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Insights into neuroepigenetics through human histonedeacetylase PET imagingHsiao-Ying Wey,1* Tonya M. Gilbert,1* Nicole R. Zürcher,1 Angela She,2

Anisha Bhanot,1 Brendan D. Taillon,1 Fredrick A. Schroeder,1 Changing Wang,1

Stephen J. Haggarty,2 Jacob M. Hooker1†

Epigenetic dysfunction is implicated in many neurological and psychiatric diseases, including Alzheimer’s dis-ease and schizophrenia. Consequently, histone deacetylases (HDACs) are being aggressively pursued as ther-apeutic targets. However, a fundamental knowledge gap exists regarding the expression and distribution ofHDACs in healthy individuals for comparison to disease states. Here, we report the first-in-human evaluation ofneuroepigenetic regulation in vivo. Using positron emission tomography with [11C]Martinostat, an imaging probeselective for class I HDACs (isoforms 1, 2, and 3), we found that HDAC expression is higher in cortical gray matterthan in white matter, with conserved regional distribution patterns within and between healthy individuals. Amonggray matter regions, HDAC expression was lowest in the hippocampus and amygdala. Through biochemical profil-ing of postmortem human brain tissue, we confirmed that [11C]Martinostat selectively binds HDAC isoforms 1, 2,and 3, the HDAC subtypes most implicated in regulating neuroplasticity and cognitive function. In human stem cell–derived neural progenitor cells, pharmacologic-level doses of Martinostat induced changes in genes closelyassociated with synaptic plasticity, including BDNF (brain-derived neurotrophic factor) and SYP (synaptophysin),as well as genes implicated in neurodegeneration, including GRN (progranulin), at the transcript level, in concertwith increased acetylation at both histone H3 lysine 9 and histone H4 lysine 12. This study quantifies HDAC expres-sion in the living human brain and provides the foundation for gaining unprecedented in vivo epigenetic infor-mation in health and disease.

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INTRODUCTION

Disorders of the central nervous system (CNS), including Alzheimer’sdisease (AD), schizophrenia, depression, and addiction, are increasinglyrecognized to involve dysregulation of epigenetic machinery. Among all,histone deacetylases (HDACs)—a family of chromatin-modifying en-zymes that dynamically regulates gene transcription—are the most fre-quently implicated (1, 2). A subset of HDACs has already been linkedto neuronal development, synaptic plasticity, and cognition (3, 4). Forexample, postmortem human brain tissue analyses and in vivo rodentstudies exposed HDAC1, HDAC2, and HDAC3 as antagonists of learn-ing and memory and contributors to AD and mood disorders (3, 5–9).Genetic manipulations or pharmacologic inhibition of aberrant HDAC2and HDAC3 activity rescued behavioral defects in rodent models ofboth AD and mood disorders (6, 7, 10–14). HDAC inhibitors were alsoproposed as a targeted treatment of frontotemporal lobar degeneration,owing to mutations that cause haploinsufficiency of the progranulin-encoding gene GRN (14). Collectively, these studies implicate a directrelationship between the levels of class I HDACs (isoforms 1, 2, and 3)and neuronal function.

In addition to the overall level of HDAC expression within thebrain, spatially localized variation of HDACs is also highly impactfulin neuronal plasticity, memory, and behavior. For example, intra-hippocampal injection of short hairpin RNA against Hdac2 selectivelynormalized HDAC2 levels and restored neuroplasticity-associated

1Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Mas-sachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.2Chemical Neurobiology Laboratory, Departments of Neurology and Psychiatry, Mas-sachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.*These authors contributed equally to this work.†Corresponding author. Email: hooker@nmr.mgh.harvard.edu

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gene transcription, synaptic density, and cognitive behavior in a mousemodel of AD (6). In contrast to the high level of hippocampal HDAC2in animal models and postmortem human tissue from AD patients,deficient HDAC2 expression was observed in the frontal cortex of post-mortem AD tissue, highlighting the importance of tightly regulated lo-calized HDAC expression (15). Analogously, focal genetic deletion ofHdac3 in the hippocampus and the nucleus accumbens enhanced long-term memory and acquisition of cocaine-associated place preferencein mice, respectively (5, 16). Although understanding of the full com-pendium of genes under HDAC-dependent regulation in definedregions of the brain is incomplete, HDAC2 chromatin immuno-precipitation studies in hippocampal tissue have identified severalimmediate-early genes (for example, BDNF and CDK5) involved inlearning and memory, as well as multiple genes involved in synapticplasticity (for example, SYP and SYT1) as downstream targets (3, 6, 17).Collectively, these studies provide support that localized HDAC ex-pression levels drive pivotal epigenetic mechanisms that modulateneuronal function.

Although there is strong evidence for localized HDAC dysfunctionin CNS disease, epigenetic models cannot recapitulate dynamic human-environment interactions and therefore may not accurately reflectin vivo human biology. Moreover, until now, there has been no meth-od to visualize in vivo epigenetic mechanisms in humans. We developedthe positron emission tomography (PET) epigenetic imaging agent,[11C]Martinostat, previously described in rodents and nonhuman pri-mates (NHPs) (12, 18, 19). Our previous work in rodents demonstratedthe specific and reversible binding properties of [11C]Martinostat andthat the agent engaged recombinant class I HDACs (isoforms 1, 2, and3) and class IIb HDAC (isoform 6) with low nanomolar affinities (18).Because [11C]Martinostat demonstrated excellent brain penetrance,

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it was used to determine whether clinically relevant HDAC inhibitors,such as suberoylanilide hydroxamic acid (SAHA) and CI-994, crossedthe blood-brain barrier and exhibited target occupancy in rodents (12).Most recently, we performed studies in NHPs to characterize the ki-netic properties of [11C]Martinostat and to estimate nondisplaceablebinding of [11C]Martinostat with pharmacologic blockades in prepara-tion for human studies (19). In addition to the brain, [11C]Martinostatshowed high specific binding and fast binding kinetics appropriate forPET imaging in heart, pancreas, spleen, and kidneys (18, 19). Here, wetranslate [11C]Martinostat for clinical research use and quantify hu-man epigenetic regulation.

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RESULTS

In vivo human PET imaging reveals conserved regional HDACexpression patterns in the healthy brainTo visualize HDAC expression in the living human brain, we performed[11C]Martinostat PET imaging on eight healthy volunteers (four malesand four females; mean age ± SD, 28.6 ± 7.6 years) (table S1). The uptakeof [11C]Martinostat reached a maximum at ~30 min after injection andshowed minimal decrease during the 90-min scan (fig. S1). The reten-tion of radioactivity is a unique feature of [11C]Martinostat, which allowsfor a stable quantification of HDAC expression levels. Regional heter-ogeneity, such as different levels of [11C]Martinostat uptake betweengray and white matter tissues, was observed at the individual subjectlevel (Fig. 1). Quantitative analysis using compartmental modelingon individual subjects’ dynamic PET data allowed us to determine thedistribution volume (VT), a measure of radiotracer binding that is nor-malized to the activity present in circulating blood, and rate constantsdescribing the pharmacokinetics of [11C]Martinostat (fig. S1 and tablesS2 and S3). VT values were stable beyond 50 min, with less than 10%variability when compared to the 90-min data (fig. S2).

Regional standardized uptake values from 60 to 90 min after radio-tracer administration (SUV60-90 min), an image-based indicator ofbinding to HDACs (Fig. 2A), correlated positively with VT values(Fig. 2B). The image-based SUV60-90 min had less intersubject variabil-ity [coefficient of variation (CV) is 11.2 to 19.2% across brain regions]than the blood data–derived VT values (CV is 22.0 to 39.2% acrossbrain regions) (Fig. 2B). SUV60-90 min may therefore be an appropriatesurrogate outcome measurement for VT and can be used in futurestudies to eliminate arterial blood sampling and reduce sample sizebecause of its smaller variation. As with all surrogate measures, vali-dation relative to a full treatment of the data using arterial blood ineach patient population will be required.

Group-level analyses showed that the average gray matter SUV60-90 min

was nearly double that of white matter (Fig. 2C, fig. S3, and table S4),and heterogeneous binding was observed among gray matter regionsexamined. Besides the white matter, the lowest [11C]Martinostat up-take was observed in the hippocampus and amygdala, and the highest wasobserved in the putamen and cerebellum (Fig. 2C, fig. S3, and table S4).To facilitate intersubject comparison of regional HDAC distribution, wenormalized regional SUV60-90 min to individual subjects’ white matterSUV60-90 min as SUV60-90 min ratios (SUVR60-90 min). SUVR60-90 min

showed that the regional distribution patterns of [11C]Martinostat bind-ing were consistent in all subjects (Fig. 2C) and on consecutive scans insingle subjects. In preliminary test/retest scans (3 hours apart) in thesame individual, SUVR60-90 min showed less than 3% variability (fig. S4).

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Ex vivo biochemistry of postmortem tissue confirmsthat Martinostat binds to HDAC1, HDAC2, and HDAC3in the healthy brainTo assess regional differences in [11C]Martinostat binding in thehuman brain, we biochemically profiled postmortem brain tissuefrom gray matter regions [superior frontal gyrus (SFG), dorsolateralprefrontal cortex, hippocampus, and anterior cingulate] and a whitematter region [corpus callosum (CC)] (table S5). Quantitative proteinlevels of HDAC1, HDAC2, HDAC3, and HDAC6 were determined byWestern blotting (Fig. 3A). Significantly lower amounts of HDAC2and HDAC3 were found in the CC relative to the SFG (Fig. 3B). Nosignificant differences in HDAC expression were noted among thedorsolateral prefrontal cortex, hippocampus, or anterior cingulate—allgray matter regions (Fig. 3C). The average expression levels of HDAC2,HDAC3, and HDAC6 were similar in the SFG (0.12 to 0.16 pmol/mgtotal protein), with the notable exception of HDAC1 (1.7 pmol/mgtotal protein). Although high HDAC1 expression was observed acrossall brain regions tested (Fig. 3), we cannot exclude the possibility thatthese values are driven by postmortem neuronal death (20, 21).

HDAC2 and HDAC3 expression level differences between the SFGand the CC could not be attributed to nuclear density, according to

Fig. 1. [11C]Martinostat images of all subjects show high corticalbinding and distinct gray-white matter differences. (A) [11C]Martinostat

(injected dose, 4.7 mCi; specific activity, 1.1 mCi/nmol) images averagedfrom 60 to 90 min after radiotracer injection (SUV60-90 min; SUV = radio-activity per injected dose per body weight) from a representative sub-ject overlaid on anatomical magnetic resonance (MR) image. (B) [11C]Martinostat SUVR60-90 min images of individual subjects. To facilitate in-tersubject comparison of regional HDAC distribution, we normalized re-gional SUV60-90 min to an individual subject’s white matter SUV60-90 min asSUV60-90 min ratios (SUVR60-90 min). The SUVR60-90 min images were also co-registered with an MNI152 standard human atlas brain.

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quantification of the number of nuclei per field of view in postmortembaboon brain tissue (fig. S5). We observed that the CC had anincreased number of nuclei compared to the SFG, which suggestedthat lower HDAC expression in the CC was not due to a depletionof cells in this brain region (fig. S5). As nuclear size (area per nu-cleus) was smaller in the CC than in the SFG, the total nuclear areaper field of view was equivalent between these regions, further re-futing that HDAC expression levels are driven by nuclear density.

Thermal shift assays evaluate target engagement, such that inhib-itor binding increases the thermal stability of a target protein, ascompared to a vehicle control (22, 23). To determine the HDAC iso-

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form selectivity of Martinostat, thermalshift assays were performed with clarifiedhuman brain homogenate and increasingconcentrations of Martinostat. Martino-stat stabilized HDAC1, HDAC2, andHDAC3 in both the SFG and the CC atnanomolar concentrations (Fig. 4A, withindividual biological replicates in fig.S6). No significant stabilization of eitherHDAC6 or HDAC8 (negative control)was observed. The former suggests differ-ences between the accessibility of endoge-nous HDAC6 complex and Martinostatbinding, relative to recombinant protein(18). To assess heterogeneity in HDACisoform selectivity across gray matter re-gions, we compared SFG binding to thedorsolateral prefrontal cortex, hippo-campus, and anterior cingulate. On thebasis of thermal stabilization data, Marti-nostat exhibited a relatively uniformbinding profile in gray matter with targetengagement observed at concentrationsaround and above 0.160 mM (Fig. 4B,with individual biological replicates infigs. S7 to S9).

Competition autoradiography wasperformed in postmortem baboon braintissue to compare the specific binding of[11C]Martinostat in gray andwhitematter.[11C]Martinostat binding in white matterwas more biased by nonspecific uptakethan in gray matter (Fig. 4C). Together,our in vivo imaging and ex vivo bio-chemistry data indicate that [11C]Martino-stat binds to a subset of class I HDACs(isoforms 1, 2, and 3) across the humanand baboon brains.

In vitro biochemistry of humanneural progenitor cells revealsdownstream targets ofMartinostat-bound HDACsTo link [11C]Martinostat uptake withdownstream HDAC substrate signalingand gene expression, we treated humanstem cell–derived neural progenitor cells

with increasing concentrations of Martinostat. Acetylation levels ofestablished class I HDAC substrates, histone H3 lysine 9 (H3K9) andhistone H4 lysine 12 (H4K12), were determined using Western blotting(3, 24). Treatment with 2.5 and 5.0 mMMartinostat increased H3K9 andH4K12 acetylation levels as compared to vehicle control (Fig. 5A).Treatment with 5.0 mM Martinostat elevated acetylation to a levelequivalent to or greater than 10 mM SAHA (Fig. 5A). Messenger RNA(mRNA) transcript levels of memory-related (3, 6, 24), neuroplasticity-related (3), and neurological disease–related genes (17) were measuredthrough quantitative polymerase chain reaction (qPCR). Treatment with2.5 and/or 5.0 mM Martinostat increased brain-derived neurotrophic

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, wherex, y, and z indicate the coordinate of each image plane shown. (B) Correlation of regional VT values, derivedfrom a two-tissue compartmental model using metabolite-corrected arterial plasma as an input function andSUV60-90 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 andSUV 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. Eachdashed line represents SUVR60-90 min from a single subject (n = 8).

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factor (BDNF), early growth responseprotein 1 (EGR1), cyclin-dependent ki-nase 5 (CDK5), synaptotagmin (SYT1),synaptophysin (SYP), and progranulin(GRN) expression compared to vehiclecontrol, but not frataxin (FXN) (Fig.5B). Treatment with 2.5 mM Martinostatelevated BDNF and SYP (about 20- and10-fold, respectively) to a level equivalentto or greater than 10 mM SAHA (Fig.5B). Together, these results indicatethat Martinostat engages the subsetHDACs that deacetylate targets includ-ing H3K9 and H4K12, to regulatedownstream genes important for neuro-plasticity (BDNF, EGR1, CDK5, SYT1,SYP, and GRN).

DISCUSSION

This first-in-human epigenetic imagingstudy with [11C]Martinostat establishesthat HDACs are highly expressed through-out the healthy brain with region-specific

distribution, including distinct differences between gray and whitematter and differences between cortical and subcortical gray matterregions. On the basis of our previous in vitro profiling with recombi-nant HDACs (18) and our ex vivo profiling with postmortem humanand baboon brain tissues, the [11C]Martinostat signal in the brain ori-

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ginated from binding class I HDACs (isoforms 1, 2, and 3), which arerelevant to cognition, memory, and mood regulation (3, 13, 16, 25).Notably, Martinostat stabilized these isoforms at a concentration of~0.1 mM, which is consistent with the imaging-derived dissociationconstant (Kd) for [

11C]Martinostat in the NHP brain (18). In contrast

Fig. 3. HDAC2 and HDAC3 expression lev-els are higher in cortical gray matter than

in white matter. Whole-cell lysates wereprepared from postmortem human SFG andCC (n = 3 replicate donor pools with two do-nors per pool), as well as dorsolateral pre-frontal cortex (DLPFC), hippocampus(Hipp), and anterior cingulate (Ant Cing)(n = 3 replicate pools with three donorsper pool). (A) Equivalent amounts of totalprotein were compared to human recom-binant HDAC standards through Westernblotting. #The HDAC2 recombinant standardwas tagged with glutathione S-transferase(GST), resulting in increased molecular weight.(B and C) Comparison of HDAC expressionbetween white matter (CC) and gray mat-ter (SFG) regions (B) and among differentgray matter regions (C). HDAC immuno-reactive band intensity values were normalizedto glyceraldehyde-3-phosphate dehydrogenase(GAPDH) intensity values. HDAC expressionlevels were calculated per milligram of totalextracted protein. Solid lines representmean ex-pression values. Donor pools are denoted byblack, gray, and open circles. P values weredetermined by unpaired t test (B) and ordi-nary one-way analysis of variance (ANOVA)(a = 0.05 with Tukey’s multiple comparisonscorrection) (C).

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with previous in vitro recombinant inhibition data (18), Martinostatdid not appear to stabilize HDAC6 in the brain regions that we as-sessed, although it is worth noting that the recombinant assay provideda more than fivefold lower median inhibitory concentration forHDAC6 when compared to isoforms 1, 2, and 3. At high concen-

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trations, a-tubulin acetylation may beincreased by Martinostat, thus impli-cating potential HDAC6 binding attherapeutic-relevant concentrations.

Our imaging data revealed that in vivoHDAC expression is higher in corticalgray matter than in white matter, whichwas confirmed for HDAC2 and HDAC3by postmortem human tissue analyses.We postulate that HDAC complexes ineach brain tissue type may affect the se-lectivity of Martinostat and other HDACinhibitors, including those currently usedas U.S. Food and Drug Administration–approved drugs. HDAC complex–directed selectivity of HDAC inhibitorshas been shown previously through che-moproteomic approaches (26, 27), andadditional work will be required to elu-cidate the HDAC complexes most repre-sented by the [11C]Martinostat signal.

Beyond regional differences in HDACdistribution, the most striking observationwas the consistency of [11C]Martinostatbinding patterns between individual sub-jects. Because epigenetic machinery, andthus HDAC expression, is a highly dy-namic process, we did not fully expect aspatially conserved pattern of HDACexpression between individuals. Thisresult not only suggests that HDAC ex-pression is tightly regulated and mayrepresent a state function, but also re-iterates the importance of localized levelsof HDACs as they directly relate to genetranscription (1). We anticipate that re-gional [11C]Martinostat uptake differ-ences between healthy and diseasedindividuals will be detectable given theconversed baseline expression that wehave measured. The use of [11C]Marti-nostat imaging may eventually enableprecision medicine approaches for dis-ease stratification and treatment basedon epigenetic aberrations in the humanbrain. As hippocampal HDAC2 over-expression has been found in postmor-tem brain tissue from AD patients (6),[11C]Martinostat PET imaging holdsgreat potential for detecting aberranthippocampal HDAC expression and as-sessing novel HDAC therapeutics in ADpatients.

Because we envision and will apply [11C]Martinostat to measureHDAC expression in patient populations, it is critical that the out-come measurements are reliable, reproducible, and noninvasive. Bycomparing the standard deviation of the mean of VT and SUV60-90 min

across brain regions, we found that intersubject variability was smaller

Fig. 4. Martinostat engages HDAC1, HDAC2, and HDAC3 in the human brain. (A) Whole-cell lysateswere 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 mM). Thermal stabilization of HDACs 1, 2, 3, 6, and 8 was compared through West-ern 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 theblack arrow (19). See fig. S6 for original Western blotting data. (B) Whole-cell lysates were prepared frompostmortem human SFG (n = 3 replicate donor pools with two donors per pool), as well as dorsolateralprefrontal cortex, hippocampus, and anterior cingulate (n = 3 replicate donor pools with three donorsper pool). Thermal shift assays were performed with increasing concentrations of Martinostat (0, 0.16,0.80, 4.0, 20, and 100 mM). Thermal stabilization of HDACs 1, 2, 3, 6, and 8 was compared throughWestern blotting with scaled immunoreactive band intensity values represented as an averaged heatmap (n = 3). See figs. S7 to S9 for original Western blotting data. (C) Baboon brain (n = 1) was sectioned toinclude gray matter and white matter regions in the same slice. Tissue was coincubated with ~100 mCi of[11C]Martinostat and either 0 or 2 mM nonradiolabeled Martinostat. Grayscale autoradiographic imageswere 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). Dataare means ± SD (n = 22 0-mM slices, n = 10 2-mM slices; one image per slice; one region of interest perbrain region). P values were determined by ordinary two-way ANOVA (a = 0.05 with Sidak’s multiple com-parisons correction).

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using SUV60-90 min analysis than VT. These results support the use ofSUV60-90 min in future studies to eliminate arterial blood samplingwhen patient enrollment would be limited by the invasiveness andrisk of this procedure. Perhaps as important, PET studies with [11C]Martinostat may be sufficiently powered with a smaller sample sizewhen SUV60-90 min is chosen as the outcome measurement instead ofVT. However, validation studies will be required to evaluate whether

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SUVs are appropriate surrogates for VT values in different patientpopulations.

To begin to connect HDAC imaging with [11C]Martinostat to generegulation in the human brain, we compared mRNA transcript levelchanges elicited by pharmacologically relevant doses of Martinostat inhuman stem cell–derived neural progenitor cells. The concentrationsof Martinostat used to treat neural progenitor cells were ~1000-foldhigher than tracer-level doses used for in vivo [11C]Martinostat imag-ing. Tracer-level doses are intended to achieve low occupancy and thusshould not perturb HDAC enzyme activity and downstream gene ex-pression. However, by using pharmacologically relevant doses for neuralprogenitor cell studies, the downstream targets of Martinostat-boundHDACs were revealed and provide insight into imaging signal in-terpretations. For example, these data suggest that in regions where[11C]Martinostat binding in the human brain is lowest, such as thehippocampus, the levels of HDAC-regulated genes, such as BDNF, areelevated. The hippocampus was previously shown to be consistentlyenriched in BDNF (17, 28–31).

Besides genes implicated in memory and neuroplasticity, Martinostatenhanced the mRNA expression of GRN encoding the glycoproteinprogranulin. GRN mutations are a major cause of autosomal domi-nant frontotemporal lobar degeneration (14). The demonstration herethat Martinostat treatment increases GRN mRNA levels supports thevalue of HDAC-targeted therapies as a disease-modifying treatmentfor this type of dementia. Moreover, because HDAC inhibitors arethe subject of current clinical investigation for frontotemporal lobardegeneration, measuring HDAC expression in the human brain with[11C]Martinostat imaging may provide a critically needed tool fordetermining optimal doses of therapeutics and for patient stratificationshould levels of HDACs change in the disease state.

We recognize several limitations in our current study. First, the im-aging data presented here are from a cohort of eight healthy subjects andthus we cannot characterize changes in “normal”HDAC expression (forexample, as a function of age). Future studies will expand our imag-ing cohort to include more healthy subjects as well as multiple HDACdysfunction-associated patient populations, including AD and schizo-phrenia, to investigate the in vivo relevance of HDAC expression inneurological and psychiatric diseases. Another limitation is that quan-titative HDAC levels in postmortem brain tissue are relative to recombi-nant HDAC standards and do not reflect the absolute values of HDACexpression in the living brain, as postmortem HDAC levels may beaffected by artifacts such as postmortem interval. Additionally, owingto the low throughput of thermal shift assays with Western blot–baseddetection and limited tissue availability, we found it necessary to poolmultiple postmortem brain samples into three biological replicates,rather than analyze individual thermal shift assays for each donor,which may have revealed a higher variability of Martinostat selectivity.Last, neural progenitor cell studies uncovered only a subset of down-stream Martinostat-bound HDAC substrates and gene targets. Futurestudies using acetyl proteomic profiling, RNA sequencing, and chemo-proteomics are needed to fully understand the biological pathwaysdetected by [11C]Martinostat.

In conclusion, this first-in-human epigenetic imaging study revealsthat HDACs are highly expressed throughout the healthy brain with aconserved regional distribution between individuals. Our study un-covers region-specific variations in HDAC inhibitor binding, whichwe postulate is due to differences between the HDAC complex iden-tities in those regions. Together, our neuroimaging and biochemical

Fig. 5. Martinostat increases histone acetylation and gene expressionlevels in human neural progenitor cells. Human neural progenitor cells

were treated with DMSO (Veh), Martinostat (MSTAT; 0.5, 2.5, or 5.0 mM),and SAHA (10 mM) for 24 hours. (A) Whole-cell lysates were prepared (n =3). #Because treatment with 5.0 mM Martinostat was toxic to cells, whole-cell lysates from three replicates were combined into one pool to obtainsufficient protein for this dose. Equivalent amounts of total protein werecompared through Western blotting. Histone acetylation immunoreactiveband intensity values were normalized to GAPDH intensity values. Data aremeans ± SD (n = 3). P values compare drug treatments to Veh, de-termined by repeated-measures two-way ANOVA (a = 0.05 with Dun-nett’s multiple comparisons correction). (B) RNA was extracted (n = 3)and converted into complementary DNA (cDNA). mRNA transcript levels ofmemory/neuronal plasticity–related (BDNF, EGR1, CDK5, SYT1, and SYP) andmonogenic neurological disorder–related (GRN and FXN) genes were com-pared through qPCR and normalized to GAPDH mRNA levels. Data aremeans ± SEM (n = 3 cDNA per condition with three technical qPCR replicatesper cDNA). P values compare drug treatments to Veh, determined byrepeated-measures two-way ANOVA (a = 0.05 with Dunnett’s multiplecomparisons correction).

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experiments provide a critical foundation for how to quantify epigeneticactivity in the living brain and in turn accomplish HDAC inhibition inthe CNS as a therapy for human brain disorders.

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MATERIALS AND METHODS

Study designOur main research objective was to quantify in vivo regional HDACexpression in the healthy human brain, using [11C]Martinostat PET.As a first-in-human PET imaging study, a cohort of eight individualswas included to evaluate intrasubject and intersubject variability of[11C]Martinostat uptake. These are critical information for appropri-ate power calculations when designing future studies. No data fromthe eight subjects were excluded as outliers for [11C]Martinostat up-take values. Regional VT and SUVs were the image-based endpointsassessed. We furthered our imaging findings through ex vivo biochem-istry to ascribe the HDAC subtype selectivity of Martinostat using hu-man and NHP brain tissues. Thermal shift and HDAC expressionlevel assays included three biological replicates, with lysates pooledfrom two to three human donors per replicate. Nuclear density andautoradiographic assays included a minimum of four NHP brainslices per region from one baboon (Papio anubis). For these assays,we excised a contiguous section of baboon brain spanning a gray andwhite matter boundary to remove external variables from our analyses.The availability of an intact baboon brain is very rare; thus, we wereonly able to access one biological replicate through multiple slices.We also furthered our imaging findings through in vitro analyses ofMartinostat-dependent substrate acetylation and gene expression levels.Acetylation and mRNA profiling assays included three biological repli-cates of human neural progenitor cells. Imaging and biochemical studieswere not blinded.

ParticipantsEight participants (four females and four males; mean age ± SD,28.6 ± 7.6 years) were included in this study (eIND #123154). Partic-ipants were healthy volunteers with no history of hepatic, renal, neu-rological, or psychiatric disease and were not taking any prescriptionmedication, as evaluated by medical examinations. Participants hadnot smoked tobacco products within the past 5 years and were notusing any illicit drugs, as assessed by a urine drug test (Discover 12Panel Test Card, American Screening Corp). Additionally, a serumpregnancy test (Sure-Vue serum hCG-STAT, Fisher HealthCare) wasperformed for female participants to ensure no pregnancy at the timeof the scan. Participants provided written informed consent to take partin the study, which was approved by the Institutional Review Board andthe Radioactive Drug Research Committee at Massachusetts GeneralHospital. Volunteers were compensated for their participation in the study.

Radiosynthesis of [11C]Martinostat[11C]Martinostat was synthesized as described in SupplementaryMethods.

MR/PET imagingParticipants had no magnetic resonance imaging (MRI) or PET con-traindications to safely undergo brain imaging. An arterial line (A-line)was placed in the radial artery of one arm, and an intravenous catheterwas placed in the antecubital vein of the other arm. A licensed nuclear

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medicine technologist administered [11C]Martinostat into the intra-venous catheter as a manual bolus, and an experienced nurse practition-er drew blood samples from the A-line during the scan to determineplasma radioactivity and radioactive metabolites. Participants wereinstructed to remain still for the total duration of each scan. PET andMRI images were acquired on a 3T Siemens TIM Trio with a BrainPETinsert (Siemens). A PET-compatible circularly polarized transmit coiland an eight-channel receive array coil were used for MRI data acqui-sition. A high-resolution anatomical scan using a multi-echo MPRAGE(magnetization-prepared rapid acquisition gradient echo) sequence [rep-etition time (TR), 2530 ms; echo time 1 (TE1), 1.64 ms; TE2, 3.49 ms;TE3, 5.35 ms; TE4, 7.21 ms; inversion time (TI), 1200 ms; flip angle, 7°;and isotropic resolution, 1 mm] was acquired.

Dynamic PET image acquisition was initiated concomitant withthe start of intravenous bolus injection of ~5 mCi (4.8 ± 0.4 mCifor the eight scans) [11C]Martinostat to the subject. PET data were ac-quired for 90 min, stored in list mode format, and binned into 28 framesof progressively longer duration (10 s × 8, 20 s × 3, 30 s × 2, 60 s ×1, 120 s × 1, 180 s × 1, 300 s × 8, and 600 s × 4). The correspondingimages were reconstructed using the three-dimensional ordinary Poissonordered-subset expectation maximization (3D OP-OSEM) algorithmwith detector efficiency, decay, dead time, attenuation, and scatter cor-rections applied. The attenuation correction map was derived using aStatistical Parametric Mapping (SPM)–based, pseudo–computed to-mography method (32), which combines segmentation and atlas-basedapproaches. Simultaneously collected MR sequences consisting of anecho-planar imaging readout were used to measure subject motionduring the scan and an MR-based motion correction was applied tothe PET data (33). The final PET images were reconstructed into 153slices with 256 × 256 pixels and a 1.25-mm isotropic voxel size, in theunits of radioactivity concentrations (becquerels per milliliter) andSUVs (mean radioactivity per injected dose per weight). Three subjectscompleted a second PET scan, which was accomplished 3 hours afterthe first scan on the same day, using identical imaging methods.

Image analysesDynamic PET data were motion-corrected to a late time point image(frame 20; 39 to 44 min after radiotracer injection) of the time seriesusing rigid body linear registration (6 df) implemented in FSL [FMRIB(Oxford Centre for Functional MRI of the Brain) Software Library](MCFLIRT) (34). A PET mean image from the motion-corrected timeseries was calculated for each subject and registered and resampled to thesubject’s T1-weighted structural scan (MPRAGE) using spmregister fromFreeSurfer (http://surfer.nmr.mgh.harvard.edu) (35). The PET meanimage was further registered to the Montreal Neurological Institute(MNI) space using a linear [FLIRT (FMRIB’s linear image registrationtool)] and a nonlinear [FNIRT (FMRIB’s nonlinear image registra-tion tool)] algorithm implemented in FSL (http://fsl.fmrib.ox.ac.uk/fsl) (36). Finally, dynamic PET images (in both radioactivity concen-tration and SUV units) were normalized to the MNI space, using acombined transformation matrix derived from the PET mean image,for further analyses.

Kinetic modeling was performed using PMOD 3.4 (PMOD Tech-nologies Ltd). Twenty-eight VOIs were defined according to the Auto-mated Anatomical Labeling human brain atlas distributed with PMOD(37). A two-tissue compartmental model was applied to the regionaltime-activity curves (TACs) extracted from the VOIs and using themetabolite-corrected arterial plasma as input function to derive VT

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and microparameters describing the pharmacokinetics of the radio-tracer (table S3). The following equation was used for compartmentalmodel fitting:

dCNDðtÞdt

¼ K1CP tð Þ � k2 þ k3ð ÞCND tð Þ þ k4CS tð Þ

dCSðtÞdt

¼ k3CND tð Þ � k4CS tð Þ

where CP is the arterial input function, CND represents the nondis-placeable compartment, CS represents the specific binding compart-ment, and CND + CS is the radioactivity that we measured with PET.

The minimum scan duration required for stable VT value estima-tion was also evaluated (fig. S2). An averaged SUV image (SUV60-90 min)was calculated from 60 to 90 min after radiotracer injection. Regionalcortical VT and SUV60-90 min values were combined for cortical lobesusing a weighted average to reduce the total number of VOIs (resultingin a total of 14 VOIs). Voxel-wise, group mean, and standard deviationmaps of the SUV60-90 min were calculated and overlaid on an MNI152template brain after spatial smoothing with a 6-mm full width at half max-imum Gaussian filter (Fig. 2A). In addition, SUV60-90 min values were nor-malized to individual subjects’ white matter SUV60-90 min (SUVR60-90 min)to evaluate intersubject variability for different VOIs (Fig. 2B).

Human tissue samplesPostmortem frozen human brain tissue was obtained from the Na-tional Institutes of Health (NIH) NeuroBioBank; specifically, tissuewas obtained from the Harvard Brain Tissue Resource Center, Univer-sity of Miami Brain Endowment Bank, Human Brain and Spinal FluidResource Center, and Brain Tissue Donation Program at the Universityof Pittsburgh Medical Center. For all donors, informed consent was ob-tained from next of kin. Donor brains had a neuropathology diagnosisof normal (table S5). Tissue lysates were prepared as described in Sup-plementary Methods.

HDAC expression levelsKnown concentrations of recombinant HDAC enzymes (Reaction Bi-ology Corp KDA-21-365, KDA-21-277, and KDA-21-213; Abcamab82071) were diluted in twofold increments and compared to 12 mgof total protein from human brain lysates (n = 3 replicate poolsper region) through Western blotting. Notably, recombinant HDAC2contained a GST tag, which increased its detected size. Immuno-reactive band intensity was quantified with ImageJ (Image Proces-sing and Analysis in Java, NIH) (38). Standard curves were calculatedfor each recombinant HDAC isoform with GraphPad Prism software,and the concentrations of HDACs per lane of lysate were determined.

Histone acetylation changes in human neural progenitorcells with Martinostat and SAHAHuman induced pluripotent stem cell–derived neural progenitor cellsfrom a healthy control subject fibroblast cell line GM08330 (Coriell In-stitute for Medical Research) were generated as described in (39) andcultured as described in (40) and Supplementary Methods. Cell pelletsfrom human neural progenitor cells (n = 3 per condition) were lysedin radioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts#BP-115) with EDTA-free protease inhibitors (Sigma #4693159001)

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and rocked at 4°C for 30 min. The lysate was centrifuged at 14,000 rpmat 4°C for 25 min, and the supernatant was collected. Protein quanti-fication was determined by a bicinchoninic acid assay (Thermo Scien-tific #23227). Lysates were diluted to 800 ng/ml in RIPA buffer andstored at −80°C until ready for use. H3K9 and H4K12 acetylation levelswere measured byWestern blotting. Mean immunoreactive band inten-sities from each replicate were quantified with ImageJ. Gene expressionchanges were determined as described in Supplementary Methods.

Statistical analysesStatistical tests were performed using GraphPad Prism (Prism6, GraphPadSoftware Inc.). For PET imaging analyses, a nonparametric Friedmantest (a = 0.05 with Dunn’s multiple comparisons correction) was carriedout to compare SUV60-90 min between brain regions (Fig. 2 and tableS4). A Pearson correlation analysis was performed between VT andSUV60-90 min values for the 14 VOIs (Fig. 2B) to evaluate whether animage-based outcome measurement (SUV60-90 min) is an appropriatesurrogate to that estimated with the full kinetic modeling data (VT). Dif-ferences in postmortem HDAC expression levels as well as differencesin nuclear density, size, and total area between the SFG and the CCwere evaluated with an unpaired t test (Fig. 3B and fig. S5). Differencesin postmortem HDAC expression levels between the dorsolateral pre-frontal cortex, hippocampus, and anterior cingulate were evaluated withan ordinary one-way ANOVA (a = 0.05 with Tukey’s multiple compar-isons correction) (Fig. 3C). Differences in histone acetylation andgene expression levels as compared to vehicle were evaluated with arepeated-measures two-way ANOVA (a = 0.05 with Dunnett’s mul-tiple comparisons correction) (Fig. 5). In autoradiographic assays, dif-ferences between [11C]Martinostat baseline and blocking intensityvalues, in gray matter and white matter, were evaluated with an ordi-nary two-way ANOVA (a = 0.05 with Sidak’s multiple comparisonscorrection) (Fig. 4C).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/351/351ra106/DC1MethodsFig. S1. TACs and compartmental model fitting (two-tissue compartmental model) results forsuperior 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.

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Acknowledgments: We are grateful to U. Mahmood, S. Stufflebeam, and O. Johnson-Akeju forconsenting participants, and to E. Pierce and O. Johnson-Akeju for placing the arterial line in par-ticipants. We thank J. Sore, G. Gautam, K. Phan, and S. To for technical assistance in radiotracersynthesis and G. Arabasz, S. Hsu, M. Wentworth, and R. Butterfield for assistance with MR/PETimaging. We also thank G. Van de Bittner and M. Riley for assistance with autoradiographic

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experiments and L. Rogers for technical assistance with HDAC density experiments. Postmortemtissue was obtained from the NIH NeuroBioBank (requests #100 and #250). Funding: This re-search received funding from the National Institute on Drug Abuse of the NIH under grantnumbers R01DA030321 (to J.M.H.) and K99DA037928 (to H-Y.W.). This research was alsosupported by the Harvard/MGH Nuclear Medicine Training Program from the Department ofEnergy under grants DE-SC0008430 (to H-Y.W., T.M.G., and C.W.) and HHSN-271-2013-00030C(to the Harvard Brain Tissue Resource Center). This research was carried out at the AthinoulaA. Martinos Center for Biomedical Imaging at Massachusetts General Hospital, using resourcesprovided by the Center for Functional Neuroimaging Technologies, P41EB015896, a P41 Bio-technology Resource Grant supported by the National Institute of Biomedical Imaging and Bio-engineering, NIH. This work was conducted with support from Harvard Catalyst and the HarvardClinical and Translational Science Center (National Center for Research Resources and the Na-tional Center for Advancing Translational Sciences, NIH Award UL1 TR001102) and financial con-tributions from Harvard University and its affiliated academic healthcare centers. Additionalsupport was provided by the Bluefield Project to Cure Frontotemporal Dementia. This work alsoinvolved the use of instrumentation supported by the NIH Shared Instrumentation GrantProgram, specifically grants S10RR017208, S10RR026666, S10RR022976, S10RR019933, andS10RR023401. Author contributions: H-Y.W., T.M.G., S.J.H., C.W., and J.M.H. designed the study.H-Y.W., N.R.Z., and A.B. collected in vivo human imaging data. H-Y.W. and N.R.Z. analyzed in vivohuman imaging data. T.M.G., B.D.T., and F.A.S. collected ex vivo human biochemical data. T.M.G.collected ex vivo NHP biochemical data. T.M.G. and J.M.H. analyzed ex vivo human and NHP

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biochemical data. A.S. collected in vitro human NPC data. A.S., S.J.H., and T.M.G. analyzed in vitrohuman NPC data. H-Y.W., T.M.G., N.R.Z., and A.S. performed statistical analyses. H-Y.W., T.M.G.,N.R.Z., A.S., A.B., F.A.S., C.W., S.J.H., and J.M.H. wrote and edited the manuscript. Competinginterests: The content is solely the responsibility of the authors and does not necessarily re-present the official views of Harvard Catalyst, Harvard University, and its affiliated academichealthcare centers, or the NIH. Intellectual property (IP) has been filed around [11C]Martinostatby J.M.H., C.W., and F.A.S. A portion of this IP has been licensed. S.J.H. has financial interests inRodin Therapeutics and is an inventor on HDAC inhibitor-related IP licensed to this entity thatis unrelated to the present study. Data and materials availability: Tissues were provided bythe Harvard Brain Tissue Resource Center, University of Miami Brain Endowment Bank, HumanBrain and Spinal Fluid Resource Center, and Brain Tissue Donation Program at the University ofPittsburgh Medical Center.

Submitted 4 November 2015Accepted 7 July 2016Published 10 August 201610.1126/scitranslmed.aaf7551

Citation: H.-Y. Wey, T. M. Gilbert, N. R. Zürcher, A. She, A. Bhanot, B. D. Taillon, F. A. Schroeder,C. Wang, S. J. Haggarty, J. M. Hooker, Insights into neuroepigenetics through human histonedeacetylase PET imaging. Sci. Transl. Med. 8, 351ra106 (2016).

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Insights into neuroepigenetics through human histone deacetylase PET imaging

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

DOI: 10.1126/scitranslmed.aaf7551, 351ra106351ra106.8Sci Transl Med

supporting its use in monitoring and understanding brain pathologies like Alzheimer's disease.subset HDACs that regulate downstream genes important for neuroplasticity, memory, and neurodegeneration,

derived neural progenitor cells, Martinostat engaged the−ightly regulated epigenetic processes. In human stem cellhumans. The authors saw surprisingly conserved regions of HDAC expression in the healthy brain, suggesting tbrain. Martinostat was previously tested in rodents and nonhuman primates, and here, it is used for the first time in

developed and applied an HDAC imaging probe, called Martinostat, to visualize HDAC expression in the living . thereforeet albut their dynamic contribution to human disease development over time is unknown. Wey

regulates gene transcription. In neurological disorders, HDACs change expression in regions throughout the brain, Certain enzymes called histone deacetylases, or HDACs, are part of the epigenetic machinery that

Brain epigenetics revealed

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