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|Mutation Type||critical splice donor site (2 bp from exon)|
|Coordinate||59,512,323 bp (GRCm38)|
|Base Change||A ⇒ T (forward strand)|
|Gene Name||ATP/GTP binding protein 1|
|Synonym(s)||2310001G17Rik, Nna1, 1700020N17Rik, 4930445M19Rik, 2900054O13Rik, 5730402G09Rik|
|Chromosomal Location||59,445,742-59,585,227 bp (-)|
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] NNA1 is a zinc carboxypeptidase that contains nuclear localization signals and an ATP/GTP-binding motif that was initially cloned from regenerating spinal cord neurons of the mouse.[supplied by OMIM, Jul 2002]
PHENOTYPE: Homozygotes show moderate ataxia due to degeneration of Purkinje cells of the cerebellum. Also, there is gradual degeneration of retina photoreceptor cells, olfactory bulb mitral cells and some thalamic neurons. Males have abnormal sperm and are sterile. [provided by MGI curators]
|Amino Acid Change|
|Institutional Source||Beutler Lab|
Ensembl: ENSMUSP00000022040 (fasta)
|Gene Model||not available|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Embryos, gDNA|
|Last Updated||2018-05-22 9:29 AM by Anne Murray|
The drunk phenotype was identified among ENU-induced G3 mutant mice; drunk mice display a severe ataxia of gait. Cerebellar Purkinje cells from homozygous drunk mice begin to degenerate beginning at approximately 3 weeks of age until virtually all cells are lost by 7 weeks of age. Degeneration of photoreceptor cells also begins at 6 weeks of age.
Homozygous drunk and wild type mice recover at the same rate after sciatic nerve crush operation (please see Background).
Male drunk mice are sterile due to a reduced number of abnormally shaped sperm cells. The drunk stock was maintained by breeding homozygous females with heterozygous males.
|Nature of Mutation|
The drunk mutation mapped to Chromosome 13, and corresponds to a T to A transversion in the donor splice site of intron 11 (GTAGGG -> GAAGGG) in the Agtpbp1 (hereafter Nna1) gene (position 44998 in Genbank genomic region NC_000079 for linear genomic DNA sequence of Agtpbp1). Sequencing of cDNA from drunk mice demonstrates that the mutation results in skipping of exon 11, thus destroying the reading frame after codon 302 (encoding glutamine) and creating a premature stop codon at codon 318.
<--exon 10 <--exon 11 intron 11--> exon 12-->
43219 ACTTCTCAA……CCGCCTGAAG GTAGGGGTG………………ACGACATTGATTTAG 49311
300 -T--S--Q-……-P--P--E- …-T--T--L--I--* 318
correct deleted aberrant
The donor splice site of intron 11, which is destroyed by the drunk mutation, is indicated in blue lettering; the mutated nucleotide is indicated in red lettering.
The drunk mutation causes the skipping of exon 11, creating a predicted premature stop codon after the insertion of fifteen aberrant amino acids. The mutation likely results in nonsense mediated decay of the aberrant transcript.
Nna1 mRNA is expressed throughout the brain, with prominent expression in cerebellar Purkinje and granule cells, mitral cells of the olfactory bulb, thalamic neurons, dorsal root ganglia, and hippocampal CA3 neurons (1;3). In the retina, Nna1 is expressed in photoreceptor cells (3). Nna1 is highly expressed in testes, in developing and mature sperm (3), and in heart, skeletal muscle and kidney (1). No Nna1 expression was found in ovary, liver, stomach, small intestine, lung, adrenal gland, spleen and thymus (1). During development, Nna1 is expression is found in the embryonic central and peripheral nervous systems (1).
Nna1 has two nuclear localization signals, and GFP-fusions with Nna1 localize to both the nucleus and the cytoplasm in primary cortical neurons (1). However, no zinc carboxypeptidases except AEBP1 have yet been found in the nucleus [discussed in (4)]. Definitive localization of the native Nna1 is unknown.
Nna1 was first identified in a screen for inducible genes in a sciatic nerve transection paradigm and it is expressed in spinal motor neurons undergoing axon regeneration. Its role in these events is currently unknown (1). Nna1 is also mutated in the spontaneously occurring pcd (Purkinje cell degeneration) mutants. There are currently eight known phenotypic alleles of pcd, out of which four have identified genetic lesions. In each of these four cases, protein levels are dramatically reduced by the genetic lesions (2;3). drunk is a new addition to the pcd allelic series.
The hallmark feature of pcd mice, as for drunk mice, is development of an ataxic gait between three and four weeks of age, which correlates with the onset of cerebellar Purkinje cell degeneration (7). Purkinje cells (Figure 2) proceed to deteriorate rapidly and die over the subsequent two week period. Distinct areas of the cerebellum display different rates of Purkinje cell degeneration, but all eventually die. Experiments with wild type-pcd chimeras demonstrated that this phenotype is cell autonomous (8). In addition to Purkinje cells, cerebellar granule cells (Figure 2) also display progressive death, with near normal numbers at three months declining to 5% by 20 months of age (7). Selected thalamic neurons also degenerate between postnatal days 50 and 60, and in addition, degeneration of retinal photoreceptors and olfactory bulb mitral cells progresses slowly over a year (3). Male pcd homozygous mice are sterile due to a reduction in the number of spermatozoa, which are sometimes abnormally shaped and immotile as well (3).
The molecular mechanisms underlying the pcd phenotypes have been under investigation for decades, yet much is still unknown. Several groups published findings supporting a role for abnormally increased apoptosis in pcd mice. A 5-fold increase in the mRNA levels of c-fos, junB and krox-24, which are associated with neuronal apoptosis, has been detected specifically in cerebellar Purkinje cells of pcd mice at postnatal day 22, at the onset of cell death (9). These transcription factors may dictate cellular outcome by coordinating the expression of various anti- and pro-apoptotic proteins. In fact, mRNA levels of the anti-apoptotic protein Bcl-2 are reduced by 35% while those of the pro-apoptotic Bax remain unchanged in pcd mice at 22 days of age (9). Because Bcl-2 prevents apoptosis by binding and physically inhibiting Bax, a decrease in Bcl-2 may favor apoptosis by de-repressing Bax activity. Consistent with the hypothesis that pcd Purkinje cells undergo increased apoptosis, nuclear DNA fragmentation (10) and activated caspase-3 (11) have been observed in these cells.
However, genetic studies investigating the potential contribution of several cell death pathways demonstrated that Bax is not involved in Purkinje cell death of pcd mice, as pcd3jbax-/- mice still develop ataxia and lose Purkinje cells (4). The p53 pathway was also tested by generating homozygous ATM-null or Puma-null pcd mice. ATM serves to phosphorylate and activate p53 upon DNA damage, resulting in either DNA repair or apoptosis. Puma is a pro-apoptotic protein and a transcriptional target of p53. Neither combining ATM nor Puma mutations with the Nna1 mutation led to rescue of the pcd phenotype, strongly suggesting that the p53 pathway is not involved in pcd Purkinje cell death (4).
A recent report supports a role for ER stress in the pcd phenotype. The levels of endoplasmic reticulum (ER)-specific chaperone BiP, and the ER-stress related transcription factor CHOP are increased pcd mice at 23 and 26 days of age (11). Moreover, an unusual configuration of the ER with associated electron-dense particles was observed during the early characterization of pcd mice (12).
Finally, the putative carboxypeptidase substrate-binding site of Nna1 was demonstrated to be required for rescue of the pcd phenotype (13). Purkinje cell-specific transgenic mice expressing wild type Nna1 were generated and crossed with pcd3j homozygous mice. pcd3jNna1-transgenic mice showed no Purkinje cell degeneration or ataxia. Sequence comparisons of Nna1 with other carboxypeptidases and structural modeling predicted R962 as part of the catalytic site, while N970 and R971 constituted the substrate-binding site of Nna1. Significantly, Purkinje cell-specific pcd transgenic mice expressing a putative substrate-binding site mutant (Nna1N970A R971A) still displayed ataxia and Purkinje cell degeneration. These results indicate that N970 and R971 are essential for Nna1 to support Purkinje cell survival, and suggest that Nna1 is a true carboxypeptidase.
|Primers||Primers cannot be located by automatic search.|
Drunk genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition. The same primers are used for PCR amplification and for sequencing.
Drunk(F): 5’-TTCCAGTACCGCAGTGCTGAGCTGTG -3’
Drunk(R): 5’-CATGGCTCAGTGGGACTGTGGCAAG -3’
1) 94°C 2:00
2) 94°C 0:30
3) 62°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 35X
6) 72°C 5:00
7) 4°C ∞
The following sequence of 689 nucleotides (from Genbank genomic region NC_000079 for linear genomic sequence of Agtpbp1, sense strand) is amplified:
44764 ttccagt accgcagtgc tgagctgtgg tctaactctc atcatcttgt ttgaacagga
44821 gtgcttggcg gtcaggactc ttgatcctct tgtcaacaca tccagtctga taatgagaaa
44881 atgcttcccc aaaaaccgcc ttccgctccc caccattaaa agttctttcc acttccaatt
44941 gccaattatc cctgtgactg gacctgtggc ccagctctac agcttgccgc ctgaaggtag
45001 gggtgggcca tgggctgctg tgggagtccg tgactctgtt gtcgaaaatg tgtatctggt
45061 tgtttaatgt gcgcatgagg tgtgaaaata cacaaaaatt tgtcagcaat caatgtaaat
45121 attaaaagta taaggtggag acattatata agttgatgtc atacaagata gtgacattac
45181 attaagagga tgacatcaga tgctcagtat gtttgcgatt acctgtgtag ctttttaaaa
45241 gaattttaat tacacattgg tagtagcatc attattgtca ctatgtgggc atccacatat
45301 gttgtgtgtg ccatgtacat gcacatttgt gctcctgggt ctgtgagcac gtgagagtca
45361 gaggacagcc ttcagatgtt ggttcttccc tttaccaggt ggttgcaagg atcaaatgta
45421 tatcaggctt gccacagtcc cactgagcca tg
Primer binding sites are underlined; the mutated T is highlighted in red.
1. Harris, A., Morgan, J. I., Pecot, M., Soumare, A., Osborne, A., and Soares, H. D. (2000) Regenerating motor neurons express Nna1, a novel ATP/GTP-binding protein related to zinc carboxypeptidases, Mol. Cell Neurosci. 16, 578-596.
2. Chakrabarti, L., Neal, J. T., Miles, M., Martinez, R. A., Smith, A. C., Sopher, B. L., and La Spada, A. R. (2006) The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabilizes Nna1 protein, Mamm. Genome 17, 103-110.
3. Fernandez-Gonzalez, A., La Spada, A. R., Treadaway, J., Higdon, J. C., Harris, B. S., Sidman, R. L., Morgan, J. I., and Zuo, J. (2002) Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1, Science 295, 1904-1906.
4. Wang, T. and Morgan, J. I. (2007) The Purkinje cell degeneration (pcd) mouse: an unexpected molecular link between neuronal degeneration and regeneration, Brain Res. 1140, 26-40.
5. Beinfeld, M. C. (2003) Biosynthesis and processing of pro CCK: recent progress and future challenges, Life Sci. 72, 747-757.
6. Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity, Nat. Genet. 10, 135-142.
7. Mullen, R. J., Eicher, E. M., and Sidman, R. L. (1976) Purkinje cell degeneration, a new neurological mutation in the mouse, Proc. Natl. Acad. Sci. U. S. A 73, 208-212.
8. Mullen, R. J. (1977) Site of pcd gene action and Purkinje cell mosaicism in cerebella of chimaeric mice, Nature 270, 245-247.
9. Gillardon, F., Baurle, J., Wickert, H., Grusser-Cornehls, U., and Zimmermann, M. (1995) Differential regulation of bcl-2, bax, c-fos, junB, and krox-24 expression in the cerebellum of Purkinje cell degeneration mutant mice, J. Neurosci. Res. 41, 708-715.
10. Gillardon, F., Baurle, J., Grusser-Cornehls, U., and Zimmermann, M. (1995) DNA fragmentation and activation of c-Jun in the cerebellum of mutant mice (weaver, Purkinje cell degeneration), Neuroreport 6, 1766-1768.
11. Kyuhou, S., Kato, N., and Gemba, H. (2006) Emergence of endoplasmic reticulum stress and activated microglia in Purkinje cell degeneration mice, Neurosci. Lett. 396, 91-96.
12. Landis, S. C. and Mullen, R. J. (1978) The development and degeneration of Purkinje cells in pcd mutant mice, J. Comp Neurol. 177, 125-143.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Pia Viviani, Xin Du, Bruce Beutler|
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