A pro-apoptotic fragment of the p75 neurotrophin receptor is expressed in p75NTRExonIV null mice - PubMed (original) (raw)

A pro-apoptotic fragment of the p75 neurotrophin receptor is expressed in p75NTRExonIV null mice

Christine E Paul et al. J Neurosci. 2004.

Abstract

The p75 neurotrophin receptor (p75NTR) regulates neuronal survival, apoptosis, and growth. Recent studies have reported that disruption of Exon IV produces a null mouse lacking all p75NTR gene products (p75NTRExonIV-/-), whereas mice lacking p75NTR Exon III (p75NTRExonIII-/-) maintain expression of an alternatively spliced form of p75NTR (s-p75NTR). Here, we report that p75NTRExonIV-/- mice express a p75NTR gene product that encodes a truncated protein containing the extracellular stalk region together with the entire transmembrane and intracellular domains. The gene product is initiated from a cryptic Kozak consensus/initiator ATG sequence within a region of Exon IV located 3' to the pGK-Neo insertion site. Overexpression of this fragment in heterologous cells results in activation of Jun kinase and induces Pro-caspase-3 cleavage, indicating that it activates p75NTR signaling cascades. These results indicate that aspects of the p75NTRExonIV-/- phenotype may reflect a gain-of-function mutation rather than loss of p75NTR function.

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Figures

Figure 1.

Figure 1.

A p75NTR fragment is expressed in p75NTRExonIV-/- mice. A, P1 brain lysates from p75NTRExonIII and p75NTRExonIV wild-type (+/+), heterozygous (+/-), and null (-/-) littermates were separated on a 15% SDS-PAGE gel, followed by immunoblotting with αP1 (top panel) or αp75NTR (middle panel), distinct polyclonal antibodies directed against the p75NTR-ICD (see Materials and Methods). Arrowheads indicate full-length p75NTR and a ∼26 kDa band present in lysates from mice bearing the p75NTRExonIV mutant allele. Blots were reprobed for βIII-tubulin to verify equal loading. B, P1 cerebellar and heart lysates from p75NTRExonIV wild-type (+/+), heterozygous (+/-), and null (-/-) littermates were prepared and subjected to SDS-PAGE and immunoblotting as described above. C, Schematic diagram showing the genomic structure of the mouse p75NTR locus and corresponding protein domains. D, RNA was isolated from P1 brain lysates of p75NTRExonIV wild-type (+/+), heterozygous (+/-), and null (-/-) littermates. cDNA was prepared in the presence (+RT) and absence (-RT) of reverse transcriptase, followed by RT-PCR using primers for β-actin and for p75NTR-ICD. Arrows in C indicate location of primers used for this analysis. Experiments in A, B, and D were repeated three times with identical results.

Figure 2.

Figure 2.

Transcription of p75NTR fragment in p75NTRExonIV-/- mice is the result of pGK-Neo cassette insertion into the p75NTRExonIV targeting vector. A, mRNA from p75NTRExonIV wild-type (+/+), heterozygous (+/-), and null (-/-) littermates was subjected to RT-PCR using primers that target regions of p75NTR upstream or downstream of the pGK-Neo cassette insertion in the p75NTRExonIV targeting vector, as indicated in the schematic diagram. B, Alignment of mouse p75NTR cDNA sequence with p75NTR transcript cloned from p75NTRExonIV-/- mice. The putative start codon is underlined, and the putative Kozak sequence is boxed. Vertical lines indicate Exon IV-V and Exon V-VI junctions, and boxes underneath the sequence denote protein domains. C, Schematic diagram showing the protein domains of mouse p75NTR and p75NTRExonIV proteins. D, P1 brain lysates from p75NTRExonIV wild-type (+/+), heterozygous (+/-), and null (-/-) littermates and cellular lysates from 293T cells transiently transfected with plasmids encoding p75NTRExonIV (ExonIV) or the parental vector (Mock) were immunoblotted using αP1. Arrowheads indicate full-length p75NTR (top) and p75NTRExonIV (bottom) products. Experiments in A and D were repeated three times with identical results.

Figure 3.

Figure 3.

p75NTRExonIV is membrane-associated and induces p75NTR signaling events. A, P1 brains from p75NTRExonIV heterozygote (Exon IV +/-) and null (Exon IV -/-) littermates were subjected to subcellular fractionation and analyzed by immunoblot. C, Cytosol; M1, heavy membrane; M2, light membrane fraction. B, COS-7 cells were transfected with plasmids encoding p75NTRExonIV or the p75NTR intracellular domain tagged with a myristoylation sequence (Roux et al., 2001) and immunostained with αP1 and α-nucleolin. Left-hand panels show p75NTR staining, middle panels show nucleolin staining, and right-hand panels show merged images. C, PC12 cells were transfected with control plasmid or plasmid expressing p75NTRExonIV in the presence or absence of plasmid expressing Bcl-2. Staurosporine treatment was used as a positive control for caspase cleavage. Cellular lysates were analyzed by immunoblot to detect p75NTR, Bcl-2, and active caspase-3, as indicated. IκBα levels were analyzed to confirm equal loading between lanes. D, PC12 cells were left untransfected or were transfected with 2 μg of a control plasmid (pcDNA3), or expression plasmids encoding p75NTRExonIV alone or p75NTRExonIV and Bcl-2 and levels of phospho-Thr183/Tyr185-JNK and total JNK, phospho-Thr69/71-ATF-2, and total ATF-2 were assessed by immunoblotting. Experiments in _A_-C were repeated three times with identical results.

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