Mapping neurodegeneration across the Huntington's disease spectrum: a five-year longitudinal analysis of plasma neurofilament light.

BACKGROUND: Neurofilament light (NfL) has previously been highlighted as a potential biomarker for Huntington's Disease (HD) using cross-sectional analyses. Our study aim was to investigate how longitudinal trajectories of plasma NfL relate to HD disease stage. METHODS: 108 participants [78 individuals with the HD mutation, and 30 healthy controls (HC)] were included in this study. Individuals with the HD mutation were categorised separately by both HD-Integrated Staging System (HD-ISS) (Study 1) and PIN score-Approximated Staging System (PASS) (Study 2) criteria. Plasma NfL trajectories were examined using Mixed Linear Modeling (MLM); associations with symptom presentation were assessed using Spearman's rho correlations. FINDINGS: The MLM coefficients for disease stage (HD-ISS ß = 32.73, p < 0.0001; PASS ß = 33.00, p < 0.0001) and disease stage∗time (HD-ISS ß = 7.85, p = 0.004; PASS ß = 6.58, p = 0.0047) suggest these are significant contributors to plasma NfL levels. In addition, the plasma NfL rate of change varied significantly across time (HD-ISS ß = 3.14, p = 0.04; PASS ß = 2.94, p = 0.050). The annualised rate of change was 8.32% for HC; 10.55%, 12.75% and 15.62% for HD-ISS Stage ≤1, Stage 2, and Stage 3, respectively; and 12.13%, 10.46%, 10.33%, 17.52%, for PASS Stage 0, Stage 1, Stage 2, and Stage 3, respectively. Plasma NfL levels correlated with the Symbol Digit Modalities Test (SDMT) in HD-ISS Stage ≤1, and both SDMT and Total Motor Score in Stage 3 (ps < 0.01). INTERPRETATION: Our findings suggest that plasma NfL levels increase linearly across earlier disease stages, correlating with the cognitive SDMT measure. Thereafter, an increase or surge in plasma NfL levels, paired with correlations with both cognitive and motor measures, suggest a late acceleration in clinical and pathological progression. FUNDING: NIH (NS111655); the UCSD HDSA CoE; the UCSD ADRC (NIH-NIA P30 AG062429).

Longitudinal neurocognitive trajectories and risk factors in the first three months following pediatric concussion.

OBJECTIVE: To identify differential trajectories of neurocognitive outcomes following pediatric concussion and investigate predictors associated with patterns of recovery up to 3 months. METHODS: 74 participants aged 8-17 years completed attention/working memory, processing speed, and executive function measures at 2 weeks, 1 month, and 3 months post-injury. We used principal component analysis to generate a composite of information processing. Group-based trajectory modeling identified latent trajectories. Multinominal logistic regression was used to examine associations between risk factors and trajectory groups. RESULTS: We identified three trajectories of neurocognitive outcomes. The medium (54.6%) and high improving groups (35.8%) showed ongoing increase in information processing, while the low persistent group showed limited change 3 months post-injury. This group recorded below average scores on Digit Span Forward and Backward at 3 months. History of pre-injury headache was significantly associated with the persistent low scoring group, relative to the medium improving (p = 0.03) but not the high improving group (p = 0.09). CONCLUSIONS: This study indicates variability in neurocognitive outcomes according to three differential trajectories, with groups partially distinguished by preexisting child factors (history of frequent headaches). Modelling that accounts for heterogeneity in individual outcomes is essential to identify clinically meaningful indices that are indicative of children requiring intervention.

Capillary blood protein markers of posttraumatic headache in children after concussion.

OBJECTIVE: Posttraumatic headache (PTH) represents the most common acute and persistent symptom in children after concussion, yet there is no blood protein signature to stratify the risk of PTH after concussion to facilitate early intervention. This discovery study aimed to identify capillary blood protein markers, at emergency department (ED) presentation within 48 hours of concussion, to predict children at risk of persisting PTH at 2 weeks postinjury. METHODS: Capillary blood was collected using the Mitra Clamshell device from children aged 8-17 years who presented to the ED of the Royal Children's Hospital, Melbourne, Australia, within 48 hours of sustaining a concussion. Participants were followed up at 2 weeks postinjury to determine PTH status. PTH was defined per clinical guidelines as a new or worsened headache compared with preinjury. An untargeted proteomics analysis using data-independent acquisition (DIA) was performed. Principal component analysis and hierarchical clustering were used to reduce the dimensionality of the protein dataset. RESULTS: A total of 907 proteins were reproducibly identified from 82 children within 48 hours of concussion. The mean participant age was 12.78 years (SD 2.54 years, range 8-17 years); 70% of patients were male. Eighty percent met criteria for acute PTH in the ED, while one-third of participants with follow-up experienced PTH at 2 weeks postinjury (range 8-16 days). Hemoglobin subunit zeta (HBZ), cystatin B (CSTB), beta-ala-his dipeptidase (CNDP1), hemoglobin subunit gamma-1 (HBG1), and zyxin (ZYX) were weakly associated with PTH at 2 weeks postinjury based on up to a 7% increase in the PTH group despite nonsignificant Benjamini-Hochberg adjusted p values. CONCLUSIONS: This discovery study determined that no capillary blood protein markers, measured at ED presentation within 48 hours of concussion, can predict children at risk of persisting PTH at 2 weeks postinjury. While HBZ, CSTB, CNDP1, HBG1, and ZYX were weakly associated with PTH at 2 weeks postinjury, there was no specific blood protein signature predictor of PTH in children after concussion. There is an urgent need to discover new blood biomarkers associated with PTH to facilitate risk stratification and improve clinical management of pediatric concussion.

Exploring bradyphrenia in Huntington's disease using the computerized test of information processing (CTiP).

Background: Bradyphrenia, best thought of as the mental equivalent of bradykinesia, has been described in several disorders of the brain including Parkinson's disease and schizophrenia; however, little is known about this phenomenon in Huntington's Disease (HD). Objective: The aim of this study was to investigate the presence of bradyphrenia in HD using the Computerized Test of Information Processing (CTiP), an easy to administer and objective task that assesses cognitive processing speed with increasing task complexity. Methods: This study included 211 participants: Huntington's Disease Integrated Staging System (HD-ISS) Stage 0 [n = 28], Stage 1 [n = 30], Stage 2 [n = 48] and Stage 3 [n = 48], and healthy controls (HC) [n = 57]. The CTiP incorporates three subtests: Simple Reaction Time (SRT), which assesses baseline motor function; Choice Reaction Time (CRT), with an added decisional component; and Semantic Search Reaction Time (SSRT), with an added conceptual component. SRT scores were subtracted from CRT and SSRT scores to establish a motor-corrected measure of central conduction time, which was used to operationalize bradyphrenia. Results: HD-ISS and HC within-group reaction times differed significantly when comparing motor-corrected CRT vs SSRT (all ps < 0.0001). Furthermore, the magnitude of these differences increased with HD disease stage (p < 0.0001). An ROC analysis determined that motor-corrected within-subject differences significantly distinguished Stage 2 + 3 from Stage 0 + 1 (AUC = 0.72, p < 0.0001). Conclusions: We report evidence of bradyphrenia in HD that increases with disease progression. This processing deficit, which can be quantified using the CTiP, has the potential to greatly impact HD daily life and warrants additional research.

Plasma NfL as a prognostic biomarker for enriching HD-ISS stage 1 categorisation: a cross-sectional study.

BACKGROUND: The recently proposed Huntington's Disease Integrated Staging System (HD-ISS) categorises individuals with the Huntintin genetic mutation into disease progression cohorts based on quantitative neuroimaging, cognitive, and functional markers for research purposes. Unfortunately, many research studies do not collect quantitative neuroimaging data, and so the authors of the HD-ISS have subsequently provided approximated cohort thresholds based on disease and clinical data alone. However, these are rough proxies that aim to maximise stage separation, and should not be considered as 1:1 substitutes for the HD-ISS. Notably, no wet biomarker met the stringent criteria required to be considered a landmark for HD-ISS categorisation. We have previously shown that levels of plasma neurofilament light (NfL), a neuronal marker associated with axonal injury, are associated with predicted years to clinical motor diagnosis (CMD). Our objective in the current study was to determine whether HD-ISS categorisation, particularly for stages prior to CMD, could be improved with consideration of plasma NfL levels. METHODS: A total of 290 blood samples, and clinical measures, were collected from participants across all HD-ISS stages: n = 50 [Stage 0], n = 64 [Stage 1], n = 63 [Stage 2], n = 63 [Stage 3], as well as 50 healthy controls. Plasma NfL levels were measured using a Meso Scale Discovery assay. FINDINGS: Cohorts differed by age, cognitive function, CAG repeat length, and select UHDRS measures. Plasma NfL levels also differed significantly across cohorts. Approximately 50% of Stage 1 participants had plasma NfL levels indicative of predicted CMD within ten years. INTERPRETATION: Our findings suggest that plasma NfL levels may have use in enriching Stage 1 membership into sub-groups that are less than, and within, predicted 10 years until CMD. FUNDING: This work was supported by the National Institutes of Health (NS111655 to E.A.T.); the UCSD Huntington's Disease Society of America Center of Excellence; and the UCSD Shiley-Marcos Alzheimer's Disease Research Center (NIH-NIA P30 AG062429).

Rapid Detection of Lithium Concentrations in Oral Fluid.

BACKGROUND: Lithium medication is considered to be the first-line treatment for bipolar disorder as a monotherapy, and for treatment-resistant depression with lithium augmentation. However, because of potential toxicity, lithium levels must be monitored frequently. Recent studies have demonstrated a significant correlation between lithium levels measured in serum and those detected in oral fluid, suggesting that oral fluid analysis may represent an easy, noninvasive means to monitor lithium levels. The aim of this study was to evaluate the analytical performance of rapid assays for lithium measurements in oral fluid. METHODS: Levels of lithium in oral fluid from psychiatric patients (n = 108 in total) taking lithium medications were quantified using 2 rapid techniques: an automated clinical chemistry analyzer and a novel, commercially available colorimetric lithium assay. These results were compared with those obtained using inductively coupled plasma optical emission spectrometry (ICP-OES). RESULTS: The mean and median oral fluid lithium levels in this cohort were 1.43-1.61 mM and 1.32-1.52 mM, respectively, depending on the method, with the overall range, across all methods, being 0.213-4.42 mM. Linear regression analysis showed excellent agreement between the oral fluid values measured using ICP-OES and the colorimetric method (r 2 value = 0.926; P < 0.0001; slope = 1.084 ± 0.038). Similarly, excellent agreement was observed between ICP-OES and the automated method (r 2 = 0.872; P < 0.0001; slope = 1.019 ± 0.057). CONCLUSIONS: These results demonstrate that lithium levels in oral fluid can be rapidly and reliably quantified using colorimetric approaches. These findings may facilitate the development of point-of-care lithium monitoring systems for use in oral fluid.

Salivary Huntingtin protein is uniquely associated with clinical features of Huntington's disease.

Measuring Huntingtin (HTT) protein in peripheral cells represents an essential step in biomarker discovery for Huntington's Disease (HD), however to date, investigations into the salivary expression of HTT has been lacking. In the current study, we quantified total HTT (tHTT) and mutant HTT (mHTT) protein in matched blood and saliva samples using single molecule counting (SMC) immunoassays: 2B7-D7F7 (tHTT) and 2B7-MW1 (mHTT). Matched samples, and clinical data, were collected from 95 subjects: n = 19 manifest HD, n = 34 premanifest HD (PM), and n = 42 normal controls (NC). Total HTT and mHTT levels were not correlated in blood and saliva. Plasma tHTT was significantly associated with age, and participant sex; whereas salivary mHTT was significantly correlated with age, CAG repeat length and CAP score. Plasma and salivary tHTT did not differ across cohorts. Salivary and plasma mHTT were significantly increased in PM compared to NC; salivary mHTT was also significantly increased in HD compared to NC. Only salivary tHTT and mHTT were significantly correlated with clinical measures. Salivary HTT is uniquely associated with clinical measures of HD and offers significant promise as a relevant, non-invasive HD biomarker. Its use could be immediately implemented into both translational and clinical research applications.

Associations between saliva and plasma cytokines in cognitively normal, older adults.

BACKGROUND: Inflammatory responses play key roles in the development and progression of many pathological conditions, including neurodegenerative diseases. Accurate quantification of inflammatory factors in saliva would be highly advantageous, given its convenience and non-invasive nature, especially in elderly populations. METHODS: In this study, we measured levels of 10 cytokines, and the pro-inflammatory factor, YKL-40, in plasma and saliva samples from a cohort of nondemented older adults (n = 71; 62% female; 70.3 ± 6.4 years) using sensitive electrochemiluminescence-based immunoassays. RESULTS: We found that the mean levels of all cytokines were higher in saliva compared to plasma and that strong sex differences were observed for both saliva and plasma cytokines in this population. Comparing each cytokine between the two biofluids, we found that levels of interferon-gamma (IFNγ), interleukin (IL)-6 and tumor necrosis factor-alpha (TNFα) in blood were significantly correlated with their respective levels in saliva. We further observed that levels of these cytokines in blood were significantly correlated with additional cytokines in saliva, including IL-1ß, IL-10, IL-8, IL12p70 and IL-13. CONCLUSIONS: These findings show that inflammatory markers in saliva are associated with those found in circulation, suggesting shared inflammatory mechanisms between these two fluids. The higher levels of cytokines measured in saliva suggest that it might represent a better peripheral fluid to gauge inflammatory processes. Finally, our findings of robust sex differences in several salivary cytokines could have important implications for their potential use as disease biomarkers in the elderly and might be related to sex differences in the prevalence of age-related conditions.

Provider Perspectives on the Current Use of Lithium Medications and Lithium Monitoring Practices for Psychiatric Conditions.

Purpose: Despite lithium being a gold standard treatment for bipolar disorder, the percentage of patients with bipolar disorder who are prescribed lithium medication has declined in many parts of the world over the past two decades. The use of lithium is limited by its narrow therapeutic window and adverse side effects, which necessitates frequent serum lithium monitoring; hence, there is a critical need for improved ways to monitor lithium levels in psychiatric patients. We have recently shown that saliva lithium levels are highly correlated with those in blood, thereby presenting an alternative to venipuncture. Saliva sampling could open the door for at-home collections - potential that has been exemplified throughout the COVID-19 pandemic - thereby allowing samples to be collected remotely and delivered to a specific site for testing. In addition, prototype point-of-care devices have been developed by others for serum lithium monitoring, suggesting potential for a saliva lithium monitoring device. Our objective was to query the perspectives of American psychiatrists on lithium treatment practices and obstacles, the potential for at-home saliva collection and point-of-care devices, for lithium monitoring, as an alternative to pathology-based blood testing. Methods: Data was collected through an online, anonymous survey, distributed to American psychiatric societies. Results: Sixty-five respondents from 21 American states completed the survey. The majority of respondents were female, over 65 years of age, and/or had practiced for 30 years or more. The most frequent obstacles encountered with regard to lithium monitoring were adverse drug effects, and the need for monitoring. Overall, respondents believed saliva lithium monitoring and point-of-care devices would be useful, however raised concerns regarding validity and time-delay. Conclusion: Point-of-care devices and saliva lithium monitoring are promising alternatives to blood testing that would be welcomed by psychiatric societies, however, require extensive development and validation before implementation into a clinical setting.

Associations between prognostic index scores and plasma neurofilament light in Huntington's disease.

INTRODUCTION: The inclusion of premanifest Huntington's Disease (Pre-HD) subjects in clinical trials necessitates selecting those who are near transition to manifest Huntington's disease (Man-HD). We previously determined that plasma neurofilament light (NfL) levels are significantly correlated with predicted years to Man-HD onset, using established formulae. Recently, a new normalized prognostic index (PIN) score for predicting Pre-HD disease progression has been validated. Our objective was to determine whether plasma NfL levels are similarly associated with PIN score and PIN score-derived years to Man-HD onset (PIN-YTO). METHOD: 112 individuals (46 Pre-HD, 66 Man-HD) underwent blood sample collection and clinical assessment, inclusive of the Symbol Digit Modalities Test and Unified Huntington's Disease Rating Scale Total Motor Score. Plasma NfL levels were measured using a Meso Scale Discovery assay. RESULTS: Pre-HD and Man-HD cohorts differed by age (p < .0001), and CAG repeat number (p = .004), but not education level or gender. Plasma NfL levels were significantly correlated with PIN scores (r = 0.69, p < .0001) and PIN-YTO (r = -0.69, p < .0001). Plasma NfL levels were similarly correlated with predicted years to onset scores determined using Langbehn and colleague's formula (r = -0.68, p < .0001). All significant correlations endured corrections for age and CAG repeat number. A plasma NfL cut-point of <45.0 pg/ml distinguished Pre-HD participants >10 predicted years from Man-HD onset, compared to those ≤10 predicted years. CONCLUSIONS: We have extensively shown that plasma NfL levels are associated with predicted years to manifest HD onset in Pre-HD participants, and present a plasma NfL cut-point that may help exclude far-from-onset Pre-HD patients from clinical trials.

Validation of the SCAT5 and Child SCAT5 Word-List Memory Task.

The Sports Concussion Assessment Tool-5th Edition (SCAT5) and the child version (Child SCAT5) are the current editions of the SCAT and have updated the memory testing component from previous editions. This study aimed to validate this new memory component against the Rey Auditory Verbal Learning Test (RAVLT) as the validated standard. This prospective, observational study, carried out within The Royal Children's Hospital Emergency Department, Melbourne, Australia, recruited 198 participants: 91 with concussion and 107 upper limb injury or healthy sibling controls. Partial Pearson correlations showed that memory acquisition and recall on delay aspects of the SCAT5 were significantly correlated with the RAVLT equivalents when controlling for age (p < 0.001, r = 0.565 and p < 0.001, r = 0.341, respectively). Factor analysis showed that all RAVLT and SCAT5 memory components load on to the same factor, accounting for 59.13% of variance. Logistic regression models for both the RAVLT and SCAT5, however, did not predict group membership (p > 0.05). Receiver operating curve analysis found that the area under the curve for all variables and models was below the recommended 0.7 threshold. This study demonstrated that the SCAT5 and Child SCAT5 memory paradigm is a valid measure of memory in concussed children.

Saliva testing as a means to monitor therapeutic lithium levels in patients with psychiatric disorders: Identification of clinical and environmental covariates, and their incorporation into a prediction model.

OBJECTIVE: The narrow therapeutic window of lithium medications necessitates frequent serum monitoring, which can be expensive and inconvenient for the patient. Compared to blood, saliva collection is easier, non-invasive, requires less processing, and can be done without the need for trained personnel. This study investigated the utility of longitudinal salivary lithium level monitoring. METHODS: We measured salivary lithium levels using ICP-OES in n = 169 passive drool samples, collected both as single observations and longitudinally for up to 18 months, from a multi-center cohort of n = 75 patients with bipolar disorder or other psychiatric conditions. RESULTS: Saliva and serum lithium levels were highly correlated. Adjustment for daily lithium dose, diabetes, and smoking improved this relationship (r = 0.77). Using the adjusted intersubject equation and a patient's salivary lithium value, we observed a strong correlation between the predicted vs. observed serum lithium levels (r = 0.70). Most patients had highly stable saliva/serum ratios across multiple visits, with longitudinal variability significantly greater with age. Use of the intrasubject saliva/serum ratio from a single prior observation had similar predictive power to the use of the adjusted intersubject equation. However, the use of the mean intrasubject ratio from three prior observations could robustly predict serum lithium levels (predicted vs. observed r = 0.90). CONCLUSIONS: These findings strongly suggest that saliva could be used for lithium monitoring, and open the door for the development and implementation of a point-of-care salivary lithium device for use at home or the clinic. We propose that the use of saliva will dramatically improve treatment opportunities for patients with mood disorders.

Plasma neurofilament light in Huntington's disease: A marker for disease onset, but not symptom progression.

OBJECTIVE: To investigate whether plasma NfL levels correlate with clinical symptom severity in premanifest (PM) and manifest HD (HD) individuals, and whether a NfL cut-point could distinguish PM from HD patients with reasonable accuracy. METHOD: 98 participants (33 control, 26 PM, 39 HD), underwent blood sample collection and clinical assessment, using both UHDRS and non-UHDRS measures, at one academic HD Center. Years to onset (YTO), probability of disease onset in 5 years, and predicted years until 60% onset probability were also calculated. NfL levels were measured using a Meso Scale Discovery assay. RESULTS: Cohorts differed by age. NfL levels differed significantly across diagnostic groups and were significantly correlated with age. Age-adjusted NfL levels were not correlated with clinical measures in either HD or PM cohorts, but were correlated when cohorts were combined. In PM subjects, NfL levels correlated with YTO, probability of onset in 5 years, and years until 60% onset probability. Using ROC analysis, a NfL cut-point of <53.15 pg/ml distinguished HD from control; <74.84 pg/ml distinguished HD from PM. CONCLUSIONS: These findings implicate plasma NfL as a peripheral prognostic marker for premanifest-HD. Notably, we show that significant correlations between NfL and clinical symptoms are detected only when PM + HD subjects are combined, but not within HD subjects alone. To date, prior studies have investigated the clinical usefulness of NfL exclusively in merged PM + HD cohorts. Our data suggests a biasing of these previous correlations, and hence potentially limited usefulness of plasma NfL in monitoring HD symptom progression, for example, in clinical trials.

Levels of Interleukin-6 in Saliva, but Not Plasma, Correlate with Clinical Metrics in Huntington's Disease Patients and Healthy Control Subjects.

Growing evidence suggests that inflammatory responses, in both the brain and peripheral tissues, contribute to disease pathology in Huntington's disease (HD), an inherited, progressive neurodegenerative disorder typically affecting adults in their 30-40 s. Hence, studies of inflammation-related markers in peripheral fluids might be useful to better characterize disease features. In this study, we measured levels of C-reactive protein (CRP), Interleukin-6 (IL-6), interleukin 1 beta (IL-1B), and alpha-amylase (AA) in saliva and plasma from n = 125 subjects, including n = 37 manifest HD patients, n = 36 premanifest patients, and n = 52 healthy controls, using immunoassays. We found increases in salivary levels of IL-6, IL-1B and CRP across different disease groups and increased levels of IL-6 in the plasma of HD patients as compared to premanifest patients and controls. The levels of salivary IL-6 were significantly correlated with each of the other salivary markers, as well as with IL-6 levels measured in plasma. Further, salivary IL-6 and IL-1B levels were significantly positively correlated with Total Motor Score (TMS) and chorea scores and negatively correlated with Total Functional Capacity (TFC) in HD patients, whereby in healthy control subjects, IL-6 was significantly negatively correlated with Montreal Cognitive Assessment (MoCA) and the Symbol Digit Modalities test (SDM). Interestingly, the plasma levels of IL-6 did not show similar correlations to any clinical measures in either HD or control subjects. These findings suggest that salivary IL-6 is particularly relevant as a potential non-invasive biomarker for HD symptoms. The advent of an effective, dependable salivary biomarker would meet the urgent need for a less invasive means of identifying and monitoring HD disease progression.

Excitatory amino acid transporter (EAAT)1 and EAAT2 mRNA levels are altered in the prefrontal cortex of subjects with schizophrenia.

Excitatory amino acid transporter (EAAT)1 and EAAT2 mediate glutamatergic neurotransmission and prevent excitotoxicity through binding and transportation of glutamate into glia. These EAATs may be regulated by metabotropic glutamate receptor 5 (mGluR5), which is also expressed by glia. Whilst we have data from an Affymetrix™ Human Exon 1.0 ST Array showing higher levels of EAAT1 mRNA (+36%) in Brodmann's are (BA)9 of subjects with schizophrenia, there is evidence that EAAT1 and EAAT2, as well as mGluR5 levels, are altered in the cortex of subjects with the disorder. Hence, we measured mRNA levels of these genes in other cortical regions in subjects with that disorder. EAAT1, EAAT2 and mGluR5 mRNA were measured, in triplicate, using Quantitative PCR in BA10 and BA46 from subjects with schizophrenia (n = 20) and age and sex matched controls (n = 18). Levels of mRNA were normalised to the geometric mean of two reference genes, transcription factor B1, mitochondrial (TFB1M) and S-phase kinase-associated protein 1A (SKP1A), for which mRNA did not vary between diagnostic groups in either region. Normalised levels of EAAT1 and EAAT2 mRNA were significantly higher in BA10 (EAAT1: U = 58, p = 0.0002; EAAT2 U = 70, p = 0.0009), but not BA46 (EAAT1: U = 122, p = 0.09; EAAT2: U = 136, p = 0.21), from subjects with schizophrenia compared to controls. mGluR5 levels in BA10 (U = 173, p=0.85) and BA46 (U = 178, p = 0.96) did not vary by cohort. Our data suggests that region-specific increases in cortical EAAT1 and EAAT2 mRNA are involved in schizophrenia pathophysiology and that disrupted glutamate uptake in schizophrenia may be of particular significance in BA10.

Associations between catechol-O-methyltransferase (COMT) genotypes at rs4818 and rs4680 and gene expression in human dorsolateral prefrontal cortex.

Having reported associations between catechol-O-methyltransferase (COMT) genotypes at SNPs rs4818 and rs4680 with levels of soluble COMT (S-COMT) in human dorsolateral prefrontal cortex (DLPFC), we postulated that changes in the levels of cortical S-COMT could impact on behavioural abilities associated with COMT genotype through S-COMT-mediated changes in gene expression. To test this hypothesis, we have examined the relationships between COMT genotypes and gene expression measured using the Affymetrix™ Human Exon 1.0 ST Array in the DLPFC from 141 individuals, some of whom had had a psychiatric disorder. There were significant differences in levels of expression of 15 genes between individuals with a homozygous genotype at rs4818 (GG vs CC), compared to differences in levels of expression of 6 genes between homozygotes at rs4680 (GG vs AA); levels of expression of CEP128, EFCAB13, and FAM133A differed between homozygotes at both SNPs. Fourteen of the genes differentially expressed in the DLPFC according to COMT genotypes have oestrogen receptor elements and their expression could, therefore, be regulated by catecholestrogens, which are substrates for COMT that occupy and activate oestrogen receptors. In addition, the changes in gene expression between the homozygotes at rs4818 or rs4680 would be expected to impact on neuronal function, synaptic plasticity, cognition, and attention. These data would support a hypothesis that the mechanism underlying the association between COMT genotype and cognition involves differential changes in cortical gene expression.

Β-actin does not show the characteristics of a reference protein in human cortex.

Levels of a reference protein must be the same as a proportion of total protein in all tissues and, in the study of human diseases, cannot vary with factors such as age, gender or disease pathophysiology. It is increasingly apparent that there may be few, if any, proteins that display the characteristics of a reference protein within the human central nervous system (CNS). To begin to challenge this hypothesis, we used Western blotting to compare variance in levels of the "gold standard" reference protein, ß-actin, in Brodmann's area 9 from 194 subjects to variance of total transferred protein measured as intensity of Ponceau S staining. The coefficient of variance of sum intensity measurements for ß-actin levels across all donors was 47% compared to 24 and 27% for the sum intensity of Ponceau S staining measured using two different detection techniques. These data strongly suggest that the level of ß-actin, proportional to total protein, is not constant in human cortex which raises further doubt about the use of reference proteins to normalise data in human CNS studies. Considering our data, we suggest an alternative approach to presenting data from Western blotting of human CNS.

Plasma Tumor Necrosis Factor Alpha Is a Predictor of Persisting Symptoms Post-Concussion in Children.

Mild traumatic brain injury (mTBI)-associated blood proteomics have become an emerging focus in the past decade, with the U.S. Food and Drug Administration recently approving the use of a blood test to determine the necessity of a computed tomography scan after adult mTBI. We now also know that the blood proteome of children is different from that of adults, and new evidence suggests that children may take longer to recover from an mTBI. Despite this, comparatively fewer studies have analyzed changes in blood protein expression after pediatric mTBI. Concussions, an mTBI subset, often go underreported, despite the potential for post-concussive symptoms to last more than one month in up to 30% of children. In the current study, we used a multiplex immunoassay to measure blood protein expression of Apolipoprotein, enolase 2, glial fibrillary acidic protein, interleukin (IL)-1B, IL-6, IL-8, IL-10, S100 calcium-binding protein B, tau and tumor necrosis factor alpha (TNFα) at admission, one to four days, two weeks, and three months post-pediatric concussion, comparing patients with normal recovery (n = 9) with those with persisting symptoms (n = 9). We identified significant differences in IL-6 (p < 0.001) and tau (p = 0.048) protein expression across time post-injury irrespective of clinical outcome and in IL-8 protein expression (p = 0.041) across time post-injury specific to children with persisting symptoms. Significantly, we have identified an increase in TNFα protein expression at one to four days post-injury (p = 0.031) in children with persisting symptoms compared with normal recovery. To our knowledge, this is the first study to identify TNFα as a potential blood biomarker for persisting symptoms post-pediatric concussion.

Catechol-O-methyltransferase (COMT) genotypes are associated with varying soluble, but not membrane-bound COMT protein in the human prefrontal cortex.

Catechol-O-methyltransferase (COMT) is an enzyme that catalyses the O-methylation, and thereby the inactivation, of catechol-containing molecules. In humans, it has been suggested that COMT modulates cognitive ability, possibly by regulating degradation of dopamine in the prefrontal cortex. Hence, it is significant that two COMT SNPs, rs4680 (c.472 G > A, p.Val158Met) and rs4818 (c.408 C > G), have been associated with cognitive ability in humans. We have shown these SNPs to be associated with levels of muscarinic M1 receptor mRNA in human cortex, which is significant as that receptor also regulates cognitive ability. We decided to determine if COMT genotype was associated with varying levels of COMT protein, as this could be a mechanism by which COMT genotype could be associated with changes in muscarinic M1 receptor mRNA levels. Hence, we measured COMT levels in prefrontal cortex obtained postmortem from 199 subjects, some of whom had a history of schizophrenia, major depressive disorders or bipolar disorders. Our data show, independent of diagnostic status, that genotype at rs4680 and rs4818, but not at rs737865 and rs165599, is associated with differing levels of soluble COMT (S-COMT), but not membrane-bound COMT (MB-COMT). These findings suggest that the association between COMT polymorphisms and cognitive functioning could be, at least in part, due to their association with varying levels of S-COMT. This is important as, unlike MB-COMT, the substrates targeted by S-COMT are likely to be intra-cellular rather than, like dopamine, located mainly in the synaptic vesicles or the extra-cellular space.

Glutamate transporters, EAAT1 and EAAT2, are potentially important in the pathophysiology and treatment of schizophrenia and affective disorders.

Glutamate is the predominant excitatory neurotransmitter in the human brain and it has been shown that prolonged activation of the glutamatergic system leads to nerve damage and cell death. Following release from the pre-synaptic neuron and synaptic transmission, glutamate is either taken up into the pre-synaptic neuron or neighbouring glia by transmembrane glutamate transporters. Excitatory amino acid transporter (EAAT) 1 and EAAT2 are Na+-dependant glutamate transporters expressed predominantly in glia cells of the central nervous system. As the most abundant glutamate transporters, their primary role is to modulate levels of glutamatergic excitability and prevent spill over of glutamate beyond the synapse. This role is facilitated through the binding and transportation of glutamate into astrocytes and microglia. The function of EAAT1 and EAAT2 is heavily regulated at the levels of gene expression, post-transcriptional splicing, glycosylation states and cell-surface trafficking of the protein. Both glutamatergic dysfunction and glial dysfunction have been proposed to be involved in psychiatric disorder. This review will present an overview of the roles that EAAT1 and EAAT2 play in modulating glutamatergic activity in the human brain, and mount an argument that these two transporters could be involved in the aetiologies of schizophrenia and affective disorders as well as represent potential drug targets for novel therapies for those disorders.