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. 2012 Jun 8;287(24):20356-68.
doi: 10.1074/jbc.M112.349738. Epub 2012 Apr 13.

Conditional deletion of Notch1 and Notch2 genes in excitatory neurons of postnatal forebrain does not cause neurodegeneration or reduction of Notch mRNAs and proteins

Jin Zheng  1 , Hirotaka Watanabe, Mary Wines-Samuelson, Huailong Zhao, Thomas Gridley, Raphael Kopan, Jie Shen
Affiliations

Conditional deletion of Notch1 and Notch2 genes in excitatory neurons of postnatal forebrain does not cause neurodegeneration or reduction of Notch mRNAs and proteins

Jin Zheng et al. J Biol Chem. .

Abstract

Activation of Notch signaling requires intramembranous cleavage by γ-secretase to release the intracellular domain. We previously demonstrated that presenilin and nicastrin, components of the γ-secretase complex, are required for neuronal survival in the adult cerebral cortex. Here we investigate whether Notch1 and/or Notch2 are functional targets of presenilin/γ-secretase in promoting survival of excitatory neurons in the adult cerebral cortex by generating Notch1, Notch2, and Notch1/Notch2 conditional knock-out (cKO) mice. Unexpectedly, we did not detect any neuronal degeneration in the adult cerebral cortex of these Notch cKO mice up to ∼2 years of age, whereas conditional inactivation of presenilin or nicastrin using the same αCaMKII-Cre transgenic mouse caused progressive, striking neuronal loss beginning at 4 months of age. More surprisingly, we failed to detect any reduction of Notch1 and Notch2 mRNAs and proteins in the cerebral cortex of Notch1 and Notch2 cKO mice, respectively, even though Cre-mediated genomic deletion of the floxed Notch1 and Notch2 exons clearly took place in the cerebral cortex of these cKO mice. Furthermore, introduction of Cre recombinase into primary cortical cultures prepared from postnatal floxed Notch1/Notch2 pups, where Notch1 and Notch2 are highly expressed, completely eliminated their expression, indicating that the floxed Notch1 and Notch2 alleles can be efficiently inactivated in the presence of Cre. Together, these results demonstrate that Notch1 and Notch2 are not involved in the age-related neurodegeneration caused by loss of presenilin or γ-secretase and suggest that there is no detectable expression of Notch1 and Notch2 in pyramidal neurons of the adult cerebral cortex.

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Figures

FIGURE 1.
FIGURE 1.
Normal gross morphology of the cerebral cortex of Notch cKO mice. A and B, shown is Nissl staining of comparable sagittal brain sections (10 μm, paraffin) of aged Notch cKO and littermate control mice (24 months for Notch1 cKO, 24 months for Notch2 cKO, 12 months for Notch1/2 cDKO). Representative images of sagittal brain sections at low magnification (A) and the boxed areas of the neocortex and hippocampus at a higher magnification (B) are shown. Scale bar, 1 mm (A) and 200 μm (B).
FIGURE 2.
FIGURE 2.
No cerebral atrophy and neuronal loss in the neocortex of aged Notch cKO mice. A, shown is stereological measurement of the cortical volume from Notch cKO and littermate control brains (16–24 months for Notch1 cKO, 22–26 months for Notch2 cKO, 6–12 months for Notch1/2 cDKO mice). Values are presented per hemisphere. There is no significant (NS) difference in the neocortical volume between Notch cKO mice and their respective littermate controls (n = 4 per genotype, p > 0.05). B, shown is stereological quantification of neuronal number in the neocortex using NeuN staining. Similar numbers of cortical neuron are present between Notch cKO mice and the littermate control mice (n = 4 per genotype, p > 0.05). NS, not significant.
FIGURE 3.
FIGURE 3.
No astrogliosis or up-regulation of GFAP in the cerebral cortex of Notch cKO mice. A, shown is GFAP immunohistochemical analysis of Notch1 cKO, Notch2 cKO, and Notch1/2 cDKO mice using paraffin sections of Notch cKO and littermate control mice at 12–26 months old of age. Representative views of hippocampal area CA1 at comparable levels are shown. There is no increase in GFAP immunoreactivity in any of the Notch cKO mice compared with the respective controls (fNotch1/fNotch1 for Notch1 cKO, fNotch2/fNotch2 for Notch2 cKO, fNotch1/fNotch1;fNotch2/fNotch2 for Notch1/2 cDKO). GFAP immunostaining of Nicastrin (Nct) cKO and littermate control (fNct/fNct) brains at 9 months of age is also included as the positive control for reactive astrogliosis. Scale bar, 100 μm. B, shown is Western analysis of GFAP in the cerebral cortex of Notch1 or Notch2 cKO mice. The level of GFAP expression (50 kDa) is similar in cortical lysates of Notch1 cKO and littermate control mice at 23 months of age or Notch2 cKO and littermate control mice at 14 months of age. β-Actin is used as loading control.
FIGURE 4.
FIGURE 4.
Deletion of the floxed Notch sequences in the cerebral cortex of Notch1 and Notch2 cKO mice. A and B, PCR using genomic DNA isolated from the neocortex and the hippocampus as well as the tail of Notch1 cKO (A) and Notch2 cKO (B) mice is shown. PCR was performed using three primers that can amplify and distinguish the floxed, deleted, and wild-type alleles. Only one PCR product (500 bp for the floxed Notch1 allele, 383 bp for the floxed Notch2 allele) is produced in the DNA samples from the neocortex, the hippocampus, and the tail of control (fNotch1/fNotch1 for Notch1 cKO, fNotch2/fNotch2 for Notch2 cKO) mice. In cKO mice, one PCR product (500 bp for the floxed Notch1 allele, 383 bp for the floxed Notch2 allele) is produced in the tail DNA sample, whereas two PCR products representing the floxed and the deleted (600 bp for Notch1, 489 bp for Notch2) alleles are produced in DNA samples from the neocortex and hippocampus, suggesting mosaic cell populations (some carrying the floxed allele, some carrying the deleted allele) in the cortex.
FIGURE 5.
FIGURE 5.
Elimination of Notch1 and Notch2 proteins in primary cortical neuronal cultures after Cre transduction. Shown is the time course of Notch1 (A) or Notch2 (B) inactivation in fNotch1/fNotch1;fNotch2/fNotch2 cortical neuronal cultures after Cre transduction. The lentivirus carrying the cDNA encoding either a GFP/defective Cre (Cre−) or a GFP/functional Cre fusion protein (Cre+) was introduced to infect cortical neuronal cultures derived from fNotch1/fNotch1;fNotch2/fNotch2 neonate pups at DIV1. Total cell lysates were collected at the designated days (DIV2–10) and were analyzed (10 μg of lysates per lane) by Western blotting using specific antibodies for the Notch1 (rabbit polyclonal, from A. Israel) or the Notch2 (rat monoclonal, clone C651.6DbHN from the Hybridoma Bank, University of Iowa) C-terminal domain. α-Tubulin or VCP (Vasolin containing protein) was used as a loading control. Although most Notch proteins were subjected to S1 cleavage (Cleaved, ∼95 kDa), full-length (FL) Notch (∼270 kDa) could be easily detected in this culture system. In the presence of Cre, inactivation of Notch1 and Notch2 occurs rapidly, and loss of Notch1 and Notch2 proteins is complete by DIV4.
FIGURE 6.
FIGURE 6.
Unchanged levels of Notch1 and Notch2 mRNAs in the cerebral cortex of Notch cKO mice. A, Northern blots (left) and quantification (right) of Notch1 mRNAs in Notch1 cKO (n = 5) and littermate control (n = 8) mice show similar levels of Notch1 mRNAs in total RNA samples isolated from the cortex of cKO and littermate controls. B, Northern blots (left) and quantification (right) of Notch2 mRNAs in Notch2 cKO (n = 3) and littermate control (n = 3) mice show similar levels of Notch2 mRNAs in control and Notch2 cKO mice. The level of Notch mRNAs is normalized to GAPDH mRNAs, and the value of littermate controls is set as 100%. All data are expressed as the mean ± S.E. Statistical analysis was performed using two-tailed unpaired Student's t test. NS, not significant.
FIGURE 7.
FIGURE 7.
Unchanged levels of Notch1 and Notch2 proteins in the cerebral cortex of Notch cKO mice. A, shown are Western analysis (left) and quantification (right) of Notch1 proteins in the neocortex and the hippocampus of adult Notch1 cKO (n = 6) and littermate control (n = 7) mice using a Notch1 rabbit monoclonal antibody (clone D1E11, Cell Signal Technology). B, shown are Western analysis (left) and quantification (right) of Notch2 proteins in the neocortex and hippocampus of adult Notch2 cKO (n = 4) and control (n = 7) mice using a Notch2 rabbit monoclonal antibody (clone D76A6, Cell Signal Technology). No significant difference of Notch1 or Notch2 proteins was detected between cKO and control samples, indicating that despite the deletion of the floxed exons at the genomic DNA level, protein expression is not altered in cKO cortical samples and suggesting that there is little Notch expression in these excitatory pyramidal neurons of the adult cerebral cortex where Cre is expressed under the control of the αCaMKII promoter. Total protein lysates of E14.5 embryonic brains of Nestin-Cre-driven Notch1 cKO (fNotch1/fNotch1;Nestin-Cre) mice (A) or neuronal cultures (DIV8) derived from fNotch1/fNotch1;fNotch2/fNotch2 (fNotch/fNotch) postnatal pups (B) are included as controls. All values are normalized to that of VCP (Vasolin containing protein) protein, which is used as loading control. The Notch1 antibody (rabbit monoclonal) used here recognizes a nonspecific signal that is only present in the adult brain (*). All data are expressed as the mean ± S.E. Statistical analysis was performed using two-tailed unpaired Student's t test. NS, not significant.
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