Cognitive phenotype and neurodegeneration associated with Tau in Huntington's disease.

OBJECTIVE: The clinical phenotype of Huntington's disease (HD) can be very heterogeneous between patients, even when they share equivalent CAG repeat length, age, or disease burden. This heterogeneity is especially evident in terms of the cognitive profile and related brain changes. To shed light on the mechanisms participating in this heterogeneity, the present study delves into the association between Tau pathology and more severe cognitive phenotypes and brain damage in HD. METHODS: We used a comprehensive neuropsychological examination to characterize the cognitive phenotype of a sample of 30 participants with early-to-middle HD for which we also obtained 3 T structural magnetic resonance image (MRI) and cerebrospinal fluid (CSF). We quantified CSF levels of neurofilament light chain (NfL), total Tau (tTau), and phosphorylated Tau-231 (pTau-231). Thanks to the cognitive characterization carried out, we subsequently explored the relationship between different levels of biomarkers, the cognitive phenotype, and brain integrity. RESULTS: The results confirmed that more severe forms of cognitive deterioration in HD extend beyond executive dysfunction and affect processes with clear posterior-cortical dependence. This phenotype was in turn associated with higher CSF levels of tTau and pTau-231 and to a more pronounced pattern of posterior-cortical atrophy in specific brain regions closely linked to the cognitive processes affected by Tau. INTERPRETATION: Our findings reinforce the association between Tau pathology, cognition, and neurodegeneration in HD, emphasizing the need to explore the role of Tau in the cognitive heterogeneity of the disease.

Cerebrospinal Fluid Flow in Patients with Huntington's Disease.

OBJECTIVE: Investigations of cerebrospinal fluid (CSF) flow aberrations in Huntington's disease (HD) are of growing interest, as impaired CSF flow may contribute to mutant Huntington retention and observed heterogeneous responsiveness to intrathecally administered therapies. METHOD: We assessed net cerebral aqueduct CSF flow and velocity in 29 HD participants (17 premanifest and 12 manifest) and 51 age- and sex matched non-HD control participants using 3-Tesla magnetic resonance imaging methods. Regression models were applied to test hypotheses regarding: (i) net CSF flow and cohort, (ii) net CSF flow and disease severity (CAP-score), and (iii) CSF volume after correcting for age and sex. RESULTS: Group-wise analyses support a decrease in net CSF flow in HD (mean 0.14 ± 0.27 mL/min) relative to control (mean 0.32 ± 0.20 mL/min) participants (p = 0.02), with lowest flow in the manifest HD cohort (mean 0.04 ± 0.25 mL/min). This finding was explained by hyperdynamic CSF movement, manifesting as higher caudal systolic CSF flow velocity and higher diastolic cranial CSF flow velocity across the cardiac cycle, in HD (caudal flow: 0.17 ± 0.07 mL/s, cranial flow: 0.14 ± 0.08 mL/s) compared to control (caudal flow: 0.13 ± 0.06 mL/s, cranial flow: 0.11 ± 0.04 mL/s) participants. A positive correlation between cranial diastolic flow and disease severity was observed (p = 0.02). INTERPRETATIONS: Findings support aqueductal CSF flow dynamics changing with disease severity in HD. These accelerated changes are consistent with changes observed over the typical adult lifespan, and may have relevance to mutant Huntington retention and intrathecally administered therapeutics responsiveness. ANN NEUROL 2023;94:885-894.

Quantifying mutant huntingtin protein in human cerebrospinal fluid to support the development of huntingtin-lowering therapies.

Huntington's disease (HD) is caused by a cytosine adenine guanine-repeat expansion in the huntingtin gene. This results in the production of toxic mutant huntingtin protein (mHTT), which has an elongated polyglutamine (polyQ) stretch near the protein's N-terminal end. The pharmacological lowering of mHTT expression in the brain targets the underlying driver of HD and is one of the principal therapeutic strategies being pursued to slow or stop disease progression. This report describes the characterisation and validation of an assay designed to quantify mHTT in the cerebrospinal fluid of individuals with HD, for use in registrational clinical trials. The assay was optimised, and its performance was characterised with recombinant huntingtin protein (HTT) varying in overall and polyQ-repeat length. The assay was successfully validated by two independent laboratories in regulated bioanalytical environments and showed a steep signal increase as the polyQ stretch of recombinant HTTs pivoted from wild-type to mutant protein forms. Linear mixed effects modelling confirmed highly parallel concentration-response curves for HTTs, with only a minor impact of individual slopes of the concentration-response for different HTTs (typically < 5% of the overall slope). This implies an equivalent quantitative signal behaviour for HTTs with differing polyQ-repeat lengths. The reported method may be a reliable biomarker tool with relevance across the spectrum of HD mutations, which can facilitate the clinical development of HTT-lowering therapies in HD.

Cerebrospinal fluid levels of proenkephalin and prodynorphin are differentially altered in Huntington's and Parkinson's disease.

BACKGROUND: Proenkephalin (PENK) and prodynorphin (PDYN) are peptides mainly produced by the striatal medium spiny projection neurons (MSNs) under dopaminergic signaling. Therefore, they may represent candidate biomarkers in Huntington's disease (HD) and Parkinson's disease (PD), two neurodegenerative diseases characterized by striatal atrophy and/or dysfunction. METHODS: Using an in-house established liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in multiple reaction monitoring mode (MRM) we measured cerebrospinal fluid (CSF) levels of PENK- and PDYN- derived peptides in patients with HD (n = 47), PD (n = 61), Alzheimer's disease (n = 11), amyotrophic lateral sclerosis (n = 14) and in 92 control subjects. Moreover, we investigated the possible associations between biomarkers and disease severity scales in HD and PD and the effect of dopaminergic therapy on biomarker levels in PD. RESULTS: In HD, CSF PENK- and PDYN-derived peptide levels were significantly decreased compared to all other groups and were associated with disease severity scores. In PD, both biomarkers were within the normal range, but higher PDYN levels were found in dopamine-treated compared to untreated patients. In PD, both CSF PENK and PDYN did not correlate with clinical severity scales. CONCLUSIONS: CSF PENK- and PDYN-derived peptides appeared to be promising pathogenetic and disease severity markers in HD, reflecting the ongoing striatal neurodegeneration along with the loss of MSNs. In PD patients, CSF PDYN showed a limitative role as a possible pharmacodynamic marker during dopaminergic therapy, but further investigations are needed.

Alterations in metal homeostasis occur prior to canonical markers in Huntington disease.

The importance of metal biology in neurodegenerative diseases such as Huntingtin Disease is well documented with evidence of direct interactions between metals such as copper, zinc, iron and manganese and mutant Huntingtin pathobiology. To date, it is unclear whether these interactions are observed in humans, how this impacts other metals, and how mutant Huntington alters homeostatic mechanisms governing levels of copper, zinc, iron and manganese in cerebrospinal fluid and blood in HD patients. Plasma and cerebrospinal fluid from control, pre-manifest, manifest and late manifest HD participants were collected as part of HD-Clarity. Levels of cerebrospinal fluid and plasma copper, zinc, iron and manganese were measured as well as levels of mutant Huntingtin and neurofilament in a sub-set of cerebrospinal fluid samples. We find that elevations in cerebrospinal fluid copper, manganese and zinc levels are altered early in disease prior to alterations in canonical biomarkers of HD although these changes are not present in plasma. We also evidence that CSF iron is elevated in manifest patients. The relationships between plasma and cerebrospinal fluid metal are altered based on disease stage. These findings demonstrate that there are alterations in metal biology selectively in the CSF which occur prior to changes in known canonical biomarkers of disease. Our work indicates that there are pathological changes related to alterations in metal biology in individuals without elevations in neurofilament and mutant Huntingtin.

Interaction between sex and neurofilament light chain on brain structure and clinical severity in Huntington's disease.

Female Huntington's disease (HD) patients have consistently shown a faster clinical worsening than male, but the underlying mechanisms responsible for this observation remain unknown. Here, we describe how sex modifies the impact of neurodegeneration on brain atrophy and clinical severity in HD. Cerebrospinal fluid neurofilament light chain (NfL) levels were used as a biological measure of neurodegeneration, and brain atrophy was assessed by structural magnetic resonance imaging. We found that larger NfL values in women reflect higher brain atrophy and clinical severity than in men (p < 0.05 for an interaction model). This differential vulnerability could have important implications in clinical trials.

Extracellular miRNA biomarkers in neurologic disease: is cerebrospinal fluid helpful?

Aim: The aim of our work is to aggregate data from publications of cerebrospinal fluid extracellular miRNA to identify candidate diagnostic biomarkers, and those warranting further study. Materials & methods: Data were pooled from nine studies, encompassing 864 patients across 16 diseases. Unsupervised clustering grouped patients by a broad category of diseases. Results & conclusion: Compared with healthy controls, in patients with Alzheimer's disease, hsa-miR-767-5p was overexpressed (p < 0.001) and in patients with Huntington's disease, hsa-miR-361-3p was underexpressed (p < 10-4). We also define a subset of extracellular miRNA as candidate biomarkers that are robustly detected across patients, studies and diseases; thereby, warranting further study.

Kynurenine pathway metabolites in cerebrospinal fluid and blood as potential biomarkers in Huntington's disease.

Converging lines of evidence from several models, and post-mortem human brain tissue studies, support the involvement of the kynurenine pathway (KP) in Huntington's disease (HD) pathogenesis. Quantifying KP metabolites in HD biofluids is desirable, both to study pathobiology and as a potential source of biomarkers to quantify pathway dysfunction and evaluate the biochemical impact of therapeutic interventions targeting its components. In a prospective single-site controlled cohort study with standardised collection of cerebrospinal fluid (CSF), blood, phenotypic and imaging data, we used high-performance liquid-chromatography to measure the levels of KP metabolites-tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid and quinolinic acid-in CSF and plasma of 80 participants (20 healthy controls, 20 premanifest HD and 40 manifest HD). We investigated short-term stability, intergroup differences, associations with clinical and imaging measures and derived sample-size calculation for future studies. Overall, KP metabolites in CSF and plasma were stable over 6 weeks, displayed no significant group differences and were not associated with clinical or imaging measures. We conclude that the studied metabolites are readily and reliably quantifiable in both biofluids in controls and HD gene expansion carriers. However, we found little evidence to support a substantial derangement of the KP in HD, at least to the extent that it is reflected by the levels of the metabolites in patient-derived biofluids.

Brain-derived neurotrophic factor in cerebrospinal fluid and plasma is not a biomarker for Huntington's disease.

Brain-derived neurotrophic factor (BDNF) is implicated in the survival of striatal neurons. BDNF function is reduced in Huntington's disease (HD), possibly because mutant huntingtin impairs its cortico-striatal transport, contributing to striatal neurodegeneration. The BDNF trophic pathway is a therapeutic target, and blood BDNF has been suggested as a potential biomarker for HD, but BDNF has not been quantified in cerebrospinal fluid (CSF) in HD. We quantified BDNF in CSF and plasma in the HD-CSF cohort (20 pre-manifest and 40 manifest HD mutation carriers and 20 age and gender-matched controls) using conventional ELISAs and an ultra-sensitive immunoassay. BDNF concentration was below the limit of detection of the conventional ELISAs, raising doubt about previous CSF reports in neurodegeneration. Using the ultra-sensitive method, BDNF concentration was quantifiable in all samples but did not differ between controls and HD mutation carriers in CSF or plasma, was not associated with clinical scores or MRI brain volumetric measures, and had poor ability to discriminate controls from HD mutation carriers, and premanifest from manifest HD. We conclude that BDNF in CSF and plasma is unlikely to be a biomarker of HD progression and urge caution in interpreting studies where conventional ELISA was used to quantify CSF BDNF.

Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington Disease.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT: Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

Cross-sectional analysis of plasma and CSF metabolomic markers in Huntington's disease for participants of varying functional disability: a pilot study.

Huntington's Disease (HD) is a progressive, fatal neurodegenerative condition. While generally considered for its devastating neurological phenotype, disturbances in other organ systems and metabolic pathways outside the brain have attracted attention for possible relevance to HD pathology, potential as therapeutic targets, or use as biomarkers of progression. In addition, it is not established how metabolic changes in the HD brain correlate to progression across the full spectrum of early to late-stage disease. In this pilot study, we sought to explore the metabolic profile across manifest HD from early to advanced clinical staging through metabolomic analysis by mass spectrometry in plasma and cerebrospinal fluid (CSF). With disease progression, we observed nominally significant increases in plasma arginine, citrulline, and glycine, with decreases in total and D-serine, cholesterol esters, diacylglycerides, triacylglycerides, phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins. In CSF, worsening disease was associated with nominally significant increases in NAD+, arginine, saturated long chain free fatty acids, diacylglycerides, triacylglycerides, and sphingomyelins. Notably, diacylglycerides and triacylglyceride species associated with clinical progression were different between plasma and CSF, suggesting different metabolic preferences for these compartments. Increasing NAD+ levels strongly correlating with disease progression was an unexpected finding. Our data suggest that defects in the urea cycle, glycine, and serine metabolism may be underrecognized in the progression HD pathology, and merit further study for possible therapeutic relevance.

Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients.

In Huntington's disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.

Cerebrospinal fluid endo-lysosomal proteins as potential biomarkers for Huntington's disease.

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington's disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington's Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington's disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples.

Early Intrathecal T Helper 17.1 Cell Activity in Huntington Disease.

OBJECTIVE: Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. No disease-modifying therapy exists for the treatment of patients with HD. The purpose of this study was therefore to investigate early disease mechanisms that potentially could be used as a target therapeutically. METHODS: Lymphocyte activity in cerebrospinal fluid (CSF) from 4 cohorts of HTT gene expansion carriers (n = 121 in total) and controls was analyzed by techniques based on flow cytometry and enzyme-linked immunosorbent assays. RESULTS: The data of this study provide evidence of immune abnormalities before motor onset of disease. In CSF of HTT gene expansion carriers, we found increased levels of proinflammatory cytokines, including IL-17, and increased consumption of the lymphocyte growth factor IL-7 before motor onset of HD. In concordance, we observed an increased prevalence of IL-17-producing Th17.1 cells in the CSF of HTT gene expansion carriers, predominantly in pre-motor manifest individuals. The frequency of intrathecal Th17.1 cells correlated negatively with progression of HD and the level of neurodegeneration, suggesting a role of Th17.1 cells in the early disease stage. We also observed a skewing in the balance between proinflammatory and regulatory T cells potentially favoring a proinflammatory intrathecal environment in HTT gene expansion carriers. INTERPRETATION: These data suggest that Th17.1 cells are implicated in the earliest pathogenic phases of HD and suggest that treatment to dampen T -cell-driven inflammation before motor onset might be of benefit in HTT gene expansion carriers. ANN NEUROL 2020;87:246-255.

Correlations between serum and CSF pNfH levels in ALS, FTD and controls: a comparison of three analytical approaches.

Background Phosphorylated neurofilament heavy (pNfH), a neuronal cytoskeleton protein, might provide a promising blood biomarker of neuronal damage in neurodegenerative diseases (NDDs). The best analytical approaches to measure pNfH levels and whether serum levels correlate with cerebrospinal fluid (CSF) levels in NDDs remain to be determined. Methods We here compared analytical sensitivity and reliability of three novel analytical approaches (homebrew Simoa, commercial Simoa and ELISA) for quantifying pNfH in both CSF and serum in samples of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and control subjects. Results While all three assays showed highly correlated CSF measurements, Simoa assays also yielded high between-assay correlations for serum measurements (ϱ = 0.95). Serum levels also correlated strongly with CSF levels for Simoa-based measurements (both ϱ = 0.62). All three assays allowed distinguishing ALS from controls by increased CSF pNfH levels, and Simoa assays also by increased serum pNfH levels. pNfH levels were also increased in FTD. Conclusions pNfH concentrations in CSF and, if measured by Simoa assays, in blood might provide a sensitive and reliable biomarker of neuronal damage, with good between-assay correlations. Serum pNfH levels measured by Simoa assays closely reflect CSF levels, rendering serum pNfH an easily accessible blood biomarker of neuronal damage in NDDs.

Alterations in the tyrosine and phenylalanine pathways revealed by biochemical profiling in cerebrospinal fluid of Huntington's disease subjects.

Huntington's disease (HD) is a severe neurological disease leading to psychiatric symptoms, motor impairment and cognitive decline. The disease is caused by a CAG expansion in the huntingtin (HTT) gene, but how this translates into the clinical phenotype of HD remains elusive. Using liquid chromatography mass spectrometry, we analyzed the metabolome of cerebrospinal fluid (CSF) from premanifest and manifest HD subjects as well as control subjects. Inter-group differences revealed that the tyrosine metabolism, including tyrosine, thyroxine, L-DOPA and dopamine, was significantly altered in manifest compared with premanifest HD. These metabolites demonstrated moderate to strong associations to measures of disease severity and symptoms. Thyroxine and dopamine also correlated with the five year risk of onset in premanifest HD subjects. The phenylalanine and the purine metabolisms were also significantly altered, but associated less to disease severity. Decreased levels of lumichrome were commonly found in mutated HTT carriers and the levels correlated with the five year risk of disease onset in premanifest carriers. These biochemical findings demonstrates that the CSF metabolome can be used to characterize molecular pathogenesis occurring in HD, which may be essential for future development of novel HD therapies.

Fluid and imaging biomarkers for Huntington's disease.

Huntington's disease is a chronic progressive neurodegenerative condition for which there is no disease-modifying treatment. The known genetic cause of Huntington's disease makes it possible to identify individuals destined to develop the disease and instigate treatments before the onset of symptoms. Multiple trials are already underway that target the cause of HD, yet clinical measures are often insensitive to change over typical clinical trial duration. Robust biomarkers of drug target engagement, disease severity and progression are required to evaluate the efficacy of treatments and concerted efforts are underway to achieve this. Biofluid biomarkers have potential advantages of direct quantification of biological processes at the molecular level, whilst imaging biomarkers can quantify related changes at a structural level in the brain. The most robust biofluid and imaging biomarkers can offer complementary information, providing a more comprehensive evaluation of disease stage and progression to inform clinical trial design and endpoints.

Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington's disease.

Huntington's disease (HD) is a genetic progressive neurodegenerative disorder, caused by a mutation in the HTT gene, for which there is currently no cure. The identification of sensitive indicators of disease progression and therapeutic outcome could help the development of effective strategies for treating HD. We assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF) and blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. Among HD mutation carriers, NfL concentrations in plasma and CSF correlated with all nonbiofluid measures more closely than did CSF mHTT concentration. Longitudinal analysis over 4 to 8 weeks showed that CSF mHTT, CSF NfL, and plasma NfL concentrations were highly stable within individuals. In our cohort, concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. In silico modeling indicated that mHTT and NfL concentrations in biofluids might be among the earliest detectable alterations in HD, and sample size prediction suggested that low participant numbers would be needed to incorporate these measures into clinical trials. These findings provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD and could be integrated into both clinical trials and the clinic.