iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease

Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent

c-Fos Activated Phospholipid Synthesis Is Required for Neurite Elongation in Differentiating PC12 Cells

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation

Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice

<div><p>Background</p><p>Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in <i>PANK2</i>, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in <i>PANK2</i> reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.</p><p>Objective, Methods, Results and Conclusions</p><p>Cellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.</p></div

GFP expression driven by the CMV or Syn promoter.

<p>(A) Western blotting analysis of GFP in tissues from animals injected with AAV9-Syn-GFP. GAPDH was used as the loading control. Immunohistochemistry of (B) brown adipose tissue and paraspinal muscle and (C) central nervous system regions. GFP expression (brown) was driven by the CMV or the Syn promoter. AAV particles were injected as described in ‘Material and Methods’ and mice were analyzed 4 weeks thereafter. The results are representative of 2 or more animals. The magnifications are indicated.</p

Nudt7 and Nudt7cyt activity and localization.

<p>(A) Nudt7 hydrolyzes the phosphodiester bond in acyl-CoA species producing acyl-phosphopantetheine and 3’,5’-ADP. (B) Enzymatic activities of purified mouse Nudt7 (closed circles) and Nudt7cyt (open circles). Data are reported as the mean ± the range. (C-H) HEK293 cells were transfected with expression plasmids encoding mCherry fused to the N-terminus of Nudt7 (C-E) or Nudt7cyt (F-H) as described in ‘Material and Methods’. mCherry-Nudt7 and mCherry-Nudt7cyt proteins are shown in magenta (C, F) and cell nuclei are stained with Hoechst 33342 in blue (D, E, G, H). Co-transfection with a plasmid encoding green fluorescent protein (GFP) with a C-terminal SKL (GFP-SKL) (D, G) designates the peroxisomes shown in green. GFP-SKL colocalizes with Nudt7 (E, shown in white) but not with Nudt7cyt (H). The mCherry-Nudt7, mCherry-Nudt7cyt and GFP-SKL proteins were visualized by live-cell confocal fluorescent microscopy. Scale bar, 10 μm.</p