Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma

Transdifferentiation (TD) is a recent advancement in somatic cell reprogramming. The direct conversion of TD eliminates the pluripotent intermediate state to create cells that are ideal for personalized cell therapy. Here we provide evidence that TD-derived induced neural stem cells (iNSCs) are an efficacious therapeutic strategy for brain cancer. We find that iNSCs genetically engineered with optical reporters and tumouricidal gene products retain the capacity to differentiate and induced apoptosis in co-cultured human glioblastoma cells. Time-lapse imaging shows that iNSCs are tumouritropic, homing rapidly to co-cultured glioblastoma cells and migrating extensively to distant tumour foci in the murine brain. Multimodality imaging reveals that iNSC delivery of the anticancer molecule TRAIL decreases the growth of established solid and diffuse patient-derived orthotopic glioblastoma xenografts 230- and 20-fold, respectively, while significantly prolonging the median mouse survival. These findings establish a strategy for creating autologous cell-based therapies to treat patients with aggressive forms of brain cancer

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

Cytosolic PanK1β partially associates with clathrin-coated and recycling endosomes.

<p>HEK293 cells were transfected with expression plasmids encoding mPanK1β fused to ZsGreen1 (green, a, c, d, f, g, i, j, l, m, o), together with different plasmids encoding the fluorescent protein mCherry fused to distinct markers for different subcellular vesicles: human clathrin LCB (magenta, b, c), human Rab5 (magenta, e, f), human Rab11 (magenta, h, i), firefly luciferase peroxisomal targeting signal (magenta, k, l) and rat Lamp1 (magenta, n, o). Rab5 designates early endosomes, Rab11 designates recycling endosomes and Lamp1 designates lysosomes. Cells were visualized using live-cell confocal imaging. Merged images show co-localization as indicated by white pixels resulting from both magenta and green pseudocolored contributions. Insets in merged images show details at higher magnification. Results are representative of 2 or more independent experiments. Scale bar, 10 µm.</p

Compartmentalization of Mammalian Pantothenate Kinases

<div><p>The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.</p> </div

Identification of the NLS of human PanK2.

<p>(A) Schematic diagram of the full length hPanK2(1–570) and derivatives named hPanK2(1–210), hPanK2(1–150), hPanK2(1–52), hPanK2(82–570), hPanK2(95–570), hPanK2(82–210), and hPanK2(82–94) fused with ZsGreen1. The indicated numbers are the amino acid residues encoded by the constructs. (B) HEK293 cells were transfected with the indicated hPanK2 sequences fused with ZsGreen1 and the fusion proteins (green) were visualized by live cell confocal microscopy. Cells were counterstained with MitoTracker Red CMXRos (mito, red), WGA-Alexa 647 (white) and Hoetsch 33342 (blue) to visualize mitochondria, plasma membrane and nuclei, respectively. Merged images (b, d, f, h, j, l, n, p) show the co-localization indicated by yellow pixels containing both red and green pseudocolored contributions. Results are representative of at least 2 experiments. Scale bar, 10 µm.</p

Leptomycin B blocks the nuclear export of human PanK2.

<p>(A) HeLa cells were transfected with the expression plasmid pAA303 encoding hPanK2(268–275) fused to ZsGreen1, or with plasmid pAA338 encoding the full length mPanK2 fused to ZsGreen1. Leptomycin B (LMB) (20 nM) and cycloheximide (50 µg/ml) were added 24 hours later. Fluorescent cells were imaged at times indicated (upper panels, black and white). Pseudo-colored micrographs are shown to better visualize the fluorescence intensity levels (lower panel, color). (B). Scoring of fluorescent cells (LMB, n = 249 cells; Control, n = 284 cells) for subcellular distribution of hPanK2(268–275)-ZsGreen1 and (C) scoring of fluorescent cells (LMB, n = 129 cells; Control, n = 143 cells) for subcellular distribution of mPanK2-ZsGreen1 after LMB treatment as nuclear “N”, [nuclear and cytoplasmic] “NC” or cytoplasmic “C”. Significance was determined using unpaired Students t-test. ***p<0.001. Scale bar, 10 um.</p

Identification of NLS in the human and mouse PanK1α isoforms.

<p>HEK293 cells were transfected with expression plasmids encoding PanK1α partial sequences fused to ZsGreen1. (A) Schematic diagram of human and mouse PanK1α proteins and their derivatives named: hPanK1α(1–235), hPanK1α(1–217), hPanK1α(218–233), mPanK1α(1–185), mPanK1α(9–185), mPanK1α(61–185), mPanK1α(168–185), mPanK1α(9–150), mPanK1α(61–150), mPanK1α(1–60) and mPanK1α(1–8). The numbers indicate amino acid positions included in the fusion proteins. Dashed lines represent internal deletions. The NLS are indicated in dark gray. (B and C) Functional analysis of predicted NLS of PanK1α. hPanK1α expression constructs (panel B, a–f, green) or mPanK1α constructs (panel C, a-p, green) were co-transfected with an expression plasmid encoding lamin A/C fused to mCherry (LmnA/C, magenta), which designates the nuclear membrane, and before imaging, cells were counterstained with wheat germ agglutinin (WGA)-Alexa 647 (white) and Hoetsch 33342 (blue) to visualize the plasma membrane and the nucleus, respectively. The results are representative of at least 2 independent experiments. Scale bar, 10 µm.</p

Localization of PanK1α within the nucleolus and with the perichromosomal region during mitosis.

<p>HEK293 cells were transfected with expression plasmids encoding mPanK1α(1–185) fused to ZsGreen1 (panels A and B, a, c, d, f, green). (A) Cells were co-transfected with plasmid pAA076 encoding human fibrillarin fused to mCherry (Fibrillarin, magenta, b, c) or plasmid pAA079 encoding human B23 fused to mCherry (B23, magenta, e, f). Fibrillarin designates the dense fibrillar component and B23 designates the granular component of nucleoli. Cell nuclei were visualized by staining with Hoechst 33342 (blue). Cells were visualized using live-cell confocal imaging. Co-localization in merged images are indicated by white pixels containing both magenta and green pseudocolored contributions. Insets in merged images show details at higher magnification. (B) HEK293 cells were co-transfected with with a plasmid encoding human heterochromatin protein 1α fused to mCherry (HP1a, b, c, e, f, magenta). Cells were visualized using live-cell confocal imaging. During interphase, mPanK1α associated with nucleoli and HP1a associated with relaxed heterochromatin, thus designating the nuclear region. In mitotic cells, the nuclear envelope is absent and mPanK1α associated with condensed chromosomes, while HP1a was dissipated throughout the cytoplasm. Dashed lines delimit cell borders (note that mitotic cells are spherical and detached from the substratum). Results are representative of at least 2 independent experiments. Results obtained using hPanK1α were the same. Scale bar, 10 µm.</p

Mutagenesis of the NLS and NES of human PanK2(82–570).

<p>(A) Schematic diagram of human PanK2 without the MTS and the mutant derivatives named: hPanK2-noNLS-mCherry and hPanK2-noNES-mCherry, each containing disrupted NLS or NES motifs, respectively. Arginine (R) or leucine (L) residues were replaced by the alanine (A) as indicated. (B) HeLa cells were transfected with expression plasmids encoding hPanK2(82–570)-mCherry (a, b), hPank2(82-570-noNLS)-mCherry (c, d), hPanK2(82-570-noNES)-mCherry (e, f) or mCherry alone (g,h). After 48 hours cells were stained with Hoetsch 33342 to visualize nuclei (green, b, d, f, h) and analyzed by live-cell confocal microscopy. Results are representative of at least 2 independent experiments. Scale bar, 10 µm.</p