A post-transcriptional regulatory mechanism restricts expression of the paraneoplastic cerebellar degeneration antigen cdr2 to immune privileged tissues

Abstract

Paraneoplastic cerebellar degeneration (PCD) is believed to be an autoimmune disorder initiated by the ectopic expression of a neuron-specific protein in breast and ovarian tumors. PCD antisera was used previously to identify several cerebellar degeneration-related (cdr) genes encoding putative PCD antigens. We have found that the cdr2 gene, which encodes a cytoplasmic leucine zipper protein of unknown function, is expressed in PCD-associated tumors, whereas other cdr genes are not; thus, cdr2 encodes the PCD tumor antigen. To determine whether the expression pattern of cdr2 is consistent with its proposed role in PCD, we have isolated the mouse homolog and examined both the mRNA and protein distribution in adult tissues. We have found that cdr2 mRNA is expressed in almost all tissues, whereas the protein is expressed only in the brain and testis. Within the brain, both the cdr2 mRNA and immunoreactivity are confined primarily to neurons in the cerebellum and brainstem, the regions most affected in PCD. These results suggest first that the tissue-specific expression of cdr2 is regulated at a post-transcriptional level. Moreover, because the brain and testis are considered to be immune-privileged sites, the expression pattern of cdr2 is compatible with the autoimmune model of PCD pathogenesis.

Fig. 2.

Above. Immunoreactivity of PCD ovarian tumors with PCD antisera. Serial sections of a paraffin-embedded PCD ovarian tumor (tumor 2 from Fig. 1) stained with either biotinylated normal human serum (A) or biotinylated PCD antisera (B). PCD antisera displays a characteristic cytoplasmic reactivity in the tumor tissue and not in the surrounding connective tissue seen in the bottom of the photomicrograph.C, Detection of the PCD ovarian tumor antigen by Western blot. PCD antisera was immunoreactive with a protein ofM_r 56 kDa (arrowhead) present in both human Purkinje (lane 1) and PCD tumor (lane 2) protein extracts. The lower reactive band in the tumor extract is IgG, determined by probing the same blot with the anti-human IgG secondary antibody alone (data not shown).

Fig. 1.

RT-PCR analysis of PCD-associated ovarian tumors. Total or poly(A⁺) RNA purified from tumor tissue or human cerebellar poly(A⁺) RNA was used as templates for the RT. Gene-specific primers corresponding to the coding region of either cdr2 or cdr3 were used for PCR amplification of the first strand cDNA. Reactions were performed both in the presence and absence of RT to control for DNA contamination. A β-actin primer pair was also used as a control for RNA integrity (data not shown). Although the transcripts of the expected size for both cdr2 and cdr3 were detectable in human cerebellum, only the cdr2 transcript was detected in the PCD tumors.

Fig. 4.

Nucleotide and predicted amino acid sequence of the adult mouse brain cdr2 cDNA. Amino acids are numbered on theleft and the nucleotides on the right. The in-frame stop codon upstream of the presumptive initiating methionine and a polyadenylation signal are underlined. The translational stop codon is indicated with anasterisk. The 455 amino acid protein of predicted M_r = 52 kDa is 87% identical to the human sequence. The amino acids that are not conserved areunderlined.

Fig. 5.

Expression of the cdr2 mRNA in adult mouse tissues. A, A Northern blot of total RNA prepared from the indicated tissues was hybridized with a ³²P-labeled cDNA probe made from the 3′-UTR of mouse cdr2 (top panel). The bottom panel shows hybridization of a GAPDH probe to the same blot as a control for loading of RNA. The mouse tissues used in the analysis were cerebellum (Cb), cerebral cortex (Cx), heart (Ht), lung (Lu), liver (Li), kidney (Kd) spleen (Sp), ovary (Ov), and testis (Ts). The relative positions of 28S (5.1 kb) and 18S (2.0 kb) rRNA are shown. A single cdr2 transcript of 2.8 kb was detected in all tissues tested, with the exception of the liver.B, RT-PCR analysis of cdr2 expression in cerebellum versus spleen RNA was performed, as described in Figure 1, using primers flanking the PCD epitope or β-actin primers. The cdr2 transcript was also detected in heart and testis by this assay (data not shown).

Fig. 6.

Detection of the PCD antigen in adult mouse tissues. A, Affinity-purified PCD antisera was used to probe a Western blot of the indicated protein extracts (top panel, abbreviated as in Fig. 3). The blot was stripped of antibody and reprobed with a monoclonal antibody to β-tubulin as a protein-loading and transfer control (bottom panel); the lower band is mouse Ig heavy chain. The cdr2 affinity-purified antibody recognizes a M_r 56 kDa antigen only in brain and testis.B, Two-dimensional gel electrophoresis of protein extracts from cerebellum (i) and testis (ii). Proteins were resolved by their isoelectric points in the horizontal direction (the direction and end points of the pH gradient are shown above) and by their molecular weights in the vertical direction. The major species detected by PCD antisera in cerebellum and testis comigrate in both dimensions.

Fig. 7.

Analysis of cdr2 expression by in situ hybridization (A, C,E) and immunohistochemistry (B,D, F). Sections of adult mouse brain (A, B), spleen (C, D), and testis (E,F) were hybridized with a ³³P-labeled cdr2 riboprobe or reacted with either affinity-purified or native PCD antisera. Dark-field photomicrographs reveal that the cdr2 mRNA is detected in cerebellar Purkinje neurons, many brainstem neurons (A), splenic cortical cells (C), and cells of the outermost layers of the seminiferous tubules in the testis (E). There was no clear pattern of expression in the cerebral cortex, and no hybridization was observed with cdr2 sense probes in any of the tissues examined (data not shown). Immunoreactivity with affinity-purified or native PCD antisera was detected in cerebellar Purkinje neurons, brainstem neurons (B, left andright panels, respectively), scattered neurons of the cerebral cortex (data not shown), and spermatogonia in the testis (F). Immunoreactivity was absent in spleen (D), which shows only background reactivity when compared with a normal human serum control (data not shown).pc, Purkinje cells; gcl, granule cell layer; ml, molecular layer; io, inferior olive; rp, red pulp; ctx, cortex; spg, spermatogonia; spc, spermatocytes.