Zebrafish calsyntenins mediate homophilic adhesion through their amino-terminal cadherin repeats.

The calsyntenins are atypical members of the cadherin superfamily that have been implicated in learning in Caenorhabditis elegans and memory formation in humans. As members of the cadherin superfamily, they could mediate cell-cell adhesion, although their adhesive properties have not been investigated. As an initial step in characterizing the calsyntenins, we have cloned clstn1, clstn2 and clstn3 from the zebrafish and determined their expression in the developing zebrafish nervous system. The three genes each have broad, yet distinct, expression patterns in the zebrafish brain. Each of the ectodomains mediates homophilic interactions through two, amino-terminal cadherin repeats. In bead sorting assays, the calsyntenin ectodomains do not exhibit homophilic preferences. These data support the idea that calsyntenins could either act as adhesion molecules or as diffusible, homophilic or heterophilic ligands in the vertebrate nervous system.

The clustered protocadherins Pcdhα and Pcdhγ form a heteromeric complex in zebrafish.

The clustered protocadherin genes encode a diverse collection of neuronal cell surface receptors. These genes have been proposed to play roles in axon targeting, synaptic development and neuronal survival, although their specific cellular roles remain poorly defined. In zebrafish there are four clustered protocadherin genes, two pcdhα clusters and two pcdhγ clusters, that give rise to over 100 distinct proteins, each with a distinct ectodomain (EC). The zebrafish is an excellent model in which to address the function of protocadherins during neural development, as the embryos are transparent, develop rapidly, and are amenable to experimental manipulation. As a first step to investigating the clustered protocadherins during zebrafish development, we have generated antibodies against the common cytodomains of zebrafish Pcdhγ. We compare the distribution of Pcdhγ with Pcdhα and find a similar pan-neuronal pattern, with strong labeling of neurons within all major regions of the central nervous system. Pcdhα and Pcdhγ are particularly enriched in the developing visual system, with strong labeling found in the synaptic layers of the retina, as well as the optic tectum. Consistent with studies in mouse, we find that Pcdhα and Pcdhγ are present in a complex, as they can be co-immunoprecipitated from zebrafish larval extracts. This interaction is direct and occurs through the ECs of these proteins. Using standard bead aggregation assays, we find no evidence for intrinsic adhesive ability by either Pcdhγ or Pcdhα, suggesting that they do not function as cell adhesion molecules.

Differential expression, alternative splicing, and adhesive properties of the zebrafish δ1-protocadherins.

Protocadherins comprise the largest family within the cadherin superfamily of cell surface receptors. Here, we characterize the δ1-protocadherin subfamily during the development of the zebrafish nervous system. In zebrafish, there are five δ1-protocadherins: pcdh1a, pcdh1b, pcdh7a, pcdh7b, andpcdh9. Each protocadherin gene is highly homologous to its human ortholog. While the expression pattern in the developing CNS is similar for each δ1-protocadherin, with labeling observed in all major subdivisions, the detailed patterns are distinct. In addition, we provide evidence for alternative splicing of the pcdh7b and pcdh9 genes, resulting in variation in their respective cytoplasmic domains. As protocadherins are widely regarded to act as cell adhesion molecules, we used in vitro assays of δ1-pcdh ectodomains to directly test their adhesive properties. We found no evidence for calcium-dependent, homophilic adhesion, contrasting sharply with the behavior of classical cadherins.

Differential neuroprotection from human heat shock protein 70 overexpression in in vitro and in vivo models of ischemia and ischemia-like conditions.

We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.