Proteomics and genetic studies of dystroglycan function in the nervous system

Muscular dystrophies are a group of diseases that are often caused by loss-of-function mutations affecting the dystrophin glycoprotein complex (DGC). The common feature of the diseases is muscle degeneration, which is often associated with mental retardation and various retinal defects, including ones of synaptic transmission. However, the mechanisms of the disease remain largely unknown, especially those in the central nervous system. I have focused on dystroglycan (DG), the transmembrane protein in the DGC that links the cytoskeleton to the extracellular matrix and is essential for muscle survival and brain development. I have used proteomics and Drosophila genetics to study DG function in the brain and retina.Using proteomics I found that beta-DG is directly associated with the GTPase dynamin 1 in the retina and in the brain together with alpha-DG and Grb2, and immunohistochemically beta-DG was colocalized with dynamin 1 in the outer plexiform layer where photoreceptor terminals are localized. Moreover, loss of DG in differentiated DG-null embryonic stem cells significantly increases dynamin-mediated transferrin-uptake and re-expression of DG in null cells by infection with an adenovirus containing DG reduced transferrin uptake to levels seen in wild-type cells. This result implies that one of mechanisms in muscular dystrophy might be the altered synaptic vesicle endocytosis, especially in the retina where synaptic transmission defect has been known for decades.Muscular dystrophies show not only impaired retinal synaptic transmission and several DG-related congenital muscular dystrophies also display retinal structural defects. To further understand the roles of DG in the retina, I used Drosophila eye as a model and demonstrated for the first time that DG is required cell-autonomously for photoreceptor morphogenesis in the developing visual system. Deficiency of DG in the eye causes severe disruption of retinal structure, aberrant lens formation and abolition of electroretinogram in the adult fly eye. These adult defects appear derived from autonomous photoreceptor cell (PRC) defects in the early pupa including size arrest, loss of polarity and progressive degeneration. All defects in the eye, however, can be reversed by re-expression of wild type DG in DG-deficient PRCs, suggesting DG functions cell-autonomously in PRCs and non-autonomously for lens. In the 3rd instar larvae DG is present in the apical tips and the basal membranes of PRCs, two polarized locations opposing the extracellular matrix. At the pupal stage it continues to mainly distribute at the apical rhabdomere and basal membrane of PRCs. Over-expression of DG leads to larger ommatidia but the PRC number remains unchanged, suggesting that DG is both necessary for and sufficient to promote PRC expansion. By rescue experiments, I demonstrated that the extracellular DG alone could not rescue DG-deficient eye defects, whereas the intracellular DG can substantially ameliorate PRC degeneration and structural defects while some PRCs remain disorganized, a sign of disrupted PRC planar polarity in absence of the extracellular DG. Therefore, our data suggest that the degeneration and planar polarity disruption in DG-deficient PRCs are two independent processes that appear to require the respective function of intracellular and extracellular DG. In summary, our experiments demonstrated several novel findings and provided the basis for future investigations on DG function and the molecular mechanisms of nervous system defects in muscular dystrophies

Abelson Phosphorylation of CLASP2 Modulates its Association With Microtubules and Actin

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. We previously showed that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) CLASP in Drosophila. Here we show in vertebrate cells that Abl binds to CLASP and phosphorylates it in response to serum or PDGF stimulation. In vitro, Abl phosphorylates CLASP with a Km of 1.89 µM, indicating that CLASP is a bona fide substrate. Abl-phosphorylated tyrosine residues that we detect in CLASP by mass spectrometry lie within previously mapped F-actin and MT plus end interaction domains. Using purified proteins, we find that Abl phosphorylation modulates direct binding between purified CLASP2 with both MTs and actin. Consistent with these observations, Abl-induced phosphorylation of CLASP2 modulates its localization as well as the distribution of F-actin structures in spinal cord growth cones. Our data suggest that the functional relationship between Abl and CLASP2 is conserved and provides a means to control the CLASP2 association with the cytoskeleton. © 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc

Dystroglycan and Mitochondrial Ribosomal Protein L34 Regulate Differentiation in the Drosophila Eye

Mutations that diminish the function of the extracellular matrix receptor Dystroglycan (DG) result in muscular dystrophies, with associated neuronal migration defects in the brain and mental retardation e.g. Muscle Eye Brain Disease. To gain insight into the function of DG in the nervous system we initiated a study to examine its contribution to development of the eye of Drosophila melanogaster. Immuno-histochemistry showed that DG is concentrated on the apical surface of photoreceptors (R) cells during specification of cell-fate in the third instar larva and is maintained at this location through early pupal stages. In point mutations that are null for DG we see abortive R cell elongation during differentiation that first appears in the pupa and results in stunted R cells in the adult. Overexpression of DG in R cells results in a small but significant increase in their size. R cell differentiation defects appear at the same stage in a deficiency line Df(2R)Dg 248 that affects Dg and the neighboring mitochondrial ribosomal gene, mRpL34. In the adult, these flies have severely disrupted R cells as well as defects in the lens and ommatidia. Expression of an mRpL34 transgene rescues much of this phenotype. We conclude that DG does not affect neuronal commitment but functions R cell autonomously to regulate neuronal elongation during differentiation in the pupa. We discuss these findings in view of recent work implicating DG as a regulator of cell metabolism and its genetic interaction with mRpL34, a member of a class of mitochondrial genes essential for norma