A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

The impact of metallothionein-II on microglial response to tumor necrosis factor-alpha (TNFα) and downstream effects on neuronal regeneration.

BACKGROUND: The extracellular environment plays an important role in supporting the regeneration of axons after injury. Metallothionein-II (MTII) is a metal-binding protein known for its neuroprotective effect by directly stimulating the growth of axons after injury. Previous studies have shown that MTII also modulates the response of astrocytes and microglia after injury. However, a detailed analysis describing how MTII modulates the interaction between microglia and neurons is lacking. METHODS: We introduced fluorescently labelled MTII into the cortex at the time of needlestick injury to investigate the cellular uptake of MTII using immunohistochemistry with antibodies against cell-type-specific markers. The role of MTII in modulating the effect of microglia on axon outgrowth following an inflammatory response is further investigated using a co-culture model involving primary rodent microglia pre-treated with TNFα and primary rodent cortical neurons. The axon lengths were assessed 24 h after the plating of the neurons onto treated microglia. We also utilised siRNA to knockdown the expression of LRP1, which allows us to investigate the role of LRP1 receptors in the MTII-mediated effect of microglia on axon outgrowth. RESULTS: Fluorescently labelled MTII was found to be associated with neurons, astrocytes and microglia following injury in vivo. Microglia-neuron co-culture experiments demonstrated that exogenous MTII altered the response of microglia to TNFα. The neurons plated onto the TNFα-stimulated microglia pre-treated with MTII have shown a significantly longer axonal length compare to the TNFα-stimulated microglia without the MTII treatment. This suggested that MTII reduce cytokine-stimulated activation of microglia, which would ordinarily impair neurite outgrowth. This inhibitory effect of MTII on activated microglia was blocked by siRNA-mediated downregulation of LRP1 receptor expression in microglia, suggesting that MTII acts via the LRP1 receptor on microglia. CONCLUSIONS: This study demonstrates that exogenous MTII acts via the LRP1 receptor to alter the inflammatory response of microglia following TNFα stimulation, providing a more supportive environment for axon growth.

Metallothionein promotes regenerative axonal sprouting of dorsal root ganglion neurons after physical axotomy.

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism.

Transcriptional insights on the regenerative mechanics of axotomized neurons in vitro.

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ± 1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.

Neuroprotection and regeneration by extracellular metallothionein via lipoprotein-receptor-related proteins.

Metallothionein has a well-documented protective and proregenerative effect in the mammalian brain, particularly following physical trauma and ischemia or during the onset of neurodegenerative disease. A range of mechanisms have been established for this, including metallothionein's metal binding properties and its ability to scavenge free radicals. In recent years it has become apparent that metallothionein is present in the extracellular compartment of the central nervous system and that it can interact with cell surface receptors of the lipoprotein-receptor-related protein family, including lipoprotein-receptor-related protein 1 (LRP1) and megalin. These interactions activate intracellular pathways which are consistent with many of the observed effects of metallothionein in the central nervous system, including its effects on neurons, glial cells, and cells of the immune system. The evidence describing the release, receptor interactions, and subsequent physiological consequences of metallothionein is discussed in this review.

Olfactory ensheathing cells moderate nuclear factor kappaB translocation in astrocytes.

Nuclear factor kappaB (NFκB) is a key transcriptional regulator of inflammatory genes. We investigated the modulatory effects of olfactory ensheathing cells (OECs), microglia and meningeal fibroblasts on translocation of NFκB to astrocyte nuclei. The percentage of activated astrocytes in co-cultures with OECs was significantly less than for co-cultures with microglia (p<0.001) and fibroblasts (p<0.05). Phorbol myristate acetate (PMA) and calcium ionophore stimulation of p65 NFκB translocation to nuclei provided an in vitro model of astrocyte inflammatory activation. Soluble factors released by OECs significantly moderated the astrocytic NFκB translocation induced by either PMA/calcium ionophore or microglia-derived factors (p<0.001). Insulin-like growth factor-1 may contribute to these effects, since it is expressed by OECs and also significantly moderated the astrocytic NFκB translocation (p<0.05), albeit insufficiently to fully account for the OEC-induced moderation (p<0.01). Olfactory ensheathing cells significantly moderated the increased transcription of the pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor in the activated astrocytes (p<0.01). These results suggest that transplanted OECs could improve neural repair after CNS injury by moderating astrocyte activation.