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Town T, Zolton J, Shaffner R, Schnell B, Crescentini R, Wu Y, Zeng J, DelleDonne A, Obregon D, Tan J, Mullan M. p35/Cdk5 pathway mediates soluble amyloid-beta peptide-induced tau phosphorylation in vitro. J Neurosci Res. 2002 Aug 1;69(3):362-72. PubMed.
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This study is signficant because it demonstrates that soluble extracellular Aβ can cause cdk5 activation and tau hyperphosphorylation. Also, the effects of Aβ on differentiated neurons are easier to interpret because under these conditions, other cell cycle-associated kinases that might phosphorylate tau are markedly downregulated. I suppose the cells were stably transfected with p35. I am curious to know whether this transfection alone led to increases in tau phosphorylation, or whether the changes described were seen in the presence of Aβ alone. Also, what was the end result? Did tau hyperphosphorylation lead to neuronal death?
Roskamp institute
Reply to Vincent comment, from J. Tan, T. Town and M. Mullan. To answer the first question, yes, the p35 overexpressing N2a cells were stably transfected with the pUSEamp plasmid containing the p35 cDNA sequence, and maintained in G418 throughout the course of the experiments. Transfection with p35 vector alone did lead to a modest but significant increase in tau phosphorylation versus mock vector transfection as detected by mAbs AT8, AT270 and pAbs pS199 and pS396. When comparing mock-vector transfected N2a cells to the p35-overexpressing variety, p25 levels are much higher in the latter group, suggesting that increased p25 levels associated with p35 overexpression could be responsible for this effect. That having been stated, we only observed a dose-dependent effect (from 1 to 5 microM) of sAβ1-42 in the p35-overexpressing N2a cells, and not in the mock-transfected variety. Thus, it seems that overexpression of p35 sensitizes the system to the effects of sA β. In our hands, with these relatively low doses of A β in soluble form, we did not observe significant neurotoxicity. Li-Huei Tsai’s group has shown that treatment of primary cortical neurons with a higher dose of Aβ1-42 (10-20 microM) for up to 4 days leads to p25 accumulation and neurotoxicity, which is dependent on activation of the calpain pathway (Lee, et al.). As our experiments used soluble forms of A β at lower levels for 24 hours, this may account for our inability to detect neurotoxicity. —Michael Mullan
References:
Lee MS, Kwon YT, Li M, Peng J, Friedlander RM, Tsai LH. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature. 2000 May 18;405(6784):360-4. PubMed.
In response to the comment by Tan, et al.:
The use of primary rat neurons in Tsai's study and a differentiated human neuronal line in the present study may account for the differences in sensitivity to amyloid. However the finding that soluble extracellular A β modulates intracellular neuronal cdk5 activity is important. It may explain the formation of NFT at sites remote from plaques in the P301L transgenic mice for instance (Gotz, 2001; and Lewis 2001), and perhaps in AD as well. I wonder if it would not be worth testing this idea in vivo, for instance, by infusing soluble A β throughout the brain of a wildtype mouse, and determining whether cdk5 activity is similarly affected, and if there is a pattern of differential neuronal vulnerability. That would be exciting!
References:
Götz J, Chen F, van Dorpe J, Nitsch RM. Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science. 2001 Aug 24;293(5534):1491-5. PubMed.
Lewis J, Dickson DW, Lin WL, Chisholm L, Corral A, Jones G, Yen SH, Sahara N, Skipper L, Yager D, Eckman C, Hardy J, Hutton M, McGowan E. Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science. 2001 Aug 24;293(5534):1487-91. PubMed.
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