Epsins function in Notch signaling activation

In mechanistic terms, endocytosis is the process by which plasma membrane (PM) components, together with extracellular solutes, macromolecules and particles, are internalized in the cell. Once the endocytic vesicle (or vacuole) is formed by fission of the PM, it is generally delivered to a specialized membrane compartment – the endosome – for recycling, degradation or re-routing. In cell-physiological terms, endocytosis exerts multiple functions, which are only partially known and characterized. At a minimum, it maintains PM homeostasis by counterbalancing the apposition of new membrane (due to exocytosis) and by renewing PM components. More extensively, endocytosis constantly modulates PM composition and takes an active part in a variety of normal and pathological cell processes, including cell nutrition, cell motility, mitosis, neurotransmission, immune response, and microorganism entry. In recent years, much of the effort to investigate this extensive endocytic activity has been focused upon unveiling the reciprocal interplay between endocytosis and cell signaling. Our laboratory, in collaboration with the laboratory of Pietro De Camilli (Yale University, USA) has pioneered the use of genetic models in mice to study several aspects of the endocytic function, mostly at the synapse. Recently, we generated mice models for a highly conserved gene family of multidomain adaptors – the epsin family - whose function was linked to endocytosis. By characterizing the phenotypic defects of the epsin1/2 double knockout mice, we found that epsins are essential components of the machinery required for Notch signaling activation during embryogenesis in mammals [1]. More recently, we characterized that epsins molecular action is exerted in a ubiquitin- dependent endocytic reaction that triggers the internalization of the Notch ligand, a process necessary for the activation of the Notch receptor. Our preliminary data extend epsin function to Notch signaling activation in primary keratinocytes and to VEGF signaling modulation in angiogenesis

Recruitment of Endophilin to Clathrin-Coated Pit Necks Is Required for Efficient Vesicle Uncoating after Fission

SummaryEndophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself

Zinc-finger and helix-loop-helix transcription factors regulate Purkinje neuron neurogenesis and cerebellar corticogenesis

Many regulatory genes have been pinpointed as orchestrators of cerebellar development, from the onset of neurogenesis to the patterning of the adult cerebellar cortex, with a special reference to the development of cerebellar Purkinje cells (PCs). PCs provide the sole output from cerebellar cortical circuits, where each PC integrates myriads of presynaptic inputs, both inhibitory and excitatory. In the murine cerebellar primordium PCs are generated from a pool of ventricular zone progenitors facing the fourth ventricle between embryonic day (E) 10.5 and 13.5. This progenitor pool expands in the ventricular zone (VZ) through symmetric cell division until E10.5, when a gradual switch to asymmetric cell division occurs, regulated by Notch1 (Lutolf et al., 2002) and its interactor (Masserdotti et al., 2010), the Zn-finger TF Zfp423 (Alcaraz et al., 2006; Warming et al., 2006; Croci et al., submitted). Zfp423 was recently implicated in Joubert syndrome and cerebellar vermis hypoplasia (Chaki et al., 2012). Allelic mutations of Zfp423 produce distinct alterations in PC development (Croci et al., submitted). PCs arise from a pool of progenitors positive for the basic-helix-loop-helix transcription factors (TFs) neurogenin (Ngn) 1 and 2 (Zordan et al., 2008; Lundell et al., 2009). Ngn2 regulates cell cycle progression and dendritic arbor generation in PC precursors (Florio et al., 2012). PCs also express HLH transcription factors of the Olf/EBF family. In Ebf2 -/- mutants, PC migration and survival are affected (Croci et al., 2006). Neonatal PC death is due to local downregulation of Igf1 gene expression (Croci et al., 2011). Finally, EBF2 regulates cortical patterning in the adult cerebellum, regulating its subdivision into alternate parasagittal stripes of distinct PC subtypes. Indeed, EBF2 is required to repress the zebrin II+ phenotype in postnatal PCs (Croci et al., 2006; Chung et al., 2008)

Mouse Models for Epsin Function

The vertebrate skin is a barrier-forming organ in which keratinocytes form a highly organized, stratified epithelium protecting the organism from the outside environment. One of the major regulators of this structure is Notch signaling. Notch is a transmembrane receptor interacting with ligands expressed on the surface of neighboring keratinocytes. Keratinocyte-specific deletion of Notch signaling pathway impairs epidermal differentiation, resulting in skin-barrier defects and skin carcinogenesis. Our laboratory, by a genetic approach in mice, demonstrates that combined inactivation of Epsin1 and Epsin2 genes leads to embryonic lethality around E9.5-10. The phenotype of Epn1;Epn2 double knockout is characterized by a subversion of the three main developmental programs active at this developmental stage, i.e., cardiovascular development, somitogenesis, and neural tube differentiation. Collectively, these morphological alterations resemble the developmental defects observed in mutants of Notch genes or in genes essential for the activation of the Notch signaling pathway, suggesting a crucial role of Epsin in enabling Notch signaling during embryogenesis. Intriguingly, the apparently healthy Epn1+/-;Epn2-/- shows an high incidence of squamous papillomas on their skin. Furthermore, expression of another epsin family member originally localized exclusively to surface epithelia, Epsin3, dramatically increases in the hyperplastic lesions of the three-allele mutants and in human basal carcinomas. In order to get further insight on Epn3 function we performed morphological expression analyses during mouse development. In contrast with initial reports, both in embryos and adults, we could detect various levels of Epn3 expression in several tissues, i.e., surface epithelia, neural tissue and heart. Moreover, in vitro studies performed on human keratinocytes in culture show a prominent role of this Epsin in the regulation of Notch signaling in this cell compartment

ZFP423, a transcription factor implicated in Joubert Syndrome and Cerebellar Vermis Hypoplasia, orchestrates the pace and mode of cerebellar neurogenesis

Neurogenesis is a tightly regulated process, both in the embryonic and in the adult brain. Its success depends on the ability of a germinative epithelium to establish the appropriate balance between maintaining an undifferentiated progenitor pool and giving birth to sequential generations of neurons and glia. The Zfp423 gene encodes a 30 Zn-finger transcription factor (TF) which interacts with the SMAD1- SMAD4 complex (BMP signaling), Notch intracellular domain, retinoic acid receptors and Collier/Olf-1/EBF TFs. This gene has been previosly implicated in cerebellar development. Mutations in the human ortholog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), and/ or exhibiting other signs of ciliopathy outside the central nervous system. We have been analyzing two mouse mutant lines carrying allelic in-frame deletions of Zfp423. One of them lacks Zn-finger domains 9-20 (Δ9-20), implicated in BMP and Notch signal transduction, while the other lacks a C-terminal domain (Δ28-30). Both mutants exhibit cerebellar malformations and severe ataxia. However, our results indicate that the two protein domains play sharply distinct roles in the context of cerebellar neurogenesis. In Zfp423Δ9-20/Δ9-20 mutants, GABAergic Purkinje cell (PC) neurogenesis is impaired and the PC progenitor pool in the ventricular zone is precociously depleted. Conversely, Zfp423Δ28-30/Δ28-30 mutants display a selective impairment in the development of glutamatergic cerebellar neurons

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50–60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS