Clathrin Assembly Protein AP180 and CALM Differentially Control Axogenesis and Dendrite Outgrowth in Embryonic Hippocampal Neurons

Abstract

Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The clathrin assembly proteins AP180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALMdeficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…Clathrin-coated vesicles from this recycling process that have been isolated from mammalian brain are the principal source of material for most biochemical and structural analyses. The presence of brain-specific isoforms of clathrin light chains (Jackson et al 1987;Kirchhausen et al 1987), auxilin (Umeda et al 2000), dynamin (Urrutia et al 1997), AP180 (Bushlin et al 2008), and various other proteins of the presynaptic membrane-recycling pathway may reflect features of the clathrin cycle in synapses that are not shared with classical, receptor-mediated endocytosis as studied in epithelial cells and fibroblasts.…”

Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons

“…Cultures of hippocampal neurons were prepared from embryonic day 18 rat brains as described previously (Mattson et al, 1989;Kaech and Banker, 2006;Bushlin et al, 2008). ShhN was added to neurons between 10 and 19 days in vitro (div) and experiments were performed 2-3 days later.…”

“…Transfection was carried out using a calcium-phosphate-based kit (Clontech, catalog no. 631312) (Hering and Sheng, 2003;Jiang and Chen, 2006;Bushlin et al, 2008).…”

P2‐030: Investigating the Role of Clu, Picalm, and Cr1 in Alzheimer's Disease

“…Indeed, Bushlin et al had previously provided evidence that PICALM and AP180 were involved in the normal development and growth of hippocampal neurons (Bushlin et al 2008). Schwartz's group also looked at the expression of both long and short isoforms of PICALM, and found expression of each, but following opposite trends.…”

“…There is evidence that PICALM is important in early development and growth of neurons, since PICALM deficient cells have been shown to lack normal dendrite structures (Bushlin et al 2008;Schwartz et al 2010). Conversely, AP180 lacking cells develop normal dendrites but do not show normal axonal development (Schwartz et al 2010).…”