Christopher M. de Fiebre

A novel nicotinic agonist facilitates induction of long-term potentiation in the rat hippocampus

Long-term potentiation (LTP) can be modulated by a number of neurotransmitter receptors including muscarinic and GABAergic receptor types. We have found that a novel nicotinic agonist, 2,4-dimethoxybenzylidene anabaseine (DMXB), facilitated the induction of LTP in the hippocampus in a dose-dependent and mecamylamine-sensitive manner. DMXB displaced high affinity nicotinic [125I]alpha-bungarotoxin and [3H]acetylcholine binding in rat brain. Xenopus oocyte studies demonstrated that DMXB has agonist activity at alpha 7 but not alpha 4/beta 2 nicotinic receptor subtypes. These results indicated that DMXB is a novel nicotinic agonist with apparent specificity for the alpha 7/alpha-bungarotoxin nicotinic receptor subtype and indicate that nicotinic receptor activation is capable of modulating the induction of long-term potentiation.

3-[2,4-Dimethoxybenzylidene]anabaseine (DMXB) selectively activates rat α7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner

The alpha7 nicotinic receptor agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB; GTS-21) was investigated for its ability to: (1) activate a variety of nicotinic receptor subtypes in Xenopus oocytes; (2) improve passive avoidance and spatial Morris water task performances in mecamylamine-sensitive manners in bilaterally nucleus basalis lesioned rats; and (3) elevate high-affinity [3H]acetylcholine (ACh) and high-affinity alpha-[125I]bungarotoxin binding in rat neocortex following 2 weeks of daily injections. DMXB (100 microM) activated alpha7 homo-oligomeric receptors, without significant activity at alpha2-, alpha3- and alpha4-containing subtypes. Mecamylamine blocked rat alpha7 receptors weakly if co-administered with agonist, but much more potently when pre-applied. Bilateral ibotenic acid lesions of the nucleus basalis interfered with passive avoidance and spatial memory-related behaviors. DMXB (0.5 mg/kg, i.p.) improved passive avoidance behavior in lesioned animals in a mecamylamine-sensitive manner. DMXB (0.5 mg/kg 15 min before each session) also improved performance in the training and probe components of the Morris water task. DMXB-induced improvement in the probe component but not the training phase was mecamylamine-sensitive. [3H]ACh binding was elevated after 14 days of daily i.p. injections with 0.2 mg/kg nicotine but not after 1 mg/kg DMXB. Neither drug elevated high-affinity alpha-[125I]bungarorotoxin binding over this interval.

The Subunit Specific Effects of Novel Anabaseine-Derived Nicotinic Agents

Nicotine readily crosses the blood-brain barrier and elicits a variety of behavioral and physiological actions. Generally, nicotine improves a variety of spatial-memory and avoidance tasks in animals, while enhancing delayed recall, information manipulation, focused attention and other memory-related behaviors in humans. Nicotine also alters mood and reaction time, which can be difficult to distinguish from changes in memory-related behaviors. One characteristic of nicotine-induced improvements in memory-related behaviors is that these are generally seen over narrow dose-ranges, with higher doses causing a variety of side effects that interfere with performance (Warburton, 1990). This narrow dose-response range may be due in part to the nicotinic activation of a multiplicity of receptors in the brain and periphery, which is a powerful impetus for targeting drugs to those nicotinic receptor subtypes selectively involved in learning and memory in order to treat conditions such as Alzheimer’s disease (AD).

Fusogenic properties of Sendai virosome envelopes in rat brain preparations

Sendai virosomes were characrerized with respect to their ability to bind to, fuse with, and introduce substances into several rat brain preparations. Encapsulation efficiency for Sendai virosomes was enhanced but binding to cerebral cortical P2 preparations was attenuated by addition of bovine brain phosphatidylcholine during reconstitution. A higher percentage of Sendai virosomes than phosphatidylcholine liposomes appeared to bind to, fuse with and subsequently deliver [14C]sucrose into osmotically labile pools of the P2 preparation. Fusogenic activity was estimated by measuring dequenching of fluorescently labelled N-NBD-phosphatidylethanolamine. More virosomally encapsulated [14C]sucrose was bound to the P2 fraction than introduced into osmotically labile organelles, and the fraction of vesicles undergoing fusion was intermediate between these two values. Non-encapsulated [14C]sucrose did not bind to and was not taken up by the P2 fraction in a quantifiable manner. Virosomal envelopes also bound to primary cultures of rat brain neurons and glia in an apparently saturable manner. Addition of increasing amounts of the adenoassociated virus-derived vector pJDT95 increased encapsulation efficiency, and virosomes reconstituted in the presence of 60 μg DNA retained most of their binding activity (5.4% of total label) compared to those containing [14C]sucrose alone (8.4%). These data indicate that Sendai virosomes may be useful in the delivery of substances into brain-derived tissues, potentially for the modulation of gene expression and neurotransmission.

Sendai virosomal infusion of an adeno-associated virus-derived construct containing neuropeptide Y into primary rat brain cultures

A novel neuronal gene-delivery system was investigated in primary neuron-enriched cultures with respect to driving the expression of neuropeptide Y (NPY). This delivery system consists of an adeno-associated virus-derived (AAV) plasmid, pJDT95npy, encapsulated in reconstituted Sendai virosomes. pJDT95npy contains full length rat NPY cDNA inserted downstream from the P40 promoter in a cap-gene deleted AAV-derived construct. The rep-sequences under control of the P5 and P19 promoters are intact. Virosomally encapsulated pJDT95npy drove the expression of NPY mRNAs, predominantly by P40. Total cellular NPY immunoreactivity and release in the presence of depolarization increased following pJDT95npy-transfection. Neither empty virosomes nor virosomes containing pJDT95 affected NPY mRNA expression or immunoreactivity. This study demonstrates that an AAV-derived plasmid can drive exogenous gene expression in intact neurons after infusion by Sendai virosomes.

Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with sendai virosomes versus LipofectinTM

The ability of Sendai virosomes or LipofectinTM to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. LipofectinTM-mediated transfection with pJDT95npy (10 μg) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.

A Novel Gene Delivery System for Neuroactive Peptides in Brain Tissue

The development of a gene delivery system that permits long term, stable expression in brain tissue would be an important step for treating long term neuropathological disorders such as Alzheimer’s disease. Conceivably, such a system could reduce levels of neurotoxic gene-products (e.g., amyloid precursor protein) through infusion of appropriate antisenseconstructs, or counteract disease-induced losses in neurotransmission/ trophism through exogenous gene-induced peptide synthesis.

Sendai Virosome Envelopes for the Infusion of Macromolecules Into Brain Neurons and Glia

IN1RODUCI10N The possibility of introducing macromolecules across plasma membranes directly into cells to counteract neuropathological processes is an intriguing one that has received increased attention only recently (e.g., 1,2). Of particular interest is the possibility of incorporating normal allelic genes into cells to replace defective ones or to increase the gene dosage for those proteins rendered hypofunctional by disease. Ideally, chronic conditions such as Alzheimers disease will eventually be treatable with specific replacement of those cellular constituents that are depleted by or responsible for the symptomology of the disease. While simple in principle, the mechanics of gene therapy are quite complex from both pharmacokinetic as well as pharmacodynarnic perspectives. Nevertheless, studies conducted in non-neuronal cells are promising. A variety of virally derived vectors are now known to be expressed in mammalian cells (3,4), and several cell-specific delivery systems are also available (5,6). Even a few clinical trials have been initiated with some degree of success, e.g., individuals with adenosine deaminase deficiency have been treated with their own T-cells that have been genetically altered toproduce the normal enzyme (7). Functional neuronal gene therapy, however, presents additional problems that are particular to this cell type. With respect to gene expression, many common vectors, including those derived from retroviruses, require cell-mitosis for effective chromosomal integration and expression (8). Neurons, being highly differentiated and post-mitotic, are not likely to express genes delivered in this manner. Altematively, episomally expressed vectors suffer from potential problems with variable expression during the extended life of the neuron. Another problem involves the delivery of genes into neurons. Even vectors with promoters that are both integrated into the host chromosome and expressed in post-mitotic cells, such as those derived from the adenoassociatedor herpes simplex-derived viruses (10,11), must be delivered into neurons in a manner that does not darnage them