Arturo Incao

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

A sensitized mutagenesis screen identifies Gli3 as a modifier of Sox10 neurocristopathy

Haploinsufficiency for the transcription factor SOX10 is associated with the pigmentary deficiencies of Waardenburg syndrome (WS) and is modeled in Sox10 haploinsufficient mice (Sox10(LacZ/+)). As genetic background affects WS severity in both humans and mice, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify modifiers that increase the phenotypic severity of Sox10(LacZ/+) mice. Analysis of 230 pedigrees identified three modifiers, named modifier of Sox10 neurocristopathies (Mos1, Mos2 and Mos3). Linkage analysis confirmed their locations on mouse chromosomes 13, 4 and 3, respectively, within regions distinct from previously identified WS loci. Positional candidate analysis of Mos1 identified a truncation mutation in a hedgehog(HH)-signaling mediator, GLI-Kruppel family member 3 (Gli3). Complementation tests using a second allele of Gli3 (Gli3(Xt-J)) confirmed that a null mutation of Gli3 causes the increased hypopigmentation in Sox10(LacZ/+);Gli3(Mos1/)(+) double heterozygotes. Early melanoblast markers (Mitf, Sox10, Dct, and Si) are reduced in Gli3(Mos1/)(Mos1) embryos, indicating that loss of GLI3 signaling disrupts melanoblast specification. In contrast, mice expressing only the GLI3 repressor have normal melanoblast specification, indicating that the full-length GLI3 activator is not required for specification of neural crest to the melanocyte lineage. This study demonstrates the feasibility of sensitized screens to identify disease modifier loci and implicates GLI3 and other HH signaling components as modifiers of human neurocristopathies.

Genetic evidence does not support direct regulation of EDNRB by SOX10 in migratory neural crest and the melanocyte lineage

Mutations in the transcription factor Sox10 or Endothelin Receptor B (Ednrb) result in Waardenburg Syndrome Type IV (WS-IV), which presents with deficiencies of neural crest derived melanocytes (hypopigmentation) and enteric ganglia (hypoganglionosis). As Sox10 and Ednrb are expressed in mouse migratory neural crest cells and melanoblasts, we investigated the possibility that SOX10 and EDNRB function through a hierarchical relationship during melanocyte development. However, our results support a distinct rather than hierarchical relationship. First, SOX10 expression continues in Ednrb null melanoblasts, demonstrating that SOX10 expression is not dependent on EDNRB function. Second, Ednrb expression persists in E10.5 Sox10null embryos, demonstrating that Ednrb is not dependent on SOX10 for expression in migratory neural crest cells. Third, over-expression of SOX10 in melanoblasts of mice that harbor null or hypomorphic Ednrb alleles does not rescue hypopigmentation, suggesting that SOX10 overexpression can neither complement a lack of EDNRB function nor increase Ednrb expression. Fourth, mice that are double heterozygous for loss-of-function mutations in Sox10 and Ednrb do not demonstrate synergistically increased hypopigmentation compared to mice that are single heterozygotes for either mutation alone, suggesting a lack of direct genetic interaction between these genes. Our results suggest that SOX10 does not directly activate Ednrb transcription in the melanocyte lineage. Given that SOX10 directly activates Ednrb in the enteric nervous system, our results suggest that SOX10 may differentially activate target genes based on the particular cellular context.

A Sox10 Expression Screen Identifies an Amino Acid Essential for Erbb3 Function

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10LacZ/+. Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1–4 (msp1–4), which show altered NC patterning. In homozygous msp1 embryos, Sox10LacZ expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3msp1, that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting.

Complementation of melanocyte development in SOX10 mutant neural crest using lineage-directed gene transfer

An in vitro gene complementation approach has been developed to dissect gene function and regulation in neural crest (NC) development and disease. The approach uses the avian RCAS virus to express genes in NC cells derived from transgenic mice expressing the RCAS receptor TVA, under the control of defined promoter elements. Constructs for creating TVA transgenic mice were developed using site‐specific recombination GATEWAY (GW), compatible vectors that can also be used to facilitate analysis of genomic fragments for transcriptional regulatory elements. By using these GW vectors to facilitate cloning, transgenic mouse lines were generated that express TVA in SOX10‐expressing NC stem cells under the control of the Pax3 promoter. The Pax3‐tv‐a transgene was bred onto a Sox10‐deficient background, and the feasibility of complementing genetic NC defects was demonstrated by infecting the Pax3‐tv‐a cells with an RCAS‐Sox10 expression virus, thereby rescuing melanocyte development of Sox10‐deficient NC cells. This system will be useful for assessing genetic hierarchies in NC development. Developmental Dynamics 229:54–62, 2004.

Acinar Cell Apoptosis in Serpini2-Deficient Mice Models Pancreatic Insufficiency

Pancreatic insufficiency (PI) when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman–Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq), exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome transgenic expression or by pancreatic enzyme supplementation demonstrates that these phenotypes are secondary to malnutrition in pq/pq mice.

Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1

Niemann-Pick disease, type C1 (NPC1) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. A murine model of NPC1 disease (Npc1-/-) displays a rapidly progressing form of NPC1 disease which is characterized by weight loss, ataxia, increased cholesterol storage, loss of cerebellar Purkinje neurons and early lethality. To test the potential efficacy of gene therapy for NPC1, we constructed adeno-associated virus serotype 9 (AAV9) vectors to deliver the NPC1 gene under the transcriptional control of the neuronal-specific (CamKII) or a ubiquitous (EF1a) promoter. The Npc1-/- mice that received a single dose of AAV9-CamKII-NPC1 as neonates (2.6 × 1011GC) or at weaning (1.3 × 1012GC), and the mice that received a single dose of AAV9-EF1a-NPC1 at weaning (1.2 × 1012GC), exhibited an increased life span, characterized by delayed weight loss and diminished motor decline. Cholesterol storage and Purkinje neuron loss were also reduced in the central nervous system of AAV9 treated Npc1-/- mice. Treatment with AAV9-EF1a-NPC1, as compared to AAV9-CamKII-NPC1, resulted in significantly increased survival (mean survival increased from 69 days to 166 and 97 days, respectively) and growth, and reduced hepatic-cholesterol accumulation. Our results provide the first demonstration that gene therapy may represent a therapeutic option for NPC1 patients and suggest that extraneuronal NPC1 expression can further augment the lifespan of the Npc1-/- mice after systemic AAV gene delivery.

A direct link between MITF, innate immunity, and hair graying

Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitfmi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.

613. A Comparison of CNS Transduction After Systemic versus Cranial Delivery of an AAV2/9 CamKII Promoter-eGFP Vector in Mice

Niemann-Pick type C (NPC) disease is a rare but fatal lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids within the lysosome. Clinical manifestations include ataxia, dementia and hepatosplenomegaly. 95% of cases are caused by an NPC1 gene mutation resulting in a lack of functional NPC1 protein, a putative cholesterol transport protein found in the lysosomal limiting membrane. The neuronal pathology seen in NPC1−/− mice begins with cerebellar axonal swelling, progresses to gliosis and visible lipid accumulation within lysosomes, then is followed by a marked loss of Purkinje cells. Weight loss and ataxia accompany disease progression. We have previously demonstrated that systemic delivery of an AAV9 vector designed to express the human NPC1 gene under the control of the neuronal-specific promoter, mouse calcium/calmodulin-dependent protein kinase II (CamKII), to Npc1−/− mice resulted in a modest but significant increase in survival. Transgene expression, assayed by immunohistochemistry (IHC), revealed widespread NPC1 expression within the brain, broadly correlating with the endogenous CamKII expression pattern. Delayed loss of Purkinje cells in the AAV treated Npc1−/− mice was also observed. Despite the increase in lifespan, expression of NPC1 in the Purkinje cells of the AAV treated Npc1−/− mice was limited, and the mice eventually succumbed to NPC1 disease. This result is notable due to the association between Purkinje cell loss and NPC1 disease progression. In an effort to improve neuronal transduction, particularly in Purkinje cells, we have explored various central nervous system (CNS) delivery routes with an AAV9 reporter, configured to express eGFP under the CamKII promoter. Three groups of mice (n=3 mice per group) received stereotactic-guided AAV9 CamKII-eGFP injections into either the lateral ventricles (dose: 3.8 × 1011 GC), cisterna magna (dose: 3.1 × 1011 GC), or a combination of both (dose: 3.8 × 1011 GC). After two weeks, eGFP expression was assessed using IHC and microscopy. Lateral ventricle and combination injections yielded widespread neuronal transduction throughout the brain, but expression in the cerebellum was limited and sporadic. Injections into the cisterna magna - expected to target the cerebellum - did not improve cerebellar expression compared to the other routes. Overall, aside from a heightened intensity of transgene expression, cranial injections did not appear to increase neuronal targeting compared to systemic delivery by retro-orbital injections (dose: 1×1012 GC). Based on preliminary evidence from a limited number of mice, gross differences in transduction between Npc1−/− and control mice were not observed, suggesting that the disease state at the time of assessment did not influence vector tropism. Our results will help define the optimal delivery route for future NPC1 vector optimization studies, and may be informative for others who seek to correct neurodegenerative mouse models using AAV9-mediated gene therapy.

198. Adeno-Associated Viral Gene Therapy To Treat Niemann-Pick Disease, Type C1

Niemann-Pick disease, type C1 disease (NPC1) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. NPC1 commonly manifests in childhood, and there are no approved treatments to delay, stop, or reverse the fatal neurodegeneration that is the hallmark of this disorder. New therapies for patients with NPC1 need to be developed. Defects in the NPC1 gene are the cause of this disease. A murine model of NPC1, Npcnih (also called BALB/(cNctr-Npc1m1N/J), arising from a spontaneous frame-shift mutation in the Npc1 gene has been described. Npcnih homozygotes (Npc1−/-) have an early, severe, and rapidly progressing disease, which is characterized by weight loss, ataxia, and lethality by 9 weeks of age. To test the potential efficacy of gene therapy with the goal of developing a new treatment for NPC patients, we constructed an adeno-associated virus (AAV) serotype 9 to deliver the human NPC1 gene under the transcriptional control of the neuronal-specific promoter, mouse calcium/calmodulin-dependent protein kinase II (CaMKII). Npc1−/- mice received 1×1012 GC of AAV9-CaMKII-NPC1 or an equivalent reporter control, AAV9-CaMKII-GFP, between 20 and 25 days of life delivered by retro-orbital injection. To achieve neuronal transduction, we relied upon the well-established property of AAV9 vectors to cross the blood-brain barrier and transduce neurons after systemic delivery. Relative to the untreated or AAV-GFP treated Npc1−/- mice (n=15, mean survival 66 days, SD=0.89), the Npc1−/- mice that received AAV9-CaMKII-NPC1 exhibited an increased life span (n=9, mean survival 105 days, SD=30; P<0.02) Although the AAV9-CaMKII-NPC1 treated Npc1−/- mice did not achieve a normal life expectancy or the same weight of wild-type mice, our results demonstrate, for the first time, the potential efficacy of systemic AAV gene therapy as a therapeutic option in patients with NPC1.