Arne M. Nystuen

An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kβ, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinaseβ (PI3Kβ), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

Mutations in a novel gene encoding a CRAL-TRIO domain cause human Cayman ataxia and ataxia/dystonia in the jittery mouse.

Cayman ataxia is a recessive congenital ataxia restricted to one area of Grand Cayman Island. Comparative mapping suggested that the locus on 19p13.3 associated with Cayman ataxia might be homologous to the locus on mouse chromosome 10 associated with the recessive ataxic mouse mutant jittery. Screening genes in the region of overlap identified mutations in a novel predicted gene in three mouse jittery alleles, including the first mouse mutation caused by an Alu-related (B1 element) insertion. We found two mutations exclusively in all individuals with Cayman ataxia. The gene ATCAY or Atcay encodes a neuron-restricted protein called caytaxin. Caytaxin contains a CRAL-TRIO motif common to proteins that bind small lipophilic molecules. Mutations in another protein containing a CRAL-TRIO domain, alpha-tocopherol transfer protein (TTPA), cause a vitamin E–responsive ataxia. Three-dimensional protein structural modeling predicts that the caytaxin ligand is more polar than vitamin E. Identification of the caytaxin ligand may help develop a therapy for Cayman ataxia.

The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development

The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the ‘meiotic’ or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

A Null Mutation in Inositol Polyphosphate 4-Phosphatase Type I Causes Selective Neuronal Loss in Weeble Mutant Mice

Weeble mutant mice have severe locomotor instability and significant neuronal loss in the cerebellum and in the hippocampal CA1 field. Genetic mapping was used to localize the mutation to the gene encoding inositol polyphosphate 4-phosphatase type I (Inpp4a), where a single nucleotide deletion results in a likely null allele. The substrates of INPP4A are intermediates in a pathway affecting intracellular Ca(2+) release but are also involved in cell cycle regulation through binding the Akt protooncogene; dysfunction in either may account for the neuronal loss of weeble mice. Although other mutations in phosphoinositide enzymes are associated with synaptic defects without neuronal loss, weeble shows that Inpp4a is critical for the survival of a subset of neurons during postnatal development in mice.

The transcription factor Nr2e3 functions in retinal progenitors to suppress cone cell generation.

The transcription factor Nr2e3 is an essential component for development and specification of rod and cone photoreceptors; however, the mechanism through which it acts is not well understood. In this study, we use Nr2e3(rd7/rd7) mice that harbor a mutation in Nr2e3, to serve as a model for the human retinal disease Enhanced S Cone Syndrome. Our studies reveal that NR2E3 is expressed in late retinal progenitors and differentiating photoreceptors of the developing retina and localized to the cell bodies of mature rods and cones. In particular, we demonstrate that the abnormal increase in cone photoreceptors observed in Nr2e3(rd7/rd7) mice arise from ectopic mitotic progenitor cells that are present in the outer nuclear layer of the mature Nr2e3(rd7/rd7) retina. A prolonged phase of proliferation is observed followed by abnormal retinal lamination with fragmented and disorganized photoreceptor synapses that result in a progressive loss of rod and cone function. An extended and pronounced wave of apoptosis is also detected at P30 and temporally correlates with the phase of prolonged proliferation. Approximately twice as many apoptotic cells were detected compared to proliferating cells. This wave of apoptosis appears to affect both rod and cone cells and thus may account for the concurrent loss of rod and cone function. We further show that Nr2e3(rd7/rd7) cones do not express rod specific genes and Nr2e3(rd7/rd7) rods do not express cone specific genes. Our studies suggest that, based on its temporal and spatial expression, NR2E3 acts simultaneously in different cell types: in late mitotic progenitors, newly differentiating post mitotic cells, and mature rods and cones. In particular, this study reveals the function of NR2E3 in mitotic progenitors is to repress the cone generation program. NR2E3 is thus one of the few genes known to influence the competency of retinal progenitors while simultaneously directing the rod and cone differentiation.

Nr2e3-Directed Transcriptional Regulation of Genes Involved in Photoreceptor Development and Cell-Type Specific Phototransduction

The retinal transcription factor Nr2e3 plays a key role in photoreceptor development and function. In this study we examine gene expression in the retina of Nr2e3(rd7/rd7) mutants with respect to wild-type control mice, to identify genes that are misregulated and hence potentially function in the Nr2e3 transcriptional network. Quantitative candidate gene real time PCR and subtractive hybridization approaches were used to identify transcripts that were misregulated in Nr2e3(rd7/rd7) mice. Chromatin immunoprecipitation assays were then used to determine which of the misregulated transcripts were direct targets of NR2E3. We identified 24 potential targets of NR2E3. In the developing retina, NR2E3 targets transcription factors such as Ror1, Rorg, and the nuclear hormone receptors Nr1d1 and Nr2c1. In the mature retina NR2E3 targets several genes including the rod specific gene Gnb1 and cone specific genes blue opsin, and two of the cone transducin subunits, Gnat2 and Gnb3. In addition, we identified 5 novel transcripts that are targeted by NR2E3. While mislocalization of proteins between rods and cones was not observed, we did observe diminished concentration of GNB1 protein in adult Nr2e3(rd7/rd7) retinas. These studies identified novel transcriptional pathways that are potentially targeted by Nr2e3 in the retina and specifically demonstrate a novel role for NR2E3 in regulating genes involved in phototransduction.

Genetic variations strongly influence phenotypic outcome in the mouse retina.

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2 -nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases.

Mapping of genetic modifiers of Nr2e3 rd7/rd7 that suppress retinal degeneration and restore blue cone cells to normal quantity.

The retinal degeneration 7 (rd7) mouse, lacking expression of the Nr2e3 gene, exhibits retinal dysplasia and a slow, progressive degeneration due to an abnormal production of blue opsin-expressing cone cells. In this study we evaluated three strains of mice to identify alleles that would slow or ameliorate the retinal degeneration observed in Nr2e3rd7/rd7 mice. Our studies reveal that genetic background greatly influences the expression of the Nr2e3rd7/rd7 phenotype and that the inbred mouse strains CAST/EiJ, AKR/J, and NOD.NON-H2nb1 carry alleles that confer resistance to Nr2e3rd7/rd7-induced retinal degeneration. B6.Cg-Nr2e3rd7/rd7 mice were outcrossed to each strain and the F1 progeny were intercrossed to produce F2 mice. In each intercross, 20–24% of the total F2 progeny were homozygous for the Nr2e3rd7/rd7 mutation in a mixed genetic background; approximately 28–48% of the Nr2e3rd7/rd7 homozygotes were suppressed for the degenerative retina phenotype in a mixed genetic background. The suppressed mice had no retinal spots and normal retinal morphology with a normal complement of blue opsin-expressing cone cells. An initial genome scan revealed a significant association of the suppressed phenotype with loci on chromosomes 8 and 19 with the CAST/EiJ background, two marginal loci on chromosomes 7 and 11 with the AKR/J background, and no significant QTL with the NOD.NON-H2nb1 background. We did not observe any significant epistatic effects in this study. Our results suggest that there are several genes that are likely to act in the same or parallel pathway as NR2E3 that can rescue the Nr2e3rd7/rd7 phenotype and may serve as potential therapeutic targets.

The mouse mutants recoil wobbler and nmf373 represent a series of Grm1 mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP3). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.

Patterned Neuroprotection in the Inpp4awbl Mutant Mouse Cerebellum Correlates with the Expression of Eaat4

The weeble mutant mouse has a frame shift mutation in inositol polyphosphate 4-phosphatase type I (Inpp4a). The phenotype is characterized by an early onset cerebellar ataxia and neurodegeneration, especially apparent in the Purkinje cells. Purkinje cell loss is a common pathological finding in many human and mouse ataxic disorders. Here we show that in the Inpp4awbl mutant, Purkinje cells are lost in a specific temporal and spatial pattern. Loss occurs early in postnatal development; however, prior to the appearance of climbing fibers in the developing molecular layer, the mutant has a normal complement of Purkinje cells and they are properly positioned. Degeneration and reactive gliosis are present at postnatal day 5 and progress rapidly in a defined pattern of patches; however, Inpp4a is expressed uniformly across Purkinje cells. In late stage mutants, patches of surviving Purkinje cells appear remarkably normal with the exception that the climbing fibers have been excessively eliminated. Surviving Purkinje cells express Eaat4, a glutamate transporter that is differentially expressed in subsets of Purkinje cells during development and into adult stages. Prior to Purkinje cell loss, reactive gliosis and dendritic atrophy can be seen in Eaat4 negative stripes. Our data suggest that Purkinje cell loss in the Inpp4awbl mutant is due to glutamate excitotoxicity initiated by the climbing fiber, and that Eaat4 may exert a protective effect.