Human and mouse ZFP57 proteins are functionally interchangeable in maintaining genomic imprinting at multiple imprinted regions in mouse ES cells.

Genomic imprinting is a common epigenetic phenomenon in mammals. Dysregulation of genomic imprinting has been implicated in a variety of human diseases. ZFP57 is a master regulator in genomic imprinting. Loss of ZFP57 causes loss of DNA methylation imprint at multiple imprinted regions in mouse embryos, as well as in embryonic stem (ES) cells. Similarly, mutations in human ZFP57 result in hypomethylation at many imprinted regions and are associated with transient neonatal diabetes and other human diseases. Mouse and human Zfp57 genes are located in the same syntenic block. However, mouse and human ZFP57 proteins only display about 50% sequence identity with different number of zinc fingers. It is not clear if they share similar mechanisms in maintaining genomic imprinting. Here we report that mouse and human ZFP57 proteins are functionally interchangeable. Expression of exogenous wild-type human ZFP57 could maintain DNA methylation imprint at three imprinted regions in mouse ES cells in the absence of endogenous mouse ZFP57. However, mutant human ZFP57 proteins containing the mutations found in human patients could not substitute for endogenous mouse ZFP57 in maintaining genomic imprinting in ES cells. Like mouse ZFP57, human ZFP57 and its mutant proteins could bind to mouse KAP1, the universal cofactor for KRAB zinc finger proteins, in mouse ES cells. Thus, we conclude that mouse and human ZFP57 are orthologs despite relatively low sequence identity and mouse ES cell system that we had established before is a valuable system for functional analyses of wild-type and mutant human ZFP57 proteins.

Doublecortin (DCX) mediates endocytosis of neurofascin independently of microtubule binding.

Doublecortin on X chromosome (DCX) is one of two major genetic loci underlying human lissencephaly, a neurodevelopmental disorder with defects in neuronal migration and axon outgrowth. DCX is a microtubule-binding protein, and much work has focused on its microtubule-associated functions. DCX has other reported binding partners, including the cell adhesion molecule neurofascin, but the functional significance of the DCX-neurofascin interaction is not understood. Neurofascin localizes strongly to the axon initial segment in mature neurons, where it plays a role in assembling and maintaining other axon initial segment components. During development, neurofascin likely plays additional roles in axon guidance and in GABAergic synaptogenesis. We show here that DCX can modulate the surface distribution of neurofascin in developing cultured rat neurons and thereby the relative extent of accumulation between the axon initial segment and soma and dendrites. Mechanistically, DCX acts via increasing endocytosis of neurofascin from soma and dendrites. Surprisingly, DCX increases neurofascin endocytosis apparently independently of its microtubule-binding activity. We additionally show that the patient allele DCXG253D still binds microtubules but is deficient in promoting neurofascin endocytosis. We propose that DCX acts as an endocytic adaptor for neurofascin to fine-tune its surface distribution during neuronal development.

Compartmentalizing the neuronal plasma membrane from axon initial segments to synapses.

Many membrane proteins localize to restricted domains in neurons, such as axons, dendrites, synapses, or axon initial segments. The exquisite subcellular compartmentalization of adhesion molecules, growth factor receptors, signaling receptors, voltage-gated and ligand-gated channels, and others underlies the complex functioning of neurons and ultimately vectorial propagation of signaling in neuronal circuits. This chapter discusses the cellular mechanisms for compartmentalizing the neuronal plasma membrane. Among the mechanisms contributing to protein segregation in the membrane are sorting and targeting in the Golgi/TGN, endocytosis, recycling, and degradation, and control of membrane protein diffusion. The molecular underpinnings of these cellular mechanisms are reviewed in the first part. The second part discusses the contribution of each cellular mechanism to targeting proteins to axons and dendrites, to synapses, to axon initial segments, and to Nodes of Ranvier. For most, if not all proteins and locations, all four mechanisms are in effect and additively contribute to the precise localization of membrane proteins in neurons. Since disruption of proper protein distribution results in defects in neuronal function and can lead to neurodegenerative diseases, a full understanding of the cellular mechanisms of plasma membrane compartmentalization is an important goal for the future.

Ankyrin-dependent and -independent mechanisms orchestrate axonal compartmentalization of L1 family members neurofascin and L1/neuron-glia cell adhesion molecule.

Axonal initial segments (IS) and nodes of Ranvier are functionally important membrane subdomains in which the clustering of electrogenic channels enables action potential initiation and propagation. In addition, the initial segment contributes to neuronal polarity by serving as a diffusion barrier. To study the mechanisms of axonal compartmentalization, we focused on two L1 family of cell adhesion molecules (L1-CAMs) [L1/neuron-glia cell adhesion molecule (L1/NgCAM) and neurofascin (NF)] and two neuronal ankyrins (ankB and ankG). NF and ankG accumulate specifically at the initial segment, whereas L1/NgCAM and ankB are expressed along the entire lengths of axons. We find that L1/NgCAM and NF show distinct modes of steady-state accumulation during axon outgrowth in cultured hippocampal neurons. Despite their different steady-state localizations, both L1/NgCAM and NF show slow diffusion and low detergent extractability specifically in the initial segment but fast diffusion and high detergent extractability in the distal axon. We propose that L1-CAMs do not strongly bind ankB in the distal axon because of spatial regulation of ankyrin affinity by phosphorylation. NF, conversely, is initially enriched in an ankyrin-independent manner in the axon generally and accumulates progressively in the initial segment attributable to preferential binding to ankG. Our results suggest that NF and L1/NgCAM accumulate in the axon by an ankyrin-independent pathway, but retention at the IS requires ankyrin binding.