Components of the endocannabinoid and dopamine systems are dysregulated in Huntington's disease: analysis of publicly available microarray datasets.

The ECS within the CNS

In the CNS, the OCGEs for CNR1,GPR55,NAPEPLD,PTGS2, and GDE1 were less than 0 (P < 0.05, Fig.​Fig.1A).1A). The OCGE for CNR2 was greater than 0 (P < 0.05, Fig.​Fig.1A).1A). The OCGEs for GPR18, GRP119, TRPV1, PPARA, DAGL, ABDH4, ABDH6, ABDH12, FAAH, MGLL, and NAAA were not different from 0 (Fig.​(Fig.1A).1A). CNS microarray data were further divided according to the stage of the disease into “pre-symptomatic,” “early,” “middle,” and “late” stage according to the description of motor symptom severity for the HD model used provided in the manuscript corresponding to each dataset (Fig.​(Fig.1C).1C). In vitro models were categorized as “early.” The OCGE was less than 0 during the presymptomatic (P < 0.05), early (P < 0.01), middle (P < 0.001), and late (P < 0.001) stages for CNR1; presymptomatic (P < 0.05), middle (P < 0.001), and late (P < 0.001) stages for GDE1; early, middle, and late stages for PTGS2 (P < 0.001); presymptomatic and late stages for GPR55 (P < 0.001), PPARA (P < 0.05), and NAPEPLD(P < 0.001); and middle stage for GPR18 (P < 0.05) (Fig.​(Fig.1C).1C). Further, the OCGE was greater than 0 during the middle (P < 0.05) and late (P < 0.01) stages for CNR2 and FAAH (Fig.​(Fig.1C1C).

To identify changes that occur in mouse and in vitro models of HD that recapitulate changes observed in human HD patients, we also conducted a comparison of OCGEs among human datasets, mouse models of HD, and in vitro models of HD for the ECS (Fig.​(Fig.1E).1E). OCGEs were consistently less than 0 for CNR1 and PTGS2 between human HD tissue, mouse, and in vitro models of HD (Fig.​(Fig.1E).1E). OCGEs were also less than 0 for PPARA, NAPEPLD, and GDE1between human HD tissue and mouse models of HD (Fig.​(Fig.1E).1E). OCGE values were greater than 0 for CNR2 and less than 0 for PTPN22 in human studies, but not mouse in vitro models of HD (Fig.​(Fig.1E).1E). Similarly, OCGE values were only greater than 0 for FAAH in mouse models of HD (Fig.​(Fig.1E).1E). Of all the changes in gene expression that have been reported, only change in CNR1and PTGS2 consistently occurred in mouse and in vitro models of HD, but not others. Notably, the increase observed for CNR2 is not observed consistently across studies, models of HD, and in human HD tissue.

The ECS in peripheral blood samples

Three microarrays studies reported on the expression of ECS transcripts in peripheral blood from patients with HD (Table​(Table2).2). The samples taken from individuals with HD were staged as pre-symptomatic, early-, middle-, or late-symptomatic and compared with control samples from healthy individuals. These studies sought to identify biomarkers of HD onset and progression. We found that the OCGE for the pooled data was less than 0 for CNR1, and greater than 0 for CNR2, GPR55, PPARA, NAPEPLD, PTGS2, GDE1, and PTPN22 (P < 0.05, Fig.​Fig.1B).1B). The OCGEs were not different from 0 for GPR18, GPR119, TRPV1, DAGL, ABDH4, ABDH6, ABDH12, FAAH, MGLL, and NAAA (Fig.​(Fig.1B).1B). Changes in the levels of CNR1 and CNR2 have been extensively documented in the literature, whereas the changes in GPR55, PPARA, NAPEPLD, PTGS2, GDE1, and PTPN22 have not been recognized as consistent in peripheral blood samples from HD patients. Further, when these data were compared with the overall data for the ECS in the CNS (Fig.​(Fig.1A),1A), GPR55, NAPEPLD, PTGS2, GDE1, and PTPN22 OCGEs are all decreased in the CNS and increased in peripheral blood samples (Fig.​(Fig.1B).1B). Next, data were subdivided by disease stage. The OCGE was greater than 0 during the presymptomatic and late stages for CNR2 (P < 0.001 and P < 0.05), PTGS2 (P < 0.001), GDE1 (P < 0.001); and the presymptomatic stage for GPR55 (P < 0.001), PPARA (P < 0.01), NAPEPLD (P < 0.001), and PTPN22 (P < 0.001) (Fig.​(Fig.1D).1D). The OCGE was greater than 0 during the early stage for FAAH(P < 0.001) and during the late stage for CNR1 and NAAA (P < 0.05) (Fig.​(Fig.1D).1D). By dividing the data according to disease stage, we observed that all changes in gene expression in the peripheral ECS were stage specific. Moreover, the OCGE values for FAAH and NAAA were not different from 0 in the overall analysis, but were different at specific stages of HD. Therefore, several components of the ECS could be utilized as disease stage-specific biomarkers in HD.

The DAS within the CNS

We calculated the OCGE for each gene examined in the DAS in the CNS the six datasets that met our inclusion criteria (Table​(Table2).2). We found that, in the CNS, the OCGEs for DRD2,TH,COMT,VMAT2,DAT, and PPP1R1B were less than 0 (P < 0.05, Fig.​Fig.2A).2A). The OCGEs for DRD1,DRD3,DRD4,DRD5,PAH,DDC,DBH,MAOA, and MAOB were not different from 0 (Fig.​(Fig.2A).2A). CNS microarray data were further divided according to disease stage (Fig.​(Fig.2C).2C). In vitro models were categorized as “early.” Only the OCGE for PPP1R1B was less than 0 during the early stage (P < 0.05) (Fig.​(Fig.2C).2C). The OCGE was less than 0 during the middle and late stages for DRD2 (P < 0.001 and P < 0.01), VMAT2 (P < 0.01 and P < 0.05), and DAT (P < 0.01), middle stage for DRD3 (P < 0.05), and late stage for TH (P < 0.01), DDC (P < 0.01), DBH (P < 0.05), and COMT (P < 0.01) (Fig.​(Fig.2C).2C). Further, the OCGE was greater than 0 during the middle stage for MAOA (P < 0.01) and the late stage for MAOB (P < 0.05) (Fig.​(Fig.2C).2C). We could not complete a comparison of models of HD for the DAS due to the limited number of studies that reported changes in the DAS. Importantly, we observed a disease stage-specific change in the OCGE for DRD3,DDC, DBH, MAOA, and MAOB where no difference had been observed in the overall analysis.

The ECS in HD

Of all the changes that are reported in the various microarray studies, only changes in the expression of CNR1,CNR2, and FAAH have been consistently reported and investigated in previous studies (Luthi-Carter et al. 2000; Laprairie et al. 2013, 2014). Changes in the expression of CNR1 have been reported in several HD mouse models using microarray in other studies that were not available in the GEO Profiles datasets (Luthi-Carter et al. 2000; Chan et al. 2002; Kuhn et al. 2007; Benn et al. 2010). Similarly, dysregulation of CNR1 expression has been observed using qRT-PCR and in situ hybridization (Denovan-Wright and Robertson 2000; Glass et al. 2000). Increased levels of CNR2 and FAAH mRNA have been documented via qRT-PCR and western blot in R6/2 mouse models of HD, STHdhQ111/Q111 cells, and tissue from HD patients (Blázquez et al. 2011; Bari et al. 2013; Laprairie et al. 2014). We observed increased FAAH levels in the late stages of HD, which may indicate increased AEA catabolism (Laprairie et al. 2014). Dysregulation of ECS tone may follow changes in the expression FAAH or other anabolic and catabolic cannabinoid enzymes (Bari et al. 2013).

The DAS in HD

Alterations in gene expression of components of the DAS in the basal ganglia have been linked to the pathology of HD in both postmortem human and animal models (El Khoury et al. 2012). Changes in the expression of DRD2, TH, DAT, VMAT2, and PPP1R1B (DARPP-32) have been consistently reported in previous studies (Bibb et al. 2000; Glass et al. 2000; Suzuki et al. 2001; Yohrling et al. 2003). Reduction in the expression of DRD2 is well documented in pre- and symptomatic human patients and R6/2 mice models of HD using in situ hybridization, qRT-PCR, autoradiographic, and positron emission tomography (Joyce et al. 1988; Richfield et al. 1991; Antonini et al. 1996; Augood et al., 1997; Glass et al. 2000; Bibb et al. 2000). Other microarray studies that did not provide publicly available data have also reported lower striatal DRD2 levels in several HD mouse models including: R6/2, R6/1, N171-82Q, YAC72, and HdhQ92/Q92 mice (Luthi-Carter et al. 2000; Chan et al. 2002; Kuhn et al. 2007; Benn et al. 2010). Postmortem studies of late-stage HD patients, R6/2, and YAC128 mice models of HD showed reduced levels of caudate DA and homovanillic acid, the principal DA metabolite (Bernheimer et al. 1973; Reynolds and Garrett 1986; Kish et al. 1987; Reynolds et al. 1999; Callahan and Abercrombie 2011). Changes in the expression of anabolic enzymes in the CNS that are involved in DA synthesis might play a role in the reduction of DA. The levels of TH, the enzyme that catalyzes the rate-limiting step in the biosynthesis of DA, have been demonstrated previously to decline during late stages of HD progression in HD patients and in R6/2 mice (Yohrling et al. 2003). The majority of synaptic DA is cleared by DAT and VMAT2. Similarly to the microarray studies, previous analysis of postmortem brains from late-stage HD patients has shown reduced striatal expression of DAT and VMAT2 using autoradiography (Suzuki et al. 2001). Reduction in DARPP-32 expression was previously observed in postmortem human HD and R6/2 mice using immunohistochemistry (Bibb et al. 2000; van Dellen et al. 2000).

Comparing changes in the ECS and DAS between mouse models of HD

The data presented here indicate that no single gene in the ECS was consistently up- or downregulated in all mouse models included in these analyses (Table S1). The most consistently observed change among ECS genes was downregulation of CNR1 in 81% of studies utilizing mouse models of HD. GPR18, GPR55, and PTGS2 levels were lower in 28% of studies, PPARAlevels were lower in 21% of studies, NAPEPLD and GDE1 levels were lower in 12% of studies, and FAAH levels were higher in 12% of studies (Table S1). Among genes analyzed in the DAS, DRD2, DRD3, DAT, and PPP1R1B were consistently downregulated in HD samples (Table S2). However, the limited size of this dataset means that these observations are difficult to interpret. Interstudy variability also existed between mouse models of HD. For example, FAAH was reported as upregulated in some studies of R6/1 mice (Benn et al. 2005; Desplats et al. 2006), but not others (Hodges et al. 2008). Similarly, PTGS2 and GDE1 were only downregulated in R6/1 mice and no other model examined. Some of the models used in these datasets may not have been old enough to display HD-like transcriptional dysregulation, as previous reports have suggested knock-in models of HD do not display transcriptional dysregulation until later in disease presentation (Kuhn et al. 2007). Similarly, the novel zQ175 knock-in mouse model of HD displays a subset of the transcriptional abnormalities observed in R6/1 and R6/2 mice, including reduced CNR1 expression, during that time of motor symptom onset rather than prior to motor symptom onset (Menalled et al. 2012). Although no microarray analyses have been done with zQ175 mice, this mouse model exhibits metabolic, behavioral, and motor deficits over a longer time course than the rapidly degenerating R6 lines, suggesting this model may be most useful for the study of long-term behaviors and phenotypes (Menalled et al. 2012). These data demonstrate that variability in the pattern of transcriptional dysregulation exists between different mouse models of HD. While certain changes, such as decreased levels of CNR1, are relatively robust, other changes may be model specific and more readily detected in aggressive models of HD, such as R6/1 and R6/2 mice.

Comparing data from mouse models of HD to human tissue studies

One major use of these data is determining the predictive power of animal models of HD in the rational development of therapies for patients suffering from HD. Our analysis revealed that, in general, changes in CNR1,PPARA, NAPEPLD, PTGS2, and GDE1 were consistent between mouse and human studies. This comparative analysis is not possible for the DAS due to the limited size of the dataset. When individual mouse models are compared to the observations made in human tissue studies, all models examined (Table​(Table2)2) reported the well-characterized downregulation of CNR1, but only the R6/1 and R6/2 mouse models, with some exceptions, displayed similar patterns of gene expression to those observed in human datasets (Table S1). The strong correlation between the genome-wide pattern of transcriptional dysregulation in R6 mouse models of HD and human tissue samples has been noted elsewhere (Kuhn et al. 2007). The absence of correlation in our data between mouse models of HD and human HD tissue samples may be the result of the small number of genes selectively analyzed (Kuhn et al. 2007). Here, we demonstrate that the pattern of gene expression for the ECS is largely similar between R6 mouse models and samples from human HD tissue and largely dissimilar between other mouse models of HD compared to human HD tissue. It is important to note that while no single mouse model of HD can be considered the most valid tool for assessing disease progression or the efficacy of novel therapeutics. The animal model must be carefully chosen as the most suitable tool to answer the question being posed.

Changes in the ECS and DAS that occur in peripheral blood may be useful as biomarkers in HD

HD is often described as a neurodegenerative disease rather than a disease that affects the whole body. The data presented here demonstrate that changes in gene expression occur in the peripheral blood in a disease stage-specific manner to an equal or greater extent than in the CNS. It is important to note that while changes in gene expression in peripheral blood may not occur in the CNS, but can still be followed as biomarkers of disease progression. Although the pathological implications of these changes are unknown, novel stage-specific biomarkers for HD could improve the selection and efficacy of therapies for the treatment of HD (Runne et al. 2007).

We acknowledge the funding support of the Canadian Institutes for Health Research, the Nova Scotia Health Research foundation, the Huntington disease research foundation, King AbdulAziz University and Dalhousie University. Thanks to Kathleen Murphy for assistance in preparing this manuscript.