Nayana Lahiri

A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease

Huntingtons disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. We examined the peripheral immune system and found widespread evidence of innate immune activation detectable in plasma throughout the course of HD. Interleukin 6 levels were increased in HD gene carriers with a mean of 16 years before the predicted onset of clinical symptoms. To our knowledge, this is the earliest plasma abnormality identified in HD. Monocytes from HD subjects expressed mutant huntingtin and were pathologically hyperactive in response to stimulation, suggesting that the mutant protein triggers a cell-autonomous immune activation. A similar pattern was seen in macrophages and microglia from HD mouse models, and the cerebrospinal fluid and striatum of HD patients exhibited abnormal immune activation, suggesting that immune dysfunction plays a role in brain pathology. Collectively, our data suggest parallel central nervous system and peripheral pathogenic pathways of immune activation in HD.

Hsa-miR-34b is a plasma-stable microRNA that is elevated in pre-manifest Huntington's disease

Huntingtons disease (HD) is a devastating, neurodegenerative condition, which lacks effective treatment. Normal Huntingtin (HTT) and mutant Huntingtin (mHTT) are expressed in multiple tissues and can alter transcription of microRNAs (miRs). Importantly, miRs are present in a bio-stable form in human peripheral blood plasma and have recently been shown to be useful biomarkers in other diseases. We therefore sought to identify potential miR biomarkers of HD that are present in, and have functional consequences for, neuronal and non-neuronal tissues. In a cell line over-expressing mHTT-Exon-1, miR microarray analysis was used to identify candidate miRs. We then examined their presence and bio-stability in control and HD plasma. We found that miR-34b is significantly elevated in response to mHTT-Exon-1, and its blockade alters the toxicity of mHTT-Exon-1 in vitro. We also show that miR-34b is detectable in plasma from small input volumes and is insensitive to freeze-thaw-induced RNA degradation. Interestingly, miR-34b is significantly elevated in plasma from HD gene carriers prior to symptom onset. This is the first study suggesting that plasma miRs might be used as biomarkers for HD.

HTT-lowering reverses Huntington’s disease immune dysfunction caused by NFκB pathway dysregulation

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntingtons disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntingtons disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntingtons disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntingtons disease.

Mutant huntingtin fragmentation in immune cells tracks Huntington’s disease progression

Huntingtons disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT levels in these cells have not been quantified before. A recently described time-resolved Förster resonance energy transfer (TR-FRET) immunoassay was used to quantify mutant and total HTT protein levels in leukocytes from patients with HD. Mean mHTT levels in monocytes, T cells, and B cells differed significantly between patients with HD and controls and between pre-manifest mutation carriers and those with clinical onset. Monocyte and T cell mHTT levels were significantly associated with disease burden scores and caudate atrophy rates in patients with HD. mHTT N-terminal fragments detected in HD PBMCs may explain the progressive increase in mHTT levels in these cells. These findings indicate that quantification of mHTT in peripheral immune cells by TR-FRET holds significant promise as a noninvasive disease biomarker.

Abnormal peripheral chemokine profile in Huntington's disease.

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. Immune activation is a well-established feature of the HD brain and we have previously demonstrated a widespread, progressive innate immune response detectable in plasma throughout the course of HD. In the present work we used multiplex ELISA to quantify levels of chemokines in plasma from controls and subjects at different stages of HD. We found an altered chemokine profile tracking with disease progression, with significant elevations of five chemokines (eotaxin-3, MIP-1β, eotaxin, MCP-1 and MCP-4) while three (eotaxin-3, MIP-1β and eotaxin) showed significant linear increases across advancing disease stages. We validated our results in a separate sample cohort including subjects at different stages of HD. Here we saw that chemokine levels (MCP-1 and eotaxin) correlated with clinical scores. We conclude that, like cytokines, chemokines may be linked to the pathogenesis of HD, and that immune molecules may be valuable in tracking and exploring the pathogenesis of HD.

Brain-Derived Neurotrophic Factor in Patients with Huntington's Disease

Reduced Brain-Derived Neurotrophic Factor (BDNF) levels have been described in a number of patho-physiological conditions, most notably, in Huntingtons disease (HD), a progressive neurodegenerative disorder. Since BDNF is also produced in blood, we have undertaken the measurement of its peripheral levels in the attempt to identify a possible link with HD prognosis and/or its progression. Here we evaluated BDNF level in 398 blood samples including 138 controls, 56 preHD, and 204 HD subjects. We found that BDNF protein levels were not reliably different between groups, whether measured in plasma (52 controls, 26 preHD, 105 HD) or serum (39 controls, 5 preHD, 29 HD). Our experience, and a re-analysis of the literature highlighted that intra-group variability and methodological aspects affect this measurement, especially in serum. We also assessed BDNF mRNA levels in blood samples from 47 controls, 25 preHD, and 70 HD subjects, and found no differences among the groups. We concluded that levels of BDNF in human blood were not informative (mRNA levels or plasma protein level) nor reliable (serum protein levels) as HD biomarkers. We also wish to warn the scientific community in interpreting the significance of changes measured in BDNF protein levels in serum from patients suffering from different conditions.

Increased central microglial activation associated with peripheral cytokine levels in premanifest Huntington's disease gene carriers

Previous studies have shown activation of the immune system and altered immune response in Huntingtons disease (HD) gene carriers. Here, we hypothesized that peripheral and central immune responses could be concurrent pathophysiological events and represent a global innate immune response to the toxic effects of mutant huntingtin in HD gene carriers. We sought to investigate our hypothesis using [(11)C]PK11195 PET as a translocator protein (TSPO) marker of central microglial activation, together with assessment of peripheral plasma cytokine levels in a cohort of premanifest HD gene carriers who were more than a decade from predicted symptomatic conversion. Data were also compared to those from a group of healthy controls matched for age and gender. We found significantly increased peripheral plasma IL-1β levels in premanifest HD gene carriers compared to the group of normal controls (P=0.018). Premanifest HD gene carriers had increased TSPO levels in cortical, basal ganglia and thalamic brain regions (P<0.001). Increased microglial activation in somatosensory cortex correlated with higher plasma levels of IL-1β (rs=0.87, P=0.013), IL-6 (rs=0.85, P=0.013), IL-8 (rs=0.68, P=0.045) and TNF-α (rs=0.79; P=0.013). Our findings provide first in vivo evidence for an association between peripheral and central immune responses in premanifest HD gene carriers, and provide further supporting evidence for the role of immune dysfunction in the pathogenesis of HD.

A SNP in the HTT promoter alters NF-[kappa]B binding and is a bidirectional genetic modifier of Huntington disease

Cis-regulatory variants that alter gene expression can modify disease expressivity, but none have previously been identified in Huntington disease (HD). Here we provide in vivo evidence in HD patients that cis-regulatory variants in the HTT promoter are bidirectional modifiers of HD age of onset. HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A) variant on the HD disease allele was associated with delayed age of onset in familial cases, whereas the presence of the rs13102260 (A) variant on the wild-type HTT allele was associated with earlier age of onset in HD patients in an extreme case–based cohort. Our findings suggest a previously unknown mechanism linking allele-specific effects of rs13102260 on HTT expression to HD age of onset and have implications for HTT silencing treatments that are currently in development.

Expressed Alu repeats as a novel, reliable tool for normalization of real-time quantitative RT-PCR data

We describe a novel strategy for mRNA normalization in quantitative real-time PCR that is based on expressed Alu repeat amplification as a measure for the mRNA fraction. We show that expressed Alu repeat amplification is a fast, accurate normalization tool that can be successfully used for quantification of selected mRNA in the human transcriptome. This result is particularly important for clinical diagnosis and biomarker validation studies based on mRNA detection in human blood.

Stability of white matter changes related to Huntington's disease in the presence of imaging noise: a DTI study

Movement artifacts and other sources of noise are a matter of concern particularly in the neuroimaging research of movement disorders such as Huntington’s disease (HD). Using diffusion weighted imaging (DWI) and fractional anisotropy (FA) as a compound marker of white matter integrity, we investigated the effect of movement on HD specific changes in magnetic resonance imaging (MRI) data and how post hoc compensation for it affects the MRI results. To this end, we studied by 3T MRI: 18 early affected, 22 premanifest gene-positive subjects, 23 healthy controls (50 slices of 2.3 mm thickness per volume, 64 diffusion-weighted directions (b = 1000 s/mm2), 8 minimal diffusion-weighting (b = 100 s/mm2)); and by 1.5 T imaging: 29 premanifest HD, 30 controls (40 axial slices of 2.3 mm thickness per volume, 61 diffusion-weighted directions (b = 1000 s/mm2), minimal diffusion-weighting (b = 100 s/mm2)). An outlier based method was developed to identify movement and other sources of noise by comparing the index DWI direction against a weighted average computed from all other directions of the same subject. No significant differences were observed when separately comparing each group of patients with and without removal of DWI volumes that contained artifacts. In line with previous DWI-based studies, decreased FA in the corpus callosum and increased FA around the basal ganglia were observed when premanifest mutation carriers and early affected patients were compared with healthy controls. These findings demonstrate the robustness of the FA value in the presence of movement and thus encourage multi-center imaging studies in HD.