Purkinje cell degeneration (pcd) Phenotypes Caused by Mutations in the Axotomy-Induced Gene, Nna1

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Science  08 Mar 2002:
Vol. 295, Issue 5561, pp. 1904-1906
DOI: 10.1126/science.1068912

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The classical recessive mouse mutant, Purkinje cell degeneration (pcd), exhibits adult-onset degeneration of cerebellar Purkinje neurons, retinal photoreceptors, olfactory bulb mitral neurons, and selected thalamic neurons, and has defective spermatogenesis. Here we identify Nna1 as the gene mutated in the original pcd and two additionalpcd alleles (pcd 2J andpcd 3J). Nna1 encodes a putative nuclear protein containing a zinc carboxypeptidase domain initially identified by its induction in spinal motor neurons during axonal regeneration. The present study suggests an unexpected molecular link between neuronal degeneration and regeneration, and its results have potential implications for neurodegenerative diseases and male infertility.

Strains of mice harboring mutations that affect the nervous system have provided important insights into normal and aberrant neural development and function (1–3). Although the genes responsible for many of the classical neurological mutants have been identified, thePurkinje cell degeneration (pcd) gene remains elusive. Unlike many mouse mutants in which neuronal loss occurs during development (2, 3), pcd is unusual in that neurodegeneration occurs after weaning, when most synaptic circuitries are established. Thus, the identification of the gene responsible for the pcd phenotype could provide a key molecular entry point into pathways or processes underlying other neurodegenerative disorders in humans.

The original pcd mutation (referred to here as “pcd 1J”, to distinguish it from later-occurring pcd mutations) arose spontaneously in the C57BR/cdJ strain and exhibits ataxia resulting from loss of cerebellar Purkinje cells during early adulthood (4, 5). Populations of thalamic neurons also degenerate between postnatal days 50 and 60 in pcd 1J (6,7), whereas degeneration of retinal photoreceptors and olfactory bulb mitral cells progresses slowly over a year (8–11). Also, adult malepcd 1J mice are infertile because they have reduced numbers of sperm that are abnormally shaped and nonmotile (5, 12). Two additional pcd alleles,pcd 2J and pcd 3J, arose spontaneously in SM/J and BALB/cByJ mouse strains, respectively. Thepcd 2J allele was maintained in C57BL/6J congenic lines for >15 backcross generations. The phenotypes ofpcd 1J and pcd 3Jhomozygotes were nearly identical, and allelic tests between them were positive (data not shown). However, the pcd 2Jmutants develop ataxia later than pcd 1J mice and are more mildly affected. Thalamic neurons do not degenerate inpcd 2J homozygotes on the original SM/J or the congenic C57BL/6J backgrounds, even at one year of age (data not shown). Also, many pcd 2J homozygous males are fertile.

To refine the position of pcd on mouse chromosome 13, we used a previously reported CAST/Ei ×pcd 1J cross (13) and set up additional crosses (CAST/Ei × pcd 1J, MOLG/Dn × pcd 1J, and CAST/Ei ×pcd 3J). We analyzed a total of 419 offspring (838 meiotic recombination events), 198 in the CAST/Ei timespcd 1J cross, 86 in the MOLG/Dn timespcd 1J cross, and 135 in the CAST/Ei timespcd 1J cross, with polymorphic DNA markers (14). Two critical crossovers defined the telomeric boundary at D13Mit157, and a third marked the centromeric boundary at D13Mit167 (Fig. 1). Thus, thepcd candidate region was refined to a genomic segment of 0.61 ± 0.33 megabases.

Figure 1

Genetic mapping of pcd and gene structure of Nna1. The pcd locus was mapped to mouse chromosome 13 between D13Mit167 andD13Mit157. Three crossovers (two telomeric, “XX” and one centromeric, “X”) defined the boundaries of the pcdcandidate region. The pcd cosyntenic region of human (9q21.33–9q22.1) contains six predicted genes (indicated by lines and their 3′ ends indicated by arrows); Ntrk2 andFlj21613 flank Nna1. Filled squares, exons; ATG, initiation codon; TGA, stop codon. Exons 8 and 16 to 19 (underlined) were used as probes for Northern and in situ hybridization analyses.

Using a mouse genomic sequence database (Celera Discovery System v. 3.01), we found the pcd candidate region to be cosyntenic with a region on human 9q21.33–9q22.1, which contains six identified or predicted genes. A previously identified gene, Nna1[NNA1 or KIAA1035 in humans (15)], spans >100 kb in the mouse pcd region (Fig. 1).Nna1 was first identified in a screen for inducible genes in a sciatic nerve transection paradigm, and it is expressed in developing and adult brain, spinal motor neurons undergoing axon regeneration, testis, and heart (15). Nna1 is a putative nuclear protein that contains a zinc carboxypeptidase domain and is structurally most related to the adipocyte enhancer binding protein 1 (AEBP1) (15).

To determine if Nna1 is altered in pcd mice, we performed Northern analyses (16) on RNA isolated from cerebellum, cerebral cortex, testis, and heart of wild-type and homozygous adult pcd 1J,pcd 2J, and pcd 3J animals. In all three pcd alleles, Nna1 messenger RNA (mRNA) was either dramatically reduced or altered in structure (data not shown) (Fig. 2), whereas mRNAs of two genes immediately flanking Nna1 were unchanged (Web fig.1, available onScience Online

Figure 2

Northern blot analysis of Nna1 in brain and testis of pcd 1J,pcd 2J, and pcd 3J mice. Total RNA from brain and testis of adult mutant (−/−) and wild-type (+/+) littermates were analyzed by Northern blotting with the sequential use of Nna1-specific and loading control probes.Nna1 probes for exon 8 (nucleotide 798–1007 of GenBank accession number NM_023328) and exons 16 to 19 (nucleotide 2408–2976 of NM_023328) were used. Forpcd 1J testis (asterisk), a probe (nucleotide 2260–3106 of GenBank accession number NM_023328) was used. Loading control probes were from Gapdh (pcd 1Jtestis, nucleotide 143–363 of NM_008084) and HO-2 (all other panels, nucleotide 872–1067 of GenBank accession number,J05405).

In pcd 3J, Nna1 mRNA in brain and testis was reduced in abundance and size when a probe derived from exons 16 to 19 was used (Fig. 2). However, the Nna1 mRNA was undetectable when an exon 8 probe was used. The absence of exon 8 inpcd 3J brain and testis was confirmed with the use of in situ hybridization. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis established that a splice junction was formed between exons 5 and 9, thereby introducing a stop codon that truncates Nna1 (Fig. 3, A and B). Genomic analysis of pcd 3J revealed a deletion of ∼12.2-kb between intron 5 and exon 8 that causes the aberrant mRNA (Fig. 3, C and D).

Figure 3

Mutation in pcd 3J. (A) Diagram of genomic deletion and resulting alternative splicing. In pcd 3J−/−, a ∼12.2-kb genomic deletion occurred between intron 5 and exon 8 (nucleotide 876 of NM_023328). This leads to alternative splicing between exon 5 (nucleotide 665 of NM_023328) and exon 9, thereby introducing a stop codon and yielding a truncated protein. (B) Forward sequence of a RT-PCR product from pcd 3J−/− brain. Primers used for RT-PCR and sequencing were from exon 4 (5′-GAGCGAGTTTCTTAGTTGCC-3′) and exon 11 (5′-CATCAACAAGCTCGGGGAAAAGG-3′). Box indicates splice junction between exons 5 and 9, forming a stop codon. (C) Genomic Southern blot analysis ofpcd 3J. The exon 4 probe was amplified with the use of primers as follows: 5′-GTCCACTACGTGAAGATAGG-3′ and 5′-GTTACAAAGATGGCTCTAGGGTC-3′. The exon 8 probe was as in Fig. 2. (D) Forward sequence of a genomic PCR product of pcd 3J−/− showing deletion junction. Primers from intron 5 (5′-CTGTAGTGCAGGTCCTGCCTGC-3′) and exon 8 were used for PCR and sequencing.

In pcd 2J, Nna1 mRNA was undetectable using the exon 16 to 19 probe in cerebellum, brain, and heart but was present at reduced levels in testis (data not shown) (Fig. 2). All exons of Nna1 are intact in pcd 2J. However, we identified an ∼7.8-kb insertion in intron 13 of theNna1 gene (Fig. 4A). This 7.8 kb insertion (GenBank accession number AF457126) appears to be a long-period interspersed sequence nor a intracisternal A particle (LINE and IAP, respectively), but rather contains repetitive sequences nearly identical to an ∼ 7.8 kb segment of Mus musculus α/δ T cell receptor locus on chromosome 14 (GenBank accession number AE008685). Intragenic suppression by transposable elements is well known and underlies another classic cerebellar mouse mutant, vibrator[(17), and references therein].

Figure 4

Mutation in pcd 2J. (A) Diagram of the ∼7.8-kb insertion within intron 13 inpcd 2J. Boxes indicate the duplicated target hexamers, CCTTAG (Fig. 4C), that result from the insertion. (B) Long-distance PCR analyses of intron 13 ofpcd 2J (22). Arrows in (A) are the two primers used (Ex13F, 5′-CCAAGACATCGAGAGGCTGATAC-3′, and Ex14R, 5′-GAATTACTTTGCGCAGATTCC-3′). (C) Genomic Southern blot analyses of thepcd 2J mutation. Exon 13 and 14 probes were obtained with the use of primer pairs Ex13F and Int13R4 (5′-GAGAACAGCAACAATGAAGCG-3′) and Ex14R and Int13F4 (5′-GAGCCTTCCGTGGTAGCTAACG-3′), respectively.

In pcd 1J, the Nna1 mRNA was undetectable in all tissues examined except for testis where it was reduced approximately 20-fold, substantially more than inpcd 2J (data not shown) (Fig. 2). Presumably, the level of Nna1 in pcd 2J testis is sufficient to support spermatogenesis, whereas the level is inadequate in pcd 1J. As the sequence of the Nna1mRNA is identical to that of wild-type and there are no overt exonic mutations (data not shown), we expect that thepcd 1J mutation is in a regulatory region ofNna1. We propose, in accordance with standard rules (18), that the pcd mutations hereafter be assigned the following gene symbols: Nna1pcd-1J ,Nna1pcd-2J , andNna1pcd-3J .

Chimera studies in mice have shown that pcd behaves as a cell-autonomous allele (19). To determine if Nna1is expressed in the cell types affected in pcd, we performed in situ hybridization (16) on one-month-old wild-type mice. Nna1 was expressed throughout the brain (Fig. 5, F, G, and H), retina (Fig. 5I),and testis (Fig. 5J) [see also (15)]. Notably, prominent expression of Nna1 mRNA was detected in Purkinje cells of cerebellum (Fig. 5F, inset), mitral cells of the olfactory bulb (Fig. 5H), neurons in the thalamus (Fig. 5G), and photoreceptors of the retina (Fig. 5I), which all degenerate in pcd(5). Nna1 was also expressed in mature and developing sperm (Fig. 5J), consistent with the defective spermatogenesis in pcd (5, 12).

Figure 5

In situ hybridization of Nna1 in brain, eye, and testis. (A), (B), (C), (D), (E), and (K) show bright-field microscopy of 1-month-old wild-type mouse. (F), (G), (H), (I), and (J) show corresponding dark-field microscropy using an antisense probe derived from exons 16-19. (A) and (F), cerebellum; (B) and (G), thalamus; (C) and (H), olfactory bulb; (D) and (I), retina; (E) and (J), testis; (L) and (M), hippocampus ofpcd 3J+/+ (L) andpcd 3J−/− (M) hybridized with probe derived from exons 16 to 19. Hippocampus of pcd 3J+/+ (N) and pcd 3J−/− (O) hybridized with an antisense probe derived from exon 8. Inset in (F), higher magnification showing Purkinje cells (arrows) that are strongly positive. In (B) and (G), thalamic neurons areNna1-positive. In (C) and (H), abbreviations are as follows: GLOM, glomerular layer; MCL, mitral cell layer (arrows); GRL, granule cell layer. Mitral cells are Nna1-positive. In (D) and (I): RPE, retinal pigment epithelium; OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, retinal ganglion layer (arrow). The inner segments (arrowheads) of photoreceptors areNna1-positive. In (E) and (J), the region containing Sertoli cells and spermatogonia (arrowheads) areNna1-negative, whereas the region containing more mature components of spermatogenesis and sperm (asterisks) areNna1-positive. In (L) and (M), the exon 16 to 19 probe exhibits similar hybridization patterns in hippocampus ofpcd 3J+/+ and pcd 3J−/−. In (N) and (O), the exon 8 probe does not hybridize to thepcd 3J−/−hippocampus. Scale bar for (A), (B), (F), (G), and (K) through (O), 75 μm; bar for (C) and (H), 40 μm; bar for (D), (E), (I), and (J), 20 μm.

Although no abnormalities have been described within the hippocampus or cerebral cortex of pcd mice (20), neurons in these structures robustly express Nna1 (data not shown) (Fig. 5L) [see also (15)]. Thus, absolute levels ofNna1 expression do not predict neuronal vulnerability. This may result from genetic redundancy because Caenorhabditis elegans harbors two Nna1-related genes (15), and human and mouse genomes contain genes encoding proteins with similar structures to Nna1.

The identification of Nna1 as the gene mutated inpcd mice provides an unexpected link between neuronal degeneration and regeneration. Because axotomy triggers induction ofNna1 in spinal motor neurons (15), pcdmice may provide a valuable paradigm to investigate molecular mechanisms of axonal regeneration. Indeed, some of the cytological features of degenerating thalamic neurons in pcd resemble those of axotomized neurons (6). It remains to be elucidated whether mutation or altered expression of NNA1 or NNA1-related genes underlies or modifies other hereditary or sporadic neurodegenerative diseases. Lastly, because Nna1 harbors a zinc carboxypeptidase domain that is essential in AEBP1 (21), it may be a target for modifying neuronal survival and/or male fertility.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: jian.zuo{at}


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