nap is an 8 amino acid peptide , which was identified as an active neuroprotective fragment of activity - dependent neuroprotective protein ( adnp ) . nap was found to be neuroprotective in various in vivo and in vitro models of neurodegeneration . nap treatment was shown to reduce two major pathological markers  tau hyperphosphorylation [ 35 ] and caspase-3 activation / apoptosis [ 6 , 7 ] . hyperphosphorylated and aggregated tau , originally detected in alzheimer 's disease ( ad ) brains by grundke - iqbal and colleagues , is a hallmark of a group of diseases , generally referred to as tauopathies  which differ from each other by genetic background and by additional pathological and phenotypic characteristics . the first study that reported disassembly of microtubules from ad brain due to the abnormal hyperphosphorylation of tau was by iqbal et al . . hyperphosphorylated tau loses its microtubule affinity causing a change in microtubule dynamics towards disassembly [ 1116 ] and further accumulation of aggregated tau . alonso et al . originally showed that ad abnormal hyperphosphorylation of tau causes not only loss of function but also the gain of toxic function , with hyperphosphorylated tau blocking microtubule assembly in the presence of normal tau   and promoting the formation of normal tau containing tangles . the most prevalent tauopathy is alzheimer 's disease ( ad ) , and other tauopathies include a selection of frontotemporal dementia / degeneration , with pure tauopathies like progressive supranuclear palsy ( psp ) . one of the hallmarks of tauopathies is the accumulation of neurofibrillary tangles ( nfts ) . nft accumulation correlates with the severity of dementia and memory loss [ 2022 ] and with neuronal degeneration in ad and psp [ 23 , 24 ] . recent studies associated the spread of tauopathies with propagation of prion - like protein inclusions   and suggested transsynaptic spread of tau . however , several studies showed that soluble defective tau is also correlated with cognitive deficits [ 2729 ] , causing synaptic loss and gliosis before nfts formation . tau is also a substrate of multiple caspases , which cleave it and promote its pathologic aggregation [ 3133 ] . cleaved and hyperphosphorylated tau is found in deposits in ad brains , and it was found that truncated tau promotes apoptosis . the relationship between tau hyperphosphorylation and cleavage is not completely understood . though some evidence suggests that caspase cleavage of tau is not necessary for tau hyperphosphorylation , this question should be further clarified [ 35 , 36 ] . caspases are known to play a role in ad ( for review , see ) , and caspase effects on modifications of tau are of a great interest . furthermore , treatment of an ad mouse model with a broad - spectrum caspase inhibitor was lately shown to reduce tau pathology but not amyloid- ( a ) pathology . to date , we have tested nap effects on either tau hyperphosphorylation or on caspase activation in separate models , showing inhibition of tau pathology or inhibition of caspase activation ( or other apoptotic markers ) ( table 1 ) . the data emerging from these studies raise the question whether there is a dependency between the effects of nap on the two pathologies . in the current study , we used an in vitro model of ischemia in which nap treatment reduced apoptosis . as animal models of ischemia exhibit tau hyperphosphorylation [ 4042 ] , we investigated whether the in vitro ischemia model mimics the in vivo situation . thus , the aim of the current study was to identify a possible interaction between caspase activity and tau hyperphosphorylation , and to test the effect of nap on both events under ischemic conditions . all procedures involving animals were approved by the animal care committee of tel - aviv university and further approved by the israel health authorities . primary neuronal cultures were produced from cortices obtained from 1 - 2-day - old sprague - dawley rat pups . pups were decapitated , their skulls were removed , and their cortices were transferred to a petri dish field with hbss - hepes buffer . neurons were produced with worthington 's papain dissociation kit ( worthington , cat.lk003150 ) according to the manufacturer 's instructions . the following antibodies were used for immunefluorescence : neun ( neuronal nuclei , chemicon , cat . mab377 ) , diluted 1  :  100 , and gfap ( glial fibrillary acidic protein , abcam , cat . the following antibodies were used for immunoblot analysis : p - tau ( anaspec , cat . 28017 ) 0.25  g / ml , total tau ( mbl international , cat . at-5004 ) 0.5  g / ml , and active caspase-3 ( abcam , cat . cells were seeded in 24 well plates on poly - d lysine- ( pdl- ) coated coverslips ( sigma - aldrich , cat . p-6407 ) at a density of 0.510  cells / well . at 56 div , the cultures were fixed using a 4% paraformaldehyde ( pfa ) solution . antigen retrieval , if needed , was performed by boiling the sample in sodium citrate buffer ( ph = 6 ) for 20 minutes . nonspecific antigen binding was blocked by incubation in a 2% bovine serum albumin ( bsa ) solution , then primary antibody ( ab ) diluted in primary antibody diluent ( biotech applications , cat . cells were washed three times in 2% solution of bsa and incubated in secondary antibody solution for 30 minutes in the dark . after an additional wash , pbs + dapi ( 4,6-diamidino-2-phenylindole , a fluorescent stain that binds strongly to a t - rich regions in dna , invitrogen ) was applied for 5 minutes , in order to stain cell nuclei . the coverslips were put on an antifading buffer on a carrier slides . for analysis of culture purity , we used 3 cultures produced independently . we used zeiss fluorescence microscope and camera to obtain 10 pictures ( 20 magnification ) from each culture . neun - positive and gfap - negative cells were considered neuronal and were expressed as % of the total dapi count . cells were seeded on pdl - coated plates employing two different plating conditions ( corning ) : ( 1 ) 10  cm petri dishes , 610 cells per dish ( for protein extraction ) , and ( 2 ) 48 well plates 0.510 cells / well ( for viability tests ) . after 5 - 6 div , the condition media ( cm cultures were washed twice with 37c pbs , and the experimental media was added ( pbs for the ogd groups , fresh media for the control group ) . treatments were added to the experimental media ( qvd - oph , a broad - spectrum caspase inhibitor , 20  m diluted in dmso ( biovision , cat . the culture dishes were placed in an ischemic chamber ( billups - rothenberg , inc . , cat . mic 101 ) which was filled with a 5%co2/95%n2 gas mixture and sealed , and the chamber was placed in an incubator for 2 hours ; at 37c . two experimental modes were applied , in the first one ( used for nap protecting concentration curve ) , gassing was for 25 minutes followed by sealing for 2 hours , in the second paradigm ( used for calibration of caspase inhibition and protein analysis ) , gassing was for 5 minutes only . po2 - 250 ) and did not exceed 1.2% any time during the ogd period . at the end of the ogd period , cells were seeded in 48 well plates at a density of 0.510 cells / well ( as described above ) . following the ogd period , the condition media ( cm ) which was removed at the initiation of the ogd treatment ( see above ) was reapplied to the corresponding treatments . absorbance at 490  nm was measured in a spectramax 190 plate reader , ( molecular devices , inc . ) . the results were analyzed using softmax pro software version / year ( molecular devices , inc . ) and expressed as % of the control . data are presented as means / medians  se . immediately after ogd treatment ( following gassing mode of 5 minutes of gassing and 2 hours ogd period ) , experimental media was discarded , and the dishes were washed twice with pbs . protein was extracted using ripa buffer with anti - proteases ( roche , cat . 23223 , 23224 ) with bovine serum albumin ( bsa ) as a standard protein . equal amounts of protein diluted in sample buffer were separated by sodium dodecyl sulfate ( sds ) polyacrylamide gel electrophoresis ( 12% gels or 420% gradient gels , nuview , cat . membranes were blocked with 5% bsa in tris - buffered saline containing 0.1% tween-20 ( tbst ) and then incubated overnight with primary antibody . membranes were washed 3 times with tbst and incubated with secondary ab for 1 hour at rt . pierce elc substrate or pierce super signal ecl was used to visualize proteins ( cat . signal density was detected with the dnr bioimaging systemsminibis pro ( dnr bio - imaging systems ltd . ) and totallab tl-100 software ( nonlinear dynamics ltd . ) . spss 19.0 for windows ( http://www-01.ibm.com/software/analytics/spss/ ) was used for statistical analysis . for viability assessment , data were analyzed using a one - way anova with 5% selected the level of significance , and post hoc scheffe and lsd corrections . the effect of nap was evaluated in 14 independent experiments , of which in 4 experiments qvd - oph effect was also tested , and the levels of significance were evaluated using anova with post hoc lsd . after 5 - 6 div ( days in vitro ) , 95.9  0.96% ( sem ) of the cells ( counted using nuclear dapi stain ) were positive for the neuronal neun marker and negative for the astrocytic marker , gfap . a representative immunefluorescence stain in figure 1 shows neuronal cells stained with neun in red , and astrocytes stained with gfap in green . cell viability was evaluated following a 25-minute gassing period and an additional 2 hours of ogd . the results were calculated and expressed as % of the control group ( figure 2 ) . under this paradigm , ~70% cell death was observed . treatment with nap increased cell viability immediately following ogd ( f = 17.667 , df = 7 , p post hoc pairwise comparisons ( lsd ) indicated that nap concentrations of 10  m and 10  m significantly protected against ischemia ( figure 2 , p  0.05 ) .   using the broad spectrum caspase inhibitor qvd - oph , we tested the extent of apoptotic death following 5-minute of gassing and 2 hours of ogd . cell viability was evaluated and compared to the control group ( figure 3 ) . under this paradigm , ~55% cell death was observed . here , qvd - oph ( 210  m ) treatment significantly increased cell viability to the control levels . in the same experiment , nap in the same experiment , nap ( 10  m ) treatment rescued from the apoptotic cell death ( f = 16.090 , df = 7 , p post hoc pairwise comparisons did not detect any significant differences between the caspase inhibitor , nap , and control no - ischemia groups . the levels of active caspase-3 were evaluated immediately after the ogd period using immunoblots with a specific active caspase-3 antibody and further quantified and normalized to actin . as shown in a representative blot in figure 4(a ) , ogd treatment increased active caspases-3 levels . this increase was prevented by nap treatment ( 10  m ) or qvd - oph ( caspase inhibitor ) treatment ( 210  m ) . a significant increase in active caspase-3 expression was induced by ogd and partially but significantly prevented by nap treatment ( f = 7.880 , df = 2 ,  p = 0.004 ) . phospho - tau ( p - tau ) levels were detected by immunoblot , with a p - tau - specific antibody . p - tau levels , were quantified , normalized to total tau levels and expressed as % of the p - tau levels in the control culture ( figure 5(b ) ) . phospho - tau levels were significantly increased following the ogd insult . both nap treatment ( 10  m ) and qvd - oph ( caspases inhibitor ) treatment ( 210  m ) prevented p - tau increase ( anova with post hoc lsd , p  0.05 ) , ( f = 5.633 , df = 2 , p = 0.012 ) . focusing on the protective properties of nap , two central characteristics of neurodegeneration have been shown to be inhibited by nap treatment both in vivo and in vitro : tau hyperphosphorylation [ 35 ] and apoptosis [ 6 , 7 , 51 ] . the main goal of the current study was to look for an association between these two pathological cascades . our experimental setup used primary neuronal cultures ( 95.9  0.96% purity ) , subjected to 2 hours of ogd with no reperfusion period . under this paradigm , apoptosis was found to be responsible for all cell death in that it was inhibited by qvd - oph . our results showed that inhibition of caspase activity prevented tau hyperphosphorylation , leading us to conclude that in the current experimental and cellular conditions , caspase activation is an upstream event to tau hyperphosphorylation . we further evaluated the effect of nap on cell viability , caspase-3 activation , and tau hyperphosphorylation . viability assays showed that nap treatment rescued cells from apoptosis as demonstrated by the reduction in active caspases-3 following nap treatment . nap treatment also prevented tau hyperphosphorylation after the ogd . combining the effect of nap on active caspases-3 and tau hyperphosphorylation , it seems likely that caspases-3 or an upstream pathway is targeted by nap activity in isolated neuronal cells that are metabolically stressed . a careful look should be given to the concentrations by which nap had a significant protective effect . previous data , including ischemia - reperfusion experiments conducted in primary neuronal and neuroglial cultures , exhibited protection from cytochrome - c release , microtubule breakdown and reduction in map2 intensity using nap concentrations of 10  m10  m [ 39 , 52 ] . though most studies used femtomolar concentration of nap , pascual and guerri   used concentration of 10 m to show nap protection in a model neurons cocultured with astrocytes obtained from prenatal ethanol - exposed fetuses . in our model , protection was detected when treating with nap at concentrations 10 m. the differences in the potent nap concentration could be explained by the combination of the acute insult applied to a relatively pure culture of immature neurons . immature neurons ( 24 div ) in a pure neuronal culture ( 99% purity ) were shown to go through spontaneous apoptotic death which was prevented when astrocyte condition media was added to the neuronal cultures . in comparison to neuroglial mixed culture , pure neuronal cultures were also found to be more sensitive to excitotoxic insult that also plays a role in the ischemia induced cell death . we have previously shown that neuronal protection in the absence of glia required increased concentrations of nap as compared to mixed neuroglial cultures . furthermore , the original discovery of the nap containing protein , adnp , was as a glial protein providing neuroprotection . adnp is essential for central nervous system development , expressed in specific brain tissue in the adult brain , and is also deregulated and may be overexpressed following acute brain damage   and in animal models of neurodegeneration [ 57 , 58 ] . adnp - like immunoreactivity is secreted from glial cells   further providing neuronal protection [ 4 , 53 , 60 ] . in this respect , nap was further shown to promote neurite outgrowth [ 61 , 62 ] provide microtubule stabilization   and protect neurons in a broad spectrum of neuropathologies models . importantly , nap also showed glial protection in vitro   and in vivo . in summary , we demonstrated that nap reduces caspase-3 activation , and tau hyperphosphorylation , suggesting that caspase-3 or upstream pathways may be targets for nap activity . we believe these data provide additional insights regarding the molecular mechanism for nap 's neuroprotective activity and the intricate interaction between microtubules / tau and apoptotic mechanisms .
the peptide drug candidate nap ( davunetide ) has demonstrated protective effects in various in vivo and in vitro models of neurodegeneration . 
 nap was shown to reduce tau hyperphosphorylation as well as to prevent caspase-3 activation and cytochrome-3 release from mitochondria , both characteristic of apoptotic cell death . 
 recent studies suggest that caspases may play a role in tau pathology . 
 the purpose of this study was to evaluate the effect of nap on tau hyperphosphorylation and caspase activity in the same biological system . 
 our experimental setup used primary neuronal cultures subjected to oxygen - glucose deprivation ( ogd ) , with and without nap or caspase inhibitor . 
 cell viability was assessed by measuring mitochondrial activity ( mts assay ) , and immunoblots were used for analyzing protein level . 
 it was shown that apoptosis was responsible for all cell death occurring following ischemia , and nap treatment showed a concentration - dependent protection from cell death . 
 ischemia caused an increase in the levels of active caspase-3 and hyperphosphorylated tau , both of which were prevented by either nap or caspase - inhibitor treatment . 
 our data suggest that , in this model system , caspase activation may be an upstream event to tau hyperphosphorylation , although additional studies will be required to fully elucidate the cascade of events .