Investigation of genetic determinants of cognitive change in later life.

Cognitive decline is a major health concern and identification of genes that may serve as drug targets to slow decline is important to adequately support an aging population. Whilst genetic studies of cross-sectional cognition have been carried out, cognitive change is less well-understood. Here, using data from the TOMMORROW trial, we investigate genetic associations with cognitive change in a cognitively normal older cohort. We conducted a genome-wide association study of trajectories of repeated cognitive measures (using generalised estimating equation (GEE) modelling) and tested associations with polygenic risk scores (PRS) of potential risk factors. We identified two genetic variants associated with change in attention domain scores, rs534221751 (p = 1 × 10-8 with slope 1) and rs34743896 (p = 5 × 10-10 with slope 2), implicating NCAM2 and CRIPT/ATP6V1E2 genes, respectively. We also found evidence for the association between an education PRS and baseline cognition (at >65 years of age), particularly in the language domain. We demonstrate the feasibility of conducting GWAS of cognitive change using GEE modelling and our results suggest that there may be novel genetic associations for cognitive change that have not previously been associated with cross-sectional cognition. We also show the importance of the education PRS on cognition much later in life. These findings warrant further investigation and demonstrate the potential value of using trial data and trajectory modelling to identify genetic variants associated with cognitive change.

Plasma neurofilament light levels show elevation two years prior to diagnosis of amyotrophic lateral sclerosis in the UK Biobank.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease with profound unmet need. In patients carrying genetic mutations, elevations in neurofilament light (NfL) have been shown to precede symptom onset, however, the natural history of NfL in general ALS patients is less characterized. METHODS: We performed a secondary analysis of the UK Biobank Pharma Proteomics Project (UKB-PPP), a subset of the UK Biobank, a population-based cohort study in the United Kingdom, to examine plasma NfL levels in 237 participants subsequently diagnosed with ALS. We applied logistic and Cox proportional hazards regression to compare cases to 42,752 population-based and 948 age and sex-matched controls. Genetic information was obtained from exome and genotype array data.Results and Conclusions: We observed that NfL was 1.42-fold higher in cases vs population-based controls. At two to three years pre-diagnosis, NfL levels in patients exceeded the 95th percentile of age and sex-matched controls. A time-to-diagnosis analysis showed that a 2-fold increase in NfL levels was associated with a 3.4-fold risk of diagnosis per year, with NfL being most predictive of case status at two years (AUC = 0.96). Participants with genetic variation that might put them at risk for familial disease (N = 46) did not show a different pattern of association than those without (N = 191). DISCUSSION: Our findings show that NfL is elevated and discriminative of future ALS diagnosis up to two years prior to diagnosis in patients with and without genetic risk variants.

A natural history study to track brain and spinal cord changes in individuals with Friedreich's ataxia: TRACK-FA study protocol.

INTRODUCTION: Drug development for neurodegenerative diseases such as Friedreich's ataxia (FRDA) is limited by a lack of validated, sensitive biomarkers of pharmacodynamic response in affected tissue and disease progression. Studies employing neuroimaging measures to track FRDA have thus far been limited by their small sample sizes and limited follow up. TRACK-FA, a longitudinal, multi-site, and multi-modal neuroimaging natural history study, aims to address these shortcomings by enabling better understanding of underlying pathology and identifying sensitive, clinical trial ready, neuroimaging biomarkers for FRDA. METHODS: 200 individuals with FRDA and 104 control participants will be recruited across seven international study sites. Inclusion criteria for participants with genetically confirmed FRDA involves, age of disease onset ≤ 25 years, Friedreich's Ataxia Rating Scale (FARS) functional staging score of ≤ 5, and a total modified FARS (mFARS) score of ≤ 65 upon enrolment. The control cohort is matched to the FRDA cohort for age, sex, handedness, and years of education. Participants will be evaluated at three study visits over two years. Each visit comprises of a harmonized multimodal Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) scan of the brain and spinal cord; clinical, cognitive, mood and speech assessments and collection of a blood sample. Primary outcome measures, informed by previous neuroimaging studies, include measures of: spinal cord and brain morphometry, spinal cord and brain microstructure (measured using diffusion MRI), brain iron accumulation (using Quantitative Susceptibility Mapping) and spinal cord biochemistry (using MRS). Secondary and exploratory outcome measures include clinical, cognitive assessments and blood biomarkers. DISCUSSION: Prioritising immediate areas of need, TRACK-FA aims to deliver a set of sensitive, clinical trial-ready neuroimaging biomarkers to accelerate drug discovery efforts and better understand disease trajectory. Once validated, these potential pharmacodynamic biomarkers can be used to measure the efficacy of new therapeutics in forestalling disease progression. CLINICAL TRIAL REGISTRATION: ClinicalTrails.gov Identifier: NCT04349514.

Comparative analytical performance of multiple plasma Aß42 and Aß40 assays and their ability to predict positron emission tomography amyloid positivity.

INTRODUCTION: This report details the approach taken to providing a dataset allowing for analyses on the performance of recently developed assays of amyloid beta (Aß) peptides in plasma and the extent to which they improve the prediction of amyloid positivity. METHODS: Alzheimer's Disease Neuroimaging Initiative plasma samples with corresponding amyloid positron emission tomography (PET) data were run on six plasma Aß assays. Statistical tests were performed to determine whether the plasma Aß measures significantly improved the area under the receiver operating characteristic curve for predicting amyloid PET status compared to age and apolipoprotein E (APOE) genotype. RESULTS: The age and APOE genotype model predicted amyloid status with an area under the curve (AUC) of 0.75. Three assays improved AUCs to 0.81, 0.81, and 0.84 (P < .05, uncorrected for multiple comparisons). DISCUSSION: Measurement of Aß in plasma contributes to addressing the amyloid component of the ATN (amyloid/tau/neurodegeneration) framework and could be a first step before or in place of a PET or cerebrospinal fluid screening study. HIGHLIGHTS: The Foundation of the National Institutes of Health Biomarkers Consortium evaluated six plasma amyloid beta (Aß) assays using Alzheimer's Disease Neuroimaging Initiative samples. Three assays improved prediction of amyloid status over age and apolipoprotein E (APOE) genotype. Plasma Aß42/40 predicted amyloid positron emission tomography status better than Aß42 or Aß40 alone.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Selective inhibition of KCC2 leads to hyperexcitability and epileptiform discharges in hippocampal slices and in vivo.

GABA(A) receptors form Cl(-) permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K(+)/Cl(-) cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl(-) levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in E(GABA) values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg(2+) conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factor ex vivo and in vivo.

Novel methodology to identify TRPV1 antagonists independent of capsaicin activation.

TRPV1 was originally characterized as an integrator of various noxious stimuli such as capsaicin, heat, and protons. TRPV1-null mice exhibit a deficiency in sensing noxious heat stimuli, suggesting that TRPV1 is one of the main heat sensors on nociceptive primary afferent neurons and a candidate target for heat hypersensitivity in chronic pain. Several different potent and selective TRPV1 antagonists have been developed by more than 50 companies since the characterization of the receptor in 1997. A consequence of this competitive interest is the crowding of patentable chemical space, because very similar in vitro screening assays are used. To circumvent this issue and to expand our understanding of TRPV1 biology, we sought to take advantage of recent advancements in automated patch-clamp technology to design a novel screening cascade. This SAR-driving assay identified novel modulators that blocked the depolarization-induced activation of outwardly-rectifying TRPV1 currents independent of agonist stimulation, and we correlated the pharmacology to three other innovative assays for higher-throughput screening. Ultimately, we have identified a screening paradigm that would have good predictive value for future TRPV1 drug discovery projects and novel chemical space with a higher probability of gaining intellectual property coverage.

Molecular basis of funny current (If) in normal and failing human heart.

I(f) overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of I(f)-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of I(f) overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode I(f)-channels, have been identified in the heart. The present study was designed to characterize HCN isoform expression in failing and non-failing hearts. Ventricular and atrial samples were obtained from normal or failing hearts explanted from patients with end-stage ischemic cardiomyopathy. I(f) was recorded in patch-clamped left ventricular myocytes. mRNA and protein expression of HCN subunits were measured in both atria and ventricles of control and diseased hearts. HCN2 and HCN4 were detected in human myocardium. Both mRNA and protein levels of HCN2/4 were significantly augmented in failing ventricles (p<0.01 for mRNA, p<0.05 for protein). These results are consistent with the electrophysiological data showing that, in failing ventricular myocytes, I(f) is of larger amplitude and activates at less negative potential. Changes in mRNA and protein expression of both HCN2/4 isoforms in atrial specimens from patients with heart failure mirrored those observed in ventricles (p<0.001 for mRNA, p<0.05 for protein). No disease-dependent alteration was detected for MiRP1, the putative beta-subunit of the I(f)-channel. In conclusion, HCN4 is the predominant channel subtype in normal human heart, and its expression is further amplified by disease. HCN upregulation likely contributes to increased I(f) and may play a role in ventricular and atrial arrhythmogenesis in heart failure.

Potassium channel subunit remodeling in rabbits exposed to long-term bradycardia or tachycardia: discrete arrhythmogenic consequences related to differential delayed-rectifier changes.

BACKGROUND: Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits. METHODS AND RESULTS: We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by approximately 50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits. CONCLUSIONS: Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.

Atrial fibrillation-associated minK38G/S polymorphism modulates delayed rectifier current and membrane localization.

BACKGROUND: Atrial fibrillation (AF) is a common acquired arrhythmia with multi-factorial pathogenesis. Recently, a single nucleotide polymorphism (SNP, A/G) at position 112 in the KCNE1 gene, resulting in a glycine/serine amino acid substitution at position 38 of the minK peptide, was associated with AF occurrence (AF more frequent with minK38G); however, the functional effect of this SNP is unknown. METHODS AND RESULTS: We used patch clamp recording, confocal microscopy and protein biochemistry to study the effect of this SNP on delayed-rectifier current expression and mathematical simulation to identify potential functional consequences. The density of slow delayed rectifier current (I(Ks)) resulting from co-expression with KvLQT1 was smaller with minK38G (e.g. at +10 mV: 50+/-7 pA/pF in Chinese hamster ovary (CHO) cells, 45+/-14 pA/pF for COS-7 cells) compared to minK38S (93+/-17 pA/pF, 104+/-23 pA/pF, respectively, P<0.05 for each). I(Ks) kinetics and voltage-dependence were unaffected. Currents resulting from co-expression of human ether-a-go-go-related gene (HERG) were similar for minK38G and minK38S, e.g. upon repolarization from +10 to -50 mV: tail currents 23+/-4 pA/pF versus 22+/-5 pA/pF (P=ns). KvLQT1 membrane immunofluorescence was less in CHO cells co-expressing minK38G versus minK38S, and surface expression of KvLQT1, as determined by labelling with streptavidin/biotin, was increased with minK38S co-expression. Computer simulations with a human atrial action potential model predicted that the minK38G SNP would slightly prolong the atrial action potential and reduce the frequency for alternans behaviour. In the presence of reduced repolarization reserve, these effects were enhanced and under specific conditions early afterdepolarizations occurred. CONCLUSIONS: The minK38G isoform is associated with reduced I(Ks), likely due to decreased KvLQT1 membrane expression. This study reveals a novel amino acid determinant of the minK-KvLQT1 interaction, and if the role of minK38G in AF is confirmed, would suggest mechanistic heterogeneity in genetic determinants of AF.

Novel targets for cardiac antiarrhythmic drug development.

Cardiac arrhythmias remain a major source of morbidity and mortality in developed countries. Antiarrhythmic drug therapy was traditionally the mainstay of cardiac arrhythmia treatment; however, drug therapy of cardiac arrhythmias has been plagued by incomplete efficacy and by potentially serious adverse reactions, of which the most worrisome has been a potential for malignant proarrhythmia and related effects to increase cardiac mortality. This article reviews the principal arrhythmia mechanisms and their ionic determinants, and discusses potential innovative approaches to new antiarrhythmic drug development, including the consideration of novel ionic targets, potential biophysical approaches and non-channel components involved in composing the arrhythmic substrate.

Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model.

BACKGROUND: The hyperpolarization-activated cation current I(f) contributes significantly to sinoatrial node pacemaker function and possibly to ectopic arrhythmogenesis. Little is known about the expression of corresponding hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits in normal hearts and HCN remodeling by diseases, like congestive heart failure (CHF), associated with disturbances of cardiac rhythm. METHODS AND RESULTS: We assessed expression of HCN1, 2 and 4 in normal mongrel dogs and dogs subjected to 2-week ventricular tachypacing-induced CHF. Competitive RT-PCR, Western blot and immunohistochemistry were used to quantify HCN subunit mRNA and protein expression in the right atrium (RA) and sinoatrial node. CHF approximately doubled sinus node recovery time, indicating suppressed sinus node pacemaker function. HCN expression under control conditions was HCN4 > HCN2 >> HCN1. HCN2 and HCN4 expression was greater at both protein and mRNA levels in sinoatrial node than RA. CHF significantly decreased sinus node HCN expression at both mRNA and protein levels (HCN2 by 78% and 82%; HCN4 by 42% and 77%, respectively). RA HCN2 expression was unaltered by CHF, but HCN4 was significantly upregulated (by 209%). CONCLUSIONS: HCN4 is the dominant subunit in canine sinoatrial node and RA; strong sinus node HCN expression likely contributes to its pacemaker function; downregulation of HCN4 and HCN2 expression contribute to CHF-induced sinus node dysfunction; and upregulation of atrial HCN4 may help to promote atrial arrhythmia formation. These findings provide novel information about the molecular basis of normal and disease-related impairments of cardiac impulse formation.

Transmural expression of transient outward potassium current subunits in normal and failing canine and human hearts.

The transient outward current (I(to)), an important contributor to transmural electrophysiological heterogeneity, is significantly remodelled in congestive heart failure (CHF). The molecular bases of transmural I(to) gradients and CHF-dependent ionic remodelling are incompletely understood. To elucidate these issues, we studied mRNA and protein expression of Kv4.3 and KChIP2, the principal alpha and beta subunits believed to form I(to), in epicardial and endocardial tissues and in isolated cardiomyocytes from control dogs and dogs with CHF induced by 240 beats min(-1) ventricular tachypacing. CHF decreased I(to) density in both epicardium and endocardium (by 73 and 55% at +60 mV, respectively), without a significant change in relative current density (endocardium/epicardium 0.11 control, 0.17 CHF). There were transmural gradients in mRNA expression of both Kv4.3 (endocardium/epicardium ratio 0.3 under control conditions) and KChIP2 (endocardium/epicardium ratio 0.2 control), which remained in the presence of CHF (Kv4.3 endocardium/epicardium ratio 0.4; KChIP2 0.4). There were qualitatively similar protein expression gradients in human and canine cardiac tissues and isolated canine cardiomyocytes; however, the KChIP2 gradient was only detectable with a highly selective monoclonal antibody and closely approximated the I(to) density gradient. Kv4.3 mRNA expression was reduced by CHF, but KChIP2 mRNA was not significantly changed. CHF decreased Kv4.3 protein expression in canine cardiac tissues and cardiomyocytes, as well as in terminally failing human heart tissue samples, but KChIP2 protein was not down-regulated in any of the corresponding sample sets. We conclude that both Kv4.3 and KChIP2 may contribute to epicardial-endocardial gradients in I(to), and that I(to) down-regulation in human and canine CHF appears due primarily to changes in Kv4.3.

Post-transcriptional alterations in the expression of cardiac Na+ channel subunits in chronic heart failure.

Clinical and experimental evidence has recently accumulated about the importance of alterations of Na(+) channel (NaCh) function and slow myocardial conduction for arrhythmias in infarcted and failing hearts (i.e., heart failure, HF). The present study evaluated the molecular mechanisms of local alterations in the expression of NaCh subunits which underlie Na(+) current (I(Na)) density decrease in HF. HF was induced in five dogs by sequential coronary microembolization and developed approximately 3 months after the last embolization (left ventricle (LV), ejection fraction = 27 +/- 7%). Five normal dogs served as a control group. Ventricular cardiomyocytes were isolated enzymatically from LV mid-myocardium and I(Na) was measured by whole-cell patch-clamp. The mRNA encoding the cardiac-specific NaCh alpha-subunit Na(v)1.5, and one of its auxiliary subunits beta 1 (NaCh beta 1), were analyzed by competitive reverse transcription-polymerase chain reaction. Protein levels of Na(v)1.5, NaCh beta 1 and NaCh beta 2 were evaluated by western blotting. The maximum density of I(Na)/C(m) was decreased in HF (n = 5) compared to control hearts (33.2 +/- 4.4 vs. 50.0 +/- 4.9 pA/pF, mean +/- S.E.M., n = 5, P < 0.05). The steady-state inactivation and activation of I(Na) remained unchanged in HF compared to control hearts. The levels of mRNA encoding Na(v)1.5, and NaCh beta 1 were unaltered in FH. However, Na(v)1.5 protein expression was reduced about 30% in HF, while NaCh beta 1 and NaCh beta 2 protein were unchanged. We conclude that experimental HF in dogs results in post-transcriptional changes in cardiac NaCh alpha-subunit expression.

Canine ventricular KCNE2 expression resides predominantly in Purkinje fibers.

Mutations in minK-related peptide 1 (MiRP1), the product of the KCNE2 gene, have been associated with malignant ventricular arrhythmia syndromes related to impaired repolarization. MiRP1 interacts with a variety of ion-channel alpha-subunits, dysfunction of which could account for arrhythmia syndromes; however, the observation of very low-level expression of MiRP1 in ventricular tissue has led to doubts about its relevance. The specialized His-Purkinje system plays a key role in cardiac electrophysiology and is an important contributor to ventricular arrhythmias related to abnormal repolarization. We examined the relative abundance of MiRP1 in canine Purkinje versus ventricular tissue and found much greater expression at both mRNA and protein levels in Purkinje tissue. Thus, the cardiac Purkinje system is a strong candidate to play a role in arrhythmic syndromes due to MiRP1 abnormalities.

Molecular basis of species-specific expression of repolarizing K+ currents in the heart.

There are important species-specific differences in K+ current profiles and arrhythmia susceptibility, but interspecies comparisons of K+ channel subunit expression are lacking. We quantified voltage-gated K+ channel (Kv) subunit mRNA and protein in rabbits, guinea pigs, and humans. Kv1.4, Kv4.2, and Kv4.3 mRNA was present in rabbits but undetectable in guinea pigs. MinK mRNA concentration in guinea pigs was almost threefold greater versus humans and 20-fold versus rabbits. MinK protein expression in guinea pigs was almost twofold that in humans and sixfold that in rabbits. KvLQT1 mRNA concentration was greatest in humans, and protein expression in humans was increased by approximately 2- and approximately 7-fold compared with values in rabbits and guinea pigs, respectively. The ether-a-go-go-related gene (ERG1) mRNA was more concentrated in humans, but ERG1 protein expression could not be compared across species because of epitope sequence differences. We conclude that important interspecies differences in cardiac K+ channel subunit expression exist and may contribute to the following: 1) lack of a transient outward current in the guinea pig (alpha-subunit transcription absent in the guinea pig heart); 2) small slow delayed rectifier current and torsades de pointes susceptibility in the rabbit (low-level minK expression); and 3) large slow component of the delayed rectifier current in the guinea pig (strong minK expression).