Abstract
Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.
Highlights
Pantothenate Kinase-associated Neurodegeneration (PKAN, OMIM #234200) belongs to the heterogeneous group of neurodegenerative diseases known as Neurodegeneration with Brain Iron Accumulation (NBIA) [1,2], characterized by a massive iron accumulation in the basal ganglia, progressive dystonia and parkinsonism, cognitive decline and psychiatric disturbances
The human PKAN neuronal model was obtained by differentiation of neuronal precursor cells (NPC) transduced with neurogenin-2 (Ngn2), as previously described [18]
After three weeks of differentiation, the iPS-derived neurons were analyzed in immunofluorescence with two neuronal markers: microtubule-associated protein 2 (Map2) and vesicular glutamate transporter 1 (Vglut1) (Figure 1a)
Summary
Pantothenate Kinase-associated Neurodegeneration (PKAN, OMIM #234200) belongs to the heterogeneous group of neurodegenerative diseases known as Neurodegeneration with Brain Iron Accumulation (NBIA) [1,2], characterized by a massive iron accumulation in the basal ganglia, progressive dystonia and parkinsonism, cognitive decline and psychiatric disturbances. Disease diagnosis is carried out with brain MR T2-weighted or magnetic susceptibility (T2 *, SWI) images, capable of identifying iron accumulation frequently associated with the characteristic ‘eye-of-the-tiger’ sign. It consists of atrophy and T2 hypointensity of bilateral globus pallidus associated with a small hyperintensity in its anterior and medial part; signal drop is due to iron accumulation and hyperintense portion is caused by gliosis and spongiosis [3]. The PANK2 protein represents one of the four isoforms present in the human genome and localizes in the mitochondrial intermembrane space [8], while the other PANK proteins (PANK1a and b, PANK3 and PANK4) are found in the cytosol [9,10]
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