ISOPRENOID METABOLISM IN THE RETINA

先天性胆固醇合成错误(Chol)包括一组严重的、通常是致命的代谢紊乱， 其中Smith-Lemli-Opitz综合征(SLOS)是人类第四大最常见的隐性疾病 最知名的。先前的工作已经建立了SLOS大鼠模型，该模型显示出进行性视网膜 光感受器比视网膜色素上皮细胞或M？ler神经胶质细胞更敏感的变性。而完全相同的 发病机制尚不清楚，最初的生化缺陷涉及7-羟色胺的低效转化。 脱氢胆固醇(7DHC)由3b-羟基甾醇-D7-还原酶(3b-羟基甾醇-D7-还原酶，3b-羟基甾醇-D7-还原酶，3b-羟基固醇-D7-还原酶)催化 DHCR7基因产物)。我们提出，这会导致多种后遗症，包括基因改变。 表达，脂质和蛋白质氧化，半胱氨酸天冬氨酸氨基转移酶激活，以及被扰乱的膜结构，然后 导致进行性细胞功能障碍和死亡。7DHC是已知的最不稳定氧的脂质，而且很容易 形成“氧合甾醇”(其中一些对细胞有极强的毒性)，而7DHC衍生的氧化甾醇往往是 比胆固醇衍生的氧化甾醇具有更强的细胞毒性。视网膜的自然环境(高氧分压， 铁和入射光)为氧甾醇的形成提供了理想的条件。我们假设 阻断DHCR7后，视网膜/RPE 7DHC水平明显升高，导致原位氧固醇化， 导致进行性功能障碍和光感受器死亡，而RPE和M？ller胶质细胞相对 幸免于难。初步数据有力地支持了这一假设，我们将通过三个具体目标对其进行进一步检验：1) 使用转化的视网膜来源的细胞系，我们将检查7DHC来源的氧化甾醇是否存在差异 在培养中改变光感受器的基因表达和活性，与RPE和M？ller胶质细胞相比，还将检查 抗氧化剂的保护作用；2)我们将检查光感受器是否对 玻璃体内注射7DHC-VS Chol来源的氧固醇与体内其他类型的视网膜细胞的比较；3)我们 将研究细胞类型特定的胆固醇生物合成中断对视网膜结构和 在体内发挥作用，选择性地敲除视杆细胞、RPE或M？ler胶质细胞中的Dhcr7。这将显著推进我们的 了解SLOS相关的视网膜变性机制，并提供新的见解 为这类疾病开发更有效的治疗干预措施。

Inborn errors in cholesterol (Chol) biosynthesis comprise a group of severe, often lethal, metabolic disorders, of which the Smith-Lemli-Opitz Syndrome (SLOS)¿ the fourth most prevalent recessive human disease¿ is the most well-known. Prior work has established a SLOS rat model, which exhibits a progressive retinal degeneration in which photoreceptors seem more susceptible than RPE cells or M¿ller glia. While the exact disease mechanism is not yet known, the initial biochemical defect involves inefficient conversion of 7- dehydrocholesterol (7DHC) to Chol, which is catalyzed by DHCR7 (3b-hydroxysterol-D7-reductase, the DHCR7 gene product). We have proposed that this leads to multiple sequelae, including altered gene expression, lipid and protein oxidation, caspase activation, and perturbed membrane structure, which then result in progressive cellular dysfunction and demise. 7DHC is the most oxygen-labile lipid known, and readily forms "oxysterols" (some of which are extraordinarily toxic to cells), and 7DHC-derived oxysterols tend to be more cytotoxic than are Chol-derived oxysterols. The native environment of the retina (high oxygen tension, iron, and incident light) presents ideal conditions for oxysterol formation. We hypothesize that the demonstrable rise in retina/RPE 7DHC levels with blockade of DHCR7 leads to in situ oxysterol formation, resulting in progressive dysfunction and death of photoreceptors, while RPE and M¿ller glia are relatively spared. Preliminary data strongly support this hypothesis, which we will test further via three Specific Aims: 1) Using transformed, retina-derived cell lines, we will examine whether 7DHC-derived oxysterols differentially alter gene expression and viability of photoreceptors, vs. RPE and M¿ller glia, in culture, and also will examine the protective effects of antioxidants; 2) We will examine whether photoreceptors are more sensitive to intravitreally injected 7DHC- vs. Chol-derived oxysterols compared to other retinal cell types in vivo; and 3) We will examine the impact of cell-type specific disruption of cholesterol biosynthesis on retinal structure and function in vivo, selectively knocking out Dhcr7 in rods, RPE, or M¿ller glia. This will markedly advance our understanding of the SLOS-associated retinal degeneration mechanism, as well as provide new insights into the development of more effective therapeutic interventions for such diseases.