Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

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

A Mutation in the Gene Encoding Mitochondrial Mg2+ Channel MRS2 Results in Demyelination in the Rat

The rat demyelination (dmy) mutation serves as a unique model system to investigate the maintenance of myelin, because it provokes severe myelin breakdown in the central nervous system (CNS) after normal postnatal completion of myelination. Here, we report the molecular characterization of this mutation and discuss the possible pathomechanisms underlying demyelination. By positional cloning, we found that a G-to-A transition, 177 bp downstream of exon 3 of the Mrs2 (MRS2 magnesium homeostasis factor (Saccharomyces cerevisiae)) gene, generated a novel splice acceptor site which resulted in functional inactivation of the mutant allele. Transgenic rescue with wild-type Mrs2-cDNA validated our findings. Mrs2 encodes an essential component of the major Mg2+ influx system in mitochondria of yeast as well as human cells. We showed that the dmy/dmy rats have major mitochondrial deficits with a markedly elevated lactic acid concentration in the cerebrospinal fluid, a 60% reduction in ATP, and increased numbers of mitochondria in the swollen cytoplasm of oligodendrocytes. MRS2-GFP recombinant BAC transgenic rats showed that MRS2 was dominantly expressed in neurons rather than oligodendrocytes and was ultrastructurally observed in the inner membrane of mitochondria. Our observations led to the conclusion that dmy/dmy rats suffer from a mitochondrial disease and that the maintenance of myelin has a different mechanism from its initial production. They also established that Mg2+ homeostasis in CNS mitochondria is essential for the maintenance of myelin

Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

BACKGROUND: Inositol 1,4,5trisphosphate (IP(3)) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab)) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab) alleles are phenotypically normal. However, the presence of one Plcd3(mNab) allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3(mNab) mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab) mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING

ANIMAL MODELS FOR THE STUDY OF LEISHMANIASIS IMMUNOLOGY

Leishmaniasis remains a major public health problem worldwide and is classified as Category I by the TDR/WHO, mainly due to the absence of control. Many experimental models like rodents, dogs and monkeys have been developed, each with specific features, in order to characterize the immune response to Leishmania species, but none reproduces the pathology observed in human disease. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue targets (mice footpad, ear, or base of tail) are being infected, and different numbers (“low” 1×102 and “high” 1×106) of metacyclic promastigotes have been inoculated. Recently, new approaches have been proposed to provide more meaningful data regarding the host response and pathogenesis that parallels human disease. The use of sand fly saliva and low numbers of parasites in experimental infections has led to mimic natural transmission and find new molecules and immune mechanisms which should be considered when designing vaccines and control strategies. Moreover, the use of wild rodents as experimental models has been proposed as a good alternative for studying the host-pathogen relationships and for testing candidate vaccines. To date, using natural reservoirs to study Leishmania infection has been challenging because immunologic reagents for use in wild rodents are lacking. This review discusses the principal immunological findings against Leishmania infection in different animal models highlighting the importance of using experimental conditions similar to natural transmission and reservoir species as experimental models to study the immunopathology of the disease

The gene encoding sarcoplasmic reticulum calcium ATPase-1 (Atp2a1) maps to distal mouse chromosome 7

Schleef M, SimonChazottes D, Lengeling A, et al. The gene encoding sarcoplasmic reticulum calcium ATPase-1 (Atp2a1) maps to distal mouse chromosome 7. MAMMALIAN GENOME. 1996;7(10):788

Erythropoietic protoporphyria in the house mouse. A recessive inherited ferrochelatase deficiency with anemia, photosensitivity, and liver disease.

A viable autosomal recessive mutation (named fch, or ferrochelatase deficiency) causing jaundice and anemia in mice arose in a mutagenesis experiment using ethylnitrosourea. Homozygotes (fch/fch) display a hemolytic anemia, photosensitivity, cholestasis, and severe hepatic dysfunction. Protoporphyrin is found at high concentration in erythrocytes, serum, and liver. Ferrochelatase activity in various tissues is 2.7-6.3% of normal. Heterozygotes (+/fch) are not anemic and have normal liver function; they are not sensitive to light exposure; ferrochelatase activity is 45-65% of normal. Southern blot analysis using a ferrochelatase cDNA probe reveals no gross deletion of the ferrochelatase gene. This is the first spontaneous form of erythropoietic protoporphyria in the house mouse. Despite the presence in the mouse of clinical and biochemical features infrequent in the human, this mutation may represent a model for the human disease, especially in its severe form