Huntington's disease-like-2 (HDL2), discovered and genetically defined by the Margolis group, is an autosomal dominant neurodegenerative disorder, clinically indistinguishable from Huntington's disease (HD). Like HD, the neuropathology of HDL2 is characterized by cortical and striatal neurodegeneration and the presence of neuronal protein aggregates. HDL2 is caused by a CTG/CAG expansion on chromosome 16q24. Normal alleles contain 6-28 triplets, while pathogenic repeats range from 40-59 triplets, again remarkably similar to HD. In the CTG orientation, the repeat falls in the gene junctophilin-3 (JPH3). A series of pathological, animal model, and cell model studies by the Margolis lab suggest that HDL2 pathogenesis is multifactorial, involving both gain- and loss-of-function mechanisms. Here we propose to investigate the pathogenic role of loss of JPH3 protein. JPH3 is one of a conserved family of proteins that, along with JPH4, brings endoplasmic reticulum into close contact with plasma membrane (PM). In neurons, this contact brings together ER ryanodine receptors, calcium channels, and potassium channels that as a complex regulate slow after-hyperpolarization current and hence neuronal function. Based on our findings that JPH3 transcript and protein is reduced in human HDL2 post-mortem brain samples, and that Jp3 KO mice exhibit motor abnormalities, we hypothesize that the HDL2 pathogenesis stems at least partially from loss of JPH3 expression, which is sufficient to cause dysfunctional Ca homeostasis and altered neuronal excitability and function. Our goal here is to test this hypothesis using sensitive calcium imaging and electrophysiological measures.
In Aim 1, we will determine the impact of loss of JP3/JPH3 on calcium homeostasis and neuronal function, and the electrophysiological parameters that can best detect this, in primary striatal neurons and coronal sections from Jp3 KO mice, and in human iPS cells differentiated into mature striatal medium spiny neurons (MSNs) in which JPH3 is reduced.
In Aim 2, we will determine the impact of the HDL2 expansion mutation on Ca homeostasis and neuronal function, and whether this effect is at least partly the consequence of loss of JPH3, using a series of human HDL2 and control iPSC lines differentiated into MSNs. The experiments in these aims will help determine the role of JPH3, and more generally the integrity of the PM-ER junction, in neuronal function and dysfunction, and in the pathogenesis of neurological disorders. We anticipate that the tools, methods, and data generated through the proposed experiments will enable us to compete for long-term funding to systematically explore HDL2 pathogenesis, and the more general role of the ER-PM junction in neurological disorders. Finally, the study is a collaboration between investigators with complementary expertise at Johns Hopkins and Howard University, and provides the opportunity for underrepresented students at Howard to gain access to the full resources of both institutions while participating in cutting-edge biomedical research.
The mutation that causes the neurodegenerative disease Huntington's disease like-2 is a CTG/CAG repeat expansion in the gene junctophilin-3. Junctophilin-3 is involved in maintaining normal neuronal calcium homeostasis and synaptic function. We propose to use animal and cell models to determine if the HDL2 mutation causes abnormalities in calcium homeostasis and synaptic function in neurons by decreasing the amount of available junctophilin-3 protein.
