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Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1-/- phenotype and forms complexes with wildtype PS1 and nicastrin.

Brautigam H, Moreno CL, Steele JW, Bogush A, Dickstein DL, Kwok JB, Schofield PR, Thinakaran G, Mathews PM, Hof PR, Gandy S, Ehrlich ME - Sci Rep (2015)

Bottom Line: Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form.Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation.These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

ABSTRACT
The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1(∆exon8) reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1- or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1- mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions.

No MeSH data available.


Related in: MedlinePlus

PS1∆exon8 is expressed as early as embryonic day 10.(A) Genotyping analysis of embryos by PCR for the human PS1∆exon8 transgene. (B) Genotyping results for murine PS1. The upper band corresponds to the PS1(+) allele while the bottom band corresponds to the mutant, KO allele. (C) PS1∆exon8 mRNA expression from embryonic mouse brain extracts by RT-PCR at E10 and E16. Expression is seen as early as E10. (D) Immunoblot of embryonic mouse brain extracts at E10 and E16 and adult mouse brain hippocampal extracts at 18 months of age using monoclonal human specific anti-N-terminus PS1, NT.1 antibody to confirm transgene protein expression. The bottom immunoblot is an actin loading control.
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f1: PS1∆exon8 is expressed as early as embryonic day 10.(A) Genotyping analysis of embryos by PCR for the human PS1∆exon8 transgene. (B) Genotyping results for murine PS1. The upper band corresponds to the PS1(+) allele while the bottom band corresponds to the mutant, KO allele. (C) PS1∆exon8 mRNA expression from embryonic mouse brain extracts by RT-PCR at E10 and E16. Expression is seen as early as E10. (D) Immunoblot of embryonic mouse brain extracts at E10 and E16 and adult mouse brain hippocampal extracts at 18 months of age using monoclonal human specific anti-N-terminus PS1, NT.1 antibody to confirm transgene protein expression. The bottom immunoblot is an actin loading control.

Mentions: To confirm that deletion of exon 8 causes a loss of PS1 activity in vivo, we assessed whether PS1∆exon8 was able to rescue mouse PS1- lethality. We hypothesized that if PS1∆exon8 were truly a complete loss-of-function mutation, PS1∆exon8 would not rescue PS1 KO lethality in embryonic mice. For the initial experiments, two hPS1∆exon8 (+/−) heterozygote mice were bred and 20 positive PS1∆exon8 transgenic mice were then individually bred to a C57Bl6/J mouse. None of the resulting 20 mice were homozygous, as each of those breedings produced Ntg mice. Also, viable mice with the hPS1∆exon8 (+/−)/mPS1(−/−) genotype were not produced from over 50 pups that were genotyped, and therefore E16-18 embryos from timed matings were examined. Human PS1∆exon8 expression from some of these initial breedings is shown in Fig. 1A. Also, each embryo was genotyped for the presence of the PS1∆exon8 transgene and endogenous PS1 (Fig. 1B). Brains from hPS1∆exon8 (+) pups were assayed for transgenic mRNA and exhibited expression as early as E10 (Fig. 1C). This unique band visible in hPS1∆exon8 mice, showed a clear increase in PS1∆exon8 expression from embryonic day 10 to 16, and expression remained stable throughout adulthood (Fig. 1C,D). At E16, mPS1 (−/−) (KO) embryos (Fig. 2A) were grossly indistinguishable from hPS1∆exon8(+/−)/mPS1(−/−) (KO) embryos (Fig. 2B). The mutant phenotype included a shortened rostro-caudal body axis, brain hemorrhage, and skeletal abnormalities. However, hPS1∆exon8(+/−)/mPS1(+/−) embryos carrying a single copy of mPS1, showed no abnormalities (Fig. 2C) compared to a mPS1(+/+) (Ntg) embryo (Fig. 2D). We confirmed the presence of hemorrhage in the intermediate zone near the lateral ventricle in the mPS1(−/−) KO embryo (Fig. 2E,I) and hPS1∆exon8 (+)/mPS1(−/−) embryo (Fig. 2F,J), while hPS1∆exon8(+) with a single endogenous mouse PS1 allele mPS1(+/−) (Fig. 2G,K) and the mPS1(+/+) (Ntg) embryos (Fig. 2H,L) showed no hemorrhage. To phenotype the adult transgenic mouse, we utilized mice carrying a single copy of hPS1∆exon8 on a mPS1(+/+) genetic background.


Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1-/- phenotype and forms complexes with wildtype PS1 and nicastrin.

Brautigam H, Moreno CL, Steele JW, Bogush A, Dickstein DL, Kwok JB, Schofield PR, Thinakaran G, Mathews PM, Hof PR, Gandy S, Ehrlich ME - Sci Rep (2015)

PS1∆exon8 is expressed as early as embryonic day 10.(A) Genotyping analysis of embryos by PCR for the human PS1∆exon8 transgene. (B) Genotyping results for murine PS1. The upper band corresponds to the PS1(+) allele while the bottom band corresponds to the mutant, KO allele. (C) PS1∆exon8 mRNA expression from embryonic mouse brain extracts by RT-PCR at E10 and E16. Expression is seen as early as E10. (D) Immunoblot of embryonic mouse brain extracts at E10 and E16 and adult mouse brain hippocampal extracts at 18 months of age using monoclonal human specific anti-N-terminus PS1, NT.1 antibody to confirm transgene protein expression. The bottom immunoblot is an actin loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660297&req=5

f1: PS1∆exon8 is expressed as early as embryonic day 10.(A) Genotyping analysis of embryos by PCR for the human PS1∆exon8 transgene. (B) Genotyping results for murine PS1. The upper band corresponds to the PS1(+) allele while the bottom band corresponds to the mutant, KO allele. (C) PS1∆exon8 mRNA expression from embryonic mouse brain extracts by RT-PCR at E10 and E16. Expression is seen as early as E10. (D) Immunoblot of embryonic mouse brain extracts at E10 and E16 and adult mouse brain hippocampal extracts at 18 months of age using monoclonal human specific anti-N-terminus PS1, NT.1 antibody to confirm transgene protein expression. The bottom immunoblot is an actin loading control.
Mentions: To confirm that deletion of exon 8 causes a loss of PS1 activity in vivo, we assessed whether PS1∆exon8 was able to rescue mouse PS1- lethality. We hypothesized that if PS1∆exon8 were truly a complete loss-of-function mutation, PS1∆exon8 would not rescue PS1 KO lethality in embryonic mice. For the initial experiments, two hPS1∆exon8 (+/−) heterozygote mice were bred and 20 positive PS1∆exon8 transgenic mice were then individually bred to a C57Bl6/J mouse. None of the resulting 20 mice were homozygous, as each of those breedings produced Ntg mice. Also, viable mice with the hPS1∆exon8 (+/−)/mPS1(−/−) genotype were not produced from over 50 pups that were genotyped, and therefore E16-18 embryos from timed matings were examined. Human PS1∆exon8 expression from some of these initial breedings is shown in Fig. 1A. Also, each embryo was genotyped for the presence of the PS1∆exon8 transgene and endogenous PS1 (Fig. 1B). Brains from hPS1∆exon8 (+) pups were assayed for transgenic mRNA and exhibited expression as early as E10 (Fig. 1C). This unique band visible in hPS1∆exon8 mice, showed a clear increase in PS1∆exon8 expression from embryonic day 10 to 16, and expression remained stable throughout adulthood (Fig. 1C,D). At E16, mPS1 (−/−) (KO) embryos (Fig. 2A) were grossly indistinguishable from hPS1∆exon8(+/−)/mPS1(−/−) (KO) embryos (Fig. 2B). The mutant phenotype included a shortened rostro-caudal body axis, brain hemorrhage, and skeletal abnormalities. However, hPS1∆exon8(+/−)/mPS1(+/−) embryos carrying a single copy of mPS1, showed no abnormalities (Fig. 2C) compared to a mPS1(+/+) (Ntg) embryo (Fig. 2D). We confirmed the presence of hemorrhage in the intermediate zone near the lateral ventricle in the mPS1(−/−) KO embryo (Fig. 2E,I) and hPS1∆exon8 (+)/mPS1(−/−) embryo (Fig. 2F,J), while hPS1∆exon8(+) with a single endogenous mouse PS1 allele mPS1(+/−) (Fig. 2G,K) and the mPS1(+/+) (Ntg) embryos (Fig. 2H,L) showed no hemorrhage. To phenotype the adult transgenic mouse, we utilized mice carrying a single copy of hPS1∆exon8 on a mPS1(+/+) genetic background.

Bottom Line: Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form.Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation.These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

ABSTRACT
The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1(∆exon8) reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1- or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1- mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions.

No MeSH data available.


Related in: MedlinePlus