Large-scale transcriptomic analysis reveals that pridopidine reverses aberrant gene expression and activates neuroprotective pathways in the YAC128 HD mouse

Huntington Disease (HD) is a progressive and neurological disorder caused by an autosomal dominant CAG trinucleotide expansion in the Htt gene [1], characterized by psychiatric, cognitive and motor disturbances, manifesting usually between 40 and 50 years of age and worsening until death [2]. Htt plays a role in facilitating axonal transport of brain-derived neurotrophic factor (BDNF) in the corticostriatal pathway of the motor circuit in wild-type animals (Fig. 1a and b) [3]. Consistently, in animal models of HD, mHtt disrupts several neuronal functions including corticostriatal communication [4] and cortical release of BDNF [5] (Fig. 1c). Breakdown of corticostriatal transmission reduces synaptic activity of striatal neurons [6] and influences downstream signal transduction within the striatum. In addition to the deficiencies in BDNF-TrkB signaling previously reported in mouse models of HD [7, 8], cyclic AMP (cAMP) signaling is disrupted in the striatum of presymptomatic R6/2 HD mice [9].

Pridopidine, a small molecule in development for the treatment of HD, improved motor function in HD patients in two large, double-blind, placebo-controlled studies (HART and MermaiHD) as exhibited by UHDRS–Total Motor Score (TMS), but did not meet primary endpoint of changes from baseline to week 12 in Modified Motor Score [10, 11]. Pridopidine is a high affinity sigma-1 receptor [12] ligand and exerts low-binding affinity towards additional CNS receptors, such as Dopamine D2, Adrenergic a2C, Serotonin 5HT-1A and Histamine H3 [13, 14]. Further, an in-vivo PET imaging study in rats confirmed that pridopidine occupies the sigma-1 receptor at low doses (3 and 15 mg/kg), and the D2R only at higher doses (60 mg/kg). Pridopidine normalizes endoplasmic reticulum (ER) calcium levels in YAC128 corticostriatal co-cultures [15], mediated by the sigma-1 receptor (Fig. 1d). The sigma-1 receptor also mediates pridopidine-induced BDNF in rat neuroblastoma cells [15]. In the striatum of R6/2 HD mice [16, 17], pridopidine treatment increases BDNF protein levels in the striatum (Fig. 1d). Finally, a gene expression analysis in WT rat striatum demonstrates pridopidine induces differential expression (DE) of genes enriched for the BDNF, D1R, and glucocorticoid receptor pathways, presumably mediated via sigma-1 receptor activation.

The effect of different doses of chronic pridopidine treatment, initiated at pre-symptomatic stages, on gene expression and transcript splicing in the context of HD was evaluated using single nucleotide resolution RNA sequencing in YAC128 mice, examining specificity of effects across brain regions.

It has recently been demonstrated that 30 mg/kg of pridopidine rescues motor behavioral deficits in YAC128 mouse model of HD [19]. To identify potential mechanisms by which pridopidine confers motor benefits, this study focuses on pridopidine induced changes in transcription across multiple brain regions and dose regimens. To characterize the functional relevance of transcriptomic changes, RNAseq data was analyzed for expression signaling, as well as splice variant modifications in pre-specified pathways, as well as across the genome unbiasedly. Testing was performed on WT and YAC128 striatum, cortex, and hippocampus after treatment with vehicle, 10 or 30 mg/kg pridopidine from a presymptomatic stage through disease progression. Both doses of pridopidine had a significant effect on gene and transcript levels in the striatum, with modest to unobserved effects in the cortex and hippocampus. However, the transcriptional effect of pridopidine in YAC128 striatum is dose-dependent. The two doses tested herein suggest linearity of the effect, which future studies employing additional doses will serve to shed further light on. While this study cannot directly query whether pridopidine’s behavioral benefits are transcriptionally mediated, the fact that pridopidine’s main transcriptomic effect is detected in the striatum supports this hypothesis. Moreover, the dose dependent functional expression signals induced by pridopidine track well with the dose-dependent behavioral benefits induced it induces at parallel experimental conditions (Garcia-Miralles et al., 2017). Lastly, genes with perturbed expression in YAC128 pathology are oppositely modulated by pridopidine in the striatum, far more so than expected by chance.

The transcriptional footprint of pridopidine demonstrates a reversal of the disease-specific gene expression and alternative splicing. The disease mechanisms reversed by pridopidine include critical neuroprotective pathways such as BDNF, D1R and glucocorticoid pathways previously reported. qPCR confirmed differential expression of many genes in these pathways. In addition, pridopidine induced gene expression triggered by cAMP transduction, also supported by modulation of downstream transcription factors (e.g. CREB1). Together, these findings provide robust data to demonstrate pridopidine restores mechanisms impaired in HD, specifically in the striatum.

Previous studies reported rescue of several aspects of HD, including phenotype and behavior, in the YAC128 mouse through BDNF overexpression [35]. Dexamethasone, a glucocorticoid that activates the GR pathway, also dampens disease progression in a HD animal model [36]. Both 10 and 30 mg/kg pridopidine treatment in YAC128 mice significantly induced the BDNF, GR and D1R pathways in the striatum, hippocampus, and cortex, consistent with prior reports in WT rat [30]. As pridopidine does not directly bind GR (internal data, not shown), it suggests that the upregulation of the GR pathway may be indirect. Pridopidine increases dopamine efflux in the striatum [37], which may explain the observed upregulation of expression for D1R pathway genes after pridopidine treatment.

In the WT striatum, dopamine is a central regulator of cAMP activity in both D1 and D2 receptor-expressing neurons, namely, medium spiny neurons, where D1Rs and D2Rs have opposing effects on cAMP levels [38]. Previous studies of HD postmortem brain tissue and animal models have shown that cAMP signaling becomes deregulated in the striatum of humans and animal HD models [9, 39, 40]. Restoration of cAMP levels reduced mHtt aggregates in the striatum of R6/2 HD mice [40], underscoring the importance of rescuing striatal cAMP signaling. In the YAC128 striatum, we observed downregulation of cAMP pathway genes, which are upregulated after treatment with pridopidine (Figs. 5, 6, and 8).

In agreement with Garcia-Miralles et al. [19], we noted reversal of compromised expression of dopamine receptor genes (Drd1 and Drd2) after pridopidine treatment in the YAC128 striatum. However, reversal of Drd1 and Drd2 expression was only nominally significant after treatment with either the 10 or 30 mg/kg dose of pridopidine [19]. Therefore, pridopidine could partly restore dopamine-cAMP signaling via compensatory mechanisms. One possibility is that pridopidine induces post-translational regulation of the D1R protein. PSD-95 has been shown to increase D1R surface level expression [41], regulate D1R internalization, and D1R-cAMP signaling [42, 43]. In the striatum, wild-type HTT binds to postsynaptic density protein 95 and promotes its clustering (PSD-95) [42, 44], whereas mHTT lacks binding affinity to PSD-95 [44]. In agreement with increased D1R activity, compromised expression of D1R-regulated genes Nr4a1 and Nr4a3 is rescued after treatment with either 10 or 30 mg/kg pridopidine in the YAC128 striatum (Adj. p-val < 0.05, Fig. 5).

In addition to dopamine signaling, other signal transduction pathways regulate cAMP response targets, which may also explain the putative effect of pridopidine on cAMP signaling. For example, we identified two additional cAMP-related DEGs (Npas4 and Gpr3) upregulated after treatment with pridopidine in the YAC128 striatum (Additional file 6: Figure S1). Activity-dependent NPAS4 has been shown to upregulate both BDNF and Gpr3 expression in cultured excitatory and inhibitory neurons, respectively [45]. GPR3 is a constitutive activator of cAMP signaling via adenylyl cyclase (AC) [46, 47]. Interestingly, Npas4 gene expression is compromised in the YAC128 striatum, but rescued after pridopidine treatment with either dose (10 or 30 mg/kg, Adj. p-val < 0.05, Fig. 5). In addition, Gpr3 gene expression is downregulated in the YAC128 striatum, whereas striatal expression of Gpr3 is upregulated after either 10 or 30 mg/kg pridopidine treatment in YAC128 mice (Additional file 5: Figure S5, Adj. p-val < 0.05). Taken together, this suggests that pridopidine could partly rescue dysregulated cAMP signaling by modulating Npas4 and Gpr3 gene expression. In addition to the GPR3-cAMP pathway, BDNF-TrkB signaling has also been shown to activate cAMP response element binding (CREB) protein activity and thus facilitate gene expression in cultured striatal neurons [26]. In other words, pridopidine may induce transcription by binding to S1R, which leads to enhanced BDNF activity, in turn activating gene and splice-variant expression. This alternative mechanism is also supported by the fact that pridopidine induces BDNF release in neuroblastoma cells [30].

Recently, it was demonstrated that treatment with 30 mg/kg of pridopidine rescues motor deficiencies in YAC128 mice, whereas no effect was detected in YAC128 animals treated with 10 mg/kg of pridopidine [19]. In agreement with this observation, we report a broader effect of 30 mg/kg pridopidine on gene expression in the YAC128 striatum compared to the 10 mg/kg dose, but also report that either dose reverses disease associated gene expression. The effect of 30 mg/kg pridopidine on motor function diminishes during the progression of the disease [19], and the lowest locomotor performance is observed between 10 and 12 months of age when mice are very ill. Given that RNA samples for this study were collected during the decline of motor activity in 30 mg/kg pridopidine-treated YAC128 mice, it is difficult to correlate improvement in motor deficit and pridopidine-induced gene expression in the striatum of YAC128 animals. Moreover, our study showed that pridopidine induces a robust gene expression signal when treatment begins early in disease course. This may suggest that in humans, pridopidine may be more effective if started in early disease stages. For both of these reasons, a longitudinal study with earlier time points would better illuminate the link between gene expression and motor behavior after treatment with pridopidine in YAC128 mice.

In conclusion, pridopidine reverses HD associated changes in transcription at the pathway, gene and splice-variant level. Pathways with transcriptomic aberrations in the YAC128 mouse that are restored to WT levels by pridopidine treatment include BDNF, D1R, GR, cAMP, and calcium signaling. These pathways together are known to interact, and likely positively feed into each other downstream of pridopidine treatment, to relieve HD associated motor symptoms. Beneficial effects when treatment is initiated early, before symptoms are manifest, tracks with trends observed in clinical trials. Studying the effect of pridopidine at multiple time points over the course of treatment against transcriptomic aberrations in YAC128 will reveal additional regulatory dynamics. The results in this study, all taken together, support exploring pridopidine’s role as a therapeutic for neuroprotection in HD and similar neurological movement disorders.