Postnatal Conditional Deletion of Bcl11b in Striatal Projection Neurons Mimics the Transcriptional Signature of Huntington's Disease

Abstract

The dysregulation of striatal gene expression and function is linked to multiple diseases, including Huntington's disease (HD), Parkinson's disease, X-linked dystonia-parkinsonism (XDP), addiction, autism, and schizophrenia. Striatal medium spiny neurons (MSNs) make up 90% of the neurons in the striatum and are critical to motor control. The transcription factor, Bcl11b (also known as Ctip2), is required for striatal development, but the function of Bcl11b in adult MSNs in vivo has not been investigated. We conditionally deleted Bcl11b specifically in postnatal MSNs and performed a transcriptomic and behavioral analysis on these mice. Multiple enrichment analyses showed that the D9-Cre-Bcl11b^tm1.1Leid transcriptional profile was similar to the HD gene expression in mouse and human data sets. A Gene Ontology enrichment analysis linked D9-Cre-Bcl11b^tm1.1Leid to calcium, synapse organization, specifically including the dopaminergic synapse, protein dephosphorylation, and HDAC-signaling, commonly dysregulated pathways in HD. D9-Cre-Bcl11b^tm1.1Leid mice had decreased DARPP-32/Ppp1r1b in MSNs and behavioral deficits, demonstrating the dysregulation of a subtype of the dopamine D2 receptor expressing MSNs. Finally, in human HD isogenic MSNs, the mislocalization of BCL11B into nuclear aggregates points to a mechanism for BCL11B loss of function in HD. Our results suggest that BCL11B is important for the function and maintenance of mature MSNs and Bcl11b loss of function drives, in part, the transcriptomic and functional changes in HD.

Keywords: BCL11B; Bcl11b conditional knockout; CTIP2; Huntington’s disease; induced pluripotent stem cells; striatal medium spiny neurons; transcriptomics.

Figure 1

Identification of transcriptome portraits of Bcl11b deletion and WT cells by RNA-seq analysis in Cre+ and Cre− MSNs populations from the striata of D9-Cre mice. (A) D9-Cre-Bcl11b^tm1.1Leid mice specifically knocked down expression Bcl11b in MSNs by using a D9-Cre under the control of regulatory elements of the mouse Ppp1r1b gene-encoding DARPP-32. D9-Cre-Bcl11b^tm1.1Leid and Cre-negative mice (4-months-old) were analyzed using immunohistochemistry (IHC) with a BCL11B antibody. Scale bar is 500 mm. (B) PCA plots using the rlog-transformed values indicate a significant difference in the transcriptome Bcl11b deleting MSNs, and controls. (C) Scatter plot shows that Bcl11b gene expression is much less in D9-Cre-Bcl11b^tm1.1Leid mice than Cre– control mice. (D) Volcano plot shows differences in Cre+ and Cre− gene expression. Genes with an adjusted p-value below 0.01 with absolute log2 fold ratio greater than 1 are highlighted. Genes in red are relatively decreased in expression in the Cre− population (i.e., enriched in the WT population), those in green are relatively increased in expression in the Bcl11b^tm1.1Leid mice, and those in grey are equally distributed among the two populations. (E) Heatmap of relative normalized count values across samples. Top 20 up- and downregulated genes that have the highest product of log fold-change and base mean are reported, respectively. Top downregulated genes include: Bcl11b, free fatty acid receptor (Ffar3), spermatogenesis and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), beta tropomyosin (Tmp2), myosin IIIB (Myo3b), wnt family member 8B (Wnt8b), R-spondin-1 (Rspo1), Obscurin (Obscn), glutathione peroxidase 6 (Gpx6), dermokine (Dmkn), serine/threonine-protein kinase receptor, R3, activin A receptor-like type 1 (Acvrl1), anoctamin-2 (Ano2), kelch-like protein 1 (Klhl1), synaptotagmin-2 (Syt2), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-2 (Plch2), Sec14l3 (uncharacterized protein), and C-type lectin domain family 12 member A (Clec12a).

Figure 2

Transcriptional profile of Bcl11b deletion is highly correlated with HD mouse models and postmortem human HD tissue. (A) Gene perturbation enrichment analysis. (B) Disease perturbation enrichment analysis. (C) Drug perturbation enrichment analysis.

Figure 3

Significantly enriched KEGG terms and IPA signaling. (A) KEGG term enrichment analysis of gene signatures altered by Bcl11b deletion highlighted the axon guidance, dopaminergic synapses, adrenergic, estrogen, cAMP, MAPK, insulin, oocytes, and glutamatergic signaling. (B) IPA signaling pathway highlights for dopamine DARPP-32 feedback cAMP signaling.

Figure 4

Gene regulatory network analysis reveals critical up-stream TFs from the gene signatures altered by Bcl11b deletion. Gene regulatory network analysis. (A) Differentially expressed TFs that are upregulated (A) or downregulated (B) in Bcl11b-deletion cells. (C) All differentially expressed TFs in Bcl11b-deletion cells.

Figure 5

Bcl11b deficiency leads to a reduced number of MSNs without microgliosis. Representative striatal images immunostained with cell type-specific markers, and graphs detailing corresponding quantification. (A). IHC of Bcl11b^tm1.1Leid and control mice IHC immunostained with NeuN (neurons) and quantification. (B). IHC of Bcl11b^tm1.1Leid and control mice immunostained with DARPP-32 (MSNs) and quantification. (C). IHC of Bcl11b^tm1.1Leid and control mice with Iba1 (microglia). Scale bars, 200 mm. Graphs show the number of NeuN, DARPP-32, and Iba1-positive cells in the striatum. Each point represents an individual mouse. All data are shown as mean  ±  SEM (WT n  =  5; Bcl11b-KO n = 5.) Two-tailed unpaired t-test, * p < 0.05.

Figure 6

Bcl11b deficiency in adult mice partly recapitulate HD-associated motor phenotype. (A). Spontaneous locomotor activity measured in the Bcl11b deficiency mice. (B). Balance beam: from left to right; numbers of frames crossed in 2 min and times to cross 30 frames. (C). Vertical pole: times to turn (left) and times to descend (right) were recorded after placing the mice upwards to the pole. Three trials were conducted, and data represent the mean ± SEM (WT n = 11, D9-Cre-Bcl11b^tm1.1Leid mice n = 18). Two-tailed unpaired t-test, * p < 0.05; ** p < 0.01. (D). Schematic diagram of catalepsy position (left). Catalepsy time after Haloperidol treatment (right). Two-Way ANOVA, with Bonferroni as post-hoc test * p < 0.05.

Figure 7

Human MSNs derived from HD patient iPSCs reveals mislocalization of BCL11B into nuclear aggregates. (A). Isogenic HD72 and C116 MSNs differentiated from iPSCs immunostained with DARPP-32. (B). Top genes dysregulated in D9-Cre-Bcl11b^tm1.1Leid mice follow similar trends in expression as measured by RT-PCR. KCNC3 and WNT10A are upregulated in HD72-MSNs, compared to control C116. Like the D9-Cre-Bcl11b^tm1.1Leid mice transcriptomics, SLIT3 was downregulated in HD-MSNs. (C). IHC with BCL11B antibody show more large nuclear aggregates in the HD72-MSNs than in C116-MSNs. (D). Quantification of the BCL11B foci per nuclear area in isogenic HD72 and C116-MSNs. * p < 0.05; **** p < 0.0001, Mann Whitney test.