D. Joshua Cameron

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

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.

Further mapping of 10q26 supports strong association of HTRA1 polymorphisms with 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.

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

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.

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

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.

Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice

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.

Alzheimer’s-Related Peptide Amyloid-β Plays a Conserved Role in Angiogenesis

Alzheimer’s disease research has been at an impasse in recent years with lingering questions about the involvement of Amyloid-β (Aβ). Early versions of the amyloid hypothesis considered Aβ 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 Aβ regulates blood vessel branching in tissues as varied as human umbilical vein and zebrafish hindbrain. High physiological concentrations of Aβ monomer induced angiogenesis by a conserved mechanism that blocks γ-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 γ-secretase activity, which may have significant implications for our understanding of Alzheimer’s pathogenesis vis-à-vis vascular changes that set the stage for ensuing neurodegeneration.

Calpain-1 and calpain-2 play opposite roles in retinal ganglion cell degeneration induced by retinal ischemia/reperfusion injury.

Calpain has been shown to be involved in neurodegeneration, and in particular in retinal ganglion cell (RGC) death resulting from increased intraocular pressure (IOP) and ischemia. However, the specific roles of the two major calpain isoforms, calpain-1 and calpain-2, in RGC death have not been investigated. Here, we show that calpain-1 and calpain-2 were sequentially activated in RGC dendrites after acute IOP elevation. By combining the use of a selective calpain-2 inhibitor (C2I) and calpain-1 KO mice, we demonstrated that calpain-1 activity supported survival, while calpain-2 activity promoted cell death of RGCs after IOP elevation. Calpain-1 activation cleaved PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and activated the Akt pro-survival pathway, while calpain-2 activation cleaved striatal-enriched protein tyrosine phosphatase (STEP) and activated STEP-mediated pro-death pathway in RGCs after IOP elevation. Systemic or intravitreal C2I injection to wild-type mice 2h after IOP elevation promoted RGC survival and improved visual function. Our data indicate that calpain-1 and calpain-2 play opposite roles in high IOP-induced ischemic injury and that a selective calpain-2 inhibitor could prevent acute glaucoma-induced RGC death and blindness.

Amyloid-β and APP Deficiencies Cause Severe Cerebrovascular Defects: Important Work for an Old Villain

Alzheimer’s disease (AD) is marked by neuritic plaques that contain insoluble deposits of amyloid-β (Aβ), yet the physiological function of this peptide has remained unclear for more than two decades. Using genetics and pharmacology we have established that Aβ 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 Aβ peptide, but not a smaller APP fragment called p3. Similar vascular defects were seen in zebrafish treated with a β-secretase inhibitor (BSI) that blocked endogenous Aβ production. BSI-induced vascular defects were also improved by treatment with human Aβ, but not p3. Our results demonstrate a direct correlation between extracellular levels of Aβ and cerebrovascular density in the developing hindbrain. These findings may be relevant to AD etiology where high levels of Aβ in the brain parenchyma precede the development of neuritic plaques and dense aberrantly-branched blood vessel networks that appear between them. The ability of Aβ 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.

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

PURPOSE Amyloid-beta (Aβ) is an endogenous peptide that becomes dysregulated in AMD and Alzheimer disease. Both of these disorders are marked by extracellular deposits that contain Aβ, highly branched capillary networks, and neurodegeneration. Although Aβ has been implicated in AMD and Alzheimer pathology for decades, its nonpathological function has remained unclear. We recently showed that high levels of monomeric Aβ 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. METHODS Transgenic zebrafish expressing enhanced green fluorescence protein (EGFP) in their endothelial cells were sedated and small intraocular injections of PBS were made into one eye and either Aβ or γ-secretase inhibitor were injected into their opposite eye. A week later, the eyes were enucleated and high resolution maps of the retina vasculature were created using confocal microscopy. Comparisons were made between the treatment groups using the general linear model ANOVA. RESULTS We found that Aβ significantly affects capillary blood vessels in the retina. Small volumes of Aβ injected into the eyes of adult zebrafish induced the formation of significantly more endothelial tip cells and capillary bridges-some with loops-near the circumferential vein. These effects were dose-dependent and increased capillary bed density, though there was no effect on larger arterial vessels. CONCLUSIONS This study reveals a previously unknown role for Aβ in regulating capillary bed density, providing new insight into the normal biological function. Aβ will help in the development of therapeutic interventions for AMD and Alzheimer disease.

10q26 Is Associated with Increased Risk of Age-Related Macular Degeneration in the Utah Population

Age-related macular degeneration (AMD) includes a wide range of phenotypes. Early AMD is mainly characterized by the presence of soft dusen in the macula without visual loss, while advanced AMD is characterized by geographic atrophy (GA or dry AMD) and neovascular AMD (wet AMD) with visual loss. Despite the rising prevalence of AMD as a result of increasing life expectancy, its underlying etiology is poorly understood and there is no specific therapy. Nutritional supplements and antagonists of vascular endothelial growth factor (VEGF) have been reported to halt the progression of visual loss in wet AMD (Bressler et al., 1988). Wet AMD is usually associated with an aggressive form of visual loss due to choroidal neovascularization (CNV). Previous reports by several groups have identified a significant association between a common missense variant (Y402H) and non-coding variants in CFH and AMD in the United States (Edwards et al., 2005; Fisher et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005; Li et al., 2006; Maller et al., 2006) and Europe (Fisher et al., 2005; Souied et al., 2005; Despriet et al., 2006; Magnusson et al., 2006; Postel et al., 2006; Sepp et al., 2006; Simonelli et al., 2006). Recently, it was reported that rs10490924 in LOC387715 is the second major locus for AMD located at 10q locus (Conley et al., 2005; Rivera et al., 2005; Haddad et al., 2006; Maller et al., 2006; Schmidt et al., 2006). In this study we performed an association study incorporating SNP data for LOC387715 and PLEKHA1 at 10q26 and for CFH at 1q31.3 in the Utah AMD cohort. Our results support previous findings that