Amanda Heslegrave

Soluble TREM-2 in cerebrospinal fluid from patients with multiple sclerosis treated with natalizumab or mitoxantrone.

Background: Microglia-mediated proteolysis of the triggering receptor expressed on myeloid cells-2 (TREM-2) produces soluble TREM-2 (sTREM-2) that can be measured in cerebrospinal fluid (CSF) samples. Loss-of-function mutations in TREM2 or in the gene encoding its adaptor protein cause the rare Nasu–Hakola disease (NHD). Multiple sclerosis (MS) is an autoimmune disease that in common with NHD is characterized by demyelination and microglial activation. Objective: To investigate the potential utility of sTREM-2 as a biomarker for MS and to follow treatment effects. Methods: sTREM-2 was analyzed in CSF samples from subjects with MS (N = 59); relapsing-remitting MS (RRMS) (N = 36), secondary progressive MS (SPMS) (N = 20) and primary progressive MS (PPMS) (N = 3), and controls (N = 27). CSF levels of sTREM-2 were also assessed before and after treatment of patients with natalizumab or mitoxantrone. Results: CSF levels of sTREM-2 were significantly increased in patients with RRMS, SPMS, and PPMS compared with controls. After natalizumab treatment, the levels of sTREM-2 were normalized to control levels. The levels of sTREM-2 were also reduced after mitoxantrone treatment. Conclusion: Increased CSF levels of sTREM-2, a new marker of microglial activation, in MS and normalization upon treatment with either natalizumab or mitoxantrone support a role for microglial activation in active MS.

Leucine-sensitive hyperinsulinaemic hypoglycaemia in patients with loss of function mutations in 3-Hydroxyacyl-CoA Dehydrogenase

BackgroundLoss of function mutations in 3-Hydroxyacyl-CoA Dehydrogenase (HADH) cause protein sensitive hyperinsulinaemic hypoglycaemia (HH). HADH encodes short chain 3-hydroxacyl-CoA dehydrogenase, an enzyme that catalyses the penultimate reaction in mitochondrial β-oxidation of fatty acids. Mutations in GLUD1 encoding glutamate dehydrogenase, also cause protein sensitive HH (due to leucine sensitivity). Reports suggest a protein-protein interaction between HADH and GDH. This study was undertaken in order to understand the mechanism of protein sensitivity in patients with HADH mutations.MethodsAn oral leucine tolerance test was conducted in controls and nine patients with HADH mutations. Basal GDH activity and the effect of GTP were determined in lymphoblast homogenates from 4 patients and 3 controls. Immunoprecipitation was conducted in patient and control lymphoblasts to investigate protein interactions.ResultsPatients demonstrated severe HH (glucose range 1.7–3.2 mmol/l; insulin range 4.8-63.8 mU/l) in response to the oral leucine load, this HH was not observed in control patients subjected to the same leucine load. Basal GDH activity and half maximal inhibitory concentration of GTP was similar in patients and controls. HADH protein could be co-immunoprecipitated with GDH protein in control samples but not in patient samples.ConclusionsWe conclude that GDH and HADH have a direct protein-protein interaction, which is lost in patients with HADH mutations causing leucine induced HH. This is not associated with loss of inhibitory effect of GTP on GDH (as in patients with GLUD1 mutations).

G‐quadruplex‐binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA‐binding proteins and through toxic dipeptide repeat proteins generated by repeat‐associated non‐ATG translation. GGGGCC repeat RNA folds into a G‐quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G‐quadruplex RNA. We investigated their effect in C9orf72 patient iPSC‐derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat‐expressing Drosophila. Therefore, small molecules that target GGGGCC repeat G‐quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS. These data provide proof of principle that targeting GGGGCC repeat G‐quadruplexes has therapeutic potential.

Novel Insights Into Fatty Acid Oxidation, Amino Acid Metabolism, and Insulin Secretion From Studying Patients With Loss of Function Mutations in 3-Hydroxyacyl-CoA Dehydrogenase

CONTEXT Mutations causing genetic defects have been described in many of the enzymes involved in mitochondrial fatty acid oxidation (FAO). Recently, mutations in the penultimate enzyme in the FAO chain have been described that result in quite different symptoms from those normally seen. Patients with mutations in 3-hydroxyacyl-CoA dehydrogenase (HADH) present with protein (leucine)-induced hyperinsulinemic hypoglycemia (HH), suggesting a link between FAO, amino acid metabolism, and insulin secretion. EVIDENCE ACQUISITION AND SYNTHESIS Peer-reviewed articles were searched in PubMed with relevance to HADH and disorders of FAO and protein sensitivity. Relevant articles were cited. Recent evidence suggests that mutations in HADH cause HH that is precipitated by protein in a similar manner to the hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by mutations in the GLUD1 gene, encoding the enzyme glutamate dehydrogenase (GDH). CONCLUSION Current data suggest that the HH observed in patients with mutations in HADH is precipitated by leucine as seen in the HI/HA syndrome. This is caused by a loss of protein/protein interaction between short-chain HADH (SCHAD, the enzyme coded for by HADH) and GDH, causing an overstimulation of GDH and a rise in cellular ATP and up-regulated insulin secretion. These observations provide new mechanistic insights into the regulation of insulin secretion by fatty acid and amino acid metabolism.

Congenital Hyperinsulinism due to mutations in HNF4A and HADH

Mutations in the HADH and HNF4A genes are rare causes of diazoxide responsive congenital hyperinsulinism (CHI). This chapter details the phenotype known to be associated with mutations in these genes. Additionally, the authors give a brief overview of the role of these genes in glucose physiology and the possible mechanisms of CHI in patients with mutations in these genes.

Do cerebrospinal fluid transfer methods affect measured amyloid β42, total tau, and phosphorylated tau in clinical practice?

Cerebrospinal fluid (CSF) neurodegenerative markers are measured clinically to support a diagnosis of Alzheimers disease. Several preanalytical factors may alter the CSF concentrations of amyloid β 1–42 (Aβ1–42) in particular with the potential to influence diagnosis. We aimed to determine whether routine handling of samples alters measured biomarker concentration compared with that of prompt delivery to the laboratory.

Cerebrospinal fluid neurogranin and TREM2 in Huntington’s disease

Biomarkers of Huntington’s disease (HD) in cerebrospinal fluid (CSF) could be of value in elucidating the biology of this genetic neurodegenerative disease, as well as in the development of novel therapeutics. Deranged synaptic and immune function have been reported in HD, and concentrations of the synaptic protein neurogranin and the microglial protein TREM2 are increased in other neurodegenerative diseases. We therefore used ELISAs to quantify neurogranin and TREM2 in CSF samples from HD mutation carriers and controls. CSF neurogranin concentration was not significantly altered in HD compared to controls, nor was it significantly associated with disease burden score, total functional capacity or motor score. An apparent increase in CSF TREM2 in manifest HD was determined to be due to increasing TREM2 with age. After age adjustment, there was no significant alteration of TREM2 in either HD group, nor any association with motor, functional or cognitive score, or brain volume quantified by MRI. Both analyses were well-powered, and sample size calculations indicated that several thousand samples per group would be needed to prove that disease-associated alterations do in fact exist. We conclude that neither neurogranin nor TREM2 is a useful biofluid biomarker for disease processes in Huntington’s disease.

Neurofilament light as a blood biomarker for neurodegeneration in Down syndrome

BackgroundDown syndrome (DS) may be considered a genetic form of Alzheimer’s disease (AD) due to universal development of AD neuropathology, but diagnosis and treatment trials are hampered by a lack of reliable blood biomarkers. A potential biomarker is neurofilament light (NF-L), due to its association with axonal damage in neurodegenerative conditions.MethodsWe measured blood NF-L concentrations in 100 adults with DS using Simoa NF-light® assays, and we examined relationships with age as well as cross-sectional and longitudinal dementia diagnosis.ResultsNF-L concentrations increased with age (Spearman’s rho = 0.789, p < 0.001), with a steep increase after age 40, and they were predictive of dementia status (p = 0.022 adjusting for age, sex, and APOE4), but they showed no relationship with long-standing epilepsy or premorbid ability. Baseline NF-L concentrations were associated with longitudinal dementia status.ConclusionsNF-L is a biomarker for neurodegeneration in DS with potential for use in future clinical trials to prevent or delay dementia.

Genetic Variants and Related Biomarkers in Sporadic Alzheimer's Disease

From a neuropathological perspective, elderly patients who die with a clinical diagnosis of sporadic Alzheimer’s disease (AD) are a heterogeneous group with several different pathologies contributing to the AD phenotype. This poses a challenge when searching for low effect size susceptibility genes for AD. Further, control groups may be contaminated by significant numbers of preclinical AD patients, which also reduces the power of genetic association studies. Here, we discuss how cerebrospinal fluid and imaging biomarkers can be used to increase the chance of finding novel susceptibility genes and as a means to study the functional consequences of risk alleles.

Neurofilament light chain and tau concentrations are markedly increased in the serum of patients with sporadic Creutzfeldt-Jakob disease, and tau correlates with rate of disease progression

Objectives A blood-based biomarker of neuronal damage in sporadic Creutzfeldt-Jakob disease (sCJD) will be extremely valuable for both clinical practice and research aiming to develop effective therapies. Methods We used an ultrasensitive immunoassay to measure two candidate biomarkers, tau and neurofilament light (NfL), in serum from patients with sCJD and healthy controls. We tested longitudinal sample sets from six patients to investigate changes over time, and examined correlations with rate of disease progression and associations with known phenotype modifiers. Results Serum concentrations of both tau and NfL were increased in patients with sCJD. NfL distinguished patients from controls with 100% sensitivity and 100% specificity. Tau did so with 91% sensitivity and 83% specificity. Both tau and NfL appeared to increase over time in individual patients, particularly in those with several samples tested late in their disease. Tau, but not NfL, was positively correlated with rate of disease progression, and was particularly increased in patients homozygous for methionine at codon 129 of PRNP. Conclusions These findings independently replicate other recent studies using similar methods and offer novel insights. They show clear promise for these blood-based biomarkers in prion disease. Future work should aim to fully establish their potential roles for monitoring disease progression and response to therapies.