BACKGROUND
hyperphosphorylation of tau, which leads to the formation of neurofibrillary tangles, is one of the two hallmarks of alzheimer disease
<cit> .
tau phosphorylation is mainly regulated by the balance of kinases/phosphatases.
several kinases are involved in tau phosphorylation including the glycogen synthase kinase 3β , map kinase , neuronal cdc2-like kinase  and cyclin dependent kinase  <dig>
<cit> .
in contrast, few phosphatases dephosphorylate tau.
phosphatase 2a , a serine/threonine phosphatase, is the most effective.
pp2a is a trimeric protein with a heterodimeric core enzyme consisting of a catalytic c subunit , and a scaffolding a subunit.
this core enzyme associates with a variable regulatory subunit to form a heterotrimeric holoenzyme
<cit> .
pp2a shows efficient enzymatic activity when it is methylated at leu-309
<cit>  and unphosphorylated at tyr-307
<cit> .
in the brain of ad patients, the activity of pp2a is compromised
<cit> .
the decrease of pp2a methylation observed in brains of ad patients
<cit>  may explain the decrease of pp2a activity and contribute to tau hyperphosphorylation
<cit> .
the activity of pp2a can be inhibited by two proteins: i1pp2a and i2pp2a, which is also called set, taf1β, or phapii
<cit> .
set is a multifunctional protein: it inhibits the acetylation of histones
<cit> , blocks dnase activity
<cit> , acts as a transcription factor
<cit> , and promotes tau phosphorylation by activating cdk5
<cit> , and inhibiting pp2a
<cit> .
in the brains of ad patients, there is an increase of set expression
<cit> .
interestingly, set is also translocated from the nucleus to the cytoplasm in the hippocampus and the temporal cortex of ad patients
<cit> .
we observed that in the ca <dig> of ad patients, the translocation of set to the cytoplasm was associated with an increase of amyloid precursor protein  cleaved in its cytoplasmic domain by a caspase
<cit> .
in in vitro models, cytoplasmic set is associated with neuronal death
<cit>  and with tau hyperphosphorylation
<cit> .
the 39 kda full-length set can be selectively cleaved resulting in a ~ 20 kda fragment in the cytosol of neurons in the brain
<cit> .
the cleavage of set protein has also been observed in primary neurons treated with kainate and in a mouse model of stroke
<cit> .
this cleavage results from the activation of an asparaginyl endopeptidase  which cuts set at asparagine asn- <dig>  generating ntf and ctf fragments and triggering dna nicking and cell death
<cit> .
both ntf and ctf are able to bind to the catalytic subunit of pp2a  inhibiting its activity and leading to tau hyperphosphorylation
<cit> .
however, it is not clear whether the cytoplasmic localization of set is always associated with its cleavage, with its over-expression, and with tau hyperphosphorylation.
it is still not clear how cytoplasmic set contributes to pp2a loss of function leading to tau hyperphosphorylation, and whether the presence of set in the cytoplasm induces low levels of methylated pp2a.
we used two models to clarify the relationship between cytoplasmic set, methylated pp2a, pp2a activity and tau hyperphosphorylation.
the first model involved the translocation of endogenous set from the nucleus to the cytoplasm in primary neurons or brain slices from wild type mice .
this translocation was induced in this model by the internalization of the jcasp peptide.
indeed, this peptide mimics the unmasked juxtamembrane cytoplasmic domain arising from the cleavage of app by caspases, which is increased in the brains of ad patients
<cit> .
moreover, this peptide is sufficient to induce in vivo both translocation of endogenous set, as occurs in the ca <dig> of wt mice following appcc overexpression, and neurodegeneration
<cit> .
the second model involved the over-expression of set by the internalization of exogenous recombinant full-length protein in brain slices from wt mice
<cit> .
in these two models, we report that cytoplasmic set induces the hyperphosphorylation of tau in the absence of detectable cleaved forms of set.
we also show that the interaction of set with pp2a impairs the methylation of pp2a and that the level of methylated pp2a that is associated with the translocation of set is also negatively correlated with the hyperphosphorylation of tau at ser- <dig>  but not at ser- <dig>  suggesting that the hyperphosphorylation of tau is regulated by different mechanisms at distinct residues.
RESULTS
internalization of jcasp peptide induces the translocation of endogenous nuclear set to the cytoplasm without cleavage or upregulation of its expression
we previously reported that the cytoplasmic internalization of the jcasp peptide by primary neurons resulted in the translocation of endogenous set to the cytoplasm and triggered pro-apoptotic signals at the cell membrane
<cit> .
thus, in this model, set promotes apoptosis, and its translocation to the cytoplasm appears to participate in the active neurodegenerative process
<cit> .
in this model, we also previously showed that the jcasp peptide fused to the penetratin vector is quickly internalized, and that endogenous set starts to move from the nucleus to the cytoplasm after 3 h, while apoptosis was observed 24 h later
<cit> .
here, we show that translocated set is still present in the cytoplasm of the cellular body and in neurites 5 h 30 min after jcasp peptide internalization .
sub-cellular fractionation and western blotting revealed the presence of a 39 kda band corresponding to endogenous set in the cytoplasm fraction of cells treated with jcasp peptide .
the proportion of translocated set was 39% of total endogenous set , which was in accordance with observations made by epifluorescence microscopy .
cleavage products of set in the nucleus and in the cytoplasm were not detected .
real-time pcr showed that the level of set transcripts was unchanged during the 5 h 30 min of jcasp peptide internalization .
hence, in this model, the translocation of set from the nucleus to the cytoplasm does not require its over-expression and/or its cleavage.
over-expression of recombinant cytoplasmic set in primary mouse neurons and mouse brain slices does not induce its cleavage
we introduced a recombinant ha-tagged set protein fused to the penetratin vector into the cytoplasm of primary neurons by direct internalization as previously described
<cit> .
we detected the 49 kda recombinant set protein by immunocytochemistry with an anti-ha antibody and analyzed its distribution by confocal microscopy.
after 3 h of internalization, the recombinant set protein, which possesses a nuclear targeting sequence, was mostly concentrated in the cell body around the nucleus and had started to move inside the nucleus .
after 5 h 30 min of internalization, the nucleus was enriched with the recombinant set protein, although recombinant set was still abundantly present in the cytoplasm .
we confirmed these observations with mouse brain slices that were treated with the recombinant set protein.
sub-cellular fractionation followed by western blotting with an anti-ha antibody revealed the presence of recombinant set in the cytoplasm .
the detected bands of low molecular weight and low intensity were unspecific bands resulting from the anti-rabbit secondary antibody .
we did not observe cleaved recombinant set protein in either sub-cellular compartment studied .
interaction between uncleaved cytoplasmic recombinant set protein and pp2a inhibits the activity of pp2a and impairs its methylation in the brain slices of adult mice
following its translocation to the cytoplasm, endogenous set interacts with the catalytic subunit of pp2a inducing its inhibition
<cit> .
we used direct internalization for 5 h 30 min to overexpress recombinant set protein  in adult mouse brain slices maintained under oxygenation conditions, as described in methods.
we first analyzed total phosphatase activity .
we used okadaic acid , a marine sponge toxin which is an inhibitor of pp2a, as a positive control
<cit> .
oa inhibited total phosphatase activity by  <dig> % ±  <dig> %  and recombinant set inhibited total phosphatase activity by  <dig> % ±  <dig> % .
hence, phosphatase activity was significantly impaired in both cases and was  <dig> % ±  <dig> % of the baseline value  for oa , and was  <dig> % ±  <dig> % for recombinant set  .
we then evaluated pp2a activity  after pp2a immunoprecipitation in brain slices treated with oa and in those containing internalized recombinant set protein.
the remaining pp2a activity was  <dig> % ±  <dig> %  in brain slices treated with oa and 84% ±  <dig> % in brain slices containing recombinant set  .
these values fit with what we observed for total phosphatase activity.
thus, these data suggest that when set is in the cytoplasm, it specifically impairs pp2a activity in a manner that does not depend on its cleavage .
the activity of pp2a depends on its methylation
<cit> .
therefore, we assessed whether the interaction between cytoplasmic recombinant set protein and endogenous pp2a impaired the methylation of pp2a, resulting in a decrease in its activity.
we used an antibody directed against total pp2a  and an antibody specifically directed against methyl-pp2a, and evaluated the ratio of methyl-pp2a to total pp2a by western blotting .
after 5 h 30 min of recombinant set protein internalization, the ratio of methyl-pp2a to total pp2a was  <dig> % ±  <dig> %  of the control ratio  in the absence of recombinant set .
the mean decrease of this ratio  was significant  suggesting that the presence of set in the cytoplasm in the absence of detectable set cleavage impairs the methylation of pp2a.
finally, we performed co-immunoprecipitation experiments to check that the internalized recombinant set protein interacted with pp2a and impaired the methylation of pp2a.
to this end, we internalized the recombinant set protein in brain slices for 5 h 30 min, and we extracted proteins from these slices and from control slices not internalized with recombinant set.
after lysis, we checked first the total level of pp2a  and methyl-pp2a  in brain slices by western blotting.
we subsequently immunoprecipitated set from slice extracts with an antibody that recognizes both endogenous and recombinant set.
we then analyzed the immunoprecipitates by western blotting with an antibody that recognizes total pp2a  and after membrane stripping, with an antibody that specifically recognizes methyl-pp2a .
only a small amount of pp2a co-immunoprecipitated with the set antibody  in extracts from brain slices lacking recombinant set, because only a small proportion of endogenous set is cytoplasmic.
more pp2a co-immunoprecipitated with set in cells containing recombinant set protein than in cells lacking the recombinant protein , showing that pp2a can bind to both recombinant and endogenous set protein.
in contrast, more methyl-pp2a co-immunoprecipitated with set in the control  than in cells containing recombinant set protein .
the mean ratio of methyl-pp2a to total pp2a was  <dig>  ±  <dig>   for the control  and  <dig>  ±  <dig>  for brain slices treated for 5 h 30 min with recombinant set protein .
thus, the level of co-immunoprecipitated methylated pp2a was significantly lower in slices internalized with set protein than in the control  .
these data confirm that cytoplasmic, uncleaved, recombinant set protein interacts with pp2a and impairs its methylation.
uncleaved set in the cytoplasm induces hyper- and abnormal phosphorylation of tau protein.
correlation with decreased methyl-pp2a levels
many phosphorylation sites of tau in paired helical filaments  have been identified in the brains of ad patients
<cit> .
inhibition of pp2a leads to the preferential hyper-phosphorylation of tau at various epitopes.
phosphorylation at ser- <dig> and ser- <dig> is more prevalent in phf-tau and fetal tau than in normal adult tau
<cit> ; therefore, we analyzed hyper-phosphorylation of tau at both these sites.
we then focused on ser- <dig> because pp2a alone regulates dephosphorylation at this site
<cit> .
we used the at <dig> antibody, which is commonly used for the immunohistological analysis of ad patient brains.
this antibody can recognize monophosphorylated tau at ser-202
<cit> .
we also focused our analysis on the abnormal phosphorylation at ser- <dig>  because the phosphorylation of this site is observed only in phf-tau and is almost undetectable in biopsy-derived adult human tau
<cit> .
we prepared lysates from brain slices internalized with jcasp peptide or recombinant set protein for 5 h 30 min and analyzed tau phosphorylation by western blotting with various antibodies  directed against the epitopes mentioned above.
we used oa, which increases tau phosphorylation at several physiological sites and induces phosphorylation at abnormal sites, as a positive control.
we used the ja peptide as a negative control; this peptide is a mutated form of the jcasp peptide in which a tyrosine is replaced with an alanine resulting in a peptide that induces neither the deleterious effect of the jcasp peptide nor set translocation
<cit> .
we observed an increase of phosphorylated tau in cells containing the jcasp peptide or recombinant set protein  whereas tau phosphorylation in cells containing the ja peptide was similar to that of the control.
we defined tau phosphorylation of the control as 100%.
hyper-phosphorylation at ser- <dig> that was induced by the recombinant set protein was  <dig>  ±  <dig> % , and that induced by recombinant set at ser- <dig> was  <dig>  ±  <dig> %  .
however the hyperphosphorylation at ser- <dig> was lower than that at ser- <dig>  except for one animal out of the seven studied.
the hyper- and/or abnormal phosphorylation of tau protein induced by recombinant set protein or jcasp peptide usually involve the same tau protein isoforms as those affected by oa.
however, in some experiments, the isoforms affected by oa differed from those affected by recombinant set protein or the jcasp peptide, as observed for the 50 kda tau isoform at ser- <dig> , ser- <dig>  and ser- <dig>  and the 70 kda tau isoform at ser- <dig> .
the level of pp2a was similar in all conditions .
interestingly, we found a significant negative correlation between the percentage of methylated pp2a and the percentage of tau protein hyper-phosphorylated at ser- <dig> that was detected with the at <dig> antibody  .
however, there was no correlation with the percentage of tau protein abnormally phosphorylated at ser- <dig>  , confirming that pp2a preferentially acts on the phosphorylation of tau at ser- <dig>
discussion
we report here that the presence of set in the cytoplasm, in absence of its over-expression or cleavage, decreases the activity of pp2a and thus stimulates tau hyperphosphorylation.
cytoplasmic set impairs the methylation of pp2a, and the level of methylated pp2a is negatively correlated with tau hyperphosphorylation at ser- <dig> but not at ser- <dig>
an increase of set expression leading to an increase of set protein has been described in the brains of ad patients, and is associated with the cytoplasmic translocation of set
<cit> .
more recently, we also observed an increase of set associated with its cytoplasmic translocation in the hippocampus of down syndrome patients and in the ca <dig> of ad patients
<cit> .
however, we cannot exclude that particular stress signals trigger the delocalization of set in the absence of its over-expression in some subregions of the brains of ad patients
<cit> .
the translocation of endogenous set was induced by a cell death signal in the jcasp model
<cit>  in absence of set over-expression.
similarly, we reported that the over-expression of appcc with a lentiviral construct in the ca <dig> of mice resulted in the translocation of endogenous set in the absence of set increase
<cit> .
phosphorylation of set at ser- <dig> by casein kinase ii causes the cytoplasmic retention of set in the neuronal cytoplasm of the brains of ad patients
<cit> .
we did not investigate the phosphorylation of set at ser- <dig> following the translocation of set from the nucleus to the cytoplasm, or after its uptake by neurons.
the role of set cleavage is not clear.
full-length set and its cleaved forms can bind to pp2a, inhibiting its activity and thus increasing tau phosphorylation
<cit> .
it is not clear whether this cleavage occurs in the nucleus or in the cytoplasm and whether both fragments remain in the same subcellular compartment.
however, transfection experiments show that full-length set is found within the nucleus as expected, whereas the ntf is distributed across cell compartments and the ctf is concentrated in the nucleus but is also present in the cytoplasm
<cit> .
a recent report showed that the level of asparaginyl endopeptidase, which cleaves set at asn- <dig>  is increased in the brains of ad patients
<cit> .
moreover, asparaginyl endopeptidase translocates from neuronal lysosomes to the cytoplasm where set can be cleaved, an event that was observed under acidic conditions in vitro and ex vivo <cit> .
the absence of acidic conditions in the ex vivo models used in this report may explain why set was not cleaved.
we cannot exclude that set is somewhat cleaved in in vivo conditions.
nonetheless, we show that cytoplasmic, full-length set is sufficient to decrease pp2a activity and induce tau hyperphosphorylation, regardless of the route of entry of set to the cytoplasm.
caspases cleave app at asp- <dig> .
an increase of appcc has already been shown in the brains of ad patients
<cit> .
appcc may be cleaved further into amyloid peptide resulting in the release of fragments containing the jcasp sequence.
app intracellular domain generated after amyloid formation, can also be cleaved by caspases, leading to the formation of analogs of the jcasp sequence.
we found that the delivery of the jcasp peptide to the cytoplasm was sufficient to induce the translocation of set and the hyper-phosphorylation of tau, similar to what we observed with the over-expression of appcc
<cit> .
recently, asp- <dig> cleavage of app in n2a cells and primary hippocampal neurons was shown to induce tau hyperphosphorylation by decreasing pp2a activity
<cit> .
the activity of pp2a is low in the brains of ad patients
<cit> .
specific inhibitors of pp2a such as i1pp2a and set, if localized in the cytoplasm, may underlie this impaired activity.
the low activity of pp2a in the brains of ad patients may be also partly related to the low level of pp2a methylated at leu- <dig> of the pp2ac
<cit> .
the methylation of this residue is also impaired in the cortex of transgenic appxps <dig> mice, in the brains of mice with mutated estrogen receptors, and after the treatment of n2a cells carrying the human app swedish mutation with oa
<cit> .
we report here that full-length set in the cytoplasm interacts with pp2a and decreases its methylation, thus impairing pp2a activity that leads to tau hyper-phosphorylation.
our co-immunoprecipitation experiments confirm the interaction of pp2a with full-length set that was reported previously
<cit> .
this interaction may impair the methylation of pp2a by inducing a conformational change that either prevents the interaction of pp2a with the leucine carboxyl methyltransferase  <dig> or stimulates its interaction with the pp2a specific methylesterase pme-1
<cit> .
alternatively, the interaction of pp2a with set may lead to a change of localization of pp2a, which may be preferentially redistributed to non-raft membrane domains where pp2a methylesterase pme- <dig> is exclusively present
<cit> .
the physiological cellular state of phosphorylation results from an equilibrium between phosphatases and kinases.
dephosphorylation at ser- <dig> and ser- <dig> is dependent on pp2a
<cit> .
interestingly, we established a negative correlation between the level of methylated pp2a and that of tau phosphorylated at ser- <dig>  confirming that the phosphorylation of the ser- <dig> site is directly dependent on pp2a activity
<cit> .
this also suggests that set, via the methylation of leu- <dig> in pp2ac, plays an important role in phosphorylation at ser- <dig>  which is consistently detected in the brains of ad patients.
in contrast, we did not observe a negative correlation between the level of methylated pp2a and tau hyperphosphorylation at ser- <dig>  despite the fact that dephosphorylation at this site depends on pp2a.
this suggests that the demethylation of pp2a that is induced by set does not substantially affect tau phosphorylation at ser- <dig>  in addition to methylation, pp2a activity can be modulated by other post-translational modifications.
we cannot exclude that the interaction of set with pp2a additionally results in the phosphorylation of pp2a which may be negatively correlated with the phosphorylation of ser- <dig>  pp2a is phosphorylated at tyr- <dig> in pp2ac by a scr kinase, and this phosphorylated form is also detected in the brains of ad patients
<cit> .
phosphorylation at this residue or at ser- <dig> in the 56α subunit, which is mediated by a pkcα, impairs pp2a activity
<cit> .
in our models, we do not know whether set induces such post-translational modifications contributing to the impairment of pp2a activity.
pp2a dephosphorylates tau, which leads to the stimulation of several tau kinases
<cit> .
in our study, cytoplasmic set inhibited pp2a; thus, the subsequent tau hyperphosphorylation observed was the result of the absence of tau dephosphorylation.
our data show that ser- <dig> becomes highly phosphorylated when cytoplasmic set inhibits pp2a activity, in contrast with the low phosphorylation at ser- <dig> in most studies.
however, phosphorylation at ser- <dig> is almost undetectable in humans at the adult stage, emphasizing the relevance of a low detectable phosphorylation, as observed in the brains of ad patients
<cit> .
ser- <dig> is principally dependent on gsk3-β
<cit>  whereas ser- <dig> is a good substrate for map kinase but not for gsk3-β
<cit> , suggesting that different sites of tau phosphorylation are regulated by specific kinases.
consequently, cytoplasmic set, via its inhibition of pp2a would be differently involved in the regulation of specific sites of phosphorylation.
in mouse brain slices, we used oa, which is an exogenous natural inhibitor of phosphatases that inhibits 70% of pp2a activity, as a positive control of tau phosphorylation.
in some cases, the pattern of the tau isoforms that were hyperphosphorylated by endogenous cytoplasmic set or cytoplasmic recombinant set protein differed from that observed with oa , which highlights the physiological relevance of our models.
moreover, both recombinant set protein and the jcasp peptide stimulated the hyperphosphorylation of the same tau isoforms , which shows that this jcasp peptide that mimics the unmasked domain of app after caspase cleavage may be a relevant model for physiopathological investigations of appcc.
in our ex vivo model, recombinant set protein was successfully internalized in the cytoplasm of adult mouse brain slices, where it impaired pp2a methylation and consequently pp2a activity, leading to an accumulation of hyperphosphorylated tau.
this model may be used to test molecules that may disrupt the interaction of set with pp2a and thus prevent the inhibition of pp2a activity
<cit> .
overall, these data suggest that when set is in the cytoplasm, it reproduces tau hyperphosphorylation observed in the brains of ad patients by impairing the methylation of pp2a.
these findings highly implicate cytoplasmic set in the progression of the disease.
CONCLUSIONS
we used two ex vivo models to show that full-length, cytoplasmic set, independent of its cleavage, inhibits pp2a activity by decreasing its methylated form, which leads to tau hyperphosphorylation.
these findings link the decrease of methylated pp2a  to the translocation of set to the neuronal cytoplasm, which are two events that are observed in the brains of ad patients.
in addition, the negative correlation between the level of methylated pp2a and some, but not all, sites of phosphorylation in tau suggests that tau phosphorylation is regulated by several mechanisms.
these data provide an important contribution to the elucidation of the mechanisms involved in ad.

 hyperphosphorylation of tau, which leads to the formation of neurofibrillary tangles, is one of the two hallmarks of alzheimer disease. tau phosphorylation is mainly regulated by the balance of kinases/phosphatases. several kinases are involved in tau phosphorylation including the glycogen synthase kinase 3β, map kinase, neuronal cdc2-like kinase and cyclin dependent kinase. in contrast, few phosphatases dephosphorylate tau. phosphatase 2a, a serine/threonine phosphatase, is the most effective. pp2a is a trimeric protein with a heterodimeric core enzyme consisting of a catalytic c subunit, and a scaffolding a subunit. this core enzyme associates with a variable regulatory subunit to form a heterotrimeric holoenzyme. pp2a shows efficient enzymatic activity when it is methylated at leu-309 and unphosphorylated at tyr-307. in the brain of ad patients, the activity of pp2a is compromised. the decrease of pp2a methylation observed in brains of ad patients may explain the decrease of pp2a activity and contribute to tau hyperphosphorylation. the activity of pp2a can be inhibited by two proteins: i1pp2a and i2pp2a, which is also called set, taf1β, or phapii. set is a multifunctional protein: it inhibits the acetylation of histones, blocks dnase activity, acts as a transcription factor, and promotes tau phosphorylation by activating cdk5, and inhibiting pp2a. in the brains of ad patients, there is an increase of set expression. interestingly, set is also translocated from the nucleus to the cytoplasm in the hippocampus and the temporal cortex of ad patients. we observed that in the ca of ad patients, the translocation of set to the cytoplasm was associated with an increase of amyloid precursor protein cleaved in its cytoplasmic domain by a caspase. in in vitro models, cytoplasmic set is associated with neuronal death and with tau hyperphosphorylation. the 39 kda full-length set can be selectively cleaved resulting in a ~ 20 kda fragment in the cytosol of neurons in the brain. the cleavage of set protein has also been observed in primary neurons treated with kainate and in a mouse model of stroke. this cleavage results from the activation of an asparaginyl endopeptidase which cuts set at asparagine asn- generating ntf and ctf fragments and triggering dna nicking and cell death. both ntf and ctf are able to bind to the catalytic subunit of pp2a inhibiting its activity and leading to tau hyperphosphorylation. however, it is not clear whether the cytoplasmic localization of set is always associated with its cleavage, with its over-expression, and with tau hyperphosphorylation. it is still not clear how cytoplasmic set contributes to pp2a loss of function leading to tau hyperphosphorylation, and whether the presence of set in the cytoplasm induces low levels of methylated pp2a. we used two models to clarify the relationship between cytoplasmic set, methylated pp2a, pp2a activity and tau hyperphosphorylation. the first model involved the translocation of endogenous set from the nucleus to the cytoplasm in primary neurons or brain slices from wild type mice. this translocation was induced in this model by the internalization of the jcasp peptide. indeed, this peptide mimics the unmasked juxtamembrane cytoplasmic domain arising from the cleavage of app by caspases, which is increased in the brains of ad patients. moreover, this peptide is sufficient to induce in vivo both translocation of endogenous set, as occurs in the ca of wt mice following appcc overexpression, and neurodegeneration. the second model involved the over-expression of set by the internalization of exogenous recombinant full-length protein in brain slices from wt mice. in these two models, we report that cytoplasmic set induces the hyperphosphorylation of tau in the absence of detectable cleaved forms of set. we also show that the interaction of set with pp2a impairs the methylation of pp2a and that the level of methylated pp2a that is associated with the translocation of set is also negatively correlated with the hyperphosphorylation of tau at ser- but not at ser- suggesting that the hyperphosphorylation of tau is regulated by different mechanisms at distinct residues.  internalization of jcasp peptide induces the translocation of endogenous nuclear set to the cytoplasm without cleavage or upregulation of its expression we previously reported that the cytoplasmic internalization of the jcasp peptide by primary neurons resulted in the translocation of endogenous set to the cytoplasm and triggered pro-apoptotic signals at the cell membrane. thus, in this model, set promotes apoptosis, and its translocation to the cytoplasm appears to participate in the active neurodegenerative process. in this model, we also previously showed that the jcasp peptide fused to the penetratin vector is quickly internalized, and that endogenous set starts to move from the nucleus to the cytoplasm after 3 h, while apoptosis was observed 24 h later. here, we show that translocated set is still present in the cytoplasm of the cellular body and in neurites 5 h 30 min after jcasp peptide internalization. sub-cellular fractionation and western blotting revealed the presence of a 39 kda band corresponding to endogenous set in the cytoplasm fraction of cells treated with jcasp peptide. the proportion of translocated set was 39% of total endogenous set, which was in accordance with observations made by epifluorescence microscopy. cleavage products of set in the nucleus and in the cytoplasm were not detected. real-time pcr showed that the level of set transcripts was unchanged during the 5 h 30 min of jcasp peptide internalization. hence, in this model, the translocation of set from the nucleus to the cytoplasm does not require its over-expression and/or its cleavage. over-expression of recombinant cytoplasmic set in primary mouse neurons and mouse brain slices does not induce its cleavage we introduced a recombinant ha-tagged set protein fused to the penetratin vector into the cytoplasm of primary neurons by direct internalization as previously described. we detected the 49 kda recombinant set protein by immunocytochemistry with an anti-ha antibody and analyzed its distribution by confocal microscopy. after 3 h of internalization, the recombinant set protein, which possesses a nuclear targeting sequence, was mostly concentrated in the cell body around the nucleus and had started to move inside the nucleus. after 5 h 30 min of internalization, the nucleus was enriched with the recombinant set protein, although recombinant set was still abundantly present in the cytoplasm. we confirmed these observations with mouse brain slices that were treated with the recombinant set protein. sub-cellular fractionation followed by western blotting with an anti-ha antibody revealed the presence of recombinant set in the cytoplasm. the detected bands of low molecular weight and low intensity were unspecific bands resulting from the anti-rabbit secondary antibody. we did not observe cleaved recombinant set protein in either sub-cellular compartment studied. interaction between uncleaved cytoplasmic recombinant set protein and pp2a inhibits the activity of pp2a and impairs its methylation in the brain slices of adult mice following its translocation to the cytoplasm, endogenous set interacts with the catalytic subunit of pp2a inducing its inhibition. we used direct internalization for 5 h 30 min to overexpress recombinant set protein in adult mouse brain slices maintained under oxygenation conditions, as described in methods. we first analyzed total phosphatase activity. we used okadaic acid, a marine sponge toxin which is an inhibitor of pp2a, as a positive control. oa inhibited total phosphatase activity by % ±  % and recombinant set inhibited total phosphatase activity by % ±  %. hence, phosphatase activity was significantly impaired in both cases and was % ±  % of the baseline value for oa, and was % ±  % for recombinant set. we then evaluated pp2a activity after pp2a immunoprecipitation in brain slices treated with oa and in those containing internalized recombinant set protein. the remaining pp2a activity was % ±  % in brain slices treated with oa and 84% ±  % in brain slices containing recombinant set. these values fit with what we observed for total phosphatase activity. thus, these data suggest that when set is in the cytoplasm, it specifically impairs pp2a activity in a manner that does not depend on its cleavage. the activity of pp2a depends on its methylation. therefore, we assessed whether the interaction between cytoplasmic recombinant set protein and endogenous pp2a impaired the methylation of pp2a, resulting in a decrease in its activity. we used an antibody directed against total pp2a and an antibody specifically directed against methyl-pp2a, and evaluated the ratio of methyl-pp2a to total pp2a by western blotting. after 5 h 30 min of recombinant set protein internalization, the ratio of methyl-pp2a to total pp2a was % ±  % of the control ratio in the absence of recombinant set. the mean decrease of this ratio was significant suggesting that the presence of set in the cytoplasm in the absence of detectable set cleavage impairs the methylation of pp2a. finally, we performed co-immunoprecipitation experiments to check that the internalized recombinant set protein interacted with pp2a and impaired the methylation of pp2a. to this end, we internalized the recombinant set protein in brain slices for 5 h 30 min, and we extracted proteins from these slices and from control slices not internalized with recombinant set. after lysis, we checked first the total level of pp2a and methyl-pp2a in brain slices by western blotting. we subsequently immunoprecipitated set from slice extracts with an antibody that recognizes both endogenous and recombinant set. we then analyzed the immunoprecipitates by western blotting with an antibody that recognizes total pp2a and after membrane stripping, with an antibody that specifically recognizes methyl-pp2a. only a small amount of pp2a co-immunoprecipitated with the set antibody in extracts from brain slices lacking recombinant set, because only a small proportion of endogenous set is cytoplasmic. more pp2a co-immunoprecipitated with set in cells containing recombinant set protein than in cells lacking the recombinant protein, showing that pp2a can bind to both recombinant and endogenous set protein. in contrast, more methyl-pp2a co-immunoprecipitated with set in the control than in cells containing recombinant set protein. the mean ratio of methyl-pp2a to total pp2a was  ±  for the control and  ±  for brain slices treated for 5 h 30 min with recombinant set protein. thus, the level of co-immunoprecipitated methylated pp2a was significantly lower in slices internalized with set protein than in the control. these data confirm that cytoplasmic, uncleaved, recombinant set protein interacts with pp2a and impairs its methylation. uncleaved set in the cytoplasm induces hyper- and abnormal phosphorylation of tau protein. correlation with decreased methyl-pp2a levels many phosphorylation sites of tau in paired helical filaments have been identified in the brains of ad patients. inhibition of pp2a leads to the preferential hyper-phosphorylation of tau at various epitopes. phosphorylation at ser- and ser- is more prevalent in phf-tau and fetal tau than in normal adult tau; therefore, we analyzed hyper-phosphorylation of tau at both these sites. we then focused on ser- because pp2a alone regulates dephosphorylation at this site. we used the at antibody, which is commonly used for the immunohistological analysis of ad patient brains. this antibody can recognize monophosphorylated tau at ser-202. we also focused our analysis on the abnormal phosphorylation at ser- because the phosphorylation of this site is observed only in phf-tau and is almost undetectable in biopsy-derived adult human tau. we prepared lysates from brain slices internalized with jcasp peptide or recombinant set protein for 5 h 30 min and analyzed tau phosphorylation by western blotting with various antibodies directed against the epitopes mentioned above. we used oa, which increases tau phosphorylation at several physiological sites and induces phosphorylation at abnormal sites, as a positive control. we used the ja peptide as a negative control; this peptide is a mutated form of the jcasp peptide in which a tyrosine is replaced with an alanine resulting in a peptide that induces neither the deleterious effect of the jcasp peptide nor set translocation. we observed an increase of phosphorylated tau in cells containing the jcasp peptide or recombinant set protein whereas tau phosphorylation in cells containing the ja peptide was similar to that of the control. we defined tau phosphorylation of the control as 100%. hyper-phosphorylation at ser- that was induced by the recombinant set protein was  ±  %, and that induced by recombinant set at ser- was  ±  %. however the hyperphosphorylation at ser- was lower than that at ser- except for one animal out of the seven studied. the hyper- and/or abnormal phosphorylation of tau protein induced by recombinant set protein or jcasp peptide usually involve the same tau protein isoforms as those affected by oa. however, in some experiments, the isoforms affected by oa differed from those affected by recombinant set protein or the jcasp peptide, as observed for the 50 kda tau isoform at ser-, ser- and ser- and the 70 kda tau isoform at ser-. the level of pp2a was similar in all conditions. interestingly, we found a significant negative correlation between the percentage of methylated pp2a and the percentage of tau protein hyper-phosphorylated at ser- that was detected with the at antibody. however, there was no correlation with the percentage of tau protein abnormally phosphorylated at ser-, confirming that pp2a preferentially acts on the phosphorylation of tau at ser- discussion we report here that the presence of set in the cytoplasm, in absence of its over-expression or cleavage, decreases the activity of pp2a and thus stimulates tau hyperphosphorylation. cytoplasmic set impairs the methylation of pp2a, and the level of methylated pp2a is negatively correlated with tau hyperphosphorylation at ser- but not at ser- an increase of set expression leading to an increase of set protein has been described in the brains of ad patients, and is associated with the cytoplasmic translocation of set. more recently, we also observed an increase of set associated with its cytoplasmic translocation in the hippocampus of down syndrome patients and in the ca of ad patients. however, we cannot exclude that particular stress signals trigger the delocalization of set in the absence of its over-expression in some subregions of the brains of ad patients. the translocation of endogenous set was induced by a cell death signal in the jcasp model in absence of set over-expression. similarly, we reported that the over-expression of appcc with a lentiviral construct in the ca of mice resulted in the translocation of endogenous set in the absence of set increase. phosphorylation of set at ser- by casein kinase ii causes the cytoplasmic retention of set in the neuronal cytoplasm of the brains of ad patients. we did not investigate the phosphorylation of set at ser- following the translocation of set from the nucleus to the cytoplasm, or after its uptake by neurons. the role of set cleavage is not clear. full-length set and its cleaved forms can bind to pp2a, inhibiting its activity and thus increasing tau phosphorylation. it is not clear whether this cleavage occurs in the nucleus or in the cytoplasm and whether both fragments remain in the same subcellular compartment. however, transfection experiments show that full-length set is found within the nucleus as expected, whereas the ntf is distributed across cell compartments and the ctf is concentrated in the nucleus but is also present in the cytoplasm. a recent report showed that the level of asparaginyl endopeptidase, which cleaves set at asn- is increased in the brains of ad patients. moreover, asparaginyl endopeptidase translocates from neuronal lysosomes to the cytoplasm where set can be cleaved, an event that was observed under acidic conditions in vitro and ex vivo. the absence of acidic conditions in the ex vivo models used in this report may explain why set was not cleaved. we cannot exclude that set is somewhat cleaved in in vivo conditions. nonetheless, we show that cytoplasmic, full-length set is sufficient to decrease pp2a activity and induce tau hyperphosphorylation, regardless of the route of entry of set to the cytoplasm. caspases cleave app at asp-. an increase of appcc has already been shown in the brains of ad patients. appcc may be cleaved further into amyloid peptide resulting in the release of fragments containing the jcasp sequence. app intracellular domain generated after amyloid formation, can also be cleaved by caspases, leading to the formation of analogs of the jcasp sequence. we found that the delivery of the jcasp peptide to the cytoplasm was sufficient to induce the translocation of set and the hyper-phosphorylation of tau, similar to what we observed with the over-expression of appcc. recently, asp- cleavage of app in n2a cells and primary hippocampal neurons was shown to induce tau hyperphosphorylation by decreasing pp2a activity. the activity of pp2a is low in the brains of ad patients. specific inhibitors of pp2a such as i1pp2a and set, if localized in the cytoplasm, may underlie this impaired activity. the low activity of pp2a in the brains of ad patients may be also partly related to the low level of pp2a methylated at leu- of the pp2ac. the methylation of this residue is also impaired in the cortex of transgenic appxps mice, in the brains of mice with mutated estrogen receptors, and after the treatment of n2a cells carrying the human app swedish mutation with oa. we report here that full-length set in the cytoplasm interacts with pp2a and decreases its methylation, thus impairing pp2a activity that leads to tau hyper-phosphorylation. our co-immunoprecipitation experiments confirm the interaction of pp2a with full-length set that was reported previously. this interaction may impair the methylation of pp2a by inducing a conformational change that either prevents the interaction of pp2a with the leucine carboxyl methyltransferase or stimulates its interaction with the pp2a specific methylesterase pme-1. alternatively, the interaction of pp2a with set may lead to a change of localization of pp2a, which may be preferentially redistributed to non-raft membrane domains where pp2a methylesterase pme- is exclusively present. the physiological cellular state of phosphorylation results from an equilibrium between phosphatases and kinases. dephosphorylation at ser- and ser- is dependent on pp2a. interestingly, we established a negative correlation between the level of methylated pp2a and that of tau phosphorylated at ser- confirming that the phosphorylation of the ser- site is directly dependent on pp2a activity. this also suggests that set, via the methylation of leu- in pp2ac, plays an important role in phosphorylation at ser- which is consistently detected in the brains of ad patients. in contrast, we did not observe a negative correlation between the level of methylated pp2a and tau hyperphosphorylation at ser- despite the fact that dephosphorylation at this site depends on pp2a. this suggests that the demethylation of pp2a that is induced by set does not substantially affect tau phosphorylation at ser- in addition to methylation, pp2a activity can be modulated by other post-translational modifications. we cannot exclude that the interaction of set with pp2a additionally results in the phosphorylation of pp2a which may be negatively correlated with the phosphorylation of ser- pp2a is phosphorylated at tyr- in pp2ac by a scr kinase, and this phosphorylated form is also detected in the brains of ad patients. phosphorylation at this residue or at ser- in the 56α subunit, which is mediated by a pkcα, impairs pp2a activity. in our models, we do not know whether set induces such post-translational modifications contributing to the impairment of pp2a activity. pp2a dephosphorylates tau, which leads to the stimulation of several tau kinases. in our study, cytoplasmic set inhibited pp2a; thus, the subsequent tau hyperphosphorylation observed was the result of the absence of tau dephosphorylation. our data show that ser- becomes highly phosphorylated when cytoplasmic set inhibits pp2a activity, in contrast with the low phosphorylation at ser- in most studies. however, phosphorylation at ser- is almost undetectable in humans at the adult stage, emphasizing the relevance of a low detectable phosphorylation, as observed in the brains of ad patients. ser- is principally dependent on gsk3-β whereas ser- is a good substrate for map kinase but not for gsk3-β, suggesting that different sites of tau phosphorylation are regulated by specific kinases. consequently, cytoplasmic set, via its inhibition of pp2a would be differently involved in the regulation of specific sites of phosphorylation. in mouse brain slices, we used oa, which is an exogenous natural inhibitor of phosphatases that inhibits 70% of pp2a activity, as a positive control of tau phosphorylation. in some cases, the pattern of the tau isoforms that were hyperphosphorylated by endogenous cytoplasmic set or cytoplasmic recombinant set protein differed from that observed with oa, which highlights the physiological relevance of our models. moreover, both recombinant set protein and the jcasp peptide stimulated the hyperphosphorylation of the same tau isoforms, which shows that this jcasp peptide that mimics the unmasked domain of app after caspase cleavage may be a relevant model for physiopathological investigations of appcc. in our ex vivo model, recombinant set protein was successfully internalized in the cytoplasm of adult mouse brain slices, where it impaired pp2a methylation and consequently pp2a activity, leading to an accumulation of hyperphosphorylated tau. this model may be used to test molecules that may disrupt the interaction of set with pp2a and thus prevent the inhibition of pp2a activity. overall, these data suggest that when set is in the cytoplasm, it reproduces tau hyperphosphorylation observed in the brains of ad patients by impairing the methylation of pp2a. these findings highly implicate cytoplasmic set in the progression of the disease.  we used two ex vivo models to show that full-length, cytoplasmic set, independent of its cleavage, inhibits pp2a activity by decreasing its methylated form, which leads to tau hyperphosphorylation. these findings link the decrease of methylated pp2a to the translocation of set to the neuronal cytoplasm, which are two events that are observed in the brains of ad patients. in addition, the negative correlation between the level of methylated pp2a and some, but not all, sites of phosphorylation in tau suggests that tau phosphorylation is regulated by several mechanisms. these data provide an important contribution to the elucidation of the mechanisms involved in ad. 
our data show a link between the translocation of set in the cytoplasm and the decrease of methylated pp2a levels leading to a decrease of pp2a activity and tau hyperphosphorylation. we used primary neurons and mouse brain slices to show that set is translocated to the cytoplasm in a manner independent of both its cleavage and over-expression. cytoplasmic recombinant full-length set in mouse brain slices induced a decrease of pp2a activity through a decrease of methylated pp2a levels. the neuronal cytoplasmic localization of set, an inhibitor of the phosphatase 2a, results in tau hyperphosphorylation in the brains of alzheimer patients through mechanisms that are still not well defined. the localization of set in the cytoplasm, either by the translocation of endogenous set or by internalization of the recombinant full-length set protein, induced tau hyperphosphorylation. the levels of methylated pp2a were negatively correlated with tau hyperphosphorylation at ser- but not with the abnormal phosphorylation of tau at ser- the presence of full-length set in the neuronal cytoplasm is sufficient to impair pp2a methylation and activity, leading to tau hyperphosphorylation. in addition, our data suggest that tau hyperphosphorylation is regulated by different mechanisms at distinct sites. the translocation of set to the neuronal cytoplasm, the low activity of pp2a, and tau hyperphosphorylation are associated in the brains of alzheimer patients. cytoplasmic setprotein phosphatase 2amethyl phosphatase 2atau hyperphosphorylationalzheimer disease
 the neuronal cytoplasmic localization of set, an inhibitor of the phosphatase 2a, results in tau hyperphosphorylation in the brains of alzheimer patients through mechanisms that are still not well defined.  we used primary neurons and mouse brain slices to show that set is translocated to the cytoplasm in a manner independent of both its cleavage and over-expression. the localization of set in the cytoplasm, either by the translocation of endogenous set or by internalization of the recombinant full-length set protein, induced tau hyperphosphorylation. cytoplasmic recombinant full-length set in mouse brain slices induced a decrease of pp2a activity through a decrease of methylated pp2a levels. the levels of methylated pp2a were negatively correlated with tau hyperphosphorylation at ser- but not with the abnormal phosphorylation of tau at ser-  the presence of full-length set in the neuronal cytoplasm is sufficient to impair pp2a methylation and activity, leading to tau hyperphosphorylation. in addition, our data suggest that tau hyperphosphorylation is regulated by different mechanisms at distinct sites. the translocation of set to the neuronal cytoplasm, the low activity of pp2a, and tau hyperphosphorylation are associated in the brains of alzheimer patients. our data show a link between the translocation of set in the cytoplasm and the decrease of methylated pp2a levels leading to a decrease of pp2a activity and tau hyperphosphorylation. this chain of events may contribute to the pathogenesis of alzheimer disease. cytoplasmic setprotein phosphatase 2amethyl phosphatase 2atau hyperphosphorylationalzheimer disease 