Successful quantification of mHTT in PBMCs from 8ml whole blood in HD.

<p>These data confirm that mHTT levels in leukocytes are higher at more advanced disease stages. Assay A (mHTT N-terminus assay) was used to selectively quantify mHTT: S/B, signal over background. A) Sample set 1 (mean of 2 replicates); (B) Sample set 2 (mean of 3 replicates).</p

Box plots showing average polyglutamine independent HTT levels across patient subgroups.

<p>(S/B: Signal / Background) No significant differences are seen between HTT species levels and disease stage, other than a difference in C-terminal HTT between Moderate and Early HD patients. (A) Distribution of Mid Region HTT (Assay C) in sample group 1. (B) Distribution of Mid Region HTT (Assay C) in sample group 2. (C) Distribution of polyglutamine-independent N terminal HTT (Assay B) in sample group 2. (D) Distribution of C terminal HTT (polyglutamine-independent Assay D) in sample group 2.</p

Association between HTT species level (S/B: Signal/Background, mean replicated samples) and disease burden score; adjusted for age, sex and CAG.

<p>(A) HTT mid region in sample set 1, (B) N-terminal HTT in sample set 2, (C) HTT mid region in sample set 2, (D) C-terminal HTT in sample set 2). Circles = premanifest, triangles = Early, squares = Moderate.</p

Schematic diagram showing the capture and detection antibody pairs for each assay.

<p>The huntingtin amino acid residue epitope to which the antibodies bind are shown (not to scale). Poly-Q, polyglutamine region; Poly-P, proline rich region. Details of the antibodies are given in the text below.</p

Results for each sample set and disease stage.

<p>Results for each sample set and disease stage.</p

Association between DBS and mHTT (S/B: Signal/Background, mean of replicated samples) in sample set 1 (A) and 2 (B).

<p>P values adjusted for age, sex and CAG.</p

Functional activity of digested 38B8 mAb in the TrkB NFAT reporter gene assays.

<p>CellSensor® TrkB-NFAT-bla CHO-K1 cells were stimulated with (A) BDNF, mAb 38B8, 38B8 Fab or 38B8 F(ab’)2 over the indicated concentration range for 5 hours (agonist mode) or (B) 38B8 Fab for 1 hour prior to 0.3 nM BDNF stimulation for 4 hours (antagonist mode) before beta-lactamase assay was performed as described in Methods. % of control (maximal BDNF concentration  =  9 nM) values were plotted for the indicated concentrations of each ligand (n = 2 ± SD for each data point).</p

mHTT-induced neuronal toxicity: 7,8-dihydroxyflavone and LM22A-4 pharmacology.

<p>Using our primary rat cortico-striatal co-culture system we stimulated with 7,8-dihydroxyflavone, LM22A-4 or BDNF over the indicated concentration range according to the mHTT-induced co-culture protocol (A), as described in Methods. % Rescue (Normalized to in-plate controls; 1 nM BDNF (100% Rescue) and vehicle (0% Rescue)) values were plotted for striatal neurons over the indicated concentrations of each ligand (n = 6 ± SD for each data point). Rat primary cortico-striatal cells (non-transfected) were stimulated with 7,8-dihydroxyflavone (B) or LM22A-4 (C) using the indicated concentrations for 15 minutes and profiled by western blot. BDNF- (10 nM) mediated TrkB phosphorylation validated the experimental system.</p

TrkB antibody panel and associated activities.

#<p>Reactivity as described by suppliers.</p

A Monoclonal Antibody TrkB Receptor Agonist as a Potential Therapeutic for Huntington’s Disease

<div><p>Huntington’s disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for <i>in vivo</i> proof of concept studies in transgenic HD models.</p></div