Research ArticleBrain Imaging

Insights into neuroepigenetics through human histone deacetylase PET imaging

See allHide authors and affiliations

Science Translational Medicine  10 Aug 2016:
Vol. 8, Issue 351, pp. 351ra106
DOI: 10.1126/scitranslmed.aaf7551
  • Fig. 1. [11C]Martinostat images of all subjects show high cortical binding and distinct gray-white matter differences.

    (A) [11C]Martinostat (injected dose, 4.7 mCi; specific activity, 1.1 mCi/nmol) images averaged from 60 to 90 min after radiotracer injection (SUV60-90 min; SUV = radioactivity per injected dose per body weight) from a representative subject overlaid on anatomical magnetic resonance (MR) image. (B) [11C]Martinostat SUVR60-90 min images of individual subjects. To facilitate intersubject comparison of regional HDAC distribution, we normalized regional SUV60-90 min to an individual subject’s white matter SUV60-90 min as SUV60-90 min ratios (SUVR60-90 min). The SUVR60-90 min images were also coregistered with an MNI152 standard human atlas brain.

  • Fig. 2. Small intersubject variation of localized regional [11C]Martinostat binding in the human brain.

    (A) Mean images (left) and standard deviation (inset, to the lower right of each composite image) of SUV60-90 min from healthy volunteers (n = 8). The images are overlaid onto the MNI152 standard brain, where x, y, and z indicate the coordinate of each image plane shown. (B) Correlation of regional VT values, derived from a two-tissue compartmental model using metabolite-corrected arterial plasma as an input function and SUV60-90 min. Data are means ± SD (n = 6 subjects), and each circle symbol represents a separate brain region (n = 14 brain regions). P value determined with Pearson correlation analysis. (C) Regional SUV60-90 min and SUV ratios (SUVR60-90 min) of cortical, subcortical, cerebellar, and white matter volumes of interest (VOIs). Individual pairs of brain regions that are significantly different from each other are listed in table S4. Each dashed line represents SUVR60-90 min from a single subject (n = 8).

  • Fig. 3. HDAC2 and HDAC3 expression levels are higher in cortical gray matter than in white matter.

    Whole-cell lysates were prepared from postmortem human SFG and CC (n = 3 replicate donor pools with two donors per pool), as well as dorsolateral prefrontal cortex (DLPFC), hippocampus (Hipp), and anterior cingulate (Ant Cing) (n = 3 replicate pools with three donors per pool). (A) Equivalent amounts of total protein were compared to human recombinant HDAC standards through Western blotting. #The HDAC2 recombinant standard was tagged with glutathione S-transferase (GST), resulting in increased molecular weight. (B and C) Comparison of HDAC expression between white matter (CC) and gray matter (SFG) regions (B) and among different gray matter regions (C). HDAC immunoreactive band intensity values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) intensity values. HDAC expression levels were calculated per milligram of total extracted protein. Solid lines represent mean expression values. Donor pools are denoted by black, gray, and open circles. P values were determined by unpaired t test (B) and ordinary one-way analysis of variance (ANOVA) (α = 0.05 with Tukey’s multiple comparisons correction) (C).

  • Fig. 4. Martinostat engages HDAC1, HDAC2, and HDAC3 in the human brain.

    (A) Whole-cell lysates were prepared from postmortem human SFG and CC (n = 3 replicate donor pools with two donors per pool). Thermal shift assays were performed with increasing concentrations of Martinostat (0, 0.0032, 0.016, 0.080, 0.40, 2.0, and 10 μM). Thermal stabilization of HDACs 1, 2, 3, 6, and 8 was compared through Western blotting with scaled immunoreactive band intensity values represented as an averaged heat map (n = 3). The imaging-derived dissociation constant (Kd) for [11C]Martinostat in NHP brain is indicated by the black arrow (19). See fig. S6 for original Western blotting data. (B) Whole-cell lysates were prepared from postmortem human SFG (n = 3 replicate donor pools with two donors per pool), as well as dorsolateral prefrontal cortex, hippocampus, and anterior cingulate (n = 3 replicate donor pools with three donors per pool). Thermal shift assays were performed with increasing concentrations of Martinostat (0, 0.16, 0.80, 4.0, 20, and 100 μM). Thermal stabilization of HDACs 1, 2, 3, 6, and 8 was compared through Western blotting with scaled immunoreactive band intensity values represented as an averaged heat map (n = 3). See figs. S7 to S9 for original Western blotting data. (C) Baboon brain (n = 1) was sectioned to include gray matter and white matter regions in the same slice. Tissue was coincubated with ~100 μCi of [11C]Martinostat and either 0 or 2 μM nonradiolabeled Martinostat. Grayscale autoradiographic images were colored using a standard lookup table (royal scale in Image J) to reflect [11C]Martinostat intensity (left). Region-specific baseline and blocking intensity values were quantitated from each slice (right). Data are means ± SD (n = 22 0-μM slices, n = 10 2-μM slices; one image per slice; one region of interest per brain region). P values were determined by ordinary two-way ANOVA (α = 0.05 with Sidak’s multiple comparisons correction).

  • Fig. 5. Martinostat increases histone acetylation and gene expression levels in human neural progenitor cells.

    Human neural progenitor cells were treated with DMSO (Veh), Martinostat (MSTAT; 0.5, 2.5, or 5.0 μM), and SAHA (10 μM) for 24 hours. (A) Whole-cell lysates were prepared (n = 3). #Because treatment with 5.0 μM Martinostat was toxic to cells, whole-cell lysates from three replicates were combined into one pool to obtain sufficient protein for this dose. Equivalent amounts of total protein were compared through Western blotting. Histone acetylation immunoreactive band intensity values were normalized to GAPDH intensity values. Data are means ± SD (n = 3). P values compare drug treatments to Veh, determined by repeated-measures two-way ANOVA (α = 0.05 with Dunnett’s multiple comparisons correction). (B) RNA was extracted (n = 3) and converted into complementary DNA (cDNA). mRNA transcript levels of memory/neuronal plasticity–related (BDNF, EGR1, CDK5, SYT1, and SYP) and monogenic neurological disorder–related (GRN and FXN) genes were compared through qPCR and normalized to GAPDH mRNA levels. Data are means ± SEM (n = 3 cDNA per condition with three technical qPCR replicates per cDNA). P values compare drug treatments to Veh, determined by repeated-measures two-way ANOVA (α = 0.05 with Dunnett’s multiple comparisons correction).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/351/351ra106/DC1

    Methods

    Fig. S1. TACs and compartmental model fitting (two-tissue compartmental model) results for superior frontal cortex and white matter.

    Fig. S2. Stability of outcome measurement (VT) as a function of scan duration.

    Fig. S3. Regional SUV60-90 min from all brain regions analyzed.

    Fig. S4. Same day test-retest reproducibility of [11C]Martinostat SUVR60-90 min.

    Fig. S5. Nuclear density, size, and total area in postmortem baboon brain tissue.

    Fig. S6. Martinostat thermal shift assay in human SFG and CC biological replicates 1, 2, and 3.

    Fig. S7. Martinostat thermal shift assay across human gray matter biological replicate 1.

    Fig. S8. Martinostat thermal shift assay across human gray matter biological replicate 2.

    Fig. S9. Martinostat thermal shift assay across human gray matter biological replicate 3.

    Table S1. Biometric information for PET imaging participants.

    Table S2. Goodness of fit for one- and two-tissue compartmental models to regional PET data.

    Table S3. Kinetic rate constants and regional VT for [11C]Martinostat.

    Table S4. Statistical comparison of [11C]Martinostat between different brain regions.

    Table S5. Sample information for postmortem human brain tissue.

  • Supplementary Material for:

    Insights into neuroepigenetics through human histone deacetylase PET imaging

    Hsiao-Ying Wey, Tonya M. Gilbert, Nicole R. Zürcher, Angela She, Anisha Bhanot, Brendan D. Taillon, Fredrick A. Schroeder, Changing Wang, Stephen J. Haggarty, Jacob M. Hooker*

    *Corresponding author. Email: hooker{at}nmr.mgh.harvard.edu

    Published 10 August 2016, Sci. Transl. Med. 8, 351ra106 (2016)
    DOI: 10.1126/scitranslmed.aaf7551

    This PDF file includes:

    • Methods
    • Fig. S1. TACs and compartmental model fitting (two-tissue compartmental model) results for superior frontal cortex and white matter.
    • Fig. S2. Stability of outcome measurement (VT) as a function of scan duration.
    • Fig. S3. Regional SUV60-90 min from all brain regions analyzed.
    • Fig. S4. Same day test-retest reproducibility of [11C]Martinostat SUVR60-90 min.
    • Fig. S5. Nuclear density, size, and total area in postmortem baboon brain tissue.
    • Fig. S6. Martinostat thermal shift assay in human SFG and CC biological replicates 1, 2, and 3.
    • Fig. S7. Martinostat thermal shift assay across human gray matter biological replicate 1.
    • Fig. S8. Martinostat thermal shift assay across human gray matter biological replicate 2.
    • Fig. S9. Martinostat thermal shift assay across human gray matter biological replicate 3.
    • Table S1. Biometric information for PET imaging participants.
    • Table S2. Goodness of fit for one- and two-tissue compartmental models to regional PET data.
    • Table S3. Kinetic rate constants and regional VT for [11C]Martinostat.
    • Table S4. Statistical comparison of [11C]Martinostat between different brain regions.
    • Table S5. Sample information for postmortem human brain tissue.

    [Download PDF]

Navigate This Article