Effects of Disease-Causing Mutations Associated with Five Bestrophinopathies on the Localization and Oligomerization of Bestrophin-1

Mutations in BEST1, the gene encoding for Bestrophin-1 (Best1), cause five, clinically distinct inherited retinopathies: Best vitelliform macular dystrophy (BVMD), adult-onset vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). Little is known regarding how BEST1 mutations cause disease and why mutations cause multiple disease phenotypes. Within the eye, Best1 is a homo-oligomeric, integral membrane protein that is exclusively localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Here, it regulates intracellular Ca2+ signaling and putatively mediates anion transport. Since defects in localization and oligomerization are known to underlie other channelopathies, we investigated how mutations causal for BVMD, AVMD, ARB, ADVIRC, and RP impact the localization and oligomerization of Best1. We generated replication-defective adenoviral vectors encoding for WT and 31 mutant forms of Best1 associated with these five diseases and expressed them in confluent, polarized Madin-Darby canine kidney and/or RPE cells. Localization was assessed via immunofluorescence and confocal microscopy. Oligomerization was examined using live-cell fluorescence resonance energy transfer (FRET) as well as reciprocal co-immunoprecipitation experiments. We report that all 31 BVMD, AVMD, ARB, ADVIRC, and RP mutants tested can reciprocally co-immunoprecipitate with and exhibit comparable FRET efficiencies to WT Best1, indicative of unimpaired oligomerization. While all RP and ADVIRC mutants were properly localized to the basolateral plasma membrane, many but not all AVMD, ARB, and BVMD mutants were mislocalized to intracellular compartments. When co-expressed with WT Best1, mislocalized mutants predominantly co-localized with WT Best1 in intracellular compartments. Studies involving four ARB truncation mutants reveal that the first 174 amino acids are sufficient to mediate oligomerization with WT Best1 and that amino acids 472-585 are not necessary for proper trafficking. We conclude that, although mislocalization is a common result of BEST1 mutation, it is not an absolute feature of any individual bestrophinopathy. Moreover, we show that some recessive mutants mislocalize WT Best1 when co-expressed, indicating that mislocalization cannot, on its own, generate a disease phenotype, and that the absence of Best1 at the plasma membrane is well tolerated.Release 17-Aug-201

Effects of extrinsic mortality on the evolution of aging: a stochastic modeling approach.

The evolutionary theories of aging are useful for gaining insights into the complex mechanisms underlying senescence. Classical theories argue that high levels of extrinsic mortality should select for the evolution of shorter lifespans and earlier peak fertility. Non-classical theories, in contrast, posit that an increase in extrinsic mortality could select for the evolution of longer lifespans. Although numerous studies support the classical paradigm, recent data challenge classical predictions, finding that high extrinsic mortality can select for the evolution of longer lifespans. To further elucidate the role of extrinsic mortality in the evolution of aging, we implemented a stochastic, agent-based, computational model. We used a simulated annealing optimization approach to predict which model parameters predispose populations to evolve longer or shorter lifespans in response to increased levels of predation. We report that longer lifespans evolved in the presence of rising predation if the cost of mating is relatively high and if energy is available in excess. Conversely, we found that dramatically shorter lifespans evolved when mating costs were relatively low and food was relatively scarce. We also analyzed the effects of increased predation on various parameters related to density dependence and energy allocation. Longer and shorter lifespans were accompanied by increased and decreased investments of energy into somatic maintenance, respectively. Similarly, earlier and later maturation ages were accompanied by increased and decreased energetic investments into early fecundity, respectively. Higher predation significantly decreased the total population size, enlarged the shared resource pool, and redistributed energy reserves for mature individuals. These results both corroborate and refine classical predictions, demonstrating a population-level trade-off between longevity and fecundity and identifying conditions that produce both classical and non-classical lifespan effects

Changes in evolved lifespan and maturation age are accompanied by corresponding shifts in juvenile energetic investments.

<p>Under classical (<b>A–C</b>) and non-classical conditions (<b>D–F</b>), the percentage of per-iteration energy devoted to somatic maintenance, reproduction, and metabolism by juveniles is shown. In both cases, the majority of energy was devoted to reproduction, followed by metabolism, followed by somatic maintenance (<b>A–F</b>). Under classical conditions, rising levels of predation, , caused juveniles to invest less in somatic maintenance (<b>A</b>), more into early peak fertility (<b>B</b>), and less into metabolism (<b>C</b>). Under non-classical conditions, larger values of caused juveniles to devote less energy to early peak fertility (<b>E</b>) and more towards somatic maintenance (<b>D</b>). Investments in metabolism were comparable for various values of predation modifier, (<b>F</b>).</p

Overview of the modeling and optimization procedures.

<p>An agent-based stochastic model was implemented in which individuals invest energy foraged from a common pool toward maturation, metabolism, mating, and maintenance and are subject to random, density-dependent predation, starvation, and aging. Maturation and intrinsic death times are inheritable traits used to determine maturation, and maintenance costs. A flowchart depicts the simulations scheme (<b>A</b>). Sample simulation solution depicting changes in observed statistics with time is shown (<b>B</b>). To find appropriate values for six simulation-invariant parameters for the classical and non-classical evolutionary response to increased predation a simulated annealing optimization approach was used (<b>C</b>). See methods for model and optimization details.</p

Higher predation impacts parameters related to density dependence under classical conditions.

<p>Under classical conditions, increased predation reduced population size (<b>A</b>) and enlarged the total energy pool that could be foraged (<b>B</b>). Average normalized birth rates increased (<b>C</b>) and, concomitant with this, average individual energy decreased (<b>D</b>).</p

Higher predation impacts parameters related to density dependence under non-classical conditions.

<p>Akin to classical conditions, higher predation under non-classical conditions resulted in smaller population sizes (<b>A</b>) and a large shared energy pool (<b>B</b>). Unlike classical conditions, however, average normalized birth rates decreased (<b>C</b>) and the average mature individual energy was increased (<b>D</b>).</p

Classical and non-classical conditions identified by simulated annealing optimization.

<p>A simulated annealing optimization scheme was used to find values for six simulation-invariant parameters that would predispose populations toward either increased or decreased maintenance in response to increased extrinsic mortality. The fit score stochastically improved over the course of the optimization (<b>A and C</b>). The optimal starvation modifier (ε), growth efficiency (), initial energy of individuals (), mating energy (), mating energy threshold (<i>mateThreshold</i>), and death cost function type () for the classical (<b>B</b>), and non-classical (<b>D</b>) effect are shown as a function of optimization duration. D_type: 0 = Sigmoidal Low, 1 = Linear Low, 2 = Asymptotic Low, 3 = Sigmoidal High, 4 = Linear High, 5 = Asymptotic High. Colored dots indicate that the intrinsic death effect was monotonic.</p

Summary of key model parameters and quantities.

<p>Populations were modeled explicitly using stochastic agents to represent individuals subject to shared resources, mating, and both extrinsic and intrinsic death.</p

Disparate effects of high predation on the evolution of lifespan and maturation age.

<p>Under classical conditions of relatively low food availability and relatively inexpensive mating costs, increased values of predation modifier, x, caused mean T_die to decrease (<b>A</b>) and mean T_mat to decrease (<b>C</b>) over time. Conversely, under the non-classical conditions of relatively abundant food but relatively expensive mating costs, higher values of predation modifier, , caused the mean T_die to increase (<b>B</b>) and the mean T_mat to increase (<b>D</b>). In (<b>E</b>) and (<b>F</b>), the population distribution of T_die is shown under classical (<b>E</b>) and non-classical (<b>F</b>) conditions at the end of 300,000 model iterations.</p