Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de Investigaciones Científicas, Universidad Pablo de Olavide, Carretera de Utrera Km 1, 41013, Sevilla, Spain.
Centro de Investigación Biomédica en Red: Enfermedades Raras, Instituto de Salud Carlos III, 41013, Sevilla, Spain.
Orphanet J Rare Dis. 2021 May 5;16(1):201. doi: 10.1186/s13023-021-01823-3.
Neurodegeneration with brain iron accumulation (NBIA) is a group of genetic neurological disorders frequently associated with iron accumulation in the basal nuclei of the brain characterized by progressive spasticity, dystonia, muscle rigidity, neuropsychiatric symptoms, and retinal degeneration or optic nerve atrophy. Pantothenate kinase-associated neurodegeneration (PKAN) is the most widespread NBIA disorder. It is caused by mutations in the gene of pantothenate kinase 2 (PANK2) which catalyzes the first reaction of coenzyme A (CoA) biosynthesis. Thus, altered PANK2 activity is expected to induce CoA deficiency as well as low levels of essential metabolic intermediates such as 4'-phosphopantetheine which is a necessary cofactor for critical proteins involved in cytosolic and mitochondrial pathways such as fatty acid biosynthesis, mitochondrial respiratory complex I assembly and lysine and tetrahydrofolate metabolism, among other metabolic processes.
In this manuscript, we examined the effect of PANK2 mutations on the expression levels of proteins with phosphopantetheine cofactors in fibroblast derived from PKAN patients. These proteins include cytosolic acyl carrier protein (ACP), which is integrated within the multifunctional polypeptide chain of the fatty acid synthase involved in cytosolic fatty acid biosynthesis type I (FASI); mitochondrial ACP (mtACP) associated with mitocondrial fatty acid biosynthesis type II (FASII); mitochondrial alpha-aminoadipic semialdehyde synthase (AASS); and 10-formyltetrahydrofolate dehydrogenases (cytosolic, ALD1L1, and mitochondrial, ALD1L2).
In PKAN fibroblasts the expression levels of cytosolic FAS and ALD1L1 were not affected while the expression levels of mtACP, AASS and ALD1L2 were markedly reduced, suggesting that 4'-phosphopantetheinylation of mitochondrial but no cytosolic proteins were markedly affected in PKAN patients. Furthermore, the correction of PANK2 expression levels by treatment with pantothenate in selected mutations with residual enzyme content was able to correct the expression levels of mitochondrial phosphopantetheinyl-proteins and restore the affected pathways. The positive effects of pantothenate in particular mutations were also corroborated in induced neurons obtained by direct reprograming of mutant PANK2 fibroblasts.
Our results suggest that the expression levels of mitochondrial phosphopantetheinyl-proteins are severely reduced in PKAN cells and that in selected mutations pantothenate increases the expression levels of both PANK2 and mitochondrial phosphopantetheinyl-proteins associated with remarkable improvement of cell pathophysiology.
神经铁沉积伴脑铁沉积（NBIA）是一组遗传性神经疾病，常伴有基底核铁沉积，其特征为进行性痉挛、肌张力障碍、肌肉僵硬、神经精神症状以及视网膜变性或视神经萎缩。泛酸激酶相关神经变性（PKAN）是最广泛的 NBIA 疾病。它是由泛酸激酶 2（PANK2）基因突变引起的，该基因催化辅酶 A（CoA）生物合成的第一个反应。因此，改变的 PANK2 活性预计会诱导 CoA 缺乏以及必需代谢中间产物的水平降低，例如 4'-磷酸泛酰巯基乙胺，这是参与细胞质和线粒体途径的关键蛋白的必要辅因子，例如脂肪酸生物合成、线粒体呼吸复合物 I 组装以及赖氨酸和四氢叶酸代谢等代谢过程。
在本手稿中，我们研究了 PANK2 突变对来自 PKAN 患者的成纤维细胞中带有磷酸泛酰巯基乙胺辅因子的蛋白质表达水平的影响。这些蛋白质包括细胞质酰基辅酶 A 载体蛋白（ACP），它整合在涉及细胞质脂肪酸生物合成 I 型（FASI）的多功能脂肪酸合酶多肽链中；与线粒体脂肪酸生物合成 II 型（FASII）相关的线粒体 ACP（mtACP）；线粒体α-氨基己二酸半醛合酶（AASS）；以及 10-甲酰四氢叶酸脱氢酶（细胞质，ALD1L1，和线粒体，ALD1L2）。
在 PKAN 成纤维细胞中，细胞质 FAS 和 ALD1L1 的表达水平不受影响，而 mtACP、AASS 和 ALD1L2 的表达水平明显降低，这表明 PKAN 患者的线粒体但不是细胞质蛋白的 4'-磷酸泛酰巯基乙胺化明显受到影响。此外，通过用泛酸处理选定具有残留酶含量的突变体来纠正 PANK2 表达水平，能够纠正线粒体磷酸泛酰巯基蛋白的表达水平，并恢复受影响的途径。泛酸在特定突变体中的积极作用也在通过直接重编程突变型 PANK2 成纤维细胞获得的诱导神经元中得到证实。
我们的结果表明，PKAN 细胞中线粒体磷酸泛酰巯基蛋白的表达水平严重降低，在某些突变中，泛酸增加了 PANK2 和线粒体磷酸泛酰巯基蛋白的表达水平，并显著改善了细胞病理生理学。
