No abstract

Treating central nervous system (CNS) diseases is complicated by the incapability of numerous therapeutics to cross the blood–brain barrier (BBB), mainly composed of brain endothelial cells (BECs). Genetically modifying BECs into protein factories that supply the CNS with recombinant proteins is a promising approach to overcome this hindrance, especially in genetic diseases, like Niemann Pick disease type C2 (NPC2), where both CNS and peripheral cells are affected. Here, we investigated the potential of the BEC‐specific adeno‐associated viral vector (AAV‐BR1) encoding NPC2 for expression and secretion from primary BECs cultured in an in vitro BBB model with mixed glial cells, and in healthy BALB/c mice. Transduced primary BECs had significantly increased NPC2 gene expression and secreted NPC2 after viral transduction, which significantly reversed cholesterol deposition in NPC2 deficient fibroblasts. Mice receiving an intravenous injection with AAV‐BR1‐NCP2‐eGFP were sacrificed 8 weeks later and examined for its biodistribution and transgene expression of eGFP and NPC2. AAV‐BR1‐NPC2‐eGFP was distributed mainly to the brain and lightly to the heart and lung, but did not label other organs including the liver. eGFP expression was primarily found in BECs throughout the brain but occasionally also in neurons suggesting transport of the vector across the BBB, a phenomenon also confirmed in vitro. NPC2 gene expression was up‐regulated in the brain, and recombinant NPC2 protein expression was observed in both transduced brain capillaries and neurons. Our findings show that AAV‐BR1 transduction of BECs is possible and that it may denote a promising strategy for future treatment of NPC2.

Ferroportin is the only known iron exporter, and its regulation seems to be controlled at both transcriptional, post-transcriptional, and post-translational levels. The objective of the current work was to investigate how cellular iron status affects the expression of the ferroportin gene Fpn under the influence of hepcidin, known to post-translational lower the available ferroportin protein. Nerve growth factor-beta (NGF-β)-differentiated PC12 cells, used as a model of neuronal cells, were evaluated in terms of their viability and expression of ferroportin after inducing cellular iron overload with ferric ammonium citrate (FAC) or hepcidin, iron deficiency with deferoxamine (DFO), or hepcidin in combination with FAC or DFO. Ferritin mRNA was significantly upregulated following treatment with 20 mM FAC. The viability of the differentiated PC12 cells was significantly reduced after treatment with 30 mM FAC or 1.0 μM hepcidin, but when combining FAC and hepcidin treatment, the cells remained unaffected. The expression of Fpn was concurrently upregulated after treatment with FAC in combination with hepcidin. Fifty millimolar DFO also increased Fpn. Together, these data point towards a transcriptional induction of Fpn in response to changes in cellular iron levels. Epigenetic regulation of Fpn may also occur as changes in genes associated with epigenetic regulation of Fpn were demonstrated.

The original version of this article unfortunately contained mistakes on Figs. 1, 2, and 7 as some of the data were not visible. With this, the correct images are hereby published. In Table 4 footnote, "Forty millimolar40 mM" should be changed two times to "Forty millimolar (40mM)". Also layout of Tables 2-4 were changed as per request of authors. Corrected Tables are presented herewith.

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