imaging-3d-reconstruction-cerebrovascular-structures-embryonic

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Western University of Health Sciences

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the developed world and has a strong genetic predisposition. A locus at human chromosome 10q26 affects the risk of AMD, but the precise gene(s) have not been identified. We genotyped 581 AMD cases and 309 normal controls in a Caucasian cohort in Utah. We demonstrate that a single-nucleotide polymorphism, rs11200638, in the promoter region of HTRA1 is the most likely causal variant for AMD at 10q26 and is estimated to confer a population attributable risk of 49.3%. The HTRA1 gene encodes a secreted serine protease. Preliminary analysis of lymphocytes and retinal pigment epithelium from four AMD patients revealed that the risk allele was associated with elevated expression levels of HTRA1 mRNA and protein. We also found that drusen in the eyes of AMD patients were strongly immunolabeled with HTRA1 antibody. Together, these findings support a key role for HTRA1 in AMD susceptibility and identify a potential new pathway for AMD pathogenesis.

A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration.

Amino-retinoid compounds in the human retinal pigment epithelium.

ELOVL4 was first identified as a disease-causing gene in Stargardt macular dystrophy (STGD3, MIM 600110.) To date, three ELOVL4 mutations have been identified, all of which result in truncated proteins which induce autosomal dominant juvenile macular degenerations. Based on sequence homology, ELOVL4 is thought to be another member within a family of proteins functioning in the elongation of long chain fatty acids. However, the normal function of ELOVL4 is unclear. We generated Elovl4 knockout mice to determine if Elovl4 loss affects retinal development or function. Here we show that Elovl4 knockout mice, while perinatal lethal, exhibit normal retinal development prior to death at day of birth. Further, postnatal retinal development in Elovl4 heterozygous mice appears normal. Therefore haploinsufficiency for wildtype ELOVL4 in autosomal dominant macular degeneration likely does not contribute to juvenile macular degeneration in STGD3 patients. We found, however, that Elovl4+/- mice exhibit enhanced ERG scotopic and photopic a and b waves relative to wildtype Elovl4+/+ mice suggesting that reduced Elovl4 levels may impact retinal electrophysiological responses.

Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice.

Mutations in the gene ELOVL4 have been shown to cause stargardt-like macular dystrophy. ELOVL4 is part of a family of fatty acid elongases and is yet to have a specific elongase activity assigned to it. We generated Elovl4 Y270X mutant mice and characterized the homozygous mutant as well as homozygous Elovl4 knockout mice in order to better understand the function or role of Elovl4. We found that mice lacking a functional Elovl4 protein died perinatally. The cause of death appears to be from dehydration due to faulty permeability barrier formation in the skin. Further biochemical analysis revealed a significant reduction in free fatty acids longer than C26 in homozygous mutant and knockout mouse skin. These results implicate the importance of these long chain fatty acids in skin barrier development. Furthermore, we suggest that Elovl4 is likely involved in the elongation of C26 and longer fatty acids.

Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival.

Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world. The two forms of advanced AMD, geographic atrophy (GA) and choroidal neovascularization (wet AMD), represent two types of degenerative processes in the macula that lead to loss of central vision. Soft confluent drusen, characterized by deposits in macula without visual loss are considered a precursor of advanced AMD. A single nucleotide polymorphism, rs11200638, in the promoter of HTRA1 has been shown to increases the risk for wet AMD. However, its impact on soft confluent drusen and GA or the relationship between them is unclear. To better understand the role the HTRA1 polymorphism plays in AMD subtypes, we genotyped an expanded Utah population with 658 patients having advanced AMD or soft confluent drusen and 294 normal controls and found that the rs11200638 was significantly associated with GA. This association remains significant conditional on LOC387715 rs10490924. In addition, rs11200638 was significantly associated with soft confluent drusen, which are strongly immunolabeled with HTRA1 antibody in an AMD eye with GA similar to wet AMD. Two-locus analyses were performed for CFH Y402H variant at 1q31 and the HTRA1 polymorphism. Together CFH and HTRA1 risk variants increase the odds of having AMD by more than 40 times. These findings expand the role of HTRA1 in AMD. Understanding the underlying molecular mechanism will provide an important insight in pathogenesis of AMD.

HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration.

Senescent Ccl2(-/-) mice are reported to develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are also found to be associated with AMD. The authors generated Ccl2(-/-)/Cx3cr1(-/-) mice to establish a more characteristic and reproducible AMD model.

Murine ccl2/cx3cr1 deficiency results in retinal lesions mimicking human age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible visual impairment in the world. Advanced AMD can be divided into wet AMD (choroidal neovascularization) and dry AMD (geographic atrophy, GA). Drusen is characterized by deposits in the macula without visual loss and is an early AMD sign in the Caucasian population. rs11200638 in the promoter of HTRA1 has recently been shown to increases the risk for wet AMD in both Caucasian and Hong Kong Chinese populations. In order to replicate these results in a different cohort, we genotyped rs11200638 for 164 Chinese patients (90 wet AMD and 74 drusen) and 106 normal controls in a Han Mainland Chinese cohort. The genotypes were compared using chi square analysis for an additive allelic model. rs11200638 was significantly associated with wet AMD (p=5.00x10(-12)). Unlike in the Caucasian population, the risk allele of rs11200638 was not associated with drusen in our Chinese population. These findings confirm the association of HTRA1 with wet AMD.

HTRA1 variant increases risk to neovascular age-related macular degeneration in Chinese population.

10q26 is associated with increased risk of age-related macular degeneration in the Utah population.

Single nucleotide polymorphism (SNP), rs11200638, in the promoter of HTRA1 has recently been shown to increase the risk for AMD. In order to investigate the association of this HTRA1 polymorphism and the bilaterality of AMD, we genotyped rs11200638 in control, unilateral, and bilateral advanced AMD patients. The A allele for SNP rs11200638 in HTRA1, was significantly more prevalent in bilateral wet AMD and GA patients than in unilateral groups (p=.02 and p=.03, respectively). The homozygote odds ratios of bilateral wet AMD and GA are significantly greater than those seen in unilateral groups (twofold and threefold increase, respectively). This finding is consistent with the role of HTRA1 in AMD pathogenesis and will help aid in the clinical management and prognosis of AMD patients.

Association of HTRA1 polymorphism and bilaterality in advanced age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex disorder with genetic and environmental influences. The genetic influences affecting AMD are not well understood and few genes have been consistently implicated and replicated for this disease. A polymorphism (rs11200638) in a transcription factor binding site of the HTRA1 gene has been described, in previous reports, as being most significantly associated with AMD. In this paper, we investigate haplotype association and individual polymorphic association by genotyping additional variants in the AMD risk-associated region of chromosome 10q26. We demonstrate that rs11200638 in the promoter region and rs2293870 in exon 1 of HTRA1, are among the most significantly associated variants for advanced forms of AMD.

Further mapping of 10q26 supports strong association of HTRA1 polymorphisms with age-related macular degeneration.

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.

Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.

Alzheimer's disease research has been at an impasse in recent years with lingering questions about the involvement of Amyloid-Î² (AÎ²). Early versions of the amyloid hypothesis considered AÎ² something of an undesirable byproduct of APP processing that wreaks havoc on the human neocortex, yet evolutionary conservation--over three hundred million years--indicates this peptide plays an important biological role in survival and reproductive fitness. Here we describe how AÎ² regulates blood vessel branching in tissues as varied as human umbilical vein and zebrafish hindbrain. High physiological concentrations of AÎ² monomer induced angiogenesis by a conserved mechanism that blocks Î³-secretase processing of a Notch intermediate, NEXT, and reduces the expression of downstream Notch target genes. Our findings allude to an integration of signaling pathways that utilize Î³-secretase activity, which may have significant implications for our understanding of Alzheimer's pathogenesis vis-Ã -vis vascular changes that set the stage for ensuing neurodegeneration.

Alzheimer's-related peptide amyloid-Î² plays a conserved role in angiogenesis.

Vitamin A deficiency causes impaired vision and blindness in millions of children around the world. Previous studies in zebrafish have demonstrated that retinoic acid (RA), the acid form of vitamin A, plays a vital role in early eye development. The objective of this study was to describe the effects of early RA deficiency by treating zebrafish with diethylaminobenzaldehyde (DEAB), a potent inhibitor of the enzyme retinaldehyde dehydrogenase (RALDH) that converts retinal to RA. Zebrafish embryos were treated for 2 h beginning at 9 h postfertilization. Gross morphology and retinal development were examined at regular intervals for 5 days after treatment. The optokinetic reflex (OKR) test, visual background adaptation (VBA) test, and the electroretinogram (ERG) were performed to assess visual function and behavior. Early treatment of zebrafish embryos with 100 Î¼M DEAB (9 h) resulted in reduced eye size, and this microphthalmia persisted through larval development. Retinal histology revealed that DEAB eyes had significant developmental abnormalities but had relatively normal retinal lamination by 5.5 days postfertilization. However, the fish showed neither an OKR nor a VBA response. Further, the retina did not respond to light as measured by the ERG. We conclude that early deficiency of RA during eye development causes microphthalmia as well as other visual defects, and that timing of the RA deficiency is critical to the developmental outcome.

Early retinoic acid deprivation in developing zebrafish results in microphthalmia.

Amyloid-beta (AÎ²) is an endogenous peptide that becomes dysregulated in AMD and Alzheimer disease. Both of these disorders are marked by extracellular deposits that contain AÎ², highly branched capillary networks, and neurodegeneration. Although AÎ² has been implicated in AMD and Alzheimer pathology for decades, its nonpathological function has remained unclear. We recently showed that high levels of monomeric AÎ² induce blood vessel branching in embryonic zebrafish brain, and here we report that a similar mechanism may contribute to aberrant blood vessel branching in the retina of adult zebrafish.

Amyloid-Î² increases capillary bed density in the adult zebrafish retina.

Introductory editorial.

Alzheimer's disease (AD) is marked by neuritic plaques that contain insoluble deposits of amyloid-Î² (AÎ²), yet the physiological function of this peptide has remained unclear for more than two decades. Using genetics and pharmacology we have established that AÎ² plays an important role in regulating capillary bed density within the brain, a function that is distinct from other cleavage products of amyloid precursor protein (APP). APP-deficient zebrafish had fewer cerebrovascular branches and shorter vessels in the hindbrain than wild-type embryos; this phenotype was rescued by treatment with human AÎ² peptide, but not a smaller APP fragment called p3. Similar vascular defects were seen in zebrafish treated with a Î²-secretase inhibitor (BSI) that blocked endogenous AÎ² production. BSI-induced vascular defects were also improved by treatment with human AÎ², but not p3. Our results demonstrate a direct correlation between extracellular levels of AÎ² and cerebrovascular density in the developing hindbrain. These findings may be relevant to AD etiology where high levels of AÎ² in the brain parenchyma precede the development of neuritic plaques and dense aberrantly-branched blood vessel networks that appear between them. The ability of AÎ² to modify blood vessels may coordinate capillary density with local metabolic activity, which could explain the evolutionary conservation of this peptide from lobe-finned fish to man.

Article	Year
A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science (New York, N.Y.)\| PubMed ID: 17053109	2006
Amino-retinoid compounds in the human retinal pigment epithelium. Advances in experimental medicine and biology\| PubMed ID: 17249557	2006
Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice. Vision research\| PubMed ID: 17254625	2007
Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival. International journal of biological sciences\| PubMed ID: 17304340	2007
HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration. Cell cycle (Georgetown, Tex.)\| PubMed ID: 17426452	2007
Murine ccl2/cx3cr1 deficiency results in retinal lesions mimicking human age-related macular degeneration. Investigative ophthalmology & visual science\| PubMed ID: 17652758	2007
HTRA1 variant increases risk to neovascular age-related macular degeneration in Chinese population. Vision research\| PubMed ID: 17904186	2007
10q26 is associated with increased risk of age-related macular degeneration in the Utah population. Advances in experimental medicine and biology\| PubMed ID: 18188952	2008
Association of HTRA1 polymorphism and bilaterality in advanced age-related macular degeneration. Vision research\| PubMed ID: 18206206	2008
Further mapping of 10q26 supports strong association of HTRA1 polymorphisms with age-related macular degeneration. Vision research\| PubMed ID: 18207215	2008
Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice. The Journal of clinical investigation\| PubMed ID: 18654668	2008
Alzheimer's-related peptide amyloid-Î² plays a conserved role in angiogenesis. PloS one\| PubMed ID: 22792182	2012
Early retinoic acid deprivation in developing zebrafish results in microphthalmia. Visual neuroscience\| PubMed ID: 23013828	2012
Amyloid-Î² increases capillary bed density in the adult zebrafish retina. Investigative ophthalmology & visual science\| PubMed ID: 23404118	2013
Introductory editorial. Ophthalmology and eye diseases\| PubMed ID: 23861605	2009
Amyloid-Î² and APP Deficiencies Cause Severe Cerebrovascular Defects: Important Work for an Old Villain. PloS one\| PubMed ID: 24040383	2013

D. Joshua Cameron

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