Mark Franko

Site-directed antisense oligonucleotide decreases the expression of amyloid precursor protein and reverses deficits in learning and memory in aged SAMP8 mice.

beta amyloid protein (Abeta) is a 40-43 amino acid peptide derived from amyloid precursor protein (APP). Abeta has been implicated as a cause of Alzheimers disease (AD). Mice with spontaneous or transgenic overexpression of APP show the histologic hallmarks of AD and have impairments in learning and memory. We tested whether antisense phosphorothiolated oligonucleotides (AO) directed at the Abeta region of the APP gene given with or without antibody directed at Abeta could reverse the elevated protein levels of APP and the behavioral impairments seen in SAMP8 mice, a strain which spontaneously overexpresses APP. We found that intracerebroventricular (ICV) administration of antibody with either of two AOs directed at the midregion of Abeta improved acquisition and retention in a footshock avoidance paradigm, whereas two AOs directed more toward the C-terminal, a random AO, and vehicle were without effect. Three injections of the more potent AO given without antibody reduced APP protein levels by 43-68% in the amygdala, septum, and hippocampus. These results show that AO directed at the Abeta region of APP can reduce APP levels in the brain and reverse deficits in learning and memory.

Neurotoxicity of MAO metabolites of catecholamine neurotransmitters: role in neurodegenerative diseases.

The monoamine oxidase (MAO) metabolites of norepinephrine (NE) or epinephrine (EPI) and of dopamine (DA) are 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and 3,4-dihydroxyphenylacetaldehyde (DOPAL), respectively. The toxicity of these catecholamine (CA) MAO metabolites was predicted over 50 years ago. However, until our recent chemical synthesis of these CA aldehyde metabolites, the hypothesis about their toxicity could not be tested. The present paper reviews recent knowledge gained about these compounds. Topics to be reviewed include: chemical synthesis and properties of DOPEGAL and DOPAL; in vitro and in vivo toxicity of CA aldehydes; subcellular mechanisms of toxicity; free radical formation by DOPEGAL versus DOPAL; mechanisms of accumulation of CA aldehydes in Alzheimers disease (AD) and Parkinsons disease (PD) and potential therapeutic targets in Alzheimers disease and Parkinsons disease.

Aggregation of α-synuclein by DOPAL, the monoamine oxidase metabolite of dopamine

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the selective loss of dopamine (DA) neurons and the presence of α-synuclein (AS) aggregates as Lewy bodies (LBs) in the remaining substantia nigra (SN) neurons. A continuing puzzle in studying PD pathogenesis is that although AS is expressed throughout the brain, LBs and selective dopaminergic cell loss lead to characteristic clinical signs of PD, suggesting that there is a link between AS aggregation and DA metabolism. One potential candidate for this link is the monoamine oxidase (MAO) metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), as neither DA nor DA metabolites other than DOPAL are toxic to SN neurons at physiological concentrations. We tested DOPAL-induced AS aggregation in a cell-free system, in vitro in DA neuron cultures and in vivo with stereotactic injections into the SN of Sprague–Dawley rats by Western blots, fluorescent confocal microscopy and immunohistochemistry. We demonstrate that DOPAL in physiologically relevant concentrations, triggers AS aggregation in the cell-free system, and in cell cultures resulting in the formation of potentially toxic AS oligomers and aggregates. Furthermore, DOPAL injection into the SN of Sprague–Dawley rats resulted in DA neuron loss and the accumulation of high molecular weight oligomers of AS detected by Western blot. Our findings support the hypothesis that DA metabolism via DOPAL can cause both DA neuron loss and AS aggregation observed in PD.

Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins and adsorptive endocytosis.

ABSTRACT Blood-borne human immunodeficiency virus type 1 (HIV-1) crosses the blood-brain barrier (BBB) to induce brain dysfunction. How HIV-1 crosses the BBB is unclear. Most work has focused on the ability of infected immune cells to cross the BBB, with less attention devoted to the study of free virus. Since the HIV-1 coat glycoprotein gp120 can cross the BBB, we postulated that gp120 might be key in determining whether free virus can cross the BBB. We used radioactive virions which do (Env+) or do not (Env−) bear the envelope proteins to characterize the ability of HIV-1 to be taken up by the murine BBB. In vivo and in vitro studies showed that the envelope proteins are key to the uptake of free virus and that uptake was enhanced by wheat germ agglutinin, strongly suggesting that the envelope proteins induce viral adsorptive endocytosis and transcytosis in brain endothelia. Capillary depletion showed that Env+virus completely crossed the vascular BBB to enter the parenchyma of the brain. Virus also entered the cerebrospinal fluid, suggesting passage across the choroid plexus as well. About 0.22% of the intravenously injected dose was taken up per g of brain. In vitro studies showed that postinternalization membrane cohesion (membrane binding not reversed with acid wash or cell lysis) was a regulated event. Intact virus was recovered from the brain endothelial cytosol and was effluxed from the endothelial cells. These results show that free HIV-1 can cross the BBB by an event related to adsorptive endocytosis and mediated by the envelope proteins.

Molecular cloning, expression, and regulation of hippocampal amyloid precursor protein of senescence accelerated mouse (SAMP8)

Alzheimers disease (AD) is associated with increased expression of amyloid precursor protein (APP) with a consequent deposition of amyloid beta peptide (Abeta) which forms characteristic senile plaques. We have noticed that the senescence accelerated mouse (SAMP8), a strain of mouse that exhibits age-dependent defects such as loss of memory and retention at an early age of 8-12 months, also produces increased amounts of APP and Abeta similar to those observed in Alzheimers disease (AD). In order to investigate if this is due to mutations in APP similar to those observed in AD, and to develop molecular probes that regulate its expression, APP cDNA was cloned from the hippocampus of 8-month-old SAMP8 mouse. The nucleotide sequence is 99.7% homologous with that of mouse and rat, 88.7% with monkey, and 89.2% with human homologues. At the amino acid level, the homology was 99.2% and 97.6% with rodent and primate sequences, respectively. A single amino acid substitution of Alanine instead of Valine at position 300 was unique to SAMP8 mouse APP. However, no mutations similar to those reported in human familial AD were observed. When the cDNA was expressed in HeLa cells, glycosylated mature APP could be detected by immunoblotting technique. The expression could be regulated in a time- and concentration-dependent manner by using an antisense oligonucleotide specific to APP mRNA. Such regulation of APP expression may have a therapeutic application in vivo.

Effect of metformin on nitric oxide synthase in genetically obese (ob/ob) mice

Genetically obese (ob/ob) mice were employed for the study of the effect of metformin on activity and expression of nitric oxide synthase (NOS ) in vitro and in vivo. For in vitro analysis, mouse liver extracts were used. For the in vivo study, (ob/ob) and their control litter mates (ob/c) mice were injected with specified amounts of metformin and the expression of NOS in the adipose tissue and hypothalamus was measured by Western blotting. Results show that metformin exhibited a biphasic effect on NOS activity in vitro. Expression of metformin was differentially altered in the hypothalamus and adipose tissues of the normal and ob/ob animals that were treated with metformin. Further, a significant decrease in food intake occurred in the (ob/ob) mice that received metformin. This decrease in food intake was not accompanied by changes in serum glucose. At inhibitory concentrations, hypothalamic NOS expression changes differentially in normal and ob/ob mice. In normal mice, metformin stimulated NOS expression, while in ob/ob mice there was an inhibition. NOS expression increased in brown adipose tissue of metformin treated control mice, while no such increase was observed in ob/ob mice. No effect of metformin was observed in white adipose tissue of control or obese mice. Thus, metformin may produce anorectic effects through modulation of NOS.