Comprehensive Notes on Protein Misfolding in Huntington's Disease

Introduction to Huntington's Disease and Protein Misfolding

  • Huntington's disease (HD) is a fatal neurodegenerative disorder linked to misfolded proteins.

  • A novel amyloid seeding assay (ASA) was developed to detect misfolded huntingtin (HTT) protein.

  • The ASA revealed early protein misfolding in a transgenic mouse model of HD (YAC128).

  • This suggests that protein misfolding may be an early event in the pathogenesis of HD.

Background on Huntington's Disease (HD)

  • HD is an autosomal dominant neurodegenerative disorder.

  • Characterized by motor impairment, cognitive decline, and behavioral abnormalities.

  • Caused by an expansion of CAG triplet repeats in exon 1 of the huntingtin (HTT) gene.

  • A hallmark of HD is the presence of misfolded HTT fragments in neuronal intranuclear inclusions (NIIs).

  • The role of large protein aggregates like NIIs in disease pathogenesis is debated.

  • Misfolding of HTT protein is considered a key step in disease pathogenesis.

Transgenic Mouse Models of HD

  • Several transgenic mouse models emulate HD pathogenesis and symptoms.

  • R6/2 and N171-82Q mice exhibit NIIs early in pathogenesis.

  • YAC128 mice express the full-length human HTT gene.

  • YAC128 mice show motor control decline at 4 months, but NIIs appear later (18 months).

  • Neuronal loss in the striatum and cortex is seen by 12 months in YAC128 mice.

Amyloid Seeding Assays (ASAs)

  • ASAs detect misfolded proteins by using them as seeds to accelerate in vitro fibrillization of monomeric protein.

  • Kinetics of amyloid formation are measured by thioflavin T (ThT) fluorescence.

  • ASAs are sensitive, with detection limits as low as a femtogram.

  • Real-time quaking-induced conversion ASA quantifies prions in animals and detects them in the cerebrospinal fluid of human Creutzfeldt-Jakob disease patients.

  • Seeding recombinant HTT protein with R6/2 brain-derived seeds analyzes conformational diversity of misfolded HTT.

Experimental Procedures

  • Peptide Solubilization:

    • K2Q44K2 peptide (90% purity) was used.

    • A modified protocol was used to create monomeric solutions.

    • Peptide suspended in a 1:1 mixture of TFA and hexafluoroisopropanol at 2 mg/ml and stirred for 2 days.

    • Solvent removed under argon flow, and peptide film dissolved in 2 M guanidine hydrochloride at 2 mg/ml.

    • Residual aggregates removed by ultracentrifugation at 300,000 x g for 3 hours at 4°C.

  • Monitoring Amyloid Formation by ThT Assay:

    • Monomeric peptide diluted with TBS (pH 8.5) and 100 µM ThT to approximately 0.4 mg/ml.

    • Mixed with an equal volume of seeding agent solution.

    • Transferred to wells of a black 96-well flat-bottom plate with a 3-mm glass bead for mixing.

    • Final concentration: 0.2 mg/ml peptide, 50 µM ThT, and 0.2 M guanidine hydrochloride in TBS (pH 8.5).

    • Plate sealed and incubated in a SpectraMax M2 plate reader at 37°C; fluorescence recorded every 10 minutes (excitation 444 nm, emission 484 nm) after shaking.

  • Transgenic Mice:

    • R6/2, N171-82Q, and YAC128 mice were crossbred with wild-type mice.

    • Genotypes confirmed by PCR.

    • R6/2 and N171-82Q mice were aged until neurodegeneration symptoms appeared (tremors, loss of balance, hind limb clasping).

    • YAC128 mice and controls were killed at indicated ages.

    • Whole brain tissues were frozen at -80°C.

  • Human Tissue Samples:

    • Brain tissue samples obtained from Harvard Brain Tissue Resource Center and Human Brain and Spinal Fluid Resource Center at UCLA.

    • Samples from caudate, putamen, and acumens region of grade 4 HD-affected patients and normal controls.

    • Superior frontal cortex region of Alzheimer's disease (AD)-affected patient and normal control.

  • Enrichment and Partial Purification of Misfolded HTT:

    • A protocol was modified to purify misfolded HTT.

    • Brain sample thawed, suspended in homogenization buffer A with protease inhibitors.

    • Homogenized by extrusion through progressively thinner needles.

    • Centrifuged at 1000 x g for 10 minutes at 4°C; supernatant S1 saved.

    • Pellet (P1) homogenized, centrifuged, and supernatant mixed with S1 to yield a 5 weight % brain homogenate equivalent supernatant (S2).

    • S2 adjusted to 1% Sarkosyl and 0.2% DTT, incubated at room temperature for 2.5 hours with stirring.

    • Multimeric huntingtin and aggregates pelleted by ultracentrifugation at 300,000 x g for 1.5 hours at 4°C.

    • Pellet P3 suspended in TBS and ultracentrifuged to remove Sarkosyl.

    • Residual pellet (P4) suspended in TBS (pH 8.5) and passed through a 27-gauge needle for ThT assay.

  • SDS-PAGE and Western Blotting:

    • Pellet P4 adjusted to 2 mg of brain tissue equivalent (BTE) and visualized by Western blotting using anti-polyglutamine monoclonal antibody 1C2.

  • Data Analysis:

    • Lag phase calculated by identifying the first of three consecutive time points significantly above background.

    • ThT fluorescence values plotted as a function of BTE; average calculated using five time points centered at t1.25 (1.25 times the lag phase).

    • p values calculated by Student’s t-test.

Results and Findings

  • Effect of Synthetic Seeds on Polyglutamine Amyloid Formation:

    • K2Q44K2 forms amyloid following a lag phase upon incubation at 37°C.

    • Lag phase significantly reduced at seed concentrations of 0.001% or higher (400 pg of preformed seed/well).

  • Brain Homogenates Inhibit Amyloid Formation:

    • Unpurified brain homogenates inhibited amyloid formation compared to unseeded reactions.

    • WT brain homogenate inhibited amyloid formation to a greater extent than R6/2 brain homogenate.

    • Lipids (0.25 mg/ml) and protein (0.25 mg/ml BSA) significantly delayed amyloid formation.

  • Partial Purification of Misfolded HTT:

    • A protocol was adapted to enrich misfolded HTT from brain homogenates while retaining yield.

    • Incubation with Sarkosyl and DTT removes undesired proteins.

    • Lipids removed by ultracentrifugation in 10% sucrose and 0.8 M NaCl.

    • Presence of HTT protein in final pellet confirmed by Western blotting.

  • Sensitive Detection of Misfolded HTT by Amyloid Seeding:

    • Misfolded HTT from transgenic mouse models (R6/2, N171-82Q) and human HD brain tissues accelerated K2Q44K2 amyloid formation.

    • Seeding effect observed for all 11 samples relative to unseeded reactions and negative controls.

    • Amyloid formation monitored as a function of brain-derived material concentration.

    • HD ASA specifically detected misfolded HTT; AD-affected patient brain tissue did not accelerate amyloid formation.

  • Statistical Confidence in HD ASA:

    • HD ASA detected misfolded HTT with high statistical confidence (p < 0.001).

      • p < 0.001

  • Detection of Misfolded HTT in 11-week-old YAC128 Mice:

    • Misfolded HTT detected in YAC128 mice as early as 11 weeks of age.

    • Amyloid seeding observed in an age- and dose-dependent manner.

    • Age dependence more evident at 1 mg of BTE.

      • No change in HTT protein expression levels with age.

Discussion

  • A sensitive ASA for detecting misfolded HTT in brain tissues from HD mouse models and humans was developed.

  • ASA detects misfolding of HTT in the YAC128 mouse model earlier than other methods.

  • K2Q44K2 was chosen as the substrate for ASA because it is commercially available and has well-characterized amyloid formation kinetics.

  • Conditions used for the HD ASA could result in greater sensitivity or a more facile assay with further optimization.

  • The limit of detection for partially purified misfolded HTT in HD ASA is approximately 40 µg of BTE for mouse models.

  • The misfolded protein detected was formed during purification.

    • No acceleration in amyloid formation was observed for the 7.4-week-old YAC128 mouse even though the protein levels at this age are the same as those observed in older YAC128 mice.

    • There was a clear age dependence on the magnitude of the increase in ThT fluorescence observed.

  • There is no direct correlation between the presence of NIIs and neurotoxicity. HD ASA detects large NIIs or earlier intermediates in the protein misfolding process, such as oligomers.

    • Some form of misfolded HTT can be detected by 11 weeks of age. Thus, HTT misfolding takes place early in the pathogenesis of disease in the YAC128 model around the same time that the earliest behavioral symptoms begin to manifest.

  • The sensitivity of the HD ASA and its ability to detect misfolded HTT early may make the assay useful as a biomarker for HD pathogenesis in preclinical trials.

  • Drugs directed toward preventing protein misfolding should be administered early in this mouse model.