Journal: Molecular Psychiatry

Article Title: Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin

doi: 10.1038/mp.2015.81

Figure Lengend Snippet: Postmortem CSF seeding enhancement of mHTT aggregation in inducible Htt14A2.6 cells and derived cell-free lysates. ( a ) Dot blots detect amount of N-terminal (N17) in both autopsy HD and control CSF samples but expanded polyQ ( Q >36) only in HD CSF. ( b ) Normalized percentage of cells with mHTT aggregates following seeding with CSF from autopsy HD and healthy control subjects (Ctl). Diagnosis: [F(1,87.6)= 120.41, P <0.0001] incubation time: [F(1,75)=17.16, P <0.0001] interaction: [F(1,75)=36.35, P <0.0001]. See for individual values. ( c ) Percentage of cells with mHTT aggregates following seeding with CSF from an autopsy HD subject (HD#134) that has been depleted by polyQ expansion-specific antibodies at dilutions of 1/20, 1/100 and 1/500. Antibody dilution: F(3,7.29)=45.8, P <0.0001. ( d ) Amount of mHTT aggregates formed in 14A2.6 cell-free lysates seeded with HD CSF of Subject HD#134 at varying dilutions (that is, final CSF concentration=10% in the cell-free lysate standard assay, 2% in ‘1/5‘ and 0.4% in ‘1/25‘ dilutions). ( e ) Amount of aggregate protein with GFP tag in same subjects in cell-free assay. Diagnosis: [F(1,14.7)=50.79, P <0.0001]. PolyQ: 0.1 μM polyQ40. Bars indicate s.e.m. Each experiment had three to nine repeats. CSF, cerebrospinal fluid; GFP, green fluorescent protein; HD, Huntington's disease; mHTT, mutant huntingtin protein.

Article Snippet: For the immunodepletion of mHTT in CSF experiment, 100 μl CSF was incubated with 20 μl protein A/G PLUS-Agrose beads (Santa Cruz Biotechnologies, Dallas, TX, USA) that were preconjugated with varying amount of MAB1574 polyQ expansion-specific antibody (EMD Millipore, Temecula, CA, USA) for 1 h at 4 °C with gentle mixing.

Techniques: Derivative Assay, Control, Biomarker Discovery, Incubation, Concentration Assay, Cell-Free Assay, Mutagenesis

Journal: Molecular Psychiatry

Article Title: Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin

doi: 10.1038/mp.2015.81

Figure Lengend Snippet: Enhanced mHTT aggregation (relative to mock) in blinded CSF samples from living PREDICT subjects. ( a ) Amount of mHTT aggregates from cell-free lysates seeded with CSF from BACHD transgenic rats (BAC) and wild-type rats (WT). BACHD vs WT: [F(1,15.6)=117.74, P <0.0001]. ( b ) Relative (to mock control) levels of cells with mHTT aggregates (bars) with GFP tags following 24 h seeding with CSF from blinded PREDICT living samples (bracketed), and autopsy HD and controls (HD#, Ctl# converted data from ), reordered for clarity. HD subjects with clinical motor symptoms in red, CAG expansion-gene-positive subjects without clinical symptoms in yellow, healthy controls in blue. Autopsy HD subjects in light red, and autopsy controls in light blue. Group (HD, CAG expansion-gene-positive, and controls): [F(2,19.6)=37.82, P <0.0001]. HD>CAG expansion-gene-positive>Controls, all P s<0.001. ( c ) Amount of aggregate protein with GFP tags following seeding with CSF from blinded PREDICT samples, with autopsy HD (HD#) and polyQ control subjects (upper) and with CSF from 10 subjects with mixed dementia (AD, Lewy body, and frontal temporal; lower; see for original gels). ( d ) Relative amount of mHTT in cell-free lysate assay following seeding with CSF from grouped PREDICT-HD subjects with clinical symptoms (red), expansion-gene-positive subjects without clinical symptoms (yellow), and controls (blue; autopsy and PREDICT controls (CTL); and non-HD dementia controls (data from c )). ** P <0.001, *** P <0.0001. Bars indicate s.e.m. Each experiment had three to nine repeats. CSF, cerebrospinal fluid; GFP, green fluorescent protein; HD, Huntington's disease; mHTT, mutant huntingtin protein.

Article Snippet: For the immunodepletion of mHTT in CSF experiment, 100 μl CSF was incubated with 20 μl protein A/G PLUS-Agrose beads (Santa Cruz Biotechnologies, Dallas, TX, USA) that were preconjugated with varying amount of MAB1574 polyQ expansion-specific antibody (EMD Millipore, Temecula, CA, USA) for 1 h at 4 °C with gentle mixing.

Techniques: Transgenic Assay, Control, Mutagenesis