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. 2016 Aug 30;7(35):56083-56106.
doi: 10.18632/oncotarget.11270.

The HERC2 Ubiquitin Ligase Is Essential for Embryonic Development and Regulates Motor Coordination

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Free PMC article

The HERC2 Ubiquitin Ligase Is Essential for Embryonic Development and Regulates Motor Coordination

Monica Cubillos-Rojas et al. Oncotarget. .
Free PMC article

Abstract

A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.

Keywords: Angelman syndrome; Pathology Section; Purkinje cells; behavioural analysis; p53; ubiquitin.

Conflict of interest statement

All authors have not conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Generation of the Herc2530 mice
A. Schematic representation of the Herc2 wild-type allele (Herc2+) and Herc2530 allele and the designed primers to identify both alleles (left). The Herc2530 allele contains the pGT0lxr vector that expresses the fusion of β-galactosidase and neomycin transferase within intron 2. The integration of the trap was determined by genotyping using the indicated primers (right), 530KO01/530KO04 for the wild-type allele and Gal7/Gal8 for the 530 allele. B. Exon structure of Herc2+ and Herc2530 (left). RT-PCR experiments with mRNA from liver, spleen and kidney of Herc2+ and Herc2530 mice was performed using the indicated primers. C. PCR products from B were sequenced. The trap was inserted after exon 2, for which the mutant protein contains the first 24 amino acids of the HERC2 and β-galactosidase protein. D. β-galactosidase expression in Herc2530 mice. The activity of β-galactosidase was determined in the testes, brain, heart and kidney of Herc2530 mice and detected by X-gal staining. E. Scheme of HERC2 protein and the expected product from the Herc2530 allele. The P594L pathological mutation is indicated (*). RLD: RCC1-like domain.
Figure 2
Figure 2. Analysis of progeny from the Herc2+/530 cross
A. Analysis of offspring born from the intercross of Herc2+/530 mice. Ninety-one animals were genotyped by PCR of genomic DNA isolated from mouse tails. The expected frequencies for Herc2+/+ and Herc2+/530 were obtained; however, no homozygous mice (Herc2530/530) were identified. B. Analysis of embryonic lethality in Herc2530/530 mice. Embryos from Herc2+/530 pregnant females at different stages were isolated and genotyped. The Herc2+/+ and Herc2+/530 genotypes were identified, but not the Herc2530/530. The placentas without embryos could not be genotyped. C. Analysis of HERC2 protein levels during development. Lysates from brains at different stages were analyzed by immunoblotting for HERC2 and β-actin. E16 (embryonic day), P0, P5 and P15 (post-natal day 0, 5 and 15, respectively) and AD (adult animal).
Figure 3
Figure 3. p53 inactivation did not rescue the lethality of Herc2530/530 homozygous mice
Graphs of growth rate A. and survival B. from Herc2+/+ and Herc2+/530 mice. Growth and survival were analyzed in male mice (n>10) at the indicated weeks. The survival for p53−/− mice also was analyzed. C. The analysis of mice from a cross of double heterozygous Herc2+/530 p53+/− animals. The offspring was genotyped by PCR of genomic DNA with the appropriate primers, indicating that the embryonic lethal phenotype of Herc2530/530 embryos was not rescued by crossing with p53−/− mice.
Figure 4
Figure 4. Herc2+/530 mice show reduced levels of HERC2 protein
A. HERC2 protein levels were analyzed by immunoblotting using specific antibodies against HERC2 in several tissues from 8 week old mice. The levels of HERC2 were quantified (n = 8) and normalized with respect to β-actin levels. B. β-galactosidase expression in brain from Herc2530 mouse. The β-galactosidase activity was detected ubiquitously in all areas using X-gal staining. However, there were forebrain cortical and subcortical areas in which β-galactosidase labeling was the most intense (asterisks). C. The levels of HERC2 were analyzed by immunoblotting in lysates of cerebellum, cerebral cortex and diencephalon from Herc2+/+ and Herc2+/530 mice at P4 (post-natal day 4). BA, basal amygdala. CA1, pyramidal cell layer of the hippocampal cornu ammonis 1. DEn, dorsal endopiriform nucleus. DG, granular cell layer of hippocampal dentate gyrus. DM, dorsomedial nucleus of the hypothalamus. Pir, piriform cortex. PV, paraventricular thalamic nucleus. RSG, retrosplenial granular cortex. VM, ventromedial nucleus of the hypothalamus.
Figure 5
Figure 5. Herc2+/530 mice show reduced activity of HERC2
A. USP33, a substrate of ubiquitination of HERC2, was analyzed in lysates (cerebellum, cerebral cortex and diencephalon) from 8 week old mice by immunoblotting. Higher levels of USP33 were observed in all areas of Herc2+/530 mice. Levels of USP33 were quantified and normalized with respect to β-actin levels. B. Herc2+/530 mice show reduced levels of p21 mRNA. RT quantitative PCR analyses were performed in forebrain and cerebellum from Herc2+/+ and Herc2+/530 mice to quantify p21 gene expression (n = 10). The levels of expression were normalized with respect to GAPDH gene expression.
Figure 6
Figure 6. A homozygous mutation in human HERC2 that causes an Angelman-like syndrome reduces the activity of the HERC2 protein
A. Fibroblasts derived from individuals with HERC2 wild-type or HERC2 with the mutation P594L were analyzed by immunoblotting for the indicated antibodies. Levels of USP33 and p21 proteins were quantified and normalized with respect to α-tubulin levels. B. Levels of p21 mRNA were analyzed by RT quantitative PCR analysis and normalized with respect to 18S gene expression. C. The levels of HERC2, p53, p21 and α-tubulin proteins were analyzed in the presence or absence of the proteasome inhibitor MG132. D. U2OS cells were transfected with non-targeting (NT) or HERC2 siRNAs and analyzed by immunoblotting against the indicated proteins. The levels of USP33 or p21 were quantified and normalized with respect to Ran levels.
Figure 7
Figure 7. Impaired motor coordination in Herc2+/530 mice
A.-B. The number of falls from the rotarod increases in Herc2+/530 mice at 6 months of age in comparison with control littermates. C. No difference was found in p53+/− and p53−/− mice in comparison with WT. (D-E) EMG measurements of CMAP amplitudes in the MG of control and Herc2+/530 mice show normal neurotransmission efficacy in postnatal heterozygous mice. D. Representative recordings during a train of stimuli at 100 Hz in a control and a Herc2+/530mouse. E. Depression of CMAP amplitudes (normalized to the first response) during a train of stimuli of 300 ms at 100 Hz in control (n = 7) and heterozygous mice (n = 18). The train index F., corresponding to the depression at the end of the train, and PPF (Pair -Pulse Facilitation) G., are similar between groups.
Figure 8
Figure 8. Purkinje cells loss in Herc2+/530 mice
Microphotographs of coronal sections through the cerebellar cortex of 9 months old wild-type (wt, A-C, G) and 9 months old Herc2+/530 mice (+/530, D-F, H). Calbindin immunohistochemistry shows as Purkinje cell somata form a continuous cell layer in the Herc2+/+ (wt) cerebellum (A-C) However, parasagittal zones lacking of immunoreactivity throughout the Herc2+/530 cerebellum indicative of Purkinje cell loss are observed (arrows and arrowheads, in (D, F)). These symmetrical Purkinje cells deprived bands, characterized by the presence of wide spaces lacking Purkinje cell somata (H, asterisks) and dendritic debris through the molecular layer (H, mol), distribute differently according a medio-lateral gradient. In the vermis and paravermal zones the immunonegative zones are sagittaly distributed in narrow gaps (D-E, arrows); while at the hemispheres the areas devoid of Purkinje cells, also bilateral, reach a greater extension (D, F, arrowheads) in which remain some surviving Purkinje cells (F, small arrows). G, illustrates the Herc2+/+ (wt) immunostaining of normal Purkinje cells; note as dendritic trees fulfill the molecular layer (G, mol), while Purkinje cells somata align in a continuous row. PCl, Purkinje cells layer. Bars = 600 μm (A, D), 400 μm (B-C, E-F), and 30 μm (G-H)
Figure 9
Figure 9. HERC2 is present in Purkinje cells
Microphotographs of coronal sections through the cerebellar cortex of 9 months old Herc2+/+ (wt, A.-B.) and 9 months old Herc2+/530 mice (+/530, D.-E.). Epifluorescence microscopy analysis shows that HERC2 is expressed in all the adult Purkinje cells colocalizating with the general marker of Purkinje cell calbindin (CaBP) (A-E) in the cerebellar cortex (A, Cc,), and their axonal endings in the cerebellar nuclei (A, Cn, arrow). Herc2+/530 cerebellum displays parasagittal bands of Purkinje cells loss in the vermis and paravermal zones (C., arrows; D, arrowheads), and areas of extensive Purkinje cell loss in the cerebellar paraflocculus (asterisks). The arrows in E illustrate the co-expression of both proteins in the Purkinje cell dendritic tree. Bars = 750μm (C), 200 μm (A), 100μm (D), 75μm (E), and 50 μm (B).
Figure 10
Figure 10. Purkinje cell degeneration in Herc2+/530mice
Microphotographs of transmitted light A.-E. and electron microscopy F.-H. of parasagittal sections through the cerebellar cortex of 9 months Herc2+/+ (wt, A), and 2 (H) and 9 months old Herc2+/530 mice (+/530, B.-G.). Calbindin immunohistochemistry reveals the presence of rounded thickenings resembling to axonal torpedoes in Herc2+/530 Purkinje cell axons (B-C, arrows), which contrast with the fine grained morphology of normal Purkinje cells axonal plexuses (A, arrows). 1.5 μm thick sections illustrated Purkinje cells (D, arrows) limiting a zone in which disappeared Purkinje cells were substituted by glial Golgi-epithelial cells (small arrows in D and E). High magnification allows detect degenerative dark accumulations within the cytoplasm of the soma (E, arrowhead) and the dendrites (E, arrows) of the Purkinje cells. Herc2+/530 Purkinje cells cytoplasm possesses lysosomes, electron-dense debris (F-H, asterisks), and autophagosomes with different degrees of evolution (G, arrows). Pcn, Purkinje cell nucleus. Bars = 50 μm (A-D), 25 μm (E), and 1 μm (F-H).
Figure 11
Figure 11. Ultrastructural analysis of Herc2+/530 mice indicates accumulation of autophagosomes and lysosomes in Purkinje cells
Electron photomicrographs of parasagittal sections through the cerebellar vermis of 9 months old Herc2+/+ (wt, A., C.-D.) and 9 months old Herc2+/530 mice (+/530, B., E.). An important difference in the presence of autophagic (arrowheads), and lysosomal (arrows) organelles can be observed between wild-type (A) and Herc2+/530 (B) Purkinje cells cytoplasm. The difference is even most evident in the principal Purkinje cell dendrites. Thus, numerous degenerative signs are present in Herc2+/530 Purkinje cells dendrite (E), while are almost absent in wild-type ones (C-D). Pcd, Purkinje cell dendrite. Pcn, Purkinje cell nucleus. Bars = 2 μm (A-C, E), and 1 μm (D).
Figure 12
Figure 12. p62/SQSTM1 in Herc2+/530 mice
Laser confocal microphotographs of coronal sections through the cerebellar cortex of the vermis of 9 months Herc2+/+ (wt, A) and 9 months old Herc2+/530 (+/530, B) mice double labeled with HERC2 and p62 antibodies. Colocalizations of HERC2 and p62 are indicated by arrows in dendrites and cell somata A., B., and by arrowheads in the axonal torpedoes of Herc2+/530 Purkinje cells (B). Asterisks in B indicate the absence of Purkinje cell bodies. Bar = 50 μm (A-B).

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