging of Mutant Huntingtin Aggregates Using 11C-CHDI-180R PET in a Nonhuman Primate Model of Huntington Disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (CAG) trinucleotide expansion in the huntingtin (HTT) gene that encodes the mutant huntingtin protein (mHTT). Visualization and quantification of cerebral mHTT will provide a proxy for target engagement and a means to evaluate therapeutic interventions aimed at lowering mHTT in the brain. Here, we validated the novel radioligand 11C-labeled 6-(5-((5-methoxypyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one (11C-CHDI-180R) using PET imaging to quantify cerebral mHTT aggregates in a macaque model of HD. Methods: Rhesus macaques received MRI-guided intrastriatal delivery of a mixture of AAV2 and AAV2.retro viral vectors expressing an HTT fragment bearing 85 CAG repeats (85Q, n = 5), a control HTT fragment bearing 10 CAG repeats (10Q, n = 4), or vector diluent only (phosphate-buffered saline, n = 5). Thirty months after surgery, 90-min dynamic PET/CT imaging was used to investigate 11C-CHDI-180R brain kinetics, along with serial blood sampling to measure input function and stability of the radioligand. The total volume of distribution was calculated using a 2-tissue-compartment model as well as Logan graphical analysis for regional quantification. Immunostaining for mHTT was performed to corroborate the in vivo findings. Results: 11C-CHDI-180R displayed good metabolic stability (51.4% ± 4.0% parent in plasma at 60 min after injection). Regional time-activity curves displayed rapid uptake and reversible binding, which were described by a 2-tissue-compartment model. Logan graphical analysis was associated with the 2-tissue-compartment model (r 2 = 0.96, P < 0.0001) and used to generate parametric volume of distribution maps. Compared with controls, animals administered the 85Q fragment exhibited significantly increased 11C-CHDI-180R binding in several cortical and subcortical brain regions (group effect, P < 0.0001). No difference in 11C-CHDI-180R binding was observed between buffer and 10Q animals. The presence of mHTT aggregates in the 85Q animals was confirmed histologically. Conclusion: We validated 11C-CHDI-180R as a radioligand to visualize and quantify mHTT aggregated species in a HD macaque model. These findings corroborate our previous work in rodent HD models and show that 11C-CHDI-180R is a promising tool to assess the mHTT aggregate load and the efficacy of therapeutic strategies.

IKBKB reduces huntingtin aggregation by phosphorylating serine 13 via a non-canonical IKK pathway.

N-terminal phosphorylation at residues T3 and S13 is believed to have important beneficial implications for the biological and pathological properties of mutant huntingtin, where inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) was identified as a candidate regulator of huntingtin N-terminal phosphorylation. The paucity of mechanistic information on IKK pathways, together with the lack of sensitive methods to quantify endogenous huntingtin phosphorylation, prevented detailed study of the role of IKBKB in Huntington's disease. Using novel ultrasensitive assays, we demonstrate that IKBKB can regulate endogenous S13 huntingtin phosphorylation in a manner, dependent on its kinase activity and known regulators. We found that the ability of IKBKB to phosphorylate endogenous huntingtin S13 is mediated through a non-canonical interferon regulatory factor3-mediated IKK pathway, distinct from the established involvement of IKBKB in mutant huntingtin's pathological mechanisms mediated via the canonical pathway. Furthermore, increased huntingtin S13 phosphorylation by IKBKB resulted in decreased aggregation of mutant huntingtin in cells, again dependent on its kinase activity. These findings point to a non-canonical IKK pathway linking S13 huntingtin phosphorylation to the pathological properties of mutant huntingtin aggregation, thought to be significant to Huntington's disease.

Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

Role of hydraulic traits in stomatal regulation of transpiration under different vapour pressure deficits across five Mediterranean tree crops.

The differential stomatal regulation of transpiration among plant species in response to water deficit is not fully understood, although several hydraulic traits have been reported to influence it. This knowledge gap is partly due to a lack of direct and concomitant experimental data on transpiration, stomatal conductance, and hydraulic traits. We measured sap flux density (Js), stomatal conductance (gs), and different hydraulic traits in five crop species. Our aim was to contribute to establishing the causal relationship between water consumption and its regulation using a hydraulic trait-based approach. The results showed that the species-specific regulation of Js by gs was overall coordinated with the functional hydraulic traits analysed. Particularly relevant was the negative and significant relationship found between the Huber value (Hv) and its functional analogue ratio between maximum Js and gs (Jsmax/gsmax) which can be understood as a compensation to maintain the hydraulic supply to the leaves. The Hv was also significantly related to the slope of the relationship between gs and Js response to vapour pressure deficit and explained most of its variability, adding up to evidence recognizing Hv as a major trait in plant water relations. Thus, a hydraulic basis for regulation of tree water use should be considered.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

On the path from xylem hydraulic failure to downstream cell death.

Xylem hydraulic failure (HF) has been identified as a ubiquitous factor in triggering drought-induced tree mortality through the damage induced by the progressive dehydration of plant living cells. However, fundamental evidence of the mechanistic link connecting xylem HF to cell death has not been identified yet. The main aim of this study was to evaluate, at the leaf level, the relationship between loss of hydraulic function due to cavitation and cell death under drought conditions and discern how this relationship varied across species with contrasting resistances to cavitation. Drought was induced by withholding water from potted seedlings, and their leaves were sampled to measure their relative water content (RWC) and cell mortality. Vulnerability curves to cavitation at the leaf level were constructed for each species. An increment in cavitation events occurrence precedes the onset of cell mortality. A variation in cells tolerance to dehydration was observed along with the resistance to cavitation. Overall, our results indicate that the onset of cellular mortality occurs at lower RWC than the one for cavitation indicating the role of cavitation in triggering cellular death. They also evidenced a critical RWC for cellular death varying across species with different cavitation resistance.

Association of rs4711998 of IL-17A, rs2275913 of IL-17A and rs763780 IL-17F gene polymorphisms with non-segmental vitiligo in a Mexican population.

Vitiligo is the most common depigmenting disease characterized by achromic macules due to selective loss of melanocytes. The pathogenesis remains poorly elucidated, and multiple hypotheses exist regarding its pathogenesis. Evidence suggests that stress on melanocytes can result in activation of the immune system, and involvement of both activated cluster of differentiation (CD8+) cytotoxic and CD4+ T cells in the dysfunction, depigmentation, and apoptosis of melanocytes. Recent studies show that the interleukin 17 (IL-17) axis plays a central role in the pathogenesis of the disease. IL-17 is an important regulatory effector cytokine in this pathway. The aim of this study was to evaluate the association of IL-17A rs4711998 (-832A/G), IL-17A rs2275913 (-197G/A), and IL-17F rs763780 (7488A/G) with vitiligo in a Northeastern Mexican population. This was a case-control study and included 116 patients with vitiligo and 116 control subjects. Genotype characterization of IL-17A rs4711998 (-832A/G), IL-17A rs2275913 (-197G/A), and IL-17F rs763780 (7488A/G) was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. A p ≤ 0.05 was considered significant. It was observed that the combination of the genotypes GG/GA for IL-17F rs763780 (7488A/G) was associated with an increased risk for the development of vitiligo (OR 2.0943, 95% Cl 1.2375-3.5445, p = 0.0056). Regarding IL-17A rs4711998 (-832A/G) and IL-17A rs2275913 (-197G/A) genotyping, no association with vitiligo development was found. In conclusion, the SNP rs763780 in the IL-17F gene appears to be a risk factor for vitiligo development in this Mexican population and it may be useful in future studies, especially for the development of new therapies.

Developing HDAC4-Selective Protein Degraders To Investigate the Role of HDAC4 in Huntington's Disease Pathology.

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).

Biodistribution and dosimetry in human healthy volunteers of the PET radioligands [11C]CHDI-00485180-R and [11C]CHDI-00485626, designed for quantification of cerebral aggregated mutant huntingtin.

PURPOSE: Huntington's disease is caused by a trinucleotide expansion in the HTT gene, which leads to aggregation of mutant huntingtin (mHTT) protein in the brain and neurotoxicity. Direct in vivo measurement of mHTT aggregates in human brain parenchyma is not yet possible. In this first-in-human study, we investigated biodistribution and dosimetry in healthy volunteers of [11C]CHDI-00485180-R ([11C]CHDI-180R) and [11C]CHDI-00485626 ([11C]CHDI-626), two tracers designed for PET imaging of aggregated mHTT in the brain that have been validated in preclinical models. METHODS: Biodistribution and radiation dosimetry studies were performed in 3 healthy volunteers (age 25.7 ± 0.5 years; 2 F) for [11C]CHDI-180R and in 3 healthy volunteers (age 35.3 ± 6.8 years; 2 F) for [11C]CHDI-626 using sequential whole-body PET-CT. Source organs were delineated in 3D using combined PET and CT data. Individual organ doses and effective doses were determined using OLINDA 2.1. RESULTS: There were no clinically relevant adverse events. The mean effective dose (ED) for [11C]CHDI-180R was 4.58 ± 0.65 µSv/MBq, with highest absorbed doses for liver (16.9 µGy/MBq), heart wall (15.9 µGy/MBq) and small intestine (15.8 µGy/MBq). Mean ED for [11C]CHDI-626 was 5.09 ± 0.06 µSv/MBq with the highest absorbed doses for the gallbladder (26.5 µGy/MBq), small intestine (20.4 µGy/MBq) and liver (19.6 µGy/MBq). Decay-corrected brain uptake curves showed promising kinetics for [11C]CHDI-180R, but for [11C]CHDI-626 an increasing signal over time was found, probably due to accumulation of a brain-penetrant metabolite. CONCLUSION: [11C]CHDI-180R and [11C]CHDI-626 are safe for in vivo PET imaging in humans. The estimated radiation burden is in line with most 11C-ligands. While [11C]CHDI-180R has promising kinetic properties in the brain, [11C]CHDI-626 is not suitable for human in vivo mHTT PET due to the possibility of a radiometabolite accumulating in brain parenchyma. TRIAL REGISTRATION: EudraCT number 2020-002129-27. CLINICALTRIALS: gov NCT05224115 (retrospectively registered).

Modelling the Human Blood-Brain Barrier in Huntington Disease.

While blood-brain barrier (BBB) dysfunction has been described in neurological disorders, including Huntington's disease (HD), it is not known if endothelial cells themselves are functionally compromised when promoting BBB dysfunction. Furthermore, the underlying mechanisms of BBB dysfunction remain elusive given the limitations with mouse models and post mortem tissue to identify primary deficits. We established models of BBB and undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived brain-like microvascular endothelial cells (iBMEC) from HD patients or unaffected controls. We demonstrated that HD-iBMECs have abnormalities in barrier properties, as well as in specific BBB functions such as receptor-mediated transcytosis.

Identification of a Potent, Selective, and Brain-Penetrant Rho Kinase Inhibitor and its Activity in a Mouse Model of Huntington's Disease.

The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.

Validation, kinetic modeling, and test-retest reproducibility of [18F]SynVesT-1 for PET imaging of synaptic vesicle glycoprotein 2A in mice.

Alterations in synaptic vesicle glycoprotein 2 A (SV2A) have been associated with several neuropsychiatric and neurodegenerative disorders. Therefore, SV2A positron emission tomography (PET) imaging may provide a unique tool to investigate synaptic density dynamics during disease progression and after therapeutic intervention. This study aims to extensively characterize the novel radioligand [18F]SynVesT-1 for preclinical applications. In C57Bl/6J mice (n = 39), we assessed the plasma profile of [18F]SynVesT-1, validated the use of a noninvasive image-derived input function (IDIF) compared to an arterial input function (AIF), performed a blocking study with levetiracetam (50 and 200 mg/kg, i.p.) to verify the specificity towards SV2A, examined kinetic models for volume of distribution (VT) quantification, and explored test-retest reproducibility of [18F]SynVesT-1 in the central nervous system (CNS). Plasma availability of [18F]SynVesT-1 decreased rapidly (13.4 ± 1.5% at 30 min post-injection). VT based on AIF and IDIF showed excellent agreement (r2 = 0.95, p < 0.0001) and could be reliably estimated with a 60-min acquisition. The blocking study resulted in a complete blockade with no suitable reference region. Test-retest analysis indicated good reproducibility (mean absolute variability <10%). In conclusion, [18F]SynVesT-1 is selective for SV2A with optimal kinetics representing a candidate tool to quantify CNS synaptic density non-invasively.

Leaf water potential measurements using the pressure chamber: Synthetic testing of assumptions towards best practices for precision and accuracy.

Leaf water potential (ψleaf ), typically measured using the pressure chamber, is the most important metric of plant water status, providing high theoretical value and information content for multiple applications in quantifying critical physiological processes including drought responses. Pressure chamber measurements of ψleaf (ψleafPC ) are most typical, yet, the practical complexity of the technique and of the underlying theory has led to ambiguous understanding of the conditions to optimize measurements. Consequently, specific techniques and precautions diversified across the global research community, raising questions of reliability and repeatability. Here, we surveyed specific methods of ψleafPC from multiple laboratories, and synthesized experiments testing common assumptions and practices in ψleafPC for diverse species: (i) the need for equilibration of previously transpiring leaves; (ii) leaf storage before measurement; (iii) the equilibration of ψleaf for leaves on bagged branches of a range of dehydration; (iv) the equilibration of ψleaf across the lamina for bagged leaves, and the accuracy of measuring leaves with artificially 'elongated petioles'; (v) the need in ψleaf measurements for bagging leaves and high humidity within the chamber; (vi) the need to avoid liquid water on leaf surfaces; (vii) the use of 'pulse' pressurization versus gradual pressurization; and (viii) variation among experimenters in ψleafPC determination. Based on our findings we provide a best practice protocol to maximise accuracy, and provide recommendations for ongoing species-specific tests of important assumptions in future studies.