Synaptic vesicle glycoprotein 2A is affected in the CNS of Huntington’s disease mice and post-mortem human HD brain

Synaptic dysfunction is a primary mechanism underlying Huntington’s Disease (HD) progression. This study investigated changes in synaptic vesicle glycoprotein 2A (SV2A) density by means of 11 C-UCB-J microPET imaging in the central nervous system (CNS) of HD mice. METHODS: Dynamic 11 C-UCB-J microPET imaging was performed at clinically relevant disease stages (at 3, 7, 10, and 16 months, M) in the heterozygous knock-in Q175DN mouse model of HD and WT littermates ( n =16-18/genotype and time point). Cerebral 11 C-UCB-J analyses were performed to assess genotypic differences during pre-symptomatic (3M) and symptomatic (7-16M) disease stages. 11 C-UCB-J binding in the spinal cord was quantified at 16M. 3 H-UCB-J autoradiography and SV2A immunofluorescence were performed post-mortem in mouse and human brain tissue. RESULTS: 11 C-UCB-J binding was declined in symptomatic heterozygous mice compared to WT littermates in parallel with disease progression (7M: p <0.01, 16M: p <0.0001). Specific 11 C-UCB-J binding was detectable in the spinal cord, with symptomatic heterozygous mice displaying a significant reduction ( p <0.0001). 3 H-UCB-J autoradiography and SV2A immunofluorescence corroborated the in vivo measurements demonstrating lowered SV2A in heterozygous mice ( p <0.05). Finally, preliminary analysis of SV2A in post-mortem human brain suggested lower SV2A in HD gene carrier compared to nondemented control. for SV2A measurement in patients with HD during disease progression and following disease-modifying therapeutic strategies. C-UCB-J PET may provide unique insights in elucidating C-UCB-J marker for the assessment of synaptic integrity in patients with HD during disease progression following disease-modifying therapeutic strategies.


INTRODUCTION
Huntington's Disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in exon 1 of the gene encoding the huntingtin protein (1), which leads to the expression of mutated huntingtin (mHTT).
A growing body of evidence suggests that mHTT induces synaptic transmission dysfunction (4), thus synaptic dysfunction represents one of the main mechanisms underlying the progression of HD (5). Alterations in pre-synaptic proteins, including regulators of endocytosis and exocytosis of synaptic vesicles such as synaptosomeassociated protein 25 and rabphilin 3A have been reported in both clinical (6,7) and preclinical (8)(9)(10) post-mortem studies. Previous studies have demonstrated mHTT abnormally associates with synaptic vesicles resulting in impaired synaptic function (11) and changes in synaptic proteins correlate with behavioural deficits (10), Thus, alterations in synaptic proteins may represent a candidate marker to monitor HD progression (12)(13)(14).
Given the current lack of effective treatment to prevent the disease or halt its progression, synaptic markers may play an important role in the development and evaluation of novel disease-modifying therapies throughout the entire central nervous system (CNS) (15).
Among pre-synaptic proteins, the synaptic vesicle glycoprotein 2A (SV2A) is an essential vesicle membrane protein involved in neurotransmitter release and is expressed ubiquitously in synapses of the brain (16,17). Recent studies have reported that SV2A can be imaged non-invasively in non-human primates, humans, and rodents utilizing positron emission tomography (PET) with the selective and high-affinity radioligand 11 C-UCB-J (18)(19)(20). 11 C-UCB-J PET may offer a proxy to assess synaptic density in vivo given its optimal clinical and preclinical pharmacokinetics and quantification properties (20,21). Thus, it provides a quantitative measure of the synaptic changes during HD progression.
Here, for the first time, we investigated 11 C-UCB-J PET imaging to quantify cerebral SV2A levels at clinically relevant disease stages in the knock-in Q175DN mouse model for HD (22)(23)(24). Additionally, given the evidence of mHTT pathology in the spinal cord (25), we evaluated the use of 11 C-UCB-J PET imaging to detect SV2A density changes in the rodent cervical spinal cord. Finally, post-mortem measurements of SV2A were performed in the mouse as well as in a preliminary exploratory evaluation in the human brain.

C-UCB-J Dynamic MicroPET Scan
MicroPET/Computed tomography (CT) imaging was performed on two Siemens Inveon PET/CT scanners (Siemens Preclinical Solution, USA). Animal preparation was performed as previously described (20). At the start of the dynamic microPET scan, animals were injected via the tail vein with a bolus of 11 C-UCB-J (5.4 ± 1.3 MBq) over a 12-second interval (1 ml/min) using an automated pump (Pump 11 Elite, Harvard Apparatus, USA). The activity was injected in a trace dose keeping the cold mass within 2.0 μg/kg across time points for consistency. Data were acquired in list-mode format.
Following the microPET scan, a 10 min 80 kV/500 μA CT scan was performed for coregistration and attenuation correction. Detailed information on the scan parameters is reported in Supplemental Table 1. Published work from our group (20) was re-analyzed for blocking validation of 11 C-UCB-J binding in the spinal cord. Blocking was achieved by pre-treatment with levetiracetam at either 50 (n = 4) or 200 (n = 4) mg/kg, injected intraperitoneally 30 min before radioligand delivery. Representative SUV images were generated based on the interval 10-90 min.

Image Processing and Analysis
Acquired PET data were histogrammed and reconstructed into 33 frames of increasing length (12x10s, 3x20s, 3x30s, 3x60s, 3x150s, and 9x300s). For quantitative analysis, all images were reconstructed using a list-mode iterative reconstruction with spatially variant resolution modeling with 8 iterations and 16 subsets of the 3D ordered subset expectation maximization (OSEM-3D) algorithm (28). Normalization, dead time, and CT-based attenuation corrections were applied. PET image frames were reconstructed on a 128x128x159 grid with 0.776x0.776x0.796 mm 3 voxels. PET images were processed and analyzed using PMOD 3.6 software (Pmod Technologies, Zurich, Switzerland).
Spatial normalization of the PET images was done through brain normalization of the PET images to an 11 C-UCB-J PET template as we previously described (20). Using the volume-of-interest template based on the Waxholm atlas (29), time-activity curves of different regions (striatum, motor cortex, hippocampus, and thalamus) were extracted from the images. Cervical spinal cord volume-of-interest was manually delineated on the individual CT images (blinded to condition) and time-activity curves were extracted. Kinetic modeling was performed to fit the time-activity curves by a standard one-tissue compartmental model (1TCM) to determine the total volume of distribution using a noninvasive image-derived input function (IDIF) to calculate V T (IDIF) as a surrogate of V T estimate as we recently validated (20). No genotypic difference in plasma-to-whole blood ratio or plasma radiometabolites was present between genotypes, therefore no correction was applied (20).
Parametric V T (IDIF), as well as K 1 maps, were generated in PMOD through voxelwise analysis (1TCM) (20). Brain parametric maps are represented as averages for each genotype overlaid on a 3D mouse brain MR template for anatomical reference, while maps focusing on the spinal cord are represented as individual animal overlaid on CT.
Regional quantification was performed blind to genotype using Fiji software (National Institute of Health, USA). 3 H-UCB-J binding was measured in triplicate (3 slices) manually drawn the regions. Regional specific binding of 3 H-UCB-J was measured by converting the mean grey values into radioactivity density (Bq/mg) calculated using commercial tritium standards (American Radiolabeled Chemicals). Next, using 3 H-UCB-J molar activity on the experimental day, radioactivity density was converted into binding density (fmol/mg) for each region. Images at 20X and 100X magnification were acquired for quantification with a high throughput fluorescence microscope (Nikon, Japan) with NIS elements software.
Quantification was performed blind to genotype using Fiji software. Since the white matter was devoid of a specific signal, after conversion into 8-bit grayscale, an intensity threshold was set to remove the background signal in the white matter (threshold 27 out of 255) and convert images into binary data. Regions-of-interest (striatum, motor cortex, hippocampus, and thalamus for mouse; a cortical grey matter for human tissue) were manually drawn on each image, and the percentage of surface area after thresholding was measured as the positive area. Quantification was done in triplicate (3 slices) for each region and the average was used for statistical analysis.

Statistical Analysis
All data were normally distributed as assessed using the Shapiro-Wilk test.
Longitudinal PET data were analyzed with a linear mixed-model by fitting each region separately using 11 C-UCB-J V T (IDIF) or K 1 (IDIF) as dependent variables, while genotype (WT and heterozygous), time (7, 10, and 16M), and the interaction between genotype and time (genotype*time) as fixed effects, with subjects as a random effect. The comparison was performed to evaluate regional temporal and genotypic differences. Two-way ANOVA (genotype and region as variables) was applied to investigate the 3M data and postmortem analyses. One-way ANOVA was used for blocking analysis in the spinal cord, while an unpaired T-test was used to compare the genotypic difference in spinal cord SV2A PET. Pearson's correlation test was used to determine the relationship between variables. Normality and two-way ANOVA tests were performed with GraphPad Prism (v 9.0) statistical software, linear mixed-model in JMP Pro 13 (SAS), and calculation of the effect size d with G*Power software (http://www.gpower.hhu.de/). P values were corrected for multiple comparisons using the Tukey's test. Data are represented as mean ± standard deviation (SD). All tests were two-tailed and statistical significance was set at p<0.05.

SV2A Density Decreases with HD Progression
Longitudinal mean V T (IDIF) parametric maps of 11 C-UCB-J at 7, 10, and 16M, displayed a broad cerebral reduction of 11 C-UCB-J binding in symptomatic heterozygous mice compared to WT littermates ( Figure 1A). Accordingly, 11 Table 2). Notably, the reduced 11 C-UCB-J uptake was not related to altered K 1 values (delivery rate of the tracer; Supplemental  Table 3.

SV2A Levels Are Reduced in the Spinal Cord of Symptomatic Heterozygous Mice
We explored the potential application of 11 C-UCB-J PET to detect SV2A in the mouse spinal cord. 11 C-UCB-J binding quantifiable and specific as validated following pretreatment with levetiracetam (F (2,10) =78.96, p<0.0001) (Figure 3).
Next, based on clinical evidence indicating the presence of mHTT pathology in the spinal cord, we quantified 11 C-UCB-J PET in the mouse cervical spinal cord of symptomatic heterozygous mice (16M). As shown in Figure 4, 11 C-UCB-J binding was significantly reduced in the cervical spinal cord of heterozygous mice compared to WT littermates (-22.5±3.8%, p<0.0001) ( Figure 4B) with a strong association to the cortical quantification (r 2 =0.90, p<0.0001) ( Figure 4C).

DISCUSSION
This work assessed synaptic integrity using the PET radioligand 11 C-UCB-J in heterozygous mice at clinically relevant pre-and symptomatic stages of the disease. This work represents the first evidence of in vivo changes in SV2A density. In particular, changes in synaptic density were detectable at all symptomatic stages of HD with mHTT accumulation broadly affecting SV2A levels in the entire CNS.
Despite the mounting evidence indicating mHTT induces pre-synaptic transmission dysfunction during the progression of HD (4,5), to date no clinical or preclinical studies have assessed alterations in presynaptic proteins in vivo. Nonetheless, cross-sectional findings in animal models of HD suggested a reduction in different synaptic proteins in different animal models at symptomatic but not pre-symptomatic stages of the disease (8-10), in agreement with our observation in vivo in heterozygous mice as well as in vitro in both HD mice and post-mortem human tissue.
Since the development of SV2A radioligands for in vivo imaging of SV2A (18,19), preclinical and clinical investigations have been restricted on the brain despite SV2A is distributed in all grey matter, including the spinal cord (33). Thus, we evaluated the specificity of the 11 C-UCB-J signal in the cortical spinal cord using our previous levetiracetam blocking study (20), demonstrating, for the first time, the capability of SV2A PET imaging in the spinal cord of a living animal. Interestingly, Lanberg and colleagues reported a 2-to-3-fold difference in SV2A expression in the spinal cord compared to the cerebral cortex in the rat (33). In this work, we measured a 2.5-fold difference in 11 C-UCB-J binding between the spinal cord and motor cortex, in agreement with the previous rat report (33). Next, based on the clinical evidence of mHTT pathology in the spinal cord (25), we explored 11 C-UCB-J binding in the cervical spinal cord of symptomatic heterozygous mice and observed a decline in SV2A density with a similar magnitude to the brain. Altogether these observations support the exploration of SV2A PET imaging as synaptic integrity marker in spinal cord-related disorders, for instance, amyotrophic lateral sclerosis as well as spinal cord injury (SCI), currently being investigated in our group and supported by the recent evidence that levetiracetam treatment leads to functional recovery in SCI models (34).
In recent years, the field of PET imaging has significantly progressed in the identification of different striatal markers to monitor HD progression (35-41). However, since the whole CNS is affected in HD, non-invasive markers with ubiquitous brain distribution such as 11

CONCLUSION
Collectively, these findings demonstrate significant SV2A deficits in the brain and spinal cord of symptomatic heterozygous mice. 11 C-UCB-J PET imaging is a promising marker for the assessment of synaptic integrity in patients with HD during disease progression and following disease-modifying therapeutic strategies.

DISCLOSURE
This work was funded by CHDI Foundation, Inc., a non-profit biomedical research organization exclusively dedicated to developing therapeutics that will substantially improve the lives of HD-affected individuals. No other potential conflicts of interest relevant to this article exist.

KEY POINTS Question:
Is SV2A density affected during the progression of Huntington's Disease (HD)?

Pertinent findings:
In this 11 C-UCB-J positron emission tomography (PET) study, we demonstrated brain and spinal cord SV2A deficits during symptomatic disease in HD mice i, highlighting the potential of SV2A PET as a marker in the entire central nervous system (CNS). Implication for patient care: 11 C-UCB-J PET imaging offers a unique tool as CNS functional marker for HD, and yields promising application for SV2A measurement in patients with HD during disease progression and following therapeutic interventions.