Imaging activity-dependent regulation of neurexin-neuroligin interactions using trans-synaptic enzymatic biotinylation.

The functions of trans-synaptic adhesion molecules, such as neurexin and neuroligin, have been difficult to study due to the lack of methods to directly detect their binding in living neurons. Here, we use biotin labeling of intercellular contacts (BLINC), a method for imaging protein interactions based on interaction-dependent biotinylation of a peptide by E. coli biotin ligase, to visualize neurexin-neuroligin trans-interactions at synapses and study their role in synapse development. We found that both developmental maturation and acute synaptic activity stimulate the growth of neurexin-neuroligin adhesion complexes via a combination of neurexin and neuroligin surface insertion and internalization arrest. Both mechanisms require NMDA receptor activity. We also discovered that disruption of activity-induced neurexin-neuroligin complex growth prevents recruitment of the AMPA receptor, a hallmark of mature synapses. Our results provide support for neurexin-neuroligin function in synapse maturation and introduce a general method to study intercellular protein-protein interactions.

Dynamic endogenous association of neurofilament mRNAs with K-homology domain ribonucleoproteins in developing cerebral cortex.

The low, middle, and high molecular mass neurofilament subunit proteins (NF-L, NF-M, and NF-H) co-polymerize to form neurofilaments (NFs). During development, NF subunit expression is highly regulated, and in neurodegenerative disease, aberrant regulation of this expression can lead to the formation of harmful aggregates. NF expression in both development and disease is under significant post-transcriptional control, but the specific ribonucleoproteins (RNPs) involved are only poorly understood. Previously, mass spectrometry on affinity purified proteins from rat brain identified three K-homology (KH) domain RNPs - hnRNP K, hnRNP E1, hnRNP E2 - as being capable of binding NF-M RNA. In the current study, to determine whether these RNPs associate with NF mRNAs endogenously, we performed a co-immunoprecipitation assay on homogenates of postnatal and developing rat cerebral cortex. We found that all three NF mRNAs indeed associated endogenously with these RNPs and that the degree of this association changed during postnatal development, a period when NF expression is under significant post-transcriptional control. The degree of these associations changed independently of the abundance of either the RNPs or the NF messages, indicating that the RNA-protein interactions themselves are directly regulated. This study is consistent with a model whereby these RNPs and NF mRNAs are components of a dynamic post-transcriptional regulatory module that influences the cytoskeletal compositions of neurons.

Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons.

Characteristic changes in the expression of neuronal intermediate filaments (nIFs), an abundant cytoskeletal component of vertebrate axons, accompany successful axon regeneration. In mammalian regenerating PNS, expression of nIFs that are characteristic of mature neurons becomes suppressed throughout regeneration, whereas that of peripherin, which is abundant in developing axons, increases. Comparable changes are absent from mammalian injured CNS; but in goldfish and lamprey CNS, expression of several nIFs increases during axon regrowth. To obtain a broader view of the nIF response of successfully regenerating vertebrate CNS, in situ hybridization and video densitometry were used to track multiple nIF mRNAs during optic axon regeneration in Xenopus laevis. As in other successfully regenerating systems, peripherin expression increased rapidly after injury and expression of those nIFs characteristic of mature retinal ganglion cells decreased. Unlike the decrease in nIF mRNAs of regenerating PNS, that of Xenopus retinal ganglion cells was transient, with most nIF mRNAs increasing above normal during axon regrowth. At the peak of regeneration, increases in each nIF mRNA resulted in a doubling of the total amount of nIF mRNA, as well as a shift in the relative proportions contributed by each nIF. The relative proportions of peripherin and NF-M increased above normal, whereas proportions of xefiltin and NF-L decreased and that of XNIF remained the same. The increases in peripherin and NF-M mRNAs were accompanied by increases in protein. These results are consistent with the hypothesis that successful axon regeneration involves changes in nIF subunit composition conducive to growth and argue that a successful injury response differs between CNS and PNS.

Post-transcriptional control of neurofilaments in development and disease.

Tight coordination of the expression of neurofilament subunits is integral to the normal development and function of the nervous system. Imbalances in their expression are increasingly implicated in the induction of neurodegeneration in which formation of neurofilamentous aggregates is central to the pathology. Neurofilament expression can be controlled not only at the transcriptional level but also through post-transcriptional regulation of mRNA localization, stability, and translational efficiency. The critical role that post-transcriptional mechanisms play in maintaining neurofilament homeostasis is highlighted, for example, by the human disease amyotrophic lateral sclerosis, in which selective destabilization of NF-L mRNA (or failure to stabilize it) is associated with the formation of neurofilamentous aggregates - a hallmark of the disease process. This review discusses the post-transcriptional regulatory mechanisms and associated ribonucleoproteins that have been implicated to date in controlling neurofilament expression during normal development and in disrupting neurofilament homeostasis during neurodegenerative disease.

Phylogenetically conserved binding of specific K homology domain proteins to the 3'-untranslated region of the vertebrate middle neurofilament mRNA.

As axons mature, neurofilament-M (NF-M) expression rises, contributing to maturation of the axonal cytoskeleton and an expansion in axon caliber. This increase is partly due to a rise in NF-M mRNA stability. Such post-transcriptional regulation is often mediated through the binding of specific proteins to the 3'-untranslated region (3'-UTR) of mRNAs. Vertebrate NF-M 3'-UTRs are remarkably well conserved, prompting us to test whether similar proteins bind the 3'-UTRs of different vertebrate NF-Ms. Identification of such proteins could lead to insights into the regulation of NF-M expression during development and in response to trauma or disease. Ultraviolet cross-linking analysis of proteins isolated from adult frog (Xenopus laevis), mouse, and rat brains revealed three ribonucleoprotein complexes (97, 70, and 47 kDa) that were present in all species and bound specifically to NF-M 3'-UTRs. Affinity purification of NF-M 3'-UTR-binding proteins from rat brain followed by mass spectrometry and immunoprecipitation assays identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP E1 as the proteins forming the 70- and 47-kDa complexes, respectively. These RNA-binding proteins of the KH domain family recognize CU-rich motifs identical to ones present in NF-M 3'-UTRs. Ultraviolet cross-linking assays performed on Xenopus embryos at different stages of neural development demonstrated that whereas hnRNP K binding occurred at all stages, hnRNP E binding occurred only at the most mature stages of axon development. Since hnRNP E is known to stabilize mRNAs, these results raise the hypothesis that these proteins may contribute to the increases in cytoplasmic levels of NF-M mRNA that accompany axonal maturation.