Gain of function mutations occurring in the cytoskeletal gene TUBB4A (tubulin beta 4A) lead to a spectrum of hypomyelinating leukodystrophies and isolated dystonia, including Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (H-ABC). H-ABC typically results from the most common TUBB4A mutation, p. Asp249Asn (D249N). Affected individuals exhibit dystonia, gait impairment and progressive neurologic decline. 

Establish the effect of TUBB4A mutations on neurons and oligodendrocytes using murine and human cells in culture.

To explore the underlying cellular contributions of TUBB4A mutations, we examined cell-autonomous effects in neurons and oligodendrocytes (OLs) in vitro from Tubb4a^D249N/D249N mice and human induced pluripotent stem cells (iPSC) derived cultures. Murine neurons were assessed for survival, axonal length and microtubule dynamics using live cell imaging of EB3 comets. OLs were studied for expression of myelin proteins and complexity of arborization using Sholl analysis. Finally, to test cell autonomous effects of human TUBB4A^D249N, we generated iPSC derived medium striatal neurons (MSN) from patients with TUBB4A^D249N mutation. MSNs were assessed for survival assays between patient and age-matched controls.

Murine Tubb4a^D249N/D249N neurons exhibit reduced survival in vitro and decreased axonal length compared to control neurons (p<0.01). A significant decrease in the number of EB3 comets (p<0.05) were seen indicating altered microtubule dynamics. Further MSNs were successfully derived from control and TUBB4A^D249N iPSCs, which demonstrated decreased neuronal survival (p<0.05) and increased apoptosis in TUBB4A^D249N neurons. Cell-autonomous effects were also seen in murine OLs with a 50% decrease in the number of mature PLP labeled OLs along with decreased complexity in cells of Tubb4a^D249N/D249N origin.  

Initial work suggests a cell-autonomous effect on both neuronal and oligodendrocyte lineages in TUBB4A^D249N mutations. Ongoing studies are focused on understanding cellular pathways in H-ABC, in order to determine potential therapeutic approaches.