Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice

Abstract

Background: The presynaptic protein alpha-synuclein is involved in a range of neurodegenerative diseases. Here we analyze potential compensatory mechanisms in alpha-synuclein null mutant mice. Furthermore, the findings reveal problems that may be associated with inbred mouse strains.

Results: Expression profiling by cDNA array technology in a transgenic mouse model revealed striking differences only in the expression level of alpha-synuclein. This was caused by a chromosomal deletion of the alpha-synuclein locus in the C57BL/6J inbred strain used for backcrossing. However, the deletion is only present in a subpopulation of C57BL/6J mice, namely animals from Harlan. No other genes are known to be affected by the deletion, which is estimated to be smaller than 2 cM. We propose to name this strain C57BL/6S. C57BL/6S animals appear phenotypically normal. They show no upregulation of beta-synuclein or gamma-synuclein, excluding a compensatory mechanism. Also, the expression of synphilin-1 was unaffected.

Conclusions: The C57BL/6S strain should help in the understanding of the physiological function of alpha-synuclein and its involvement in synucleinopathies. Also, the findings exemplify unexpected complications that may arise during the study of transgenic models or inbred strains, in particular when combined with genome wide screening techniques.

Figure 1

α-synuclein expression levels.A. cDNA array experiment using brain polyA⁺-RNA from a transgenic mouse and a wildtype littermate at the developmental time point E18.5. From all analyzed genes (approximately 9000 ESTs), only the expression of α-synuclein (GenBank W41663) was significantly altered (indicated by an arrow; differential expression factor 7.6x). The data are represented without background substraction. Expression is quantified as units of fluorescence intensity. Diagonals indicate differential expression by 2x and 10x, respectively.B. Northern blot of brain polyA⁺-RNA (500 ng per lane) from 129/Ola, ICR, and BL6JHUK mice at the developmental time points P0, P8, P15, and adult, probed with an α-synuclein 3'UTR probe. The α-synuclein transcript size is approximately 1.5 kB. RNA samples were pooled from 4 animals (at P0), or from 2 animals (at P8 and P15) from the same litter. After stripping the membrane was re-probed with an actin probe. C. Brain homogenate from 129/Ola, ICR, and BL6JHUK mice at P0, P8, P15, and adult age was separated by 15% SDS-PAGE (20 μg protein per lane). The Western blot membrane was simultaneously probed with the monoclonal anti α-synuclein and anti α-tubulin antibodies. Recognized protein bands have apparent molecular weights of approximately 19 kDa and 55 kDa, respectively.

Figure 2

Deletion of the α-synuclein gene locus in the BL6JHUK strain.A. Semi-quantitative analysis of α-synuclein expression in BL6JHUK mice by RT-PCR, sized on a 1.5% agarose gel. A 568 bp α-synuclein band is detected after as few as 20 PCR cycles on brain total RNA of an ICR mouse at P15. No signal was detected in the BL6JHUK sample even after 35 cycles. However, when the BL6JHUK sample was contaminated with as little as 0.01% ICR RNA, the α-synuclein band could still be detected after 35 PCR cycles. The control shows amplification of a 473 bp tubulin fragment with constant 35 cycles. B. Probing for the presence of the α-synuclein gene in the ICR, 129/Ola, BL6JHUK, BL6N, BL6, and BL6Jax strains. Genomic α-synuclein DNA fragments of exon 4 and of exon 6 were amplified by PCR and the products separated on a 2% agarose gel. In a control experiment, a fragment of the NR1 gene was amplified. Fragment sizes are 130 bp, 266 bp, and 508 bp, respectively. C. Genomic PCR on BL6JHUK and BL6Jax DNA with the markers Atoh2, D6Mit122, D6Mit357, Atoh1, and D6Mit299. The D6Mit357 signal was absent from the BL6JHUK strain, whereas all other markers amplified on the two DNA samples. Atoh2 and D6Mit122 are both currently mapped to about 1 cM upstream of Snca (marker D6Mit357), Atoh1 and D6Mit299 both to about 1 cM downstream of this locus [[22]; and databases cited there]. PCR products were separated on a 2% agarose gel.

Figure 3

Developmental expression of β- and γ-synuclein in different mouse strains.A. 15% SDS-PAGE was performed on brain homogenate from the 129/Ola, ICR, and the BL6JHUK strain at time points P0, P8, P15, and adult (20 μg protein per lane). The Western blot membrane was probed with polyclonal anti β-synuclein IgG, detecting the 19 kDa protein. The lower part shows the anti α-tubulin control. B. Northern blot of brain polyA⁺-RNA (500 ng per lane) from 129/Ola, ICR, and BL6JHUK mice at different developmental stages (P0, P8, P15, and adult). Probing with a 3'UTR γ-synuclein probe detects a transcript of approximately 0.8 kB. RNA samples were pooled from 4 animals (at P0), or from 2 animals (at P8 and P15) from one litter (same membrane as in Fig. 1B, actin control identical to Fig. 1B).

Figure 4

Developmental expression of synphilin-1 in different mouse strains. A. Northern blot of brain polyA⁺-RNA (500 ng per lane) from 129/Ola, ICR, and BL6JHUK mice at different developmental stages (P0, P8, P15, and adult). Probing with a 1.5 kB synphilin-1 probe detects a transcript of approximately 4 kB. RNA samples were pooled from 4 animals (at P0), or from 2 animals (at P8 and P15) from one litter. After stripping, the membrane was re-probed with an actin probe. B. Brain homogenate from 129/Ola, ICR, and BL6JHUK mice at P0, P8, P15, and adult age separated by 7.5% SDS-PAGE (30 μg protein per lane). The Western blot membrane was sequentially probed with polyclonal anti synphilin-1 and monoclonal anti α-tubulin antibodies. The recognized proteins have apparent molecular weights of approximately 85 kDa and 55 kDa, respectively.