The three neurodevelopmental disorders (ASD, Schizophrenia, and Bipolar) showed a

nearly identical enrichment pattern, among many categories. In contrast, there was almost no enrichment for neurodegenerative disease lists. Similarly, the neuropsychiatric disorders showed no enrichment for miRNA target genes, except for major depressive disorder, where the pattern was similar to the neurodevelopmental disorders.

Figure 2.4.3. Enrichment of differentially expressed miRNA target genes by brain region for disease associated genes. Dashed line indicates significance (corrected p-value < 0.01).

Figure 2.4.4. Enrichment of differentially expressed miRNA target genes among male versus female sets for disease associated genes. Dashed line indicates significance (corrected p-value <

Finally, I explored the temporal-spatial correlation between three high-confidence autism candidate genes (PTEN, BDNF, and MECP2) and their experimentally-known regulator miRNAs (Mellios and Sur 2012). To do so, the Pearson correlation was calculated between each ASD candidate gene and its cognate miRNA by brain region across all of developmental time (Figure 2.4.5a-c). This analysis showed that miRNA-mediated gene suppression appears to be region specific, and that this region specificity is unique to each gene-miRNA pair, as significant anti-correlations were only found in certain brain regions and these regional patterns differed between the three genes. For instance, both PTEN and BDNF appear to be significantly down-regulated by their cognate miRNAs in the dorsolateral and ventrolateral prefrontal cortices, but not in other brain regions; whereas MECP2 appears to be regulated by its miRNA mainly in the cerebellum. Furthermore, different mature isoforms of the same precursor miRNA sometimes display opposite correlations (e.g. miR212-3-p vs. miR21205-p in panel C, DFC), providing strong evidence for highly-specific miRNA- mediate gene repression.

Figure 2.4.5. Temporal, spatial, and isoform-specific miRNA regulation of three autism candidate genes. a-c. Pearson correlation analysis between three autism candidate genes and their

known miRNAs by brain region across all of developmental time. d-f. Temporal profile of significant miRNA-gene expression pairs.

Assessing the temporal profile of significant miRNA-gene expression pairs shows that miRNA-mediated gene suppression also appears to be time-period specific (Figure 2.4.5d-f). For instance, miR-21-3p appears to modulate expression of PTEN throughout development in the DFC (d), whereas miR-212-3p appears to only modulate MECP2 expression in the

cerebellum after infancy (f). Taken together, these results further illuminate the critical regulatory roles that miRNAs play in modulating expression of ASD candidate genes.

2.4.5

Discussion

In summary, the work described in this chapter represents the most comprehensive assessment to date of spatio-temporal miRNA expression in the developing human brain. I identified miRNAs differentially expressed both within and between brain regions, and demonstrated that the greatest shifts in miRNA expression occur shortly after birth. However, unlike global gene expression patterns, miRNAs become more differentially expressed between brain regions over time, potentially driving regional specialization as the brain matures. Target genes under putative control by region-specific differentially expressed miRNAs are most related to the processes of transcription regulation and neurodevelopment, highlighting the central function of these miRNAs to brain transcription networks. Additionally, sex-biased expression of miRNAs increases in the prefrontal cortex around puberty, and the pathways related to sex-biased target genes are further enriched for Wnt signaling and TGF-β pathways. Common neurodevelopmental disorders with complex genetic etiologies are highly related to genes targeted by these miRNAs, but this was not found for genes related to neurodegenerative or other neuropsychiatric diseases with adult onset. Examining the specific relationship between three high confidence ASD candidate genes and their experimentally-known miRNAs showed that miRNA-mediated gene silencing appears to be highly temporally and spatially specific, and even isoform-specific miRNA regulation appears during neurodevelopment. These results highlight the importance of miRNAs in understanding the functional genomics of ASD, and suggest that future work should more closely examine the role of miRNAs in ASD molecular pathogenesis.

This study has a number of important limitations. First, the total sample size is 18 donor brains, potentially limiting the statistical power. Unfortunately, this problem is prevalent throughout human neurosciences research owing to the lack of large repositories of human post mortem brain tissue (Button et al. 2013). Therefore, it will be important for future studies to replicate and aggregate the data presented here with larger datasets when they become available. Additionally, while computational prediction of miRNA targets based on sequence homology is an effective discovery tool, individual miRNAs of interest will require

2.4.6

Conclusion

In conclusion, while thousands of genes are differentially expressed throughout human neurodevelopment, I have identified a set of miRNAs with differential spatio-temporal and sex-biased expression patterns that may regulate these expression changes. A number of the identified miRNAs are of note for their known role in neurodevelopmental processes. For instance, miR-9, which I found to be increased in expression nearly 5-fold in the hippocampus of early childhood samples as compared to infants (FDR = 0.0039), is known to be a critical regulator of neural progenitor migration and proliferation (Delaloy et al. 2011). Intriguingly, I did not observe increased miR-9 expression in any other brain region during post-natal development, which is to be expected, as the hippocampus was the only region assessed that contains neural stem cells after embryonic development (Song et al. 2002). Similarly intriguing was the finding of significant differential expression of miR-103 between the prefrontal cortex of males and females in adolescence (fold change 1.73, FDR = 0.0041). MiR-103 has been demonstrated to regulate expression of the insulin like growth factor (IGF) family of proteins (Liao and Lonnerdal 2010), of which IGF-2 is known to exhibit genomic imprinting—the phenomena of expressing an allele from either the paternal or maternal DNA but not both—and has unique, brain-region specific imprinted expression patterns (Pham 1999). This is particularly interesting given that microRNAs are one of the main mechanisms by which genomic imprinting is maintained (Delaval and Feil 2004), and imprinting mechanisms could partially account for the significant sex bias seen in neurodevelopmental disorders like ASD (Skuse 2000). These examples further support the notion that the identified miRNAs are likely critical regulators of neurodevelopmental transcriptional processes.

The targets of these differentially expressed miRNAs are highly enriched for genes related to transcriptional regulation, neurodevelopmental processes, and common neurodevelopmental disorders. Furthermore, inter-regional expression differences of miRNAs appear to increase over development. These results suggest the identified miRNAs are likely hubs of critical brain developmental and pathologic transcriptional processes.

Chapter 3. Functional Genomics Studies of