Molecular Mechanisms of Cerebellar Circuit Formation

The cerebellar cortex is a key model for cortical histogenesis as it consists of only two principle neurons, the granule cell (GC) and Purkinje cell (PC). Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. To analyze global gene expression patterns in GCs and PCs during circuit formation, we used translating ribosome affinity purification (TRAP). Metagene analysis of GC gene expression patterns revealed pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar GCs during circuit formation. During this stage, Tet genes are up-regulated and vitamin C activation of Tet enzymes increases 5-hydroxymethylcytosine (5hmC) of up-regulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1and Tet3by RNA interference in ex vivocerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in developing granule cells during the formation of cerebellar circuitry. Studies are ongoing to further define chromatin changes in pre and post-migratory GCs.

To model human cerebellar disease and circuit dysfunction, we developed methods to generate PCs from human pluripotent stem cells (hPSCs). hPSC-PCs resemble developing human PCs morphologically and when co-cultured with mouse GCs, fire action potentials detected by genetically encoded calcium indicators. A comparison of global gene expression of hPSC-PCs with developing mouse PCs reveals that differentiated hPSC-PCs are most similar to mouse PCs at postnatal day 21. These results show that hPSC-PCs are a powerful model system to complement genetic modeling of human disease in mice, and provide an approach to directly compare genetic and epigenetic pathways in mouse and human cerebellar neurons in order to analyze pathways affected by developmental and degenerative cerebellar disease.