Investigating surface signaling effectors of neurofilamenthyperphosphorylation: implications for neurodegenerative disease.

Project Summary: Neurofilament (NF) proteins are intermediate filament proteins expressed by neurons that are essential for axonal organization, support, transport, and synaptic function. NFs consist of three subunits, NF-H (Heavy), NF- M (Medium), and NF-L (Light), and their assembly and disassembly are regulated through phosphorylation by a complex network of second messenger kinases and phosphatases. NF hyperphosphorylation and aggregation are pathologies associated with several neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) and ALS/Frontotemporal dementia (FTD), and are detected in plaques and tangles in Alzheimer’s Disease (AD). NF hyperphosphorylation slows axonal transport and is implicated in neurodegeneration and loss in pre- clinical mouse models of ALS, suggesting contributions to neurodegenerative disease etiology. However, the signaling pathways that specifically disrupt NF phosphorylation dynamics and increase NF phosphorylation are unresolved. Bridging this gap in knowledge would clarify fundamental processes of NF biology and may clarify initiating pathways involved in NF pathophysiology in disease. Preliminary studies here identify a signaling pathway that operates on the cell surface that, when disrupted, leads to NF hyperphosphorylation and neurodegeneration. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is one of three vertebrate- specific enzymes that act at the plasma membrane to cleave the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane and the only one expressed in neurons. Notably, GDE2 distribution and function are disrupted in ALS, ALS/FTD, and AD, suggesting potential contributions of GDE2 failure to disease pathology. Mice lacking GDE2 (Gde2KO) show robust increases in NF protein phosphorylation and age- progressive neurodegeneration, suggesting the overarching hypothesis that GDE2 regulates signaling events at the cell surface to regulate NF phosphorylation to ensure appropriate neuronal function and survival. Preliminary gain- and loss-of-function studies in cultured neurons and in vivo suggest the model that GDE2 regulates NF phosphorylation dynamics through cleavage and inactivation of the GPI-anchored protein RECK (Reversion- inducing Cysteine-rich protein with Kazal-like motifs). This model will be tested in this proposal in three Aims. Aim 1 will utilize in vitro and in vivo models to determine if RECK mediates GDE2-dependent regulation of NF phosphorylation and survival. Aims 2 and 3 will utilize structure-function studies, proteomic screens, and functional assays to determine the surface mechanism and downstream kinase pathways by which increased RECK surface activity promotes NF phosphorylation. Outcomes from these studies are expected to provide new insight into the fundamental regulatory mechanisms of NF phosphorylation and have the potential to shed light on disease-relevant pathways that are causal for NF abnormalities associated with disease.