Caroline J. Zeiss

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110s, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially “compensated” to maintain the misfolded protein in a soluble state, become progressively unable to do so.

A null mutation of Hhex results in abnormal cardiac development,defective vasculogenesis and elevated Vegfa levels

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2) hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice

Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5–E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.

Feline Intraocular Tumors May Arise from Transformation of Lens Epithelium

Feline ocular sarcomas are malignant intraocular neoplasms that are frequently associated with a history of ocular trauma. They usually present as fibrosarcomas, but some have both epithelial and mesenchymal features. The purpose of this study was to determine the cell of origin of a subset of feline intraocular sarcomas that display a mixed epithelial-mesenchymal phenotype, with elaboration of basement membrane—type matrix. We examined the morphology and histochemical and immunohistochemical phenotypes of nine feline intraocular sarcomas. Immunohistochemistry and in situ hybridization were performed to detect expression of crystallin alpha A. In addition, tumors were examined for expression of vimentin, cytokeratin, smooth muscle actin, desmin, melan A, neural cell adhesion molecule, S-100, glial fibrillary acidic protein, nerve growth factor receptor, and collagen type IV. Animals ranged from 7 to 17 years of age—no breed or sex predilection for tumor occurrence was present. Tumors were characterized by mixed epithelial and mesenchymal phenotypes, both of which elaborated basement membrane-type material and expressed vimentin highly. On the basis of collagen type IV and crystallin alpha A immunopositivity, we established that three of nine tumors were of lens epithelial origin. Expression of desmin and smooth muscle actin identified one tumor as a leiomyosarcoma. The remainder were undifferentiated sarcomas of myofibroblastic origin. This is the first report of lens epithelial neoplasia in clinical material from any species. The history and morphologic features of feline ocular sarcomas are reminiscent of feline vaccine-induced sarcomas. These tumors may share pathophysiologic similarities unique to this species.

Mutation of POLB Causes Lupus in Mice

A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLB gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol β, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol β activity results in SLE, we constructed a mouse model of POLB that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLB allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol β activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol β in humans is an underlying cause of SLE.

β-Hydroxybutyrate Deactivates Neutrophil NLRP3 Inflammasome to Relieve Gout Flares

Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1β (IL-1β) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases β-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1β in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.

Hypothalamic prolyl endopeptidase (PREP) regulates pancreatic insulin and glucagon secretion in mice

Significance The ventromedial nucleus of the hypothalamus (VMH) plays an important role in the regulation of glucose metabolism. Here we show that prolyl endopeptidase (PREP), a serine protease, is expressed in the VMH where it functions to regulate glucose-induced insulin secretion. Experimental knockdown of central PREP induced impairment of VMH glucose sensing, resulting in reduced insulin and increased glucagon secretion by the pancreas via altered sympathetic outflow. Our data reveal PREP as a new hypothalamic player in the control of glucose homeostasis. Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prepgt/gt) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prepgt/gt and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prepgt/gt and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prepgt/gt mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus–PREP reversed the glucose-intolerant phenotype of the Prepgt/gt mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.

Designing Phenotyping Studies for Genetically Engineered Mice

A phenotyping study records physiologic or morphologic changes in an experimental animal resulting from an intervention. In mice, this intervention is most frequently genetic, but it may be any type of experimental manipulation. Accurate representation of the human condition under study is essential if the model is to yield useful conclusions. In this review, general approaches to the design of phenotyping studies are considered. These approaches take into account major sources of reduced model validity, such as unexpected phenotypic variation in mice, evolutionary divergence between mice and humans, unanticipated sources of variation, and common design errors. As poor design is the most common reason why studies fail to yield enduring results, emphasis is placed on reduction of bias, sampling, controlled study design, and appropriate statistical analysis.

Correlation of tumor phenotype with c-fms proto-oncogene expression in an in vivo intraperitoneal model for experimental human breast cancer metastasis

Although proto-oncogene expression has been shown to correlate with clinical outcome in breast carcinoma, an experimental model has not been proposed to study this phenomenon in vivo. In addition, the ability to modulate this proto-oncogene in vivo to correlate with phenotypic behavior has not been determined. Utilizing an intraperitoneal model for metastatic spread with BT20 human breast carcinoma cells, clonally expanded cells expressing five fold higher c-fms protein were compared with parent BT20 cells as well as an underexpressing clone using intrasplenic injection following left flank cut-down in female nude and Severe combined immunodeficient (SCID) mice. Athymic BALB/c nude and SCID animals were observed for clinical evidence of tumorigenicity with necropsy performed at either 50 or 80 days unless compromised earlier. Immunohistochemistry (IHC) of the harvested tumors was performed to correlate c-fms expression from its original in vitro culture to the in vivo model. At day 50, differences in primary tumor take and spread to the pelvis were already evident favoring the c-fms over-expression group with IHC of these tumors revealing significantly higher intensity of staining for c-fms, (mean H score of 205 vs. 43 in the over-expression and parent groups, respectively). At day 80, tumor take and spread was comparable; however, tumor size in the over-expression group was significantly larger than the parent and under-expressing group in both the BALB/c and SCID experiments. Modulation of c-fms proto-oncogene expression was also achieved using the anti-glucocorticoid, RU-486, via oral administration to SCID mice with subsequent correlation to IHC staining. This model thus provides tumors of significant size and organ diversity which retain their phenotype early in tumorigenesis allowing an early endpoint to assess efficacy of novel treatments.