Title: Characterization of pathogenic BicD2 mutations in vitro and in vivo
Description: Microtubule motor proteins play fundamental roles in transporting a broad range of cellular cargoes in most eukaryotic cells. While there are over 40 kinesins helping to accommodate these diverse cellular demands, there is only one major form of cytoplasmic dynein (dynein, hereafter) carrying out almost all aspects of microtubule (MT) minus end-directed cargo transport. Dynein achieves this versatility by utilizing a wide range of adaptor proteins. Bicaudal D2 (BicD2) is a dynein adaptor protein responsible for linking cytoplasmic dynein to multiple forms of subcellular cargo. These include Rab6A, which contributes to Golgi function (Grigoriev et al., 2007; Matanis et al., 2002); the nucleoporin RanBP2 (Splinter et al., 2010); and the nucleus-cytoplasmic linker LINC complex protein Nesprin-2 (Goncalves et al., 2020). The latter two proteins were found to play important specific roles in the developing brain, respectively in the oscillatory interkinetic nuclear migration (INM) behavior characteristic of Radial Glial Progenitor (RGP) cells and in the directed nuclear migration in postmitotic neurons traveling to the expanding cortical plate.

The BICD2 gene was implicated in the autosomal dominant forms of neuromuscular diseases such as Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2) (Neveling et al., 2013; Oates et al., 2013; Peeters et al., 2013; Synofzik et al., 2014) and, more recently in at least three developmental brain pathologies: polymicrogyria (Ravenscroft et al., 2016), cerebellar hypoplasia (Fiorillo et al., 2016), and Lissencephaly (Tsai et al., 2020) expanding the clinical spectrum from neuromuscular only to potentially the entire central nervous system. It is largely unknown how these diverse clinical presentations have any relations to the BicD2 mutational sites. To investigate the genotype-phenotype relationship, it is inevitable to study each point mutation molecularly and characterize the mutational effects in in vivo setting. To investigate the mutational effects of BicD2, I used five different BicD2 fragments and nine missense mutants and characterized their behavior using biochemical and cellular approaches.

Four of the missense mutations were further tested in the rat embryonic brain system, in which our lab has previously elucidated BicD2's roles during neurogenesis and post-mitotic neuronal migration. The chapters are organized by mutational effects on the BicD2-cargo interactions (Chapter 2) and the BicD2-dynein interactions (Chapter 3). In Chapter 4, I include supplemental materials to Chapters 2 and 3. In Chapter 5, I summarize, discuss, and provide my perspectives on all the mutational phenotypes found in the previous chapters. Lastly, all experimental procedures and reagents are described in Chapter 6. Here, I describe the first identification of novel gain-of-function (GoF) defects in BicD2- nuclear cargo interaction associated with two pathogenic mutations, R690C and E770G (Chapter 2). Furthermore, I characterize the GoF defects in the embryonic rat brain by in utero electroporation. The cell-type specific expression of R690C or E770G constructs revealed mutation-specific impairment of nuclear migrations in the developing cerebral cortex.

In addition, I found GoF defects in BicD2-dynein interaction associated with three pathogenic mutations, L679R, R690C, and T699, surprisingly, in the C-terminal cargo binding domain of BicD2 (Chapter 3). I demonstrate that these missense mutations cause a defect in BicD2 autoinhibition control, which in turn results in abnormally enhanced dynein association. I provide evidence for hyper-activation of BicD2 for dynein binding contributes to Golgi fragmentation, which has been associated with many neuromuscular diseases (Martinez-Menarguez et al., 2019), including SMA-LED2 (Martinez-Carrera and Wirth, 2015). The entirety of this work describes molecular defects in 9 representative BicD2 mutations in vitro and demonstrates the mutational effects in vivo. I propose that the differential mutational effects associated with the BicD2 mutations might contribute to the broad spectrum of clinical phenotypes seen in patients with BicD2 diseases.