9 November 2008. Good nutrition may not be the first thing on our minds in the candy-heavy week after Halloween, but papers in the current issue of the Journal of Neuroscience will help to refocus attention on two vitamins and their relation to Alzheimer disease. The first report proposes a mechanism by which folate deficiency, a risk factor for AD, can elevate tau phosphorylation and harm neurons. The results, from Estelle Sontag and colleagues at the University of Texas Southwestern Medical Center in Dallas, indicate that defective protein methylation in folate-starved cells destabilizes the major tau protein phosphatase (PP2A), leading to enhanced tau phosphorylation. Overexpression of a PP2A methyltransferase restores phosphatase activity and reverses both tau hyperphosphorylation and cell toxicity. Taken together with similar in vivo data, the results suggest that PP2A is a prime target of folate deficiency in neurons, and its methylation could be a site of intervention in folate-related disorders and tauopathies.
The second paper focuses on the vitamin A metabolite retinoic acid, a well-known inducer of cell differentiation. Guo-Huang Fan and colleagues at the Virginia Commonwealth University School of Medicine in Richmond treated AD mice with the compound, and found that it could reduce Aβ deposition and improve cognitive function. “People have written reviews and suggested that retinoic acid might be useful in treatment of AD, but there was no evidence—we are the first to report data in an animal model,” Fan told ARF. The compound Fan and colleagues tested, all-trans retinoic acid, is already used to treat cancer, and the researchers hope they can quickly move the compound into trials for the treatment or prevention of AD.
Folic acid and its companion vitamin B12 are necessary for proper “one carbon” metabolism through pathways that shuttle methyl groups on and off DNA, lipids, and proteins. Low folate and low B12 are risk factors for AD and are linked to high homocysteine, itself a risk factor for cardiovascular disease and AD (see ARF related news story). In spite of much evidence in favor of the idea that folate and vitamin B12 protect against AD, a recent trial showed no effect of 18 months of supplements on people with mild to moderate AD (see ARF related news story).
Those results underscore the idea that the interactions among folate, B12, homocysteine, diet, and cognition are exceedingly complex, and not clear even now, after 150 years of study (for a thorough review see McCaddon, 2006). The new data from Sontag and colleagues helps to explain how changes in methylation of a specific protein lead to one of the hallmarks of folate deprivation, enhanced tau phosphorylation.
Using the N2a neuronal cell line, first author Jean-Marie Sontag and colleagues began by looking at methylation of the major tau phosphatase, PP2A. The catalytic subunit of PP2A requires methylation by the leucine carboxyl methyltransferase-1 (LCMT-1) enzyme before it can assemble into an active holoenzyme complex of A, Bα and C subunits. In folate-deficient cells, the investigators find, LCMT-1 is downregulated, demethylated PP2A C accumulates, the unassembled Bα subunit is destabilized, and tau phosphorylation increases. Overexpression of LCMT-1 or the Bα subunit reverses tau phosphorylation and renders the cells more resistant to cell death induced by folate deprivation. Knocking down LCMT-1, on the other hand, made cells more sensitive to folate deprivation. The de novo methylation of PP2A by LCMT-1 seemed to play a unique role in the cells, because its effects were not replicated when demethylation was prevented by knocking down the PP2A methylesterase PME-1.
To ask if similar changes in PP2A regulation occurred in vivo, the researchers examined mice that had been folate-deprived for eight weeks to lower serum folate and raise homocysteine. The mice had region-specific decreases in the S-adenosylmethionine/S-adenosylhomocysteine ratios in the brain, indicative of cellular folate deprivation. In the cortex and cerebellum, PP2A methylation was reduced by 75-80 percent, and both LCMT-1 and Bα proteins were downregulated. In regions where Bα was downregulated, significant increases in tau phosphorylation at the PHF-1 and Ser422 epitopes were observed.
The authors conclude that LCMT-1 appears to be a critical intermediate in the role of folate in the CNS. “Our cellular and mouse data reinforce the strong connection between LCMT-1-dependent ABαC holoenzyme formation/stabilization and tau regulation,” they write. They further propose that offsetting the neuronal loss of LCMT-1 or Bα could be a valuable therapeutic approach for tauopathies and folate-dependent CNS disorders.
As a cancer researcher, Guo-Huang Fan was struck by reports of cell cycle reactivation in neurons in the AD brain (e.g., see ARF related news story). The idea that postmitotic neurons try to proliferate and instead die by apoptosis made him ask whether drugs that promote differentiation and prevent proliferation might stop or slow the death of neurons in AD. Retinoic acid, the active metabolite of vitamin A, is a classical differentiation-inducing factor used to treat cancers. It occurs as cis and trans isomers, and Fan says they chose all-trans retinoic acid (ATRA) because it had a better clinical effect as a cancer treatment.
When first author Yun Ding and colleagues treated five-month-old APP/PS1 double transgenic mice (B6C3-Tg) for eight weeks with ATRA, they found significantly reduced Aβ deposition in the cortex and the hippocampus. The reduction in Aβ appeared to be due to a decrease in amyloid precursor protein (APP) processing, with no change in expression. ATRA treatment caused a reduction in APP phosphorylation, which could have affected processing. ATRA also decreased tau phosphorylation, and the compound appeared to decrease the activity of the APP and tau kinase, cyclin-dependent kinase 5 (Cdk5), though there was little effect on another tau kinase, GSK3β. Treated mice showed less inflammatory activation of microglia and astrocytes, and staining for neuronal markers suggested that treatment preserved synapses and neuronal size in the transgenic mice. Finally, the researchers showed the treated mice performed better in the Morris water maze test of learning and memory.
Fan and colleagues are keen to translate their animal results into human trials as soon as possible, although no experiments are planned yet. In addition, Fan noted, almost all differentiation-inducing factors used in cancer treatment prevent the activation of Cdk5 and GSK3β, suggesting that more of these drugs may be candidates for treating AD. His lab is now testing other such compounds, and he says they will have more results coming out soon.—Pat McCaffrey.
Sontag J-M, Nunbhakdi-Craig C, Montgomery L, Arning E, Bottiglieri T, Sontag E. Folate deficiency induces in vitro and mouse brain region-specific downregulation of leucine carboxyl methyltransferase-1 and protein phosphatase 2A Balpha subunit expression that correlate with enhanced tau phosphorylation. J. Neurosci. 2008 Nov 5;28(45):11477-11487. Abstract
Ding Y, Qiao A, Wang Z, Goodwin JS, Lee E-S, Block ML, Allsbrook M, McDonald MP, Fan G-H. Retinoic acid attenuates beta-amyloid deposition and rescues memory deficits in an Alzheimer’s disease transgenic mouse model. J. Neurosci. 2008 Nov 5;28(45):11622-11634. Abstract