The knock-down of CDK5 reduces the levels of phosphorylated tau, the number of neurofibrillary tangles and blocks the rapid neuronal loss in the hippocampi of elder triple transgenic mice. Based on our results, we propose CDK5 RNAi-mediated silencing as a novel gene therapy strategy against the tau pathology associated with Alzheimer's disease.
Several studies report CDK5 inhibition by pharmacological inhibitors such as, olomoucine, roscovitine, purvalanol, flavopiridol, butyrolactone, indirubins, hymenialdisine, the paullones and the aloisines (Garrett and Fattaey, 1999
; Gray et al., 1999
; Meijer et al., 1999; Leost et al., 2000
; Sielecki et al., 2000
; Knockaert et al., 2002
; Mettey et al., 2003
). Interestingly, hymenialdisine inhibits in vivo
phosphorylation of tau at AD-specific sites in cultured insect cells (Meijer et al., 2000
). Alsterpaullone, an inhibitor for CDK5 and GSK-3β inhibits the in vivo
phosphorylation of tau at AD-specific sites in slices from mouse striatum (Leost et al., 2000
). A notable disadvantage to all these inhibitors is that these are competitive inhibitors at the ATP binding site, resulting in a lack of specificity among the many ATP-dependent kinases. However, the shRNAmiCDK5 (CDK5miR) offers specific inhibition of CDK5. This study shows that CDK5 is a valid therapeutic target. The previous chemical inhibitors lacked the specificity as compared to RNAi. Therefore this is a proof-of-concept study, which suggests that RNAi is a valid therapeutic approach but also suggests that developing more specific inhibitors of CDK5 is a worthy goal.
We observed that 3 weeks CDK5miR treatment decreased the phosphorylated tau in adult brain of wild type mice. However, the CDK5 protein levels displayed tendency to recover at 3 weeks of treatment compared to one week. This may indicate an endogenous feedback operating to maintain the CDK5 necessary for its normal function in the nervous tissue. In vivo
data of CDK5 kinetic showed normal activity after silencing of CDK5. We calculated a CDK5 KM
of 4 μM with histone as substrate. This value is comparable to the previously described KM
of 4 μM in vivo
tau (Jae Suk Ahn et al., 2004
) and KM
of 10 μM in vitro
for histone (Min Liu et al., 2008
). A 45–50% reduction in the histone phosphorylation rate with CDK5 obtained from cerebral tissue of CDK5miR treated mice was associated with a comparable reduction of CDK5 in the same tissues. Based on these data, we suggest that diminution of the amount of CDK5 available in the tissue regulates or normalizes the activity of CDK5 in the transgenic mice, resulting in amelioration of the tau pathology.
We also observed increased p35 levels in the brain of 3xTg-AD mice treated with CDK5miR. Mice that over-express human p35, CDK5, and tau display increased CDK5 activity without an evident increase in tau phosphorylation (Van den Haute et al., 2001
). These findings are consistent with our results and indicate that p35/CDK5 does not efficiently phosphorylate tau. However, recombinant p25/CDK5 phosphorylates tau with higher efficiency than p35/CDK5 in vitro
(Hashiguchi et al., 2002
). The 3xTg-AD mice treated with CDK5miR in our study had increased p35 and reduced CDK5. These circumstances may reduce p25-mediated CDK5 phosphorylation of tau. As a consequence a lower number of tangle bearing neurons were seen in treated mice, despite of their advanced age.
Recent studies show that p25/CDK5 deregulates histone deacetylase 1 (HCDAC1) activity inducing aberrant cell cycle and double-strand DNA breaks prior to neuronal death in CK-p25 mice (Kim et al., 2008
). Other reports showed that conditional knockout of CDK5 in the adult mouse brain improved performance in spatial learning tasks, enhanced hippocampal long-term potentiation, and decreased CDK5-induced NR2B (NMDA subunit) degradation by calpain (Hawasli et al. 2007
). Moreover, partial inhibition of CDK5 has been a proposed as therapeutic option (Camins et al., 2006
; Zheng et al 2005
Decreasing the availability of CDK5 could be a useful therapeutic approach. Excessive up-regulation of CDK5 by the truncated activators may contribute to neurodegeneration and alter the phosphorylation state of cytosolic and cytoskeletal proteins. Increased CDK5 activity has been implicated not only in Alzheimer's Disease (AD), but also in amyotrophic lateral sclerosis (ALS), Parkinson's disease, Niemann-Pick type C disease, ischemia and animal models with some of these conditions (Lee et al., 1999
; Patrick et al., 1999
; Nguyen et al., 2001
; Bu et al., 2002
; Lu et al., 2003
; Nguyen and Julien, 2003
; Smith et al., 2003
; Wang et al., 2003
). In summary, silencing CDK5 opens a novel gene therapy strategy to control the amount of CDK5 to reduce the tau pathology associated to Alzheimer's disease and other tauopathies.