Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase

Abstract

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G<-->A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

Figure 1. Characteristics of tambaleante mice.

Hind limbs clasping reflex (A) and rotarod performance (B) of tambaleante (tbl/tbl) and control (+/+) mice. Data show mean±s.d. *** p<0.001 (C) H&E stained sections of the cerebellum of a +/+ control mouse aged two months. (D–F) H&E sections of the cerebellum of Herc1^tbl/Herc1^tbl mice aged respectively of 1, 2, and 3 months (M), exhibiting Purkinje cell degeneration. Anti-calbindin D28-k staining of parasagittal sections of a normal (G,I) and tambaleante (H,J) mouse cortex aged 4 months. The cortex of the mutant mouse is almost completely depleted of PCs. Scale bars: (G,H) 500 µm; (I,J) 25 µm.

Figure 2. Growth and lifespan of tambaleante mice and control.

Graphs of growth (left) and survival (right) from mice tambaleante (tbl/tbl) and mice control (+/+). Growth was analyzed in mice (n>9) aged 3–12 months of age. Data show mean±s.d.

Figure 3. Molecular genetics of the Herc1^tbl locus.

(A) Genotyping 30 tbl/tbl–F2 mice (60 meiotic events) of an intersubspecific cross allowed to map the tbl locus to mouse chromosome 9 between D9Mit233 and D9Mit165. White rectangles symbolize homozygosity for the DW genotype, blue rectangles symbolize heterozygosity, and deep blue homozygosity for MBT genotype. A single mouse, with a crossover between D9Mit302 and D9Mit303 and a +/+ genotype at the tbl locus, allowed us to eliminate the Rora locus (where the mutation staggerer occurred) as a candidate. (B) The tbl candidate region contains eleven genes. BAC clone RP23-355L9 (shown as a green line) was used for transgenic rescue. (C) Sequence analysis of the cDNA from Herc1^tbl/Herc1^tbl and +/+ DW mice showed a point mutation in exon 5, resulting in a Gly⇔Glu substitution. This missense mutation is located in the RCC1-like domain (RLD) 1 of the HERC1 protein (arrow in D) and changes a highly preserved glycine in the HERC and RCC1 family of proteins (arrow in E; see also [11]). Mouse HERC family and characteristic domains are also shown (D; see text for details).

Figure 4. Genotyping analysis of tbl mutation.

The tbl mutation generates a new restriction site (GAAGA) for the MboII enzyme. PCR amplification of genomic DNA samples of wild-type (+) or Herc1^tbl haplotypes with specific primers followed by digestion of the amplification products with the MboII enzyme allows identifying a haplotype specific pattern by PAGE.

Figure 5. Rescue of the tambaleante phenotype.

(A) Genotyping analysis by PCR of genomic DNA to identify transgenic and tambaleante animals. (B) Motor coordination was tested in wild-type (+/+), homozygous (tbl/tbl), and homozygous with transgenic copies (tbl/tbl;Tg^HERC1cDNA) animals (n = 5–9). Testing began at 4 weeks of age and was conducted until week 12 to follow the progression on each phenotype. The animals were put on the rotarod until the latency to fall off reached the total time of 60 s and the percentatge (%) of animals that stayed during this time was represented. (C) Weight chart of these animals (n = 5–9) aged 3–6 months of age. Data show mean±s.d. *** p<0.001, * p<0.05. Staining with H&E (D) and immunostaining with anti-calbindin D28k antibodies (E) of cerebellum sections of these animals. Scale bars: (D,E lower pictures) 25µm; (E upper pictures) 500µm. See Materials and Methods for detailed protocols.

Figure 6. Herc1 gene expression in mice.

Herc1 is widely expressed in various tissues as indicated by RT–PCR. Gapdh expression was used as control.

Figure 7. Herc1 expression and functional analysis.

Brain (A–D), kidney (A,C) and cerebellum (D) homogenates from wild-type (+/+), heterozygous (+/tbl) and homozygous (tbl/tbl) mice were analyzed by Western-blot using specific antibodies against the indicated proteins. HERC1 (in brain and in kidney), P-T389-S6K1 (in kidney), p62/SQSTM1 and LC3-II (in brain and in cerebellum) levels were quantified (n = 4–9) and expressed as the mean±s.d. of percentage of respective control. ** p<0.01,*** p<0.001. (E) Parasagittal section of a 2-month-old tbl/tbl cerebellum double immunostained with anti-calbindin D28k antibodies to visualize PC (green) and anti-LAMP-1 (red) to identify lysosomes and autophagosomes. The arrowheads point to an area of the cerebellar cortex almost devoid of PC. In this area, LAMP-1 positive puncta are numerous, testifying for the degeneration of PC. (F) Parasagittal 1µm-thick plastic section stained with toluidine blue, illustrating four PC somata (asterisks). The arrows point to dense cytoplasmic inclusions accumulated in the cell bodies and proximal dendrites, particularly at their branching points. The arrowhead points to the first Ranvier node of a PC axon initial segment, which looks normal. (G,H) Electron-micrographs made from the same mouse showing two profiles of the proximal dendritic compartment. The one in (G) illustrates a branching zone in an early stage of the autophagic process. The dendrite contains some vacuoles bound by a smooth double membrane (arrows), enclosing whorls of membrane-like elements, and dense debris (asterisks) suspended in an electronlucent matrix. The other dendritic profile (H) corresponds to a more advanced stage in autophagy, characterized by the occurrence of extremely numerous single membrane bound vacuoles corresponding to autolysosomes, some of them of large size (asterisk). The arrow points to a giant spine emerging from the dendrite and postsynaptic to several parallel fibers varicosities. Scale bars: the bar is equal to 100 µm in (E), 24 µm in (F), 1.2 µm in (G), and 1.5 µm in (H).