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Research was undertaken to establish transplacental delivery of active genes to fetal brain by a non-viral vector, antibody-specific targeted therapeutic procedure. PEGylated immunoliposomes (PILs) containing firefly luciferase DNA under the influence of the SV40 promoter injected intravenously into near-term pregnant mice produced luminometric evidence of CNS tissue luciferase activity at 48-h post-injection in all newborn pups. In utero delivery of this pGL3 DNA was shown after a single i.v. injection in maternal and neonatal brains, spleen and lesser amounts in lungs, with only negligible background levels in negative controls exposed to unencapsulated pDNA. In addition to studies of normal wild-type mice, we similarly injected pregnant Lafora Knockout (EPM2a null-mutant) and demonstrated luciferase activity days later in the maternal and newborn pup brains of both types. Delivery of PILs containing a second reporter gene (the pSV40 beta-galactosidase transgene) transplacentally by the same procedure was also successful. Histochemical and biochemical demonstration of beta-galactosidase was documented for all mutant and non-mutant neonates. Brain areas of highest Lafora body development (such as the hippocampus and pontine nuclei) showed intraneuronal beta-glucosidase activity. We conclude that receptor-mediated transport of PIL-borne gene therapeutics across both the placental barrier as well as the fetal BBB in utero is feasible.

Dietary omega-3 fatty acid (i.e. docosohexaenoic acid (DHA)) and exercise are gaining recognition for supporting brain function under normal and challenging conditions. Here we evaluate the possibility that the interaction of DHA and exercise can involve specific elements of the synaptic plasma membrane. We found that voluntary exercise potentiated the effects of a 12-day DHA dietary supplementation regimen on increasing the levels of syntaxin 3 (STX-3) and the growth-associated protein (GAP-43) in the adult rat hippocampus region. STX-3 is a synaptic membrane-bound protein involved in the effects of DHA on membrane expansion. The DHA diet and exercise also elevated levels of the NMDA receptor subunit NR2B, which is important for synaptic function underlying learning and memory. The actions of exercise and DHA dietary supplementation reflected on enhanced learning performance in the Morris water maze as learning ability was associated with higher levels of STX-3 and NR2B. The overall findings reveal a mechanism by which exercise can interact with the function of DHA dietary enrichment to elevate the capacity of the adult brain for axonal growth, synaptic plasticity, and cognitive function., (Copyright 2009. Published by Elsevier B.V.)

Myelin is a major obstacle for axonal growth after CNS injury, to the extent that it is crucial to develop interventions to counteract postinjury growth inhibition and foster neural repair. We have studied the effects of the fluid percussion injury (FPI) model of traumatic brain injury (TBI) on protein levels of two myelin-associated molecules, myelin-associated glycoprotein (MAG) and Nogo-A, in the adult rat. We found that FPI elevated hippocampal levels of MAG and Nogo-A. Given the beneficial effects of exercise on CNS function, we evaluated the capacity of exercise to reduce these myelin-derived proteins after FPI. One week of voluntary running wheel exercise overcame the injury-related increase in MAG and Nogo-A. The action of brain-derived neurotrophic factor (BDNF) has been associated with exercise as well as with the modulation of growth inhibition in vitro. We found that the selective blockade of BDNF using the immunoadhesive chimera TrkB-IgG abolished the effects of exercise on MAG and Nogo-A. FPI reduced levels of growth-associated protein 43 (GAP-43), a marker of axonal growth, and synaptophysin (SYP), an indicator of synaptic growth. Exercise counteracted the effects of FPI on GAP-43 and SYP, while BDNF blockade abolished these effects of exercise. Protein kinase A (PKA) has been related to the ability of BDNF to overcome growth inhibition. In agreement, we found that exercise increased PKA levels and this effect was prevented by BDNF blockade. These results indicate that exercise promotes a permissive cellular environment for repair after TBI, in a process in which BDNF plays a central role.

Both neuronal activity and neurotrophin signaling play critical roles in normal CNS development. This study examined whether spontaneous retinal activity (SRA) also governs the axonal transport of endogenous brain-derived neurotrophic factor (BDNF) protein within the developing chick visual system. In previous work, we have found that during the normal period of SRA, retinal BDNF protein levels decrease by about 50% while BDNF mRNA levels remain elevated. Here, we show that the blockade of SRA with tetrodotoxin (TTX), or the blockade of axonal transport with colchicine, both reversed the normal mismatch between retinal BDNF mRNA and protein. The axonal transport of retinal-derived BDNF in segments of the optic nerve as well as tectal-derived BDNF protein transported in segments of the optic tract were both significantly reduced after very brief periods of activity blockade. These results suggest that normal SRA plays a role in regulating the axonal transport of endogenous BDNF protein.

During the assembly process of herpes simplex virus type 1 capsids, there is an essential interaction between the C-terminal tail of the scaffold proteins (22a and 21) and the major capsid protein (VP5). Recent studies of spontaneous revertant viruses that overcome a blocked maturation cleavage site of the scaffold proteins have shown that the N-terminus of VP5 is important for this interaction. One of the revertant viruses, PR7, encodes a second-site mutation at residue 69 of VP5 which unlike wild-type VP5 fails to interact with 22a and thus gives white colonies in the yeast two-hybrid assay. In the present study a small DNA fragment, encoding residues 1 to 85 of wild-type and PR7 VP5, was mutagenized using error-prone PCR. Mutagenized DNA was used in the yeast two-hybrid assay to identify mutations in wild-type VP5 that resulted in loss of 22a binding (white colonies), or in PR7 VP5 that resulted in a gain of function (blue colonies). For the loss of function experiments, using KOS VP5, a row of eight thymidine nucleotides (codons 37-40) resulted in many frameshift mutations, which led us to terminate the study without reaching a statistically significant result. For the PR7 experiment, 30 clones were identified that had single amino acid substitutions, and these mutations were localized to amino acids 27-45 and 63-84 of VP5. The most frequent mutation was a reversion back to wild-type. The next most frequent were E28K and N63S, and these gave the highest beta-galactosidase enzyme activities (indicative of PR7VP5-22a interaction), 30 and 20% of wild-type, respectively. When E28K and N63S were transferred into the wild-type VP5 background, that is, in the absence of the PR7 mutation, they gave rise to different phenotypes. The E28K mutation lost its ability to interact with the scaffold proteins as judged by this assay. Therefore, it may be acting as a compensatory mutation whose phenotype is only expressed in the presence of the original PR7 mutation. However, the N63S mutation in the wild-type VP5 background increased the interaction, as judged by the beta-galactosidase activity, by a factor of 9 relative to when the PR7 mutation was present. Even more surprising, in the absence of the PR7 mutation the enzyme activity was still greater, by a factor of 2, than that observed for wild-type VP5. This study provides further evidence that the N-terminus of VP5 is in intimate association with the C-terminus of the scaffold proteins., (Copyright 2001 Academic Press.)