To elucidate the extent of cell-autonomous transcriptional alterations in HD, we performed genome-wide expression profiling of striatal tissue from DE5 transgenic mice. Importantly, this is the first microarray study performed in an in vivo
‘striatal-specific’ genetic HD model. In this study, we demonstrate a relatively high degree of overlap of changes in gene expression in DE5 transgenic mice, a forebrain striatal-
specific mouse model of HD expressing the Htt N171 fragment and two transgenic pan-cellular R6 models, human caudate and Htt N171 fragment-expressing cultured striatal neurons (9
). Collectively, these data provide strong evidence that intrinsic effects of mutant Htt are sufficient to result in transcriptional alterations in several pathways relevant to HD pathology. Conversely, these data suggest that expression of mutant Htt in the cortex is not required for most of the transcriptional dysregulation present in mouse models of HD.
Transcriptional effects of the cortex on the striatum in HD may arise at least in part from decreased synthesis and transport of brain-derived neurotrophic factor (BDNF) (18
), and it has been suggested that cortical-specific deletion of BDNF mimics the pattern of transcriptional dysregulation in HD (12
). Importantly, DE5 transgenic mice express normal levels of cortical BDNF and striatal trkB mRNA (11
). Moreover, early embryonic deletion of BDNF may not accurately represent the pattern of decreased BDNF noted in HD. Of note, however, many of the striatal-enriched genes are also regulated by BDNF (19
), and a decrease in this trophic factor may contribute to a greater magnitude of the alterations in pan-neuronal HD models. It is also possible that the magnitude of these alterations would be greater in older DE5 mice than those utilized in this study, since DE5 mice have a normal lifespan.
Other mechanistic candidates leading to selective neuronal vulnerability in HD include mitochondrial dysfunction that induces energy deficits, increased oxidative stress and abnormalities of calcium signaling. Pathways associated with both cell- and non-cell-autonomous components include glutamate and dopamine neurotransmission (20
). We therefore compared and contrasted changes in gene expression in these canonical pathways among the different mouse models. We also included the PPAR pathway, as it potentially unites transcriptional dysregulation with mitochondrial dysfunction (21
). We anticipated that changes in gene expression associated with mitochondrial dysfunction and oxidative stress, and the responses to these stressors, would be largely cell autonomous, resulting in a high degree of overlap between the DE5 and R6/2 transgenic models. It was therefore surprising to discover that levels of a number of genes in these pathways were altered only in the DE5 transgenic line, including the qPCR-validated Atp5l
, despite a longer polyglutamine repeat and greater symptomatology in the R6 transgenic models. This suggests that mitochondrial deficits and oxidative stress are prominent features of intrinsic striatal dysfunction. The fact that other genes associated with these functions are altered in their expression in R6/2 suggests that non-cell-autonomous effects might both exacerbate and compensate for this dysfunction. Of note, however, Mras
, whose protein product may be required for neurotrophic signaling and dendritic remodeling but is also expressed in glia (22
), is uniquely highly down-regulated in the DE5 model.
In the calcium signaling pathway, the expression profiles of the three mouse models are highly shared, implying a substantial degree of cell-autonomous changes related to this pathway. This is in contrast to the glutamate and dopamine receptor signaling pathways for which the greatest number of gene expression changes occurred in R6/2 transgenic mice and very few occurred in DE5 transgenic mice. As increased sensitivity to excitotoxity appears to result from combinations of increased cortical glutamate neurotransmission and mislocalization of MSN glutamate receptor subtypes in the extra-synaptic region (reviewed in 23
), which may not be reflected by transcriptional dysregulation, these results do not obviously allow for a prediction regarding sensitivity to glutamate excitotoxicity in the DE5 mouse. Perhaps not surprisingly, although Drd2, Rgs9 and Ppp1r1b mRNAs are decreased in the striatum of DE5 transgenic mice, many more genes in the dopamine receptor signaling pathway are dysregulated in R6/2 compared with DE5 striatum, probably reflecting trans-synaptic regulation and/or compensatory responses to reduced dopaminergic input (24
As noted above, we found a substantial overlap of gene expression differences between DE5 transgenic mice and Htt171-82Q-expressing primary striatal cultures. Furthermore, we found that DE5 transgenic mice showed a similar enrichment of GO categories to those reported previously from these in vitro
studies. In particular, gene expression changes unique to DE5 transgenic mice and cultured striatal neurons, but not R6/2 mice, were associated with protein transport, axon development and RNA splicing (9
). There are actually fewer changes in the DE5 model relative to the cultured neurons. We hypothesize that this is due to the somewhat immature nature of the neurons in vitro
, and also to the fact that by definition, they are removed from their normal developmental and adult environment and changes are, therefore, both cell autonomous and independent of brain circuitry. Another previous microarray study reported transcription changes from rat clonal striata-derived cells expressing an N-terminal 548-amino acid Htt fragment under the control of a doxycycline-regulated promoter (10
). These studies revealed altered expression of genes involved in cell signaling, transcription, lipid metabolism as well as RNA processing, consistent with our findings. Overall, of the ~80 gene expression abnormalities reported, we found 6 genes, including 2 lipid-related genes, Acat2
, that were also dysregulated in the DE5 transgenic mice.
Of the altered genes that are ubiquitously expressed, there were important differences in the direction of change in a subset of transcripts that may reflect compensatory changes. For example, Ppargc1a
is a member of the PPAR-regulated pathway, and its down-regulation in MSNs is hypothesized to play a causative role in HD pathophysiology (25
). In DE5 transgenic mice, however, Ppargc1a mRNA is up-regulated, whereas it is unchanged in the R6 transgenic models in this study. Interestingly, Nr2f1
expression is also up-regulated in DE5 mice and Map3k7ip1
is down-regulated, but both are unchanged in the R6 models, which could serve to balance the impairment of Ppargc1a
-associated activity in DE5 mice and subsequent down-regulation of protective mitochondrial activity. Another example of an abnormality in the oxidative phosphorylation pathway in DE5 mice only is Ndufc1,
which follows the same pattern as Ppargc1a
(Fig. ), The functional implications, therefore, may actually be similar to those models in which Ppargc1a
is down-regulated and may indicate altered mitochondrial energy metabolism in DE5 transgenic mice.
Mutant Htt protein interacts with multiple transcription factors, most of which are not cell specific, e.g. the cAMP response element-binding protein (CREB)-binding protein, Specificity protein 1 (Sp1), TATA binding box protein, Bcl11b, repressor element-1 transcription factor/neuron restrictive silencer factor and widely expressed components of the basal polymerase II transcriptional machinery (29
). Overall, the genome-wide transcriptome changes induced by mutant Htt in DE5 cannot yet be ascribed to a particular transcription factor regulatory network (32
A final distinction worthy of consideration is the cellular localization of expression of genes of interest. The D9 promoter driving Htt expression in DE5 transgenic mice does not direct expression to striatal interneurons or to glia (36
). Striatal GABAergic/parvalbumin interneurons receive direct, convergent cortical input (37
) and are in fact highly sensitive in HD and in models of excitotoxicity (38
). In mouse HD models, Pvalb
gene expression is usually decreased; however, in the DE5 model, Pvalb
expression is increased. As noted above, PGC1α mRNA is also increased, which may be driving the Pvalb
). These data imply that in the DE5 mouse, transcriptome alterations in these interneurons are mediated by cell–cell interactions, whereas there may be more direct, cell-autonomous alterations when mutant Htt is expressed in a pan-cellular distribution. In regards to glia, it has recently been demonstrated that selective expression of mutant Htt in glia results in a distinct phenotype and further exacerbates symptoms in the N171-82Q transgenic mouse (40
). Importantly, the Nrf2-mediated protective pathway is largely localized to astrocytes (42
), so it will be important to determine in which cell types(s) alterations in expression of genes in this pathway are occurring in all HD mouse models.
In summary, our data are consistent with the notion that transcriptional dysregulation in HD MSNs in vivo
is largely cell autonomous in specific canonical pathways, and largely non-cell autonomous in others. They are also consistent with our previous findings regarding the nature and severity of the motor deficit in the DE5 transgenic mouse, particularly when directly compared with the prp-N171-82Q model (11
). Our underlying assumption is that the changes noted in the DE5 mouse striatum are cell autonomous, but can simultaneously be compensating for cell-intrinsic abnormalities. The converse is also true, i.e. compensatory pathways may also normalize some of the anticipated alterations. Conversely, in pan-neuronal models, e.g. R6/2, transcriptome alterations that may occur on a cell-autonomous basis may be ‘masked' by opposing, non-cell-autonomous effects of the mutation on the MSN, resulting in gene alterations that are unique to DE5 transgenic mice. These latter alterations may also be compensatory, and may be either beneficial or deleterious. Finally, it is possible that the response of individual changes may differ based on the Htt fragment length, although published data do not support a major role for the fragment length. Overall, this situation is perhaps most similar to what has been hypothesized for the etiology of amyotrophic lateral sclerosis (43
), in which the pathophysiological processes are initiated in highly vulnerable neuronal subtypes, but progression proceeds more rapidly via non-cell-autonomous mechanisms. Therapeutic targeting of cell- or non-cell-autonomous sites will, therefore, depend on which pathway(s) is the desired target.