Modeling antisense oligonucleotide therapy in MECP2 duplication syndrome human iPSC-derived neurons reveals gene expression programs responsive to MeCP2 levels.

Genomic copy-number variations (CNVs) that can cause neurodevelopmental disorders often encompass many genes, which complicates our understanding of how individual genes within a CNV contribute to pathology. MECP2 duplication syndrome (MDS or MRXSL in OMIM; OMIM#300260) is one such CNV disorder caused by duplications spanning methyl CpG-binding protein 2 (MECP2) and other genes on Xq28. Using an antisense oligonucleotide (ASO) to normalize MECP2 dosage is sufficient to rescue abnormal neurological phenotypes in mouse models overexpressing MECP2 alone, implicating the importance of increased MECP2 dosage within CNVs of Xq28. However, because MDS CNVs span MECP2 and additional genes, we generated human neurons from multiple MDS patient-derived induced pluripotent cells (iPSCs) to evaluate the benefit of using an ASO against MECP2 in a MDS human neuronal context. Importantly, we identified a signature of genes that is partially and qualitatively modulated upon ASO treatment, pinpointed genes sensitive to MeCP2 function, and altered in a model of Rett syndrome, a neurological disorder caused by loss of MeCP2 function. Furthermore, the signature contained genes that are aberrantly altered in unaffected control human neurons upon MeCP2 depletion, revealing gene expression programs qualitatively sensitive to MeCP2 levels in human neurons. Lastly, ASO treatment led to a partial rescue of abnormal neuronal morphology in MDS neurons. All together, these data demonstrate that ASOs targeting MECP2 benefit human MDS neurons. Moreover, our study establishes a paradigm by which to evaluate the contribution of individual genes within a CNV to pathogenesis and to assess their potential as a therapeutic target.

A Comprehensive and Integrative Approach to MeCP2 Disease Transcriptomics.

Mutations in MeCP2 result in a crippling neurological disease, but we lack a lucid picture of MeCP2's molecular role. Individual transcriptomic studies yield inconsistent differentially expressed genes. To overcome these issues, we demonstrate a methodology to analyze all modern public data. We obtained relevant raw public transcriptomic data from GEO and ENA, then homogeneously processed it (QC, alignment to reference, differential expression analysis). We present a web portal to interactively access the mouse data, and we discovered a commonly perturbed core set of genes that transcends the limitations of any individual study. We then found functionally distinct, consistently up- and downregulated subsets within these genes and some bias to their location. We present this common core of genes as well as focused cores for up, down, cell fraction models, and some tissues. We observed enrichment for this mouse core in other species MeCP2 models and observed overlap with ASD models. By integrating and examining transcriptomic data at scale, we have uncovered the true picture of this dysregulation. The vast scale of these data enables us to analyze signal-to-noise, evaluate a molecular signature in an unbiased manner, and demonstrate a framework for future disease focused informatics work.

MeCP2 regulates Gdf11, a dosage-sensitive gene critical for neurological function.

Loss- and gain-of-function of MeCP2 causes Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), respectively. MeCP2 binds methyl-cytosines to finely tune gene expression in the brain, but identifying genes robustly regulated by MeCP2 has been difficult. By integrating multiple transcriptomics datasets, we revealed that MeCP2 finely regulates growth differentiation factor 11 (Gdf11). Gdf11 is down-regulated in RTT mouse models and, conversely, up-regulated in MDS mouse models. Strikingly, genetically normalizing Gdf11 dosage levels improved several behavioral deficits in a mouse model of MDS. Next, we discovered that losing one copy of Gdf11 alone was sufficient to cause multiple neurobehavioral deficits in mice, most notably hyperactivity and decreased learning and memory. This decrease in learning and memory was not due to changes in proliferation or numbers of progenitor cells in the hippocampus. Lastly, loss of one copy of Gdf11 decreased survival in mice, corroborating its putative role in aging. Our data demonstrate that Gdf11 dosage is important for brain function.

Disruption of the ATXN1-CIC complex reveals the role of additional nuclear ATXN1 interactors in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative disease in that it is caused by a mutation in a broadly expressed protein, ATXN1; however, only select populations of cells degenerate. The interaction of polyglutamine-expanded ATXN1 with the transcriptional repressor CIC drives cerebellar Purkinje cell pathogenesis; however, the importance of this interaction in other vulnerable cells remains unknown. Here, we mutated the 154Q knockin allele of Atxn1154Q/2Q mice to prevent the ATXN1-CIC interaction globally. This normalized genome-wide CIC binding; however, it only partially corrected transcriptional and behavioral phenotypes, suggesting the involvement of additional factors in disease pathogenesis. Using unbiased proteomics, we identified three ATXN1-interacting transcription factors: RFX1, ZBTB5, and ZKSCAN1. We observed altered expression of RFX1 and ZKSCAN1 target genes in SCA1 mice and patient-derived iNeurons, highlighting their potential contributions to disease. Together, these data underscore the complexity of mechanisms driving cellular vulnerability in SCA1.

Antisense oligonucleotide therapy in a humanized mouse model of MECP2 duplication syndrome.

Many intellectual disability disorders are due to copy number variations, and, to date, there have been no treatment options tested for this class of diseases. MECP2 duplication syndrome (MDS) is one of the most common genomic rearrangements in males and results from duplications spanning the methyl-CpG binding protein 2 (MECP2) gene locus. We previously showed that antisense oligonucleotide (ASO) therapy can reduce MeCP2 protein amount in an MDS mouse model and reverse its disease features. This MDS mouse model, however, carried one transgenic human allele and one mouse allele, with the latter being protected from human-specific MECP2-ASO targeting. Because MeCP2 is a dosage-sensitive protein, the ASO must be titrated such that the amount of MeCP2 is not reduced too far, which would cause Rett syndrome. Therefore, we generated an "MECP2 humanized" MDS model that carries two human MECP2 alleles and no mouse endogenous allele. Intracerebroventricular injection of the MECP2-ASO efficiently down-regulated MeCP2 expression throughout the brain in these mice. Moreover, MECP2-ASO mitigated several behavioral deficits and restored expression of selected MeCP2-regulated genes in a dose-dependent manner without any toxicity. Central nervous system administration of MECP2-ASO is therefore well tolerated and beneficial in this mouse model and provides a translatable approach that could be feasible for treating MDS.

Losing Dnmt3a dependent methylation in inhibitory neurons impairs neural function by a mechanism impacting Rett syndrome.

Methylated cytosine is an effector of epigenetic gene regulation. In the brain, Dnmt3a is the sole 'writer' of atypical non-CpG methylation (mCH), and MeCP2 is the only known 'reader' for mCH. We asked if MeCP2 is the sole reader for Dnmt3a dependent methylation by comparing mice lacking either protein in GABAergic inhibitory neurons. Loss of either protein causes overlapping and distinct features from the behavioral to molecular level. Loss of Dnmt3a causes global loss of mCH and a subset of mCG sites resulting in more widespread transcriptional alterations and severe neurological dysfunction than MeCP2 loss. These data suggest that MeCP2 is responsible for reading only part of the Dnmt3a dependent methylation in the brain. Importantly, the impact of MeCP2 on genes differentially expressed in both models shows a strong dependence on mCH, but not Dnmt3a dependent mCG, consistent with mCH playing a central role in the pathogenesis of Rett Syndrome.