Bursts of Brain Activity May Protect Against Alzheimer's Disease

Evidence indicates that the accumulation of amyloid-beta proteins, which form the plaques found in the brains of Alzheimer's patients, is critical for the development of Alzheimer's disease, which impacts 5.4 million Americans. And not just the quantity, but also the quality of amyloid-beta peptides is crucial for Alzheimer's initiation. The disease is triggered by an imbalance in two different amyloid species -- in Alzheimer's patients 

there is a reduction in a relative level of healthy amyloid-beta 40 compared to 42.

Now Dr. Inna Slutsky of Tel Aviv University's Sackler Faculty of Medicine and the Sagol School of Neuroscience, with postdoctoral fellow Dr. Iftach Dolev and PhD student Hilla Fogel, have uncovered two main features of the brain circuits that impact this crucial balance. The researchers have found that patterns of electrical pulses (called "spikes") in the form of high-frequency bursts and the filtering properties of synapses are crucial to the regulation of the amyloid-beta 40/42 ratio. Synapses that transfer information in spike bursts improve the amyloid-beta 40/42 ratio.

This represents a major advance in understanding that brain circuits regulate composition of amyloid-beta proteins, showing that the disease is not just driven by genetic mutations, but by physiological mechanisms as well.

Cholesterol Increases Risk of Alzheimer's and Heart Disease

In the new study published this week in the on-line journal PLOS ONE, Antoneta Granic, PhD, and Huntington Potter, PhD, show that cholesterol, particularly in the LDL form, called 'bad cholesterol', causes cells in both humans and mice to divide incorrectly and distribute their already-duplicated chromosomes unequally to the next generation. The result is an accumulation of defective daughter cells with the wrong number of chromosomes and therefore the wrong number of genes. Instead of the correct two copies of each chromosome, and thus two copies of each gene, some cells acquired three copies and some only one.

Granic and Potter's study of the effects of cholesterol on cell division included a prominent finding of cells carrying three copies of the chromosome (#21 in humans and #16 in mice) that encodes the amyloid peptide that is the key component of the neurotoxic amyloid filaments that accumulate in the brains of Alzheimer patients.

Human trisomy 21 cells are significant because people with Down syndrome have trisomy 21 in all of their cells from the moment of conception, and they all develop the brain pathology and many develop the dementia of Alzheimer's disease by age 50. Earlier studies by Granic, Potter and others have shown that as many as 10% of cells in an Alzheimer patient, including neurons in the brain, have three copies of chromosome 21 instead of the usual two. Thus, Alzheimer's disease is, in some ways, a form of acquired Down syndrome. Furthermore, mutant genes that cause inherited Alzheimer's disease cause the same defect in chromosome segregation as does cholesterol, thus indicating the presence of a common cell division problem in both familial and 'sporadic' (non-familial) Alzheimer's disease.

The new research also found trisomy 21 neurons in the brains of children with what, until now, was thought to be an unrelated neurodegenerative disease (Niemann Pick type C), caused by a mutation affecting cholesterol physiology. This result suggests that neurodegeneration itself might be linked to chromosome missegregation.

Such a model is supported by the finding of Thomas Arendt, MD, and colleagues at the University of Leipzig that 90% of the neuronal cell death observed at autopsy in Alzheimer patients is due to the creation and selective loss of neurons with the wrong number of chromosomes.

Identifying the specific problem caused by cholesterol will lead to completely new approaches to therapy for many human diseases, including Alzheimer's disease, atherosclerosis and possibly cancer, all of which show signs of defective cell division. Granic and Potter already have found a potentially simple approach to preventing cholesterol from causing cells to distribute their chromosomes unequally into their new daughter cells. Specifically, when cells in culture were first treated with ethanol, the subsequent exposure to bad cholesterol was without effect on cell division: Each daughter cell received the correct number of chromosomes.

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FUTURE DIRECTIONS

          Our ultimate goal is to use newly discovered compounds directed to APP-mRNA 5'UTR to limit A(3-peptide output in cell culture systems, and subsequently to therapeutically test these compounds in transgenic mouse models for APP and A(3 over-expression. We have developed a collaboration with Dr. Steve Gullans (Director of Renal Division Research, Harvard Institutes of Medicine).
     During the course of this project, we have started a screen for APP 5'UTR-binding compounds from a unique library of 1,500 FDA preapproved drugs arranged in a convenient format for transferring to cells growing in 96 well plates. Using a new transfection-based assay, in which APP-5'UTR sequences drive the expression of luciferase and green fluorescent protein (GFP) reporter genes, our two laboratories are conducting a screen to identify new "hits" of therapeutic compounds that already have FDA approval.

     Single lead drugs, or more potent combinations, will be tested for their capacity to suppress APP translation by RNA targeting. In conducting these screens, great care has to be taken to ensure that lead compounds do not alter APLP-1 and APLP-2 and ferritin gene expression.
     This serves as a screening control designed to minimize side effects. New and powerful combinations of FDA-approved drugs will soon be available as therapy for AD patients to use at low doses. Their efficacy will be directed to suppress APP translation, an effect that subsequently would reduce neurotoxic production of A(3-peptide..

Phenserine improves cognitive performance

     Phenserine improves cognitive performance in rats and is undergoing phase II clinical trials as an acetylcholinesterase inhibitor.

     We found that phenserine imparts a double therapeutic action by targeting 5'UTR sequences in APP mRNA to suppress translation of the precursor, and reduces A(3-peptide secretion with a high degree of specificity. The finding that Df suppresses translational enhancement by 5'UTR sequences in APP mRNA is consistent with evidence for the presence of a modified IRE in the 5'UTR of APP mRNA.

    Neither Df or most anticholinesterases is considered to be completely desirable as agents for AD therapy. Desferrioxamine generates hypotension at high doses (Hallaway & Hedlund, 1992), and current anticholinesterases have been shown to be beneficial only for mild cases of AD over 1 year of usage with marked side effects to the liver .
       There is clearly a need to develop effective screens to generate more promising APP 5'UTR-directed drugs, such as phenserine, for future clinical us.

Phenserine

    Impairment of the cholinergic system is one of the most important clinical featured symptoms of AD. This process can be partially reversed by acetylcholinesterase inhibitors, including phenserine. Coincidentally, we have discovered that phenserine is a member of a class of preexisting medicinal compounds that also block APP translation by targeting the unique stemloop formed from 5'UTR sequences in APP mRNA.

       Indeed, studies of rats with forebrain cholinergic lesions have shown that phenserine decreases the level of APP production, unlike some other acetylcholinesterase inhibitors that increase the production of APP. Phenserine is an example of a small molecule (Mw = 487) that acts, at least in part, by blocking translation of APP mRNA through the regulation of the 5'UTR, to suppress APP synthesis, thus inhibiting A(3-peptide output (Dr. N. Greig, NIA, Baltimore, MD).

       We have tested the capacity of the lead compound phenserine (and other related RNA-targeting compounds) to block APP gene expression through 5'UTR sequences in APP mRNA. The iron chelator, Df, was used as a positive control, representing a small molecule that markedly suppressed APP mRNA translation via 5'UTR sequences (Molecular weight of Df = 656.8).

Desferrioxamine

     Desferrioxamine

    Autopsy samples from AD patients reveal elevated levels of iron, particularly in the neurons of the basal ganglia Iron is relevant to neurodegenerative pathogenesis in the brains of AD patients, as evidenced by their disrupted brain-iron distribution. Since we had discovered that the APP 5'UTR is a modified ironresponsive element, we decided to test whether iron chelation by Df can suppress translation conferred to a luciferase reporter by APP mRNA 5'UTR sequences. In the first step, a Luciferase-reporter construct, MS121, was prepared by inserting a PCR-generated APP-mRNA 5'UTR fragment.

    Neuroblastoma cells (SY5Y) were transfected with this APP-5'UTR-specific construct and cells were then exposed to Df (5uM), before preparation aof cell lysates and use of a luciferase assay to determine levels of reporter gene activity. As a negative control, neuroblastoma cells were transfected with a parental pGL-3 reporter plasmid. The results showed that the APP 5'UTR was clearly a target for the action of Df to suppress APP-5'UTR translational regulation.

     Desferrioxine was shown to be beneficial for Alzheimer's patients in one study. However, the use of this iron chelator is currently restricted to the treatment of patients with sickle cell disease, thalassemias, arid to counteract iron poisoning. We reasoned that Df would be a strong positive control to screen for new compounds, including anticholinesterase derivatives (phenserine), which might suppress APP mRNA translation without chelating intracellular iron.