Eric H. Schroeter

A presenilin-1-dependent |[gamma]|-secretase-like protease mediates release of Notch intracellular domain

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of γ-secretase-mediated cleavage of β-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimers disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by γ-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several γ-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of γ-secretase for the treatment of Alzheimers disease may risk toxicity caused by reduced Notch signalling.

A Ligand-Induced Extracellular Cleavage Regulates γ-Secretase-like Proteolytic Activation of Notch1

Gamma-secretase-like proteolysis at site 3 (S3), within the transmembrane domain, releases the Notch intracellular domain (NICD) and activates CSL-mediated Notch signaling. S3 processing occurs only in response to ligand binding; however, the molecular basis of this regulation is unknown. Here we demonstrate that ligand binding facilitates cleavage at a novel site (S2), within the extracellular juxtamembrane region, which serves to release ectodomain repression of NICD production. Cleavage at S2 generates a transient intermediate peptide termed NEXT (Notch extracellular truncation). NEXT accumulates when NICD production is blocked by point mutations or gamma-secretase inhibitors or by loss of presenilin 1, and inhibition of NEXT eliminates NICD production. Our data demonstrate that S2 cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch activation.

Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1.

The Notch genes encode single-pass transmembrane receptors that transduce the extracellular signals responsible for cell fate determination during several steps of metazoan development. The mechanism by which extracellular signals affect gene transcription and ultimately cell fate decisions is beginning to emerge for the Notch signalling pathway. One paradigm is that ligand binding to Notch triggers a Presenilin1-dependent proteolytic release of the Notch intracellular domain from the membrane, resulting in low amounts of Notch intracellular domain which form a nuclear complex with CBF1/Su(H)/Lag1 to activate transcription of downstream targets. Not all observations clearly support this processing model, and the most rigorous test of it is to block processing in vivo and then determine the ability of unprocessed Notch to signal. Here we report that the phenotypes associated with a single point mutation at the intramembranous processing site of Notch1, Val1,744→Gly, resemble the null Notch1 phenotype. Our results show that efficient intramembranous processing of Notch1 is indispensable for embryonic viability and proper early embryonic development in vivo.

Conditional targeted cell ablation in zebrafish: A new tool for regeneration studies

Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal‐ and spatial‐specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross‐linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein–NTR (CFP‐NTR) under control of tissue‐specific promoters allowed us to induce the death of cardiomyocytes, pancreatic β‐cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration. Developmental Dynamics 236:1025–1035, 2007.

γ-Secretase inhibitors repress thymocyte development

A major therapeutic target in the search for a cure to the devastating Alzheimers disease is γ-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Aβ42 peptides, precipitates of which are thought to cause the disease. γ-Secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of γ-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4−/CD8− state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological γ-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of γ-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.

Ectodomain shedding and intramembrane cleavage of mammalian Notch proteins is not regulated through oligomerization.

Intramembrane cleaving proteases such as site 2 protease, γ-secretase, and signal peptide peptidase hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. It has been proposed that removing the extracellular domain allows dissociation of substrate TMD, held together by the extracellular domain or loop. Using γ-secretase as a model intramembrane cleaving protease and Notch as a model substrate, we investigated whether activating and inactivating mutations in Notch modulate γ-secretase cleavage through changes in oligomerization. We find that although the Notch epidermal growth factor repeats can promote dimer formation, most surface Notch molecules in mammalian cells are monomeric as are constitutively active or inactive Notch1 proteins. Using a bacterial assay for TM dimerization, we find that the isolated TMD of Notch and amyloid precursor protein self-associate and that mutations affecting Notch cleavage by γ-secretase cleavage do not alter TMD dimerization. Our results indicate that ligand-induced reversal of controlled TMD dimerization by the Notch extracellular domain is unlikely to underlie the regulatory mechanism of intramembranous cleavage.

In vivo development of retinal ON-bipolar cell axonal terminals visualized in nyx::MYFP transgenic zebrafish

Axonal differentiation of retinal bipolar cells has largely been studied by comparing the morphology of these interneurons in fixed tissue at different ages. To better understand how bipolar axonal terminals develop in vivo, we imaged fluorescently labeled cells in the zebrafish retina using time-lapse confocal and two photon microscopy. Using the upstream regulatory sequences from the nyx gene that encodes nyctalopin, we constructed a transgenic fish in which a subset of retinal bipolar cells express membrane targeted yellow fluorescent protein (MYFP). Axonal terminals of these YFP-labeled bipolar cells laminated primarily in the inner half of the inner plexiform layer, suggesting that they are likely to be ON-bipolar cells. Transient expression of MYFP in isolated bipolar cells indicates that two or more subsets of bipolar cells, with one or two terminal boutons, are labeled. Live imaging of YFP-expressing bipolar cells in the nyx::MYFP transgenic fish at different ages showed that initially, filopodial-like structures extend and retract from their primary axonal process throughout the inner plexiform layer (IPL). Over time, filopodial exploration becomes concentrated at discrete foci prior to the establishment of large terminal boutons, characteristic of the mature form. This sequence of axonal differentiation suggests that synaptic targeting by bipolar cell axons may involve an early process of trial and error, rather than a process of directed outgrowth and contact. Our observations represent the first in vivo visualization of axonal development of bipolar cells in a vertebrate retina.

Laminar circuit formation in the vertebrate retina

Neuronal function depends on the accurate wiring between pre- and postsynaptic cells. Determining the mechanisms underlying precision in neuronal connectivity is challenging because of the complexity of the nervous system. In diverse parts of the nervous system, regions of synaptic contact are organized into distinct parallel layers, or laminae, that are correlated with distinct functions. Such an arrangement enables the development of synapse specificity to be more readily investigated. Here, we present an overview of the developmental mechanisms that are thought to underlie the formation of synaptic layers in the vertebrate retina, a highly laminated CNS structure. We will contrast the roles of activity-dependent and activity-independent mechanisms in establishing functionally discrete sublaminae in the inner retina, where circuits involving many subtypes of retinal neurons are assembled precisely. In addition, we will discuss new optical imaging approaches for elucidating how retinal synaptic lamination occurs in vivo.