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Cdk5 Protein Inhibition and Aβ42 Increase BACE1 Protein Level in Primary Neurons by a Post-transcriptional Mechanism

IMPLICATIONS OF CDK5 AS A THERAPEUTIC TARGET FOR ALZHEIMER DISEASE*
  • Katherine R. Sadleir
    Affiliations
    Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
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  • Robert Vassar
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, 303 E. Chicago Ave., Chicago, IL 60611. Fax: 312-503-7912
    Affiliations
    Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants 5T32AG00026 and F32AG033445 (to K. R. S.) and R01AG030142 (to R. V.).
    This article contains supplemental Table 1 and Figs. 1 and 2.
Open AccessPublished:January 05, 2012DOI:https://doi.org/10.1074/jbc.M111.333914
      The β-secretase enzyme BACE1 initiates production of the amyloid-β (Aβ) peptide that comprises plaques in Alzheimer disease (AD) brain. BACE1 levels are increased in AD, potentially accelerating Aβ generation, but the mechanisms of BACE1 elevation are not fully understood. Cdk5/p25 has been implicated in neurodegeneration and BACE1 regulation, suggesting therapeutic Cdk5 inhibition for AD. In addition, caspase 3 has been implicated in BACE1 elevation. Here, we show that the Cdk5 level and p25:p35 ratio were elevated and correlated with BACE1 level in brains of AD patients and 5XFAD transgenic mice. Mouse primary cortical neurons treated with Aβ42 oligomers had increased BACE1 level and p25:p35 ratio. Surprisingly, the Aβ42-induced BACE1 elevation was not blocked by Cdk5 inhibitors CP68130 and roscovitine, and instead the BACE1 level was increased greater than with Aβ42 treatment alone. Moreover, Cdk5 inhibitors alone elevated BACE1 in a time- and dose-dependent manner that coincided with increased caspase 3 cleavage and decreased Cdk5 level. Caspase 3 inhibitor benzyloxycarbonyl-VAD failed to prevent the Aβ42-induced BACE1 increase. Further experiments suggested that the Aβ42-induced BACE1 elevation was the result of a post-transcriptional mechanism. We conclude that Aβ42 may increase the BACE1 level independently of either Cdk5 or caspase 3 and that Cdk5 inhibition for AD may cause BACE1 elevation, a potentially negative therapeutic outcome.

      Introduction

      The β-secretase, β-site APP
      The abbreviations used are:
      APP
      amyloid precursor protein
      AD
      Alzheimer disease
      amyloid-β
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      FAD
      familial AD
      Z
      benzyloxycarbonyl.
      -cleaving enzyme1 (BACE1), is the enzyme that cleaves APP to initiate the production of the β-amyloid (Aβ) peptide involved in Alzheimer disease (AD) (
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      ) suggest that increased activity of BACE1 may be sufficient to induce Alzheimer disease in humans. In addition, BACE1 levels are increased ∼1.5–3-fold in the brains of AD patients, suggesting that BACE1 elevation contributes to disease (
      • Fukumoto H.
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      β-Secretase protein and activity are increased in the neocortex in Alzheimer disease.
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      Phosphorylation of the translation initiation factor eIF2α increases BACE1 levels and promotes amyloidogenesis.
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      • Berry R.
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      β-Site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques. Implications for Alzheimer disease pathogenesis.
      ). These results imply that preventing the BACE1 increase may be beneficial for AD.
      Inhibition of BACE1 should effectively lower Aβ levels in AD, but therapeutically useful BACE1 inhibitors have proved challenging to design. Moreover, concerns have been raised over potential mechanism-based side effects of BACE1 inhibition, as suggested by phenotypes observed in BACE1 null mice, including memory and emotional deficits (
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      ,
      • Ohno M.
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      BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer disease.
      ,
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      Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null mice.
      ) and hypomyelination (
      • Hu X.
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      • He W.
      • Wong P.
      • Macklin W.B.
      • Trapp B.D.
      • Yan R.
      Bace1 modulates myelination in the central and peripheral nervous system.
      ,
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      ). Blocking the BACE1 increase observed in AD could be therapeutically useful in slowing or preventing AD, while at the same time allowing normal levels of BACE1 to perform BACE1 functions and thus minimize side effects. However, the mechanisms that regulate BACE1 levels in the brain are not fully understood.
      Using immunostaining on brain sections from AD patients and mouse models of AD, we have demonstrated that BACE1 levels are elevated in neurons surrounding amyloid plaques (
      • Zhao J.
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      • Shao P.
      • Hitt B.
      • O'Connor T.
      • Logan S.
      • Maus E.
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      β-Site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques. Implications for Alzheimer disease pathogenesis.
      ), a result that was recently confirmed by another group with a different anti-BACE1 antibody (
      • Zhang X.M.
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      β-Secretase-1 elevation in transgenic mouse models of Alzheimer disease is associated with synaptic/axonal pathology and amyloidogenesis. Implications for neuritic plaque development.
      ). These results suggest that Aβ42, the main constituent of amyloid plaques, may increase BACE1 levels in neurons near plaques. As such, a feed-forward mechanism may be initiated where BACE1 elevation increases Aβ42 generation, which further raises the BACE1 level.
      Recent evidence implicates the calcium/calpain/Cdk5 kinase signaling pathway in AD pathogenesis, suggesting it could be responsible for an Aβ42-induced BACE1 increase. Aβ42 increases intracellular calcium in neurons, which activates calpain to cleave the Cdk5 regulatory subunit, p35 to p25 (
      • Lee M.S.
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      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ). Overexpression of the p25 subunit causes cytoskeletal disruption, altered phosphorylation patterns, and neurotoxicity (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ). Mice with elevated cerebral Cdk5/p25 activity were reported to have increased BACE1 levels and Aβ production (
      • Cruz J.C.
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      ,
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      ,
      • Wen Y.
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      • Herman M.
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      • Lau L.F.
      • Lahiri D.K.
      • Duff K.
      Transcriptional regulation of β-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.
      ). Based on these data suggesting a connection between Cdk5 dysregulation and AD in humans and mice, Cdk5 inhibitors are being developed as potential AD therapeutics (
      • Wen Y.
      • Planel E.
      • Herman M.
      • Figueroa H.Y.
      • Wang L.
      • Liu L.
      • Lau L.F.
      • Yu W.H.
      • Duff K.E.
      Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3β mediated by neuregulin signaling leads to differential effects on Tau phosphorylation and amyloid precursor protein processing.
      ).
      Caspase 3 activation may also increase BACE1 levels. Levels of activated caspase 3 are elevated in AD brains (
      • Louneva N.
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      ,
      • Selznick L.A.
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      • Johnson Jr., E.M.
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      In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease.
      ,
      • Stadelmann C.
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      Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer disease. Evidence for apoptotic cell death.
      ,
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      • Cotman C.W.
      Activated caspase-3 expression in Alzheimer and aged control brain. Correlation with Alzheimer pathology.
      ). Caspase 3 cleavage of the adaptor protein GGA3, which may traffic BACE1 to the lysosome for degradation, appears to result in BACE1 accumulation in vitro and in vivo (
      • Tesco G.
      • Koh Y.H.
      • Kang E.L.
      • Cameron A.N.
      • Das S.
      • Sena-Esteves M.
      • Hiltunen M.
      • Yang S.H.
      • Zhong Z.
      • Shen Y.
      • Simpkins J.W.
      • Tanzi R.E.
      Depletion of GGA3 stabilizes BACE and enhances β-secretase activity.
      ). It may also play a role in AD, as the GGA3 level appears to be decreased in the brains of Alzheimer patients (
      • Tesco G.
      • Koh Y.H.
      • Kang E.L.
      • Cameron A.N.
      • Das S.
      • Sena-Esteves M.
      • Hiltunen M.
      • Yang S.H.
      • Zhong Z.
      • Shen Y.
      • Simpkins J.W.
      • Tanzi R.E.
      Depletion of GGA3 stabilizes BACE and enhances β-secretase activity.
      ).
      Here, we investigated the potential roles of Cdk5 and caspase 3 activation in Aβ42 oligomer-induced BACE1 elevation in mouse primary neuronal cultures. We show that Aβ42 oligomers increase the BACE1 level and p25:p35 ratio in primary neurons. Although p35/25-Cdk5 signaling may be increased in AD patients and in the 5XFAD mouse model of AD, inhibition of Cdk5 activity in primary neurons did not prevent the Aβ42-induced increase in BACE1 level nor did caspase inhibition. Our results have important implications for both the molecular mechanism of the Aβ42-induced BACE1 elevation and for therapeutic approaches involving Cdk5 inhibition for AD.

      DISCUSSION

      Studies from several groups have determined that BACE1 levels are elevated in AD brain and suggest that the BACE1 increase may play a role in AD pathogenesis. Therefore, understanding the molecular mechanism of the BACE1 elevation may provide new insights of therapeutic value for AD. Cdk5 has been implicated in neurodegeneration and AD, and our results here with human post-mortem and 5XFAD transgenic mouse brain samples (Fig. 1) provide additional data supporting an association of Cdk5 with AD (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      A survey of Cdk5 activator p35 and p25 levels in Alzheimer disease brains.
      ) and corroborate previous studies reporting elevated BACE1 levels and/or activity associated with AD (
      • Fukumoto H.
      • Cheung B.S.
      • Hyman B.T.
      • Irizarry M.C.
      β-Secretase protein and activity are increased in the neocortex in Alzheimer disease.
      ,
      • Holsinger R.M.
      • McLean C.A.
      • Beyreuther K.
      • Masters C.L.
      • Evin G.
      Increased expression of the amyloid precursor β-secretase in Alzheimer disease.
      ,
      • Li R.
      • Lindholm K.
      • Yang L.B.
      • Yue X.
      • Citron M.
      • Yan R.
      • Beach T.
      • Sue L.
      • Sabbagh M.
      • Cai H.
      • Wong P.
      • Price D.
      • Shen Y.
      Amyloid β peptide load is correlated with increased β-secretase activity in sporadic Alzheimer disease patients.
      ,
      • O'Connor T.
      • Sadleir K.R.
      • Maus E.
      • Velliquette R.A.
      • Zhao J.
      • Cole S.L.
      • Eimer W.A.
      • Hitt B.
      • Bembinster L.A.
      • Lammich S.
      • Lichtenthaler S.F.
      • Hébert S.S.
      • De Strooper B.
      • Haass C.
      • Bennett D.A.
      • Vassar R.
      Phosphorylation of the translation initiation factor eIF2α increases BACE1 levels and promotes amyloidogenesis.
      ,
      • Zhao J.
      • Fu Y.
      • Yasvoina M.
      • Shao P.
      • Hitt B.
      • O'Connor T.
      • Logan S.
      • Maus E.
      • Citron M.
      • Berry R.
      • Binder L.
      • Vassar R.
      β-Site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques. Implications for Alzheimer disease pathogenesis.
      ).
      Availability of an in vitro model to investigate Aβ42-induced BACE1 elevation in neurons would facilitate elucidation of the molecular mechanisms responsible for the BACE1 increase, which may represent novel therapeutic targets. This is the first study that oligomeric and fibrillar Aβ42 elevate BACE1 in murine primary cortical neurons. Both fibrillar and oligomeric Aβ42 raised BACE1 levels in primary neurons, although oligomeric Aβ42 had a more potent effect (Fig. 2). We used our primary neuron culture model to investigate the potential involvement of Cdk5 and caspase 3 in the Aβ42-induced BACE1 elevation, two molecules that have been previously implicated in regulation of BACE1 level and AD (
      • Wen Y.
      • Yu W.H.
      • Maloney B.
      • Bailey J.
      • Ma J.
      • Marié I.
      • Maurin T.
      • Wang L.
      • Figueroa H.
      • Herman M.
      • Krishnamurthy P.
      • Liu L.
      • Planel E.
      • Lau L.F.
      • Lahiri D.K.
      • Duff K.
      Transcriptional regulation of β-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.
      ,
      • Tesco G.
      • Koh Y.H.
      • Kang E.L.
      • Cameron A.N.
      • Das S.
      • Sena-Esteves M.
      • Hiltunen M.
      • Yang S.H.
      • Zhong Z.
      • Shen Y.
      • Simpkins J.W.
      • Tanzi R.E.
      Depletion of GGA3 stabilizes BACE and enhances β-secretase activity.
      ).
      Although we showed that levels of Cdk5 and its activator proteins p25 and p35 were dysregulated in post-mortem AD brain, 5XFAD transgenic mouse brain, and Aβ42-treated primary neurons, our results suggest that increased Cdk5 levels or activity may not be responsible for the Aβ42-induced BACE1 increase, at least in primary neuron culture. Treatment of primary neurons with the Cdk5 inhibitor CP681301 at its IC50 concentration of 0.5 μm3 decreased APP phosphorylation by ∼50% but did not attenuate the BACE1 increase (Fig. 4B). Unexpectedly, CP681301 appeared to potentiate the Aβ42-induced BACE1 increase in neurons and was able to elevate BACE1 levels when administered alone. CP681301 treatment also increased caspase 3 cleavage (Fig. 5B), suggesting it may induce apoptosis at certain doses. Results similar to those of CP681301 treatment were obtained with a different, well studied Cdk5 inhibitor, roscovitine (supplemental Fig. 1), demonstrating that the effects of CP681301 were likely caused by Cdk5 inhibition and were not the result of compound-specific side effects.
      In addition, inhibition of caspase 3 cleavage with ZVAD also failed to block the BACE1 increase in Aβ42-treated primary neurons (Fig. 6). Lower concentrations (1–2 μm) of Aβ42 elevated BACE1 levels in primary neurons without increasing caspase 3 cleavage (Fig. 7), suggesting a caspase 3-independent mechanism for the Aβ42-induced increase.
      Finally, although 10 μm oligomeric or fibrillar Aβ42 treatment both significantly increased BACE1 levels, these conditions had no effect on the BACE1 mRNA level in primary neurons (Fig. 8). Similarly, treatment of primary neurons with lower concentrations (1–2 μm) of oligomeric Aβ42 did not increase BACE1 mRNA levels (data not shown). In addition, the BACE1 increase appeared to require protein synthesis (supplemental Fig. 2). Taken together, our results suggest that neither Cdk5 nor caspase 3 is responsible for the Aβ42-induced BACE1 elevation in primary neurons, but they point toward a post-transcriptional mechanism, such as an increase in BACE1 mRNA translation. Recently, we have described a translational control mechanism of BACE1 that involves phosphorylation of the translation initiation factor eIF2α (
      • O'Connor T.
      • Sadleir K.R.
      • Maus E.
      • Velliquette R.A.
      • Zhao J.
      • Cole S.L.
      • Eimer W.A.
      • Hitt B.
      • Bembinster L.A.
      • Lammich S.
      • Lichtenthaler S.F.
      • Hébert S.S.
      • De Strooper B.
      • Haass C.
      • Bennett D.A.
      • Vassar R.
      Phosphorylation of the translation initiation factor eIF2α increases BACE1 levels and promotes amyloidogenesis.
      ). In future work it will be of interest to explore the potential role of eIF2α phosphorylation in the Aβ42-induced BACE1 elevation in our primary neuron model.
      We deliberately chose to use micromolar (1–10 μm) concentrations of Aβ42 to treat primary neuron cultures in this study. It has been suggested that such concentrations are much higher than those found in the brain, even in Alzheimer patients. This may be true for the concentration of Aβ42 when averaged over the entire brain. However, we were interested in modeling the effects of high concentrations of Aβ42 in the immediate vicinity of amyloid plaques. Previously, we reported that BACE1 elevation is observed in dystrophic neurites in close proximity to plaques in the brains of AD patients and two independent APP transgenic mouse strains (
      • Zhao J.
      • Fu Y.
      • Yasvoina M.
      • Shao P.
      • Hitt B.
      • O'Connor T.
      • Logan S.
      • Maus E.
      • Citron M.
      • Berry R.
      • Binder L.
      • Vassar R.
      β-Site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques. Implications for Alzheimer disease pathogenesis.
      ). A third APP transgenic strain also exhibited BACE1 elevation around plaques (
      • Sheng J.G.
      • Price D.L.
      • Koliatsos V.E.
      The β-amyloid-related proteins presenilin 1 and BACE1 are axonally transported to nerve terminals in the brain.
      ). Given that the BACE1 elevation correlates with amyloid plaques in APP transgenic and AD brains, it is likely that the major constituent of plaques, Aβ42, rather than other APP processing products, is responsible for the BACE1 increase. The 1–10 μm Aβ42 concentrations that we used in our experiments here produced increases of BACE1 levels in primary neuron cultures comparable with those observed in APP transgenic and AD brains. Clearly, a very high concentration of fibrillar Aβ42 exists in plaques, and evidence suggests that plaques are surrounded by halos of Aβ42 oligomers associated with decreased synaptic density (
      • Koffie R.M.
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      • Hashimoto T.
      • Adams K.W.
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      • Kim M.L.
      • Lee V.M.
      • Hyman B.T.
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      Oligomeric amyloid β associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques.
      ). This synaptic loss may be responsible for memory impairment in AD (
      • Lacor P.N.
      • Buniel M.C.
      • Furlow P.W.
      • Clemente A.S.
      • Velasco P.T.
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      Aβ oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer disease.
      ,
      • Shankar G.M.
      • Bloodgood B.L.
      • Townsend M.
      • Walsh D.M.
      • Selkoe D.J.
      • Sabatini B.L.
      Natural oligomers of the Alzheimer amyloid-β protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway.
      ) before frank neuronal loss, so understanding detrimental changes in synapses and neurites exposed to high levels of oligomeric and/or fibrillar Aβ42 near plaques may suggest novel therapeutic approaches to slow memory loss.
      Inhibition of caspase 3 cleavage with ZVAD blocks the BACE1 increase induced by the apoptotic agents staurosporine and etoposide in cultured human H4 neuroglioma cells (
      • Tesco G.
      • Koh Y.H.
      • Kang E.L.
      • Cameron A.N.
      • Das S.
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      • Hiltunen M.
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      • Zhong Z.
      • Shen Y.
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      • Tanzi R.E.
      Depletion of GGA3 stabilizes BACE and enhances β-secretase activity.
      ). The inability of ZVAD to inhibit the Aβ42-induced BACE1 elevation in murine primary neurons suggests that Aβ42 may elevate BACE1 through a mechanism independent of caspase 3 cleavage. Because staurosporine and etoposide increased BACE1 level 6–9-fold (
      • Tesco G.
      • Koh Y.H.
      • Kang E.L.
      • Cameron A.N.
      • Das S.
      • Sena-Esteves M.
      • Hiltunen M.
      • Yang S.H.
      • Zhong Z.
      • Shen Y.
      • Simpkins J.W.
      • Tanzi R.E.
      Depletion of GGA3 stabilizes BACE and enhances β-secretase activity.
      ), and Aβ42 elevated BACE1 ∼150–250%, it seems plausible that apoptotic agents and Aβ42 may increase the BACE1 levels by different mechanisms, at least in cell culture.
      We report that Cdk5 level is elevated in AD brain, although other publications have reported that Cdk5 levels appear unchanged (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Borghi R.
      • Giliberto L.
      • Assini A.
      • Delacourte A.
      • Perry G.
      • Smith M.A.
      • Strocchi P.
      • Zaccheo D.
      • Tabaton M.
      Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.
      ,
      • Tandon A.
      • Yu H.
      • Wang L.
      • Rogaeva E.
      • Sato C.
      • Chishti M.A.
      • Kawarai T.
      • Hasegawa H.
      • Chen F.
      • Davies P.
      • Fraser P.E.
      • Westaway D.
      • St George-Hyslop P.H.
      Brain levels of CDK5 activator p25 are not increased in Alzheimer or other neurodegenerative diseases with neurofibrillary tangles.
      ,
      • Taniguchi S.
      • Fujita Y.
      • Hayashi S.
      • Kakita A.
      • Takahashi H.
      • Murayama S.
      • Saido T.C.
      • Hisanaga S.
      • Iwatsubo T.
      • Hasegawa M.
      Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains.
      ). This may be related to the degree of neuronal loss in the AD brain samples we analyzed, as another group reported that when normalized to synaptophysin levels, which are decreased in AD, Cdk5 level was actually increased (
      • Jacobs E.H.
      • Williams R.J.
      • Francis P.T.
      Cyclin-dependent kinase 5, Munc18a, and Munc18-interacting protein 1/X11α protein up-regulation in Alzheimer disease.
      ). In 2-month-old 5XFAD mice, which may represent an early stage of the disease, both p35 and p25 levels were elevated equally and to the same degree as Cdk5, suggesting that the Cdk5-p35/25 system may be up-regulated early in AD pathogenesis. In post-mortem AD brains, which represent a late end-stage of the disease, we observed that although p35 was not elevated, the levels of p25 were slightly raised leading to an increased p25:p35 ratio, suggesting that increased cleavage of p35 may be associated with AD (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ).
      Whether the p25:p35 ratio is increased in the brains of AD patients remains controversial. Tsai and co-workers (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      A survey of Cdk5 activator p35 and p25 levels in Alzheimer disease brains.
      ) reported an elevated p25:p35 ratio in AD, although others observe no change in ratio (
      • Borghi R.
      • Giliberto L.
      • Assini A.
      • Delacourte A.
      • Perry G.
      • Smith M.A.
      • Strocchi P.
      • Zaccheo D.
      • Tabaton M.
      Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.
      ,
      • Nguyen K.C.
      • Rosales J.L.
      • Barboza M.
      • Lee K.Y.
      Controversies over p25 in Alzheimer disease.
      ,
      • Takashima A.
      • Murayama M.
      • Yasutake K.
      • Takahashi H.
      • Yokoyama M.
      • Ishiguro K.
      Involvement of cyclin-dependent kinase5 activator p25 on Tau phosphorylation in mouse brain.
      ,
      • Tandon A.
      • Yu H.
      • Wang L.
      • Rogaeva E.
      • Sato C.
      • Chishti M.A.
      • Kawarai T.
      • Hasegawa H.
      • Chen F.
      • Davies P.
      • Fraser P.E.
      • Westaway D.
      • St George-Hyslop P.H.
      Brain levels of CDK5 activator p25 are not increased in Alzheimer or other neurodegenerative diseases with neurofibrillary tangles.
      ,
      • Taniguchi S.
      • Fujita Y.
      • Hayashi S.
      • Kakita A.
      • Takahashi H.
      • Murayama S.
      • Saido T.C.
      • Hisanaga S.
      • Iwatsubo T.
      • Hasegawa M.
      Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains.
      ) or even a decrease (
      • Yoo B.C.
      • Lubec G.
      p25 protein in neurodegeneration.
      ). It has been shown in rat brain that p25 cleavage increases with post-mortem interval (
      • Taniguchi S.
      • Fujita Y.
      • Hayashi S.
      • Kakita A.
      • Takahashi H.
      • Murayama S.
      • Saido T.C.
      • Hisanaga S.
      • Iwatsubo T.
      • Hasegawa M.
      Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains.
      ), so it has been asserted that the post-mortem interval should be short to measure an accurate p25:p35 ratio (
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      A survey of Cdk5 activator p35 and p25 levels in Alzheimer disease brains.
      ,
      • Borghi R.
      • Giliberto L.
      • Assini A.
      • Delacourte A.
      • Perry G.
      • Smith M.A.
      • Strocchi P.
      • Zaccheo D.
      • Tabaton M.
      Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.
      ). Variability of control cases and sample handling also affect the accuracy of p25:p35 ratio measurements, as post-mortem ischemia and freeze-thaw cycles may lead to cleavage of p35 (
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      A survey of Cdk5 activator p35 and p25 levels in Alzheimer disease brains.
      ). Our AD and control groups had an average post-mortem interval of 6.5 h, ranging from 3.5 to 14.75 h (see supplemental Table 1). Only the studies by Tseng et al. (
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      A survey of Cdk5 activator p35 and p25 levels in Alzheimer disease brains.
      ), reporting an increase in p25:p35, and by Taniguchi et al. (
      • Taniguchi S.
      • Fujita Y.
      • Hayashi S.
      • Kakita A.
      • Takahashi H.
      • Murayama S.
      • Saido T.C.
      • Hisanaga S.
      • Iwatsubo T.
      • Hasegawa M.
      Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains.
      ), reporting no change, had shorter average post-mortem intervals than our own. Perhaps the relatively short post-mortem interval of our sample group, along with use of protease inhibitors and careful sample handling, allowed us to detect a small increase in p25:p35 ratio in the AD group, although other studies did not.
      We did not expect Cdk5 inhibition to elevate BACE1 in primary neuron cultures, as another group reported that the compound reduced BACE1 levels in the brains of treated mice (
      • Wen Y.
      • Yu W.H.
      • Maloney B.
      • Bailey J.
      • Ma J.
      • Marié I.
      • Maurin T.
      • Wang L.
      • Figueroa H.
      • Herman M.
      • Krishnamurthy P.
      • Liu L.
      • Planel E.
      • Lau L.F.
      • Lahiri D.K.
      • Duff K.
      Transcriptional regulation of β-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.
      ). A single injection of CP681301 reduced Stat3 phosphorylation and BACE1 levels within 4 h in 4–6-day-old wild type and p25 transgenic mice (
      • Wen Y.
      • Yu W.H.
      • Maloney B.
      • Bailey J.
      • Ma J.
      • Marié I.
      • Maurin T.
      • Wang L.
      • Figueroa H.
      • Herman M.
      • Krishnamurthy P.
      • Liu L.
      • Planel E.
      • Lau L.F.
      • Lahiri D.K.
      • Duff K.
      Transcriptional regulation of β-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.
      ). This coincides with a time of high BACE1 expression in the early post-natal mouse brain. BACE1 levels peak between P0 and P8 at 8–10 times the adult level and then decrease during postnatal weeks 2 and 3 (
      • Willem M.
      • Garratt A.N.
      • Novak B.
      • Citron M.
      • Kaufmann S.
      • Rittger A.
      • DeStrooper B.
      • Saftig P.
      • Birchmeier C.
      • Haass C.
      Control of peripheral nerve myelination by the β-secretase BACE1.
      ). It is possible that Cdk5 and Stat3 may regulate BACE1 transcription during this post-natal period of high BACE1 expression, although the BACE1 increase observed in AD, APP transgenic mice, and Aβ42 treated primary neurons may be controlled by another mechanism. CP681301 was able to decrease cerebral BACE1 levels in the brains of wild type adult mice (
      • Wen Y.
      • Yu W.H.
      • Maloney B.
      • Bailey J.
      • Ma J.
      • Marié I.
      • Maurin T.
      • Wang L.
      • Figueroa H.
      • Herman M.
      • Krishnamurthy P.
      • Liu L.
      • Planel E.
      • Lau L.F.
      • Lahiri D.K.
      • Duff K.
      Transcriptional regulation of β-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.
      ); however, the effect of the compound in a mouse model of AD has not yet been reported. In addition, chronic in vivo administration of CP681301 has not been addressed, which would be necessary for its validation as an AD therapeutic. Although we currently do not understand the mechanism of the BACE1 increase following CP681301 and roscovitine treatment of primary neurons, our results suggest that in vivo evaluation of Cdk5 inhibitors should include monitoring for BACE1 elevation and caspase 3 cleavage during chronic administration. Finally, because we have only tested the effects of CP681301 treatment on primary neurons in vitro, it is possible that the mechanisms of Aβ42-induced BACE1 elevation differ between neurons in culture versus neurons in the brain so that Cdk5 may still play a role in the AD-associated BACE1 increase in vivo.
      In conclusion, we present data demonstrating that Aβ42 oligomers elevate BACE1 levels in cultured primary neurons through a post-transcriptional mechanism. Although Cdk5 and p25/p35 are dysregulated in AD brains and in the 5XFAD mouse model of AD and have been shown to influence BACE1 levels in other contexts, our data suggest that Cdk5 may not be responsible for Aβ42-induced BACE1 elevation, at least not in primary neurons in vitro. In addition, although caspase 3 cleavage is triggered by oligomeric Aβ42 and has been demonstrated to increase BACE1 in other systems, caspase 3 cleavage does not appear to be required for Aβ42-induced BACE1 elevation in primary neurons. Although Cdk5 inhibitors are under investigation as potential AD therapeutics, our data suggest that Cdk5 inhibition may potentially increase levels of BACE1 in neurons exposed to Aβ42 oligomers. Because Cdk5 inhibitors and Aβ42 synergize to elevate BACE1 levels above each treatment alone, and given the high cerebral amyloid loads of AD patients, the possibility exists that therapeutic inhibition of Cdk5 for AD may exacerbate disease progression rather than reduce it.

      Acknowledgments

      We acknowledge Dr. David A. Bennett and the Rush Memory and Aging Project (National Institutes of Health Grant R01AG17917) for the generous gift of human brain samples, Dr. Lit-Fui Lau (Pfizer, Inc.) for the donation of the selective Cdk5 inhibitor CP681301, and the laboratories of Drs. Linda Van Eldik, D. Martin Watterson, and Mary Jo Ladu for Aβ42 preparation and helpful advice.

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