The disintegrin/metalloproteinase ADAM10 is essential for the establishment of the brain cortex

Abstract

The metalloproteinase and major amyloid precursor protein (APP) alpha-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10(-/-) mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that alpha-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP alpha-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

Figure 1.

Generation of Adam10 cKO mice. A, Schematic representation of wild-type Adam10 allele (I), targeting vector (II), conditional targeted allele (floxed allele) (III), and disrupted Adam10 allele (IV). Exon 2 is indicated as a black box. LoxP and Frt recombination sites are indicated as black arrowheads and white flags, respectively. Arrows indicate the location of the primers. The expected sizes for the indicated restriction enzyme digest fragments detected by 5′, 3′, or internal hygromycin probe (Hyg) from targeted and wild-type allele are indicated below every construct with line diagrams. Positive selection marker is indicated as a gray box. B, Example of Southern blot of DNA isolated from one of the selected embryonic stem cell lines, digested with the indicated restriction enzymes, and hybridized with the different probes (5′, 3′, Hyg). The fragments detected for the wild-type (+) and targeted floxed (Fl) Adam10 allele are indicated. C, PCR analysis of DNA extracted from tail clips of embryos. The fragments detected for the wild-type (+) and the floxed (Fl) Adam10 allele and the cre recombinase are indicated. D, Western blot analysis of brain extracts from E12.5 wild-type, heterozygous, and conditional Adam10 knock-out mice using an antibody against the C terminus of ADAM10. pADAM10, Precursor of ADAM10; mADAM10, mature form of ADAM10.

Figure 2.

Histological analysis of control and Adam10 cKO mice at different developmental stages. A, Lateral and coronal view of control and Adam10 cKO embryos at E17.5 and P1. Showing intracranial hemorrhages in the cKO (b, d, f, indicated by arrows). B, Serial coronal sections at E12.5, E15.5, and E17.5 of Adam10 cKO and littermate controls were stained with hematoxylin and eosin (a–r). a, b, Coronal brain sections of control and Adam10 cKO at E12.5. c–f, Under higher magnification, there was no difference in the cortex (c, d) formation, however the size of the GE (e, f) was slightly reduced in the cKO compared with littermate controls. g, h, Coronal brain sections of control and Adam10 cKO at E15.5. The GE was prominent in the control but much less in the Adam10 cKO. i, j, Higher-power views of the boxed areas in g and h showing disruption of the cortical layering, with an indistinct boundary between the VZ and the IZ in the Adam10 cKO. k, l, Higher-power views of the GE region boxed in g and h. m, n, Coronal brain sections of control and Adam10 cKO at E17.5. The arrow in n indicates the disruption of the subcortical region of the temporal lobe in the cKO brain. o, p, Higher-power views of the cortical region boxed in m and n showing a cortical atrophy and a disrupted laminar organization, with an indistinct boundary between the cortical layers in the Adam10 cKO brain. q, r, Higher-power view of the GE region boxed in m and n showing a severely reduced GE in the Adam10 cKO. Scale bars, 100 μm.

Figure 3.

Increased neuronal differentiation and disrupted laminar organization of the cerebral cortex in Adam10 cKO mice. A, Serial coronal brain sections for E12.5, E15.5, and E17.5 of Adam10 cKO and littermate controls were stained with a monoclonal antibody against NeuN (a–f). a, b, At E12.5 the NeuN labeling patterns were the same in Adam10 cKO and control brains. c–f, Higher-power views of boxed areas in a and b. g, h, At E15.5 more NeuN-positive neurons were present in the developing cortex in the Adam10 cKO in the VZ and the GE. i–l, Higher-power views of boxed areas in g and h. m, n, At E17.5 there was less NeuN-positive staining in the Adam10 cKO and it was distributed diffusely and was not arranged into well defined areas as in the control brain. o–r, Higher-power views of boxed areas in m and n. Scale bars, 100 μm. B, BrdU birth dating revealed the determination of newly generated neurons labeled at E13.5 and collected at E17.5. Coronal sections of control and Adam10 cKO showed more intensely labeled neurons in the Adam10 cKO cortex (b), which were distributed more diffusely also in the IZ and not mainly into the well defined cortical plate (CP) as in the control brain (a). Scale bars, 100 μm.

Figure 4.

Comparison of proliferation in the Adam10 cKO and littermate control brains. a–r, Comparable coronal sections of Adam10 cKO and control at E12.5, E15.5, and E17.5 were stained with a monoclonal antibody against KI67. a, b, At E12.5 the KI67 staining pattern was unchanged in Adam10 cKO and control. c–f, Higher-power views, boxed in a and b. g, h, At E15.5 the KI67 staining pattern was similar in the cortex, whereas the GE of the Adam10 cKO contained fewer proliferating neurons than that of the control. i–l, Higher-power views, boxed in g and h. m, n, At E17.5 the KI67 staining is less in both the cortex and the GE of the Adam10 cKO. o–r, Higher-power views, boxed in m and n. Scale bars, 100 μm.

Figure 5.

Neural stem cells are depleted in E13.5 Adam10 cKO ganglionic eminence and Adam10 mutant spheres have a disrupted sphere organization. A, Neurospheres, which consist of neural stem cells, isolated from the ganglionic eminence of E13.5 control and Adam10 cKO brains at the day of plating are shown. There were more nonproliferating cells already attached to the surface of the culture flask in the cKO (b, d) compared with the control (a, c). Scale bar, 50 μm. B, Secondary neurosphere organization: Control neurospheres had an edge of dividing KI67⁺, Nestin⁺ cells and a core of mainly early differentiated gfap⁺, doublecortin⁺ cells, and Bielschowsky-stained cell processes (c, e, g, i, k), while the Adam10 cKO spheres showed a disrupted organization (d, f, h, j, l). However the apoptotic staining [activated caspase 3 (Act casp3)] pattern was the same in both mutant and control spheres (m, n). Scale bar, 20 μm. C, Quantitative estimation of B showing no differences in the amount of proliferating cells or apoptotic cells in the secondary Adam10 cKO and control spheres.

Figure 6.

Adam10 mutant spheres display an increase in neuronal differentiation at the expense of astrocytes due to a disturbed Notch-1 pathway. A, Single primary neurospheres as a whole or in single cells from control and Adam10 cKO brains were induced to differentiate and immunolabeled for neurons (MAP2), astrocytes (GFAP), oligodendrocytes (CNPase), and activated caspase-3 (a–h). Scale bar, 100 μm. B, Greater percentages of neurons expressing MAP2 and fewer GFAP-positive cells were observed differentiating from Adam10 cKO spheres compared with control spheres (***p < 0.001). C, Schematic representation of the effects of ADAM10 in cell fate decision in the developing nervous system. NSC, Neural stem cell. D, A defect in the proteolytic processing of Notch-1 in Adam10 cKO brains was demonstrated by immunoblotting P1 brain extracts from controls and Adam10 cKOs. Immunoblotting with the anti-Notch-1 mN1A antibody shows an accumulation of an approximately 120 kDa fragment (most likely the S1 furin cleavage product) and a decrease in the S2 Notch-1 fragment in Adam10-deleted brains. Blotting with an antibody (8925) against the intracellular fragment of Notch-1 reveals a dramatic reduction in the generation of the NICD. Actin blotting shows equal loading. Using the same lysates, immunoblot analysis against mouse ADAM10 shows a strong reduction in Adam10 expression in the cKO brains. TMIC, Transmembrane and intracellular domain (Furin cleavage on Notch1); pro, proform of ADAM10; mat, mature form of ADAM10. E, Downregulation of Notch-1 signaling in Adam10 cKO brain. qRT-PCR of Notch target genes Hes1, Hes5, Hey1, and Hey2. A strong reduction in the levels of Hes1, Hes5, Hey1, and Hey5 was found in the Adam10 cKO brain (**p < 0.01, ***p < 0.001).

Figure 7.

APPα processing is severely reduced in neurons derived from E14.5 Adam10 cKO embryos. A, C, Primary neurons were infected with recombinant Semliki Forest Virus driving expression of human wild-type APP. Antibody B63 recognizing the 20 carboxyterminal amino acid residues of APP was used to detect holo-APP and α- and β-secretase-generated carboxyterminal stubs from the cell extracts (c). The α-CTF is >90% decreased in the Adam10 cKO neurons (n = 8, ***p < 0.001), but also CTFβ was significantly reduced in the Adam10 cKO neurons (n = 8, ***p < 0.001). The conditioned medium (s) of the same neurons was incubated with 6E10, which recognizes sAPPα and Aβ. A dramatic decrease in sAPPα (n = 8, ***p < 0.001) and also a significant decrease in sAPPβ and total Aβ production is seen in the cKO (n = 8, *p < 0.05, ***p < 0.001, respectively). B, Quantitative estimation of A and using Aβ_1-40- and Aβ_1-42-specific ELISA, Aβ levels in conditioned medium from primary neurons of controls and Adam10 cKO were measured and normalized to the level of expression of APP holoprotein. A significant reduction in both Aβ₄₀ and Aβ₄₂ in the Adam10 cKO is shown (n = 8, **p < 0.01, ***p < 0.001, respectively). C, D, Urea-SDS-PAGE of conditioned medium of primary neurons results in an overall downregulation of all the different Aβ species in the Adam10 cKO; quantification in D (n = 3, *p < 0.05).