Defining the cellular basis of neurological dysfunction in models of ALG8 Congenital Disorder of Glycosylation - PROJECT SUMMARY Neural circuit development and function depends on precise interactions between neurons and glia. Astrocytes, the primary peri-synaptic glia, mediate synapse formation, stability, and function. Neuron-astrocyte crosstalk is facilitated by complex protein-protein interactions, and loss of these interactions contributes to circuit instability in many neurological disorders. Thus, understanding the mechanisms that regulate neuron-astrocyte communication is of broad clinical importance. Glycosylation is a posttranslational modification that regulates protein stability and binding through addition of sugar groups to specific amino acids. Mutation of genes in glycosylation pathways cause congenital disorders of glycosylation (CDGs), a group of monogenic disorders associated with neurological dysfunction, including epilepsy, autism, and cerebellar degeneration. The mechanisms underlying neurological dysfunction in CDGs remain unknown. Here, I focus on ALG8, an enzyme in the N-glycosylation pathway. To explore the molecular underpinnings of ALG8-CDG, I first needed to develop models that reflect the patient population. To this end, I generated a predicted null zebrafish line (alg8stl973) and human embryonic stem cell (hESC) lines with a missense mutation (p.Thr47Pro) found in ALG8-CDG patients. My preliminary data revealed a decrease in astrocyte numbers in the brains of alg8 mutant zebrafish with no change in total cells, and reduced proliferation of ALG8 mutant hESC-derived astrocytes. Moreover, in alg8stl973 fish, astrocyte morphological complexity is reduced. As astrocyte-synapse association is necessary for neuronal signaling, I hypothesize that defective glycosylation disrupts specification and maturation of astroglia, which in turn drives circuit imbalance and CDG-associated behavioral deficits. To address this hypothesis, I will leverage preexisting transgenic tools in zebrafish to label astrocytes and test whether changes in proliferation and/or cell death result in reduced astrocytes in alg8stl973 fish (Aim 1). Furthermore, I will use biochemistry and in vivo imaging to characterize how loss of alg8 impacts the glycosylation status of one key regulator of astrocyte morphogenesis: NrCam (Aim 2). Finally, as ALG8 is expressed in all neural cell types, I will use cell-type specific rescue in fish and co-culture of hESC-derived neural cells to determine which cell type(s) drive changes in astrocyte morphology and synaptogenesis in ALG8-CDG (Aim 3). My long-term goal is to define common molecular changes in brain development across distinct CDGs. Critically, various CDG subtypes result in common neurological symptoms, but the cellular and molecular underpinnings of these phenotypes are largely unknown. Similar to my preliminary findings in ALG8-CDG models, recent work indicates that astrogenesis is altered in a mouse model of MGAT5-CDG, a CDG with defective N-glycosylation. Thus, I anticipate that my findings will be broadly applicable to the CDG community and will enhance our fundamental understanding of how glycosylation shapes brain development.
