Although RA has been suggested as a potential therapeutic approach to prevent or decrease Aβ-associated neurodegeneration (Goodman and Pardee, 2003
; Goodman, 2006
; Maden, 2007
), the actual therapeutic role of RA in AD pathology and dementia has not yet been ascertained. Our findings indicate that ATRA treatment, for as little as 8 weeks, inhibits and possibly reverses accumulation of Aβ deposits and tau hyperphosphorylation in APP/PS1 double-transgenic mice. The ATRA-treated APP/PS1 mice showed significantly decreased levels of activated glial markers, elevated levels of neuronal markers in cortical and/or hippocampal regions, and improved spatial learning and memory, when compared with the vehicle-treated APP/PS1 mice.
The inhibitory effect of ATRA on Aβ accumulation is likely attributable to its inhibition of APP processing, because the production of APP–CTFs, the direct precursor of Aβ (Evin et al., 2003
), was attenuated by the ATRA treatment. In addition, a previous study has shown that ATRA prevents formation of fibrillar Aβ from fresh Aβ (Ono et al., 2004
), suggesting that ATRA is involved in multiple steps of Aβ deposition. APP processing can be modulated by different mechanisms, including but not limited to an altered APP expression as well as expression/function of BACE1, a major β-secretase involved in APP processing. However, we did not observe a significant difference in the expression of APP or BACE1 between the groups. This is in contrast with a previous report showing that ATRA reversed the downregulation of APP, BACE1, and APP–CTFs in the brain of rats deprived of vitamin A (Husson et al., 2006
). This discrepancy suggests that RA differentially influences APP expression under diverse conditions.
It has been shown that Thr668 phosphorylation facilitates the β-secretase cleavage of APP and increases Aβ generation (Lee et al., 2003
). Based on the observation that ATRA-treatment reversed the elevation of APP phosphorylation in APP/PS1 mice, we postulate that ATRA may prevent APP processing by inhibiting its phosphorylation. Among the several protein kinases phosphorylating APP at Thr668 in vitro
or in vivo
(Suzuki et al., 1994
; Iijima et al., 2000
; Standen et al., 2001
), CDK5 is believed to be a key kinase responsible for APP phosphorylation in neuronal cells (Iijima et al., 2000
; Liu et al., 2003
; Wen et al., 2008a
), compatible with our result showing a concomitant downregulation of CDK5 activity by ATRA treatment in the APP/PS1 transgenic mice. However, we cannot exclude the possible involvement of other pathways modulated by CDK5 in the inhibitory effect of ATRA on Aβ accumulation. For instance, p25 overexpression results in enhanced forebrain Aβ levels, likely attributable to axonal transport dysfunction (Stokin et al., 2005
; Cruz et al., 2006
). Based on the observation that ATRA treatment reduced the levels of p35, we propose that ATRA attenuates Aβ accumulation via regulating axonal transport of Aβ. In addition, p25/CDK5 has been shown to participate in transcriptional regulation of BACE1, leading to enhanced amyloidogenic processing (Wen et al., 2008b
). Unexpectedly, ATRA treatment did not affect BACE1 expression, albeit p35/CDK5 was downregulated. This discrepancy may be attributable to different animal models used.
Another interesting finding of the present study is the significant inhibition of tau hyperphosphorylation by the ATRA treatment. Although both CDK5 and GSK3β are believed to be the most important kinases that regulate tau phosphorylation in the brain (Lovestone and Reynolds, 1997
), our results demonstrated that CDK5, rather than GSK3β, was predominantly inhibited by ATRA, suggesting that ATRA attenuates tau phosphorylation primarily through the inhibition of CDK5. Compatible with this result, we observed that CDK5 phosphorylation sites were more susceptible to the ATRA treatment than GSK3β sites on tau. For instance, among the several phosphorylation sites tested, e.g., Ser235, Ser396, Ser404, Ser519, and Thr205, the phosphorylation of tau at Ser396, which is catalyzed by GSK3β rather than CDK5 (Li and Paudel, 2006
; Wang et al., 2007
), was attenuated to a less extent by the ATRA treatment than other phosphorylation sites. The mechanisms behind the inhibitory role of ATRA in CDK5 activity are largely unknown. In addition to a possible direct influence on CDK5 activation, ATRA may inhibit CDK5 through stabilizing APP. Because APP has been shown to reciprocally regulate CDK5 activity (Han et al., 2005
), ATRA-induced inhibition of APP processing observed in APP/PS1 mice may cause an enhanced stability of APP, thereby resulting in indirect inhibition of CDK5 activity and attenuation of tau phosphorylation.
Given the central role of fibrillar Aβ in the activation of microglia and astrocytes seen in AD brain (Rozemuller et al., 2005
) and in AD animal models (Frautschy et al., 1998
; Apelt and Schliebs, 2001
; Matsuoka et al., 2001
), the significant decrease in activated microglia and astrocytes seen in the ATRA-treated APP/PS1 mice can be attributed to its inhibition of Aβ accumulation. However, ATRA appears to possess an inherent anti-inflammatory function independent of Aβ (Mehta et al., 1994
; Datta et al., 2001
). Although the underlying mechanisms remain largely unclear, ATRA-mediated inhibition of nuclear factor-κB may play a role in this process (Choi et al., 2005
; Dheen et al., 2005
). Nevertheless, because brain inflammation is a risk factor for neurodegenerative disease, the anti-inflammatory effect of ATRA in the AD model mouse provides additional evidence for its therapeutic potential for AD.
We observed that ATRA treatment of the APP/PS1 mice significantly attenuated impairment of neuronal integrity compared with the vehicle treatment. SYN, a protein localized in the neuronal synaptic vesicles, has been shown to be decreased in the AD brain and correlated with the severity of cognitive deficits (Terry et al., 1991
; Masliah et al., 1993
). However, in transgenic APP mouse models, SYN is either reduced or unchanged (Irizarry et al., 1997
; Hsia et al., 1999
), likely attributable to different levels of transgenic APP and different stages of the neurodegenerative process. In this study, a significant decrease in SYN immunoreactivity was observed in the stratum lucidum of the CA3 area in the brains of the vehicle-treated APP/PS1 mice compared with the vehicle-treated wild-type mice, and a significant reversal of this decrease was observed in the ATRA-treated APP/PS1 mice. ATRA-mediated prevention of synaptic loss in the stratum lucidum of the CA3 area, in which the mossy fibers from the dentate gyrus synapse with the dendrites of the pyramidal neurons, may play a key role in rescuing deficits of learning and memory, because alterations in the distribution of mossy fibers are related to neuronal plasticity and long-term memory (Cremer et al., 1998
; Ramirez-Amaya et al., 2001
In support of the results with SYN, ATRA-treated APP/PS1 mice showed a similar rescue of loss of immunoreactivity of MAP2, a marker for neuronal cell body and dendrites, indicating that the impaired neuronal integrity observed in the control APP/PS1 mice was improved by ATRA treatment. In the brains of APP/PS1 mice, a discrete neuronal loss is associated with Aβ plaques (Calhoun et al., 1998
). Consistent with this result, we observed degenerative neurons surrounding the Aβ plaques in the APP/PS1 mice treated with vehicle. In the ATRA-treated APP/PS1 mice, Aβ deposits were significantly smaller, and, correspondingly, the extent of neuronal loss was much lower compared with the vehicle-treated APP/PS1 mice. The neuroprotective effect of ATRA seen in APP/PS1 mice is in line with a previous report showing protection against Aβ-induced injury of primary hippocampal neuronal cultures (Sahin et al., 2005
We demonstrate that ATRA treatment of APP/PS1 transgenic mice reverses cognitive deficits. As reported, excessive Aβ accumulation is associated with disturbed cognitive function in an AD mouse model (Chen et al., 2000
), and hyperphosphorylated tau leads to memory deficits and loss of functional synapses in a transgenic mouse model (Schindowski et al., 2006
). The beneficial effect of ATRA on cognitive improvement in APP/PS1 mice is likely attributable to the combined effects of decreased levels of toxic Aβ peptides, tau hyperphosphorylation, and neurodegeneration. However, we cannot exclude the possibility that ATRA improves the learning and memory in a manner independent of decreasing Aβ accumulation and tau hyperphosphorylation, because a previous study has shown that RA treatment of naturally aged mice alleviated age-related deficits in the CA1 LTP and completely alleviated their memory deficits (Etchamendy et al., 2001
). The mechanism by which ATRA regulates spatial memory has not been delineated. The cholinergic (ACh) system is a potential target of retinoids, because RA increases the levels of choline acetyltransferase (ChAT) (Berse and Blusztajn, 1995
), the enzyme that synthesizes ACh. Because the loss of ChAT-expressing neurons is characteristic of AD (Whitehouse et al., 1982
), and because ATRA overcomes the reduction in ChAT induced by Aβ peptides (Sahin et al., 2005
), it is possible that ATRA may act as a neuroprotective agent in AD by restoring ChAT levels.
Together, the present study provides evidence that ATRA is able to attenuate Aβ-associated neuropathology and memory deficits in a APP/PS1 transgenic AD mouse model. ATRA is a small molecule that readily enters tissues and is concentrated in the brain compartments when administrated systemically (Kurlandsky et al., 1995
; Le Doze et al., 2000
). As an existing U.S. Pharmacopoeia drug, its toxicology profile has been well established, so the initiation of clinical trials could be accelerated.