Sirtuins are class III histone deacetylases (HDAC) whose activation slows age-associated disease in mice through transcriptional pathways closely resembling those induced by caloric restriction (see ARF related news story). This led many, including first author Kim Green, to presume that blocking the activity of sirtuins would make things worse for mice that harbor both hallmark AD pathologies and show memory impairments at four months of age.

Instead, Green and colleagues found striking cognitive improvement in eight-month-old 3xTg-AD mice that were given nicotinamide-spiked drinking water (200 mg/kg/d), as opposed to vehicle-treated, for four months. In tests of long-term, short-term, and hippocampal-dependent spatial memory, nicotinamide rescued 3xTg-AD mice to wild-type levels, and even boosted short-term memory in non-transgenic animals. Nicotinamide-treated 3xTg-AD mice fared better than vehicle-treated counterparts in a fear conditioning test requiring the amygdala—a brain area with prominent pathology in the triple transgenics. However, both groups did equally well on an object recognition task involving the cortex, a region largely spared of Aβ pathology. Consistent with their equally good performance on the cortex-dependent task, nicotinamide-treated 3xTg-AD mice had roughly similar brain Aβ levels as did control mice—measured by ELISA on detergent-soluble and -insoluble brain homogenates, and by immunohistochemical staining of hippocampus, amygdala, and cortex.

Given nicotinamide’s lack of effect on Aβ levels, the researchers figured the compound must be improving cognition through another mechanism. Analyzing protein extracts from whole brain samples of treated and control 3xTg-AD mice, they found a 20 percent reduction in steady-state levels of human tau in the nicotinamide-treated animals. They saw no differences at several tau sites typically phosphorylated in 3xTg-AD mice by eight months (Thr212/Ser214, Ser199/202, or Thr181), but a whopping 60 percent reduction in Thr231-phospho-tau in the nicotinamide group compared with vehicle. “It's incredibly dramatic,” Green told ARF. “This thing is just wiped from the brain very specifically.”

To address whether phosphorylation at Thr231 affects stability and accumulation of tau, the researchers transfected fibroblast cells with wild-type human tau or a phospho-mimic human tau (T231E) containing a glutamic acid substitution at the Thr231 site. Compared to cells overexpressing wild-type tau, the T231E-expressing cells had reduced steady-state levels of tau (yet similar myc-actin levels). In a filter retardation assay, accumulation of insoluble tau was seen for wild-type but not T231E mutant proteins. Phosphorylation at Thr231 “seems to target tau for degradation,” Green said. “We think that somehow the nicotinamide is facilitating that degradation in the AD brain.” Interestingly, another group recently showed that inhibition of another histone deacetylase, HDAC6, specifically reduces tau phosphorylation at the very same residue (Thr231) (Ding et al., 2008).

In the current study, nicotinamide did not affect levels of cyclin-dependent kinase 5 (Cdk5) or GSK3β, two kinases that mediate tau hyperphosphorylation in 3xTg-AD mice and human brain. The researchers did, however, find that nicotinamide treatment increased levels of Cdk5’s coactivator, p25, whose upregulation may be either pathological or physiological (see ARF related news story). Relative to vehicle-treated counterparts, nicotinamide-treated 3xTg-AD mice also had more than doubled levels of di-acetyl-α-tubulin, a protein associated with increased microtubule stability, and a greater than fourfold increase in microtubule-associated protein 2c (MAP2c), which shares 80 percent homology with tau in its microtubule-binding domain.

To address, albeit indirectly, whether nicotinamide’s effects are in fact being mediated by sirtuins, the scientists crossed homozygous 3xTg-AD mice with heterozygous SIRT1 knockouts (homozygous null mice are embryonic lethal). Total human tau levels in the brain did not differ between mice with one or two copies of SIRT1, but Thr231-phospho-tau levels were about 50 percent lower in the single-copy mice, suggesting that SIRT1 deletion and nicotinamide have similar effects on tau pathology. However, SIRT1 knockdown did not lead to increased levels of α-tubulin or p25, suggesting that these proteins are regulated by other nicotinamide targets.

Considering recent reports of SIRT2 inhibitors that can prevent α-synuclein toxicity in cells and flies (see Outeiro et al., 2007 and ARF related news story) and suppress pathogenesis in a fly model of Huntington disease (Pallos et al., 2008), the new data might not seem so surprising. Though activating sirtuins is beneficial earlier in life, Green said, when it comes to age-related neurodegeneration, inhibiting brain sirtuins may activate “a protective mechanism against a whole bunch of different cytosolic aggregating proteins.”

Nailing down the role of sirtuins in AD could still prove tricky, though. In an ICAD 2008 poster (see preliminary abstract), researchers led by Frederic Calon at Laval University Medical Center, Quebec, Canada, report decreased SIRT1 protein levels in parietal cortex of AD patients but not of patients with mild cognitive impairment (MCI). They also found no change in SIRT1 levels in the cortex of 3xTg-AD mice at 12, 16, and 20 months of age. These data suggest that loss of SIRT1 occurs at relatively late stages of AD, Calon told ARF. The full paper is currently in press in the Journal of Neuropathology and Experimental Neurology.

Based on the nicotinamide data in mice, LaFerla’s group is recruiting mild to moderate AD patients for a six-month clinical trial. Of the 50 patients, half will receive placebo pills, the other half 1,500 mg of nicotinamide twice a day. This dose is the human equivalent of the 200 mg/kg per day received by the 3xTg-AD mice, Green said. A standard multivitamin contains just tens of milligrams of niacin, which seemed to protect against AD and age-related cognitive decline in a prospective study (Morris et al., 2004). The primary outcome measure in the new trial will be the Alzheimer Disease Assessment Scale-Cognitive (ADAS-Cog), which will be administered to participants at six-week intervals. In addition, the researchers will perform spinal taps at the beginning and end of the study to measure CSF phospho-tau levels. They will also monitor liver function to see if the high doses of nicotinamide are safe.—Esther Landhuis.

Reference:
Green KN, Steffan JS, Martinez-Coria H, SunX, Schreiber SS, Thompson LM, LaFerla FM. Nicotinamide Restores Cognition in Alzheimer’s Disease Transgenic Mice Via a Mechanism Involving Sirtuin Inhibition and Selective Reduction of Thr231-Phosphotau. 5 Nov 2008;28(45):11500-11510. Abstract