Neuralized family member NEURL1 is a ubiquitin ligase for the cGMP-specific phosphodiesterase 9A.

Neuralized functions as a positive regulator of the Notch pathway by promoting ubiquitination of Notch ligands via its E3 ligase activity, resulting in their efficient endocytosis and signaling. Using a yeast two-hybrid screen, we have identified a cGMP-hydrolysing phosphodiesterase, PDE9A, as a novel interactor and substrate of Neuralized E3 ubiquitin protein ligase 1 (NEURL1). We confirmed this interaction with co-immunoprecipitation experiments and show that both Neuralized Homology Repeat domains of NEURL1 can interact with PDE9A. We also demonstrate that NEURL1 can promote polyubiquitination of PDE9A that leads to its proteasome-mediated degradation mainly via lysine residue K27 of ubiquitin. Our results suggest that NEURL1 acts as a novel regulator of protein levels of PDE9A.

Rat NEURL1 3'UTR is alternatively spliced and targets mRNA to dendrites.

Neuralized, an E3 ubiquitin ligase, interacts with and positively modulates the Notch pathway by promoting ubiquitination and subsequent endocytosis of its ligands. NEURL1 mRNA is dendritically localised in the dentate gyrus of an adult rat brain, implying that it may be locally translated, but its transport mechanisms remain unstudied. Here, we report the presence of a previously unknown, shorter splice-variant of rat NEURL1 3'UTR (1477bp in length), and identify it as a potential target of nonsense-mediated decay. We show that endogenous NEURL1 mRNAs with both longer and shorter 3'UTRs are enriched in the neurites of cultured rat primary hippocampal neurons. Both NEURL1 3'UTRs can mediate transport of reporter mRNAs into dendrites in primary hippocampal neurons. By analysing the dendritic trafficking capacity of reporter mRNAs linked to various regions of longer or shorter NEURL1 3'UTR, we localise the dendritic targeting element (DTE) of spliced version of NEURL1 3'UTR to its first half, corresponding to the nucleotides 1-148 and 416-914 of the full-length 3'UTR. In contrast, the dendritic targeting capacity of the full-length NEURL1 3'UTR is abolished by splitting its 3'UTR in two halves (nt 1-914 and nt 915-1744), suggesting that slightly different DTE might mediate dendritic transport of the two transcripts.

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord.

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine "irregular" patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.

Neuralized-2: expression in human and rodents and interaction with Delta-like ligands.

Delta-Notch signaling is a universal cell-cell communication pathway crucial for numerous developmental and physiological processes. Several proteins interact with and regulate the Notch pathway, including the E3 ubiquitin ligase Neuralized (Neur) that influences the stability and activity of Notch ligands. In mammals there are two homologs of Neur, Neur1 and Neur2, that both can interact with Notch ligands Delta-like1 and Jagged1. Here, we show that Neur2, in contrast to Neur1, is highly expressed during embryonic development of the brain and several non-neural tissues and its mRNA levels subside postnatally. In the hippocampal neurons of the adult brain Neur2 transcripts, in contrast to Neur1, are excluded from the dendrites. Neur2 protein has a predominantly cytoplasmic localization. We also show that in addition to Delta-like1, Neur1 and Neur2 interact with another Notch ligand, Delta-like4.

Identification of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease gene presenilin2.

Presenilins play prominent roles in the molecular pathogenesis of Alzheimer's disease and during embryo development. We have isolated a zebrafish presenilin orthologue (pre2), which shows a high degree of sequence identity to the human PS2 protein. Zebrafish pre2 is maternally and ubiquitously expressed during early embryo development, whereas Pre2 protein expression is initiated between 6 and 12 hours post fertilisation (hpf), suggesting strict regulation of pre2 translation. pre2 expression is especially high in neural-crest-derived melanocytes.

The identity and distribution of neural cells expressing the mesodermal determinant spadetail.

BACKGROUND: The spadetail (spt) gene of zebrafish is expressed in presomitic mesoderm and in neural cells previously suggested to be Rohon-Beard neurons. The mechanism(s) generating the apparently irregular rostrocaudal distribution of spt-expressing cells in the developing CNS is unknown. RESULTS: spt-expressing neural cells co-express huC, a marker of neurons. These cells also co-express the genes islet-1, -2 and -3 but not valentino. The islet-1 gene expression, irregular distribution and dorsolateral position of spt-expressing cells in the developing CNS are characteristic of dorsal longitudinal ascending (DoLA) interneurons. Shortly after their birth, these neurons extend processes rostrally into which spt mRNA is transported. At 24 hours post fertilisation(hpf), spt-expressing neurons occur most frequently at rostral levels caudal of the 5th-formed somite pair. There is no apparent bias in the number of spt-expressing cells on the left or right sides of embryos. Extended staining for spt-transcription reveals expression in the dorsocaudal cells of somites at the same dorsoventral level as the spt-expressing neurons. There is frequent juxtaposition of spt-expression in newly formed somites and in neurons. This suggests that both types of spt-expressing cell respond to a common positional cue or that neurons expressing spt are patterned irregularly by flanking somitic mesoderm. CONCLUSIONS: spt-expressing cells in the developing CNS appear to be DoLA interneurons. The irregular distribution of these cells along the rostrocaudal axis of the spinal cord may be due to "inefficient" patterning of neural spt expression by a signal(s) from flanking, regularly distributed somites also expressing spt.