A novel strategy for delivering Niemann-Pick type C2 proteins across the blood-brain barrier using the brain endothelial-specific AAV-BR1 virus.

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.

Upregulation of Transferrin Receptor 1 (TfR1) but Not Glucose Transporter 1 (GLUT1) or CD98hc at the Blood-Brain Barrier in Response to Valproic Acid.

BACKGROUND: Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood-brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA). METHODS: Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS. RESULTS: In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression. CONCLUSIONS: VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1.

Rethinking neurodegenerative diseases: neurometabolic concept linking lipid oxidation to diseases in the central nervous system.

ABSTRACT: Currently, there is a lack of effective medicines capable of halting or reversing the progression of neurodegenerative disorders, including amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, or Alzheimer's disease. Given the unmet medical need, it is necessary to reevaluate the existing paradigms of how to target these diseases. When considering neurodegenerative diseases from a systemic neurometabolic perspective, it becomes possible to explain the shared pathological features. This innovative approach presented in this paper draws upon extensive research conducted by the authors and researchers worldwide. In this review, we highlight the importance of metabolic mitochondrial dysfunction in the context of neurodegenerative diseases. We provide an overview of the risk factors associated with developing neurodegenerative disorders, including genetic, epigenetic, and environmental factors. Additionally, we examine pathological mechanisms implicated in these diseases such as oxidative stress, accumulation of misfolded proteins, inflammation, demyelination, death of neurons, insulin resistance, dysbiosis, and neurotransmitter disturbances. Finally, we outline a proposal for the restoration of mitochondrial metabolism, a crucial aspect that may hold the key to facilitating curative therapeutic interventions for neurodegenerative disorders in forthcoming advancements.

Epigenetic Regulation of Ferroportin in Primary Cultures of the Rat Blood-Brain Barrier.

Ferroportin plays an essential role for iron transport through the blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs). To maintain the integrity of the BBB, the BCECs gain support from pericytes and astrocytes, which together with neurons form the neurovascular unit (NVU). The objectives of the present study were to investigate ferroportin expression in primary cells of the NVU and to determine if ferroportin mRNA (Fpn) expression is epigenetically regulated. Primary rat BCECs, pericytes, astrocytes, and neurons all expressed ferroportin mRNA at varying levels, with BCECs exhibiting the highest expression of Fpn, peaking when co-cultured but examined separately from astrocytes. Conversely, Fpn expression was lowest in isolated astrocytes, which correlated with high DNA methylation in their Slc40a1 promoter. To provide further evidence for epigenetic regulation, mono-cultured BCECs, pericytes, and astrocytes were treated with the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (SB), which significantly increased Fpn and ferroportin protein in BCECs and pericytes. Furthermore, 59Fe export from BCECs was elevated after treatment with VPA. In conclusion, we present first time evidence stating that Fpn expression is epigenetically regulated in BCECs, which may have implications for pharmacological induction of iron transport through the BBB.

Correction to: Hepcidin Mediates Transcriptional Changes in Ferroportin mRNA in Differentiated Neuronal-like PC12 Cells Subjected to Iron Challenge.

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.

Hepcidin Mediates Transcriptional Changes in Ferroportin mRNA in Differentiated Neuronal-Like PC12 Cells Subjected to Iron Challenge.

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-ß)-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.