The Cbln family of proteins interact with multiple signaling pathways.

Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic ß-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.

Comparison of Cbln1 and Cbln2 functions using transgenic and knockout mice.

Cerebellin precursor protein 1 (Cbln1) is the prototype of a family of secreted neuronal glycoproteins (Cbln1-4) and its genetic elimination results in synaptic alterations in cerebellum (CB) and striatum. In CB, Cbln1 acts as a bi-functional ligand bridging pre-synaptic ß-neurexins on granule cells to post-synaptic Grid2 on Purkinje neurons. Although much is known concerning the action of Cbln1, little is known of the function of its other family members. Here, we show that Cbln1 and Cbln2 have similar binding activities to ß-neurexins and Grid2 and the targeted ectopic expression of Cbln2 to Purkinje cells in transgenic mice rescues the cerebellar deficits in Cbln1-null animals: suggesting that the two proteins have redundant function mediated by their common receptor binding properties. Cbln1 and Cbln2 are also co-expressed in the endolysosomal compartment of the thalamic neurons responsible for the synaptic alterations in striatum of Cbln1-null mice. Therefore, to determine whether the two family members have similar functions, we generated Cbln2-null mice. Cbln2-null mice do not show the synaptic alterations evident in striatum of Cbln1-null mice. Thus, Cbln2 can exhibit functional redundancy with Cbln1 in CB but it does not have the same properties as Cbln1 in thalamic neurons, implying one or both utilize different receptors/mechanisms in this brain region.

The human immunodeficiency virus-1 protein Tat and its discrete fragments evoke selective release of acetylcholine from human and rat cerebrocortical terminals through species-specific mechanisms.

The effect of the human immunodeficiency virus-1 protein Tat was investigated on neurotransmitter release from human and rat cortical nerve endings. Tat failed to affect the release of several neurotransmitters, such as glutamate, GABA, norepinephrine, and others, but it evoked the release of [3H]ACh via increase of cytosolic [Ca2+]. In human nerve terminals, the Tat effect partly depends on Ca2+ entry through voltage-sensitive Ca2+ channels, because Cd2+ halved the Tat-evoked release. Activation of group I metabotropic glutamate receptors (mGluR) and mobilization of Ca2+ from IP3-sensitive intraterminal stores are also involved, because the Tat effect was prevented by mGluR antagonists 2-methyl-6-(phenylethynyl)pyridine hydrochloride and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester and by the IP3 receptor antagonists heparin and xestospongin C. Furthermore, the group I selective mGlu agonist (RS)-3,5-dihydroxyphenylglycine enhanced [3H]ACh release. In rat nerve terminals, the Tat-evoked release neither depends on external Ca2+ ions entry nor on IP3-mediated mechanisms. Tat seems to cause mobilization of Ca2+ from ryanodine-sensitive internal stores because its effect was prevented by both 8-bromo-cyclic adenosine diphosphate-ribose and dantrolene. The Tat-evoked release from human synaptosomes was mimicked by the peptide sequences Tat 32-62, Tat 49-86, and Tat 41-60. In contrast, the Tat 49-86 and Tat 61-80 fragments, but not the Tat 32-62 fragment, were active in rat synaptosomes. In conclusion, Tat elicits Ca2+-dependent [3H]ACh release by species-specific intraterminal mechanisms by binding via discrete amino acid sequences to different receptive sites on human and rat cholinergic terminals.

Presence and functional significance of presynaptic ryanodine receptors.

Ca(2+)-induced Ca(2+) release (CICR) mediated by sarcoplasmic reticulum resident ryanodine receptors (RyRs) has been well described in cardiac, skeletal and smooth muscle. In brain, RyRs are localised primarily to endoplasmic reticulum (ER) and have been demonstrated in postsynaptic entities, astrocytes and oligodendrocytes where they regulate intracellular Ca(2+) concentration ([Ca(2+)](i)), membrane potential and the activity of a variety of second messenger systems. Recently, the contribution of presynaptic RyRs and CICR to functions of central and peripheral presynaptic terminals, including neurotransmitter release, has received increased attention. However, there is no general agreement that RyRs are localised to presynaptic terminals, nor is it clear that RyRs regulate a large enough pool of intracellular Ca(2+) to be physiologically significant. Here, we review direct and indirect evidence that on balance favours the notion that ER and RyRs are found in presynaptic terminals and are physiologically significant. In so doing, it became obvious that some of the controversy originates from issues related to (i) the ability to demonstrate conclusively the physical presence of ER and RyRs, (ii) whether the biophysical properties of RyRs are such that they can contribute physiologically to regulation of presynaptic [Ca(2+)](i), (iii) how ER Ca(2+) load and feedback gain of CICR contributes to the ability to detect functionally relevant RyRs, (iv) the distance that Ca(2+) diffuses from plasma membranes to RyRs to trigger CICR and from RyRs to the Active Zone to enhance vesicle release, and (v) the experimental conditions used. The recognition that ER Ca(2+) stores are able to modulate local Ca(2+) levels and neurotransmitter release in presynaptic terminals will aid in the understanding of the cellular mechanisms controlling neuronal function.