The Key Regulator Endophilin-A

ENDOPHILINS BAR AND SH3DOMAINS ARE REQUIRED DIVERSE FUNCTIONS

Through interactions with a large number of proteins predominantly involved in endocytic processes, endophilin is seen as a hub that orchestrates the induction and stabilization of membrane curvature, bud constriction, fission, and uncoating in neurons (see Fig. 1-4B; Saheki and Camilli, 2012). In vertebrates, three members of the endophilin-A family have been identified, of which endophilin-A1 is brain specific, endophilin-A2 is ubiquitously expressed, and endophilin-A3 is enriched in brain and testis (Giachino et al., 1997; Ringstad et al., 1997). The three endophilin genes are highly similar, which allows for partial compensation if one or more endophilin alleles are absent ( see Fig. 1-4A; Milosevic et al., 2011; Murdoch et al., 2016).

In general, endophilins contain an N-terminal BAR (Bin-Amphiphysin-Rvs) domain and a C-terminal SH3 domain and form crescent-shaped homodimers (Gallop et al., 2006; Ringstad et al., 1997). The BAR domain of endophilin was shown to induce membrane curvature, to stabilize existing membrane invaginations, and to sense membrane curvature in order to recruit further proteins to the neck of CCPs (Farsad et al., 2001; Gallop et al., 2006; Masuda et al., 2006). Recent studies suggest that the organized recruitment of endophilin to the nucleation sites in neurons is facilitated by the multi-domain scaffolding protein intersectin-1 via interaction of their SH3 domains (Pechstein et al., 2015). The SH3 domain of endophilin further interacts with the proline-rich domains of dynamin (Ferguson et al., 2009; Ringstad et al., 1997) and of synaptojanin-1 (Perera et al., 2006; Schuske et al., 2003; Verstreken et al., 2003).

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Fig. 1-4: Absence of endophilin-A results in disturbed SV recycling. (A) The three mammalian endophilin-As show a high similarity. All three contain a BAR domain and an SH3 domain. (B) Endophilin-A is involved in different steps of CME via regulating the fission and uncoating process. (C) Endophilin 1/3-DKOs show increasing motor defects with age, 1/2-1/3-DKOs have a truncated life expectancy paired with major neurological and motor defects. TKOs survive few hours after birth. (D) Absence of endophilins leads to accumulations of CCVs and reduced numbers in SVs in 1/2-DKOs and even more prominently in TKOs.

Images modified from (Milosevic et al., 2011; Murdoch et al., 2016).

KNOCKOUT MODELS FOR ENDOPHILIN

Even though endophilin is active in different steps of CME/ clathrin-dependent SV reformation, particularly the uncoating process seems to be of physiological relevance, as studied in diverse animal models: Genetic studies in Drosophilia, C. elegans and mice revealed accumulations of CCVs accompanied by impaired synaptic transmission in absence of endophilins (see Fig. 1-4D; Dickman

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et al., 2005; Milosevic et al., 2011; Schuske et al., 2003; Verstreken et al., 2003). A similar phenotype has been described for synaptojanin-1 KO models (Cremona et al., 1999; Milosevic et al., 2011).

Absence of all three endophilins in mice (see Fig. 1-4C; triple-knockouts; TKOs) was shown result in a life expectancy of only few hours after birth, likely induced by impaired synaptic transmission, breathing problems, and the inability to drink milk (Milosevic et al., 2011). Hippocampal neurons of TKOs exhibit accumulations of CCVs, whereas numbers of uncoated SVs are significantly decreased (Milosevic et al., 2011). Electrophysiological recordings in those TKO cells further indicated reduced rates of sustained exocytosis, which may be a result of impaired SV recycling or indicate a potential involvement of endophilin in SV fusion and/ or SV replenishment (Milosevic et al., 2011). Double knockouts lacking endophilin A1 and A2 (1/2-DKOs) have a drastically truncated life expectancy of only 2-3 weeks, a strong growth delay, as well as major neurological and motor impairments (Milosevic et al., 2011). Mutants missing endophilin A1 and A3 (1/3-DKOs) display motor impairments and epileptic seizures with age-dependent increasing severity (Milosevic et al., 2011;

Murdoch et al., 2016). Additional heterozygous deletion of endophilin A2 further increases these symptoms. Eventually, these observations have been linked to increased apoptosis rates in neurons (Milosevic et al., 2011; Murdoch et al., 2016). Whether absence of endophilins results in hearing defects and impaired endocytosis in IHCs has been investigated in the first chapter of my studies.

FUNCTIONS OF ENDOPHILIN BEYOND ENDOCYTOSIS

It has been ascertained that not only defective endocytosis, but also disturbed protein homeostasis via upregulation of the cellular protein degradation system (autophagy) are responsible for neurodegenerative processes in endophilin mutants (Murdoch et al., 2016). This finding goes along with previous studies, in which interactions of endophilin with proteins involved in Park disease, namely parkin and LRRK2, have been identified (Cao et al., 2014; Soukup et al., 2016).

Endophilin has furthermore been shown to interact with voltage-gated Ca²⁺-channels (Chen et al., 2003). Performing co-immunoprecipitations and pull-downs, the authors detected a Ca²⁺-dependent formation of complexes formed by endophilin and Ca²⁺-channels. Therefore, they concluded that endophilin is involved in the coupling of exo- and endocytosis (Chen et al., 2003).

Lastly, we could show in work not included in this thesis that endophilin is involved in the recruitment, priming, and fusion of large dense core vesicles (LDCVs) in chromaffin cells

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(Gowrisankaran et al., unpublished). These neuroendocrine cells are a favored model system to study neurosecretion, as key players in Ca²⁺-induced neurotransmitter release are identical in chromaffin cells and in neurons (De Camilli and Jahn, 1990; Neher, 2006). We could show that exocytosis in endophilin TKOs is reduced while numbers of LDCVs and cell size as well as SV recycling are unaffected. However, distances of LDCVs to the cell membrane, where release takes place, were found to be increased in adrenal gland tissue of TKOs pointing towards deficits in SV replenishment.

In a multi-methodological approach, we could show that this novel role of endophilin in LDCV recruitment and release is, at least in parts, achieved by the interaction of endophilin and intersectin.