the senile plaques and neurofibrillary tangles ( nfts ) , which are composed of self - polymerized amyloid- peptide ( a ) and hyperphosphorylated tau proteins , respectively , are the two major pathological hallmarks in brains of ad .
pathogenic a aggregates initiate a cascade of molecular events that foster widespread neurodegeneration , although the exact pathogenesis from a aggregation to neurodegeneration is not clear .
a originates from proteolysis of the amyloid precursor protein ( app ) by the sequential enzymatic actions of beta - site amyloid precursor protein - cleaving enzyme 1 ( bace1 ) , -secretase , and -secretase , a protein complex with presenilin 1 ( ps1 ) at its catalytic core .
-secretase adam10 conversely cleaves app within the eventual a sequence to preclude a generation , and it yields the n - terminal protein of app ( soluble app- [ sapp ] ) .
sapp decreases a generation by directly associating with bace1 , and so it may be a potential agent to ameliorate imbalances in app processing.4 , 5 overexpression of adam10 markedly reduces a plaque load and soluble a , restoring learning deficits in a double - transgenic alzheimer mouse model .
neurotrophins ( neurotrophic factors [ ntfs ] ) are important in the  development , differentiation , and regeneration of brain neurons . in  recent years
, some neurotrophins , such as nerve growth factor  ( ngf ) and brain - derived neurotrophic factor ( bdnf ) , were explored as candidate therapeutics for ad.8 , 9 human acidic fibroblast growth factor ( hafgf ) has neuroprotective functions similar to ntfs. exogenous administration of hafgf has been shown to  prevent degeneration and apoptosis of neurons in epileptic seizure.10 , 11 hafgf is involved in the regulation of synaptic plasticity , processes attributed to learning and memory through improving cholinergic nerve functions .
hafgf showed a neuroprotective effect against brain injury resulting from focal ischemia - reperfusion in rats,13 , 14 and it repaired human spinal cord injury in a clinical trial.15 , 16 it is reported that the concentration of acid fibroblast growth factor ( afgf ) is increased in the serum and cerebrospinal fluid of patients with ad . however , due to its large molecular size of 17  kda , the 154-amino acid protein hafgf is unable to freely pass through biological membranes and the blood - brain barrier ( bbb ) .
novel strategies were used to address this bottleneck problem , such as fusing the target protein with tat - ptd and utilizing intranasal delivery.19 , 20 our previous study showed that modified protein tat - hafgf14154 could penetrate the bbb and was distributed in hippocampus and cortex via intravenous injection .
following intranasal administration , the distribution of tat - hafgf14154 was observed at 15  min in the brains of rats .
tat - ptd could increase the concentration of hafgf in brain , and tat - hafgf14154 improved cognition and reduced a plaques more significantly than hafgf14154 in senescence - accelerated mouse prone-8 ( samp8 ) and app / ps1 mice.22 , 23 meanwhile , liposomes have gained increasing attention as a promising strategy for brain - targeted drug delivery due to their large delivery capabilities , capacity for  surface decoration , low toxicity , and biocompatibility with biodegradability .
studies have shown that liposomes easily cross the bbb through absorptive mediated transcytosis . in the current study , tat - hafgf14154
was encapsulated with cationic liposomes , and it was delivered to the app / ps1 double - transgenic mouse model for ad via intranasal administration to investigate neuroprotection .
the molecular mechanism related to neuroprotection of tat - hafgf14154 was also explored in primary cortical neurons of rats injured by a142 .
results demonstrated that tat - hafgf14154 ameliorated the ad phenotype by increasing adam10 expression at the transcriptional level through the pi3k - creb - ire1/xbp1 pathway , thereby enhancing adam10 enzyme activity .
the highest entrapment efficiency was achieved with a 1:2 ratio of tat - hafgf14154 and liposomes incubated at 25c or 30c for 0.5  hr ( 97% ; figure  s1a ) .
these conditions did not alter the electrophoretic mobility profile of tat - hafgf14154 on sds - page ( figure  s1b ) .
after a single intranasal administration , afgf was detected in the olfactory bulb ( ob ) and entorhinal cortex ( ec ) of 7-month - old c57bl/6 mice ( figure  s1c ) . both tat - hafgf14154 ( 600  g / kg , t600 ) and tat - hafgf14154-loaded cationic liposomes ( 600  g / kg , l  + t600 ) significantly enhanced the levels of afgf in the ob ( p  < 0.05 for t600 , p  < 0.01 for l  + t600 versus vehicle ; figure  s1d ) ; only l  + t600 enhanced the levels of afgf significantly in the ec ( p  < 0.01 versus vehicle ; figure  s1e ) .
intranasal treatment of tat - hafgf14154-loaded cationic liposomes demonstrated a higher efficiency for the delivery of afgf compared with tat - hafgf14154 alone , as evidenced by the significantly elevated levels of afgf in the ec ( p  < 0.01 for l  + t600 versus t600 ; figure  s1e ) .
tat - hafgf14154-loaded cationic liposomes were delivered intranasally to 7-month - old app / ps1 mice every 2  days for 6  weeks , followed by a morris water maze ( mwm ) test in the fifth week and nest construction ( nc ) and open field ( of ) tests in the sixth week ( figure  1a ) .
representative movement tracks of mice on day 5 in the place navigation component of the mwm are shown in figure  1b .
both t600 and l  + t600 significantly decreased the escape latency compared with vehicle ( p  < 0.001 for t600 and l  + t600 ; figure  1c ) .
mice treated with l  + t600 exhibited a shorter escape latency compared to those treated with t600 ( p  < 0.01 ; figure  1c ) during the 6  days of testing .
t600 and l  + t600 also decreased the path length in app / ps1 mice compared with vehicle during the 6  days of tests ( p  < 0.001 for t600 and l  + t600 ; figure  1d ) .
l  + t600 exhibited a notably shorter path length from day 3 ( p  <  0.05 on day 3 , p  < 0.01 on days 4 and 5 ; figure  1e ) , whereas this was only evident with t600 on day 5 ( p  < 0.05 ; figure  1e ) compared with vehicle .
after finishing the navigation test on day  5 , a spatial probe test was conducted 24  hr later .
movement tracks , the incidence of crossing the removed - platform area , and percentage of time spent in target quadrant were recorded ( figures 1f1h ) .
l  +  t600 significantly increased the incidence of crossing the removed - platform area ( p  < 0.01 ; figure  1 g ) and time spent in the target quadrant ( p  < 0.01 ; figure  1h ) compared with vehicle .
there were no differences in the swimming speed of mice among all  groups in the place navigation and spatial probe tests ( figure  s2a ) .
these results indicate that tat - hafgf14154-loaded cationic liposomes significantly attenuated cognitive behavior deficits and that the liposome tat - hafgf14154 preparation was more potent than tat - hafgf14154 alone.figure  1tat - hafgf14154-loaded cationic liposomes attenuated cognitive behavioral deficits in app / ps1 mice(a ) schematic of the protocol followed for the experiments .
aliquots of 0.9% saline ( vehicle ) , tat - hafgf14154 ( 600  g / kg , t600 ) , and tat - hafgf14154-loaded cationic liposomes ( 600  g / kg , l  + t600 ) were delivered intranasally to 7-month - old app / ps1 mice every 2  days for 6  weeks ( n  = 8) .
mwm was conducted in the fifth week , and nest construction ( nc ) and of tests were in the sixth week .
representative movement tracks of mice on day 5 ( b ) , escape latencies ( c ) , and path length ( d and e ) over the 5  days are shown .
( f  h ) following the completion of the navigation test , a spatial probe test was conducted 24  hr later .
representative movement tracks ( f ) , percentage of time mice spent in the target quadrant ( g ) , and times of crossing the removed - platform area ( h ) were assessed .
all values are presented as means   sem ( n  = 8 ; * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , two - way anova with bonferroni post hoc test for c  e , one - way anova with bonferroni s post hoc test for g and h ) .
, intranasal administration ; mwm , morris water maze ; of , open field ; ns .
tat - hafgf14154-loaded cationic liposomes attenuated cognitive behavioral deficits in app / ps1 mice ( a ) schematic of the protocol followed for the experiments .
aliquots of 0.9% saline ( vehicle ) , tat - hafgf14154 ( 600  g / kg , t600 ) , and tat - hafgf14154-loaded cationic liposomes ( 600  g / kg , l  + t600 ) were delivered intranasally to 7-month - old app / ps1 mice every 2  days for 6  weeks ( n  = 8) .
mwm was conducted in the fifth week , and nest construction ( nc ) and of tests were in the sixth week .
representative movement tracks of mice on day 5 ( b ) , escape latencies ( c ) , and path length ( d and e ) over the 5  days are shown .
( f  h ) following the completion of the navigation test , a spatial probe test was conducted 24  hr later .
representative movement tracks ( f ) , percentage of time mice spent in the target quadrant ( g ) , and times of crossing the removed - platform area ( h ) were assessed .
all values are presented as means   sem ( n  = 8 ; * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , two - way anova with bonferroni post hoc test for c  e , one - way anova with bonferroni s post hoc test for g and h ) .
i.n . , intranasal administration ; mwm , morris water maze ; of , open field ; ns . , no significance . next
, non - cognitive behaviors were examined in app / ps1 mice , including nc and of tests in the sixth week after intranasal administration .
body weights of mice had no significant change in the whole experimental period ( figure  s2b ) .
it is reported that app / ps1 mice fail to construct nests and display an increased locomotor activity compared with wild - type mice.28 , 29 the nesting scores of the ps1/app mice improved significantly with both t600 and l  + t600 treatments ( p  < 0.01 for t600 , p  < 0.01 for l  + t600 ; figures s3a and s3b ) . in the of test , l  + t600 significantly decreased time spent ( p  < 0.05 ; figure  s3c ) and crossing times ( p  < 0.01 ; figure  s3d ) in the central area , and it shortened the total distance of movement ( p  < 0.01 ; figure  s3e ) compared with vehicle .
l  + t600 treatment was significantly more effective than t600 in the of test ( p  < 0.05 in figure  s3c and p  < 0.01 in figure  s3e )
. overload of a plaque is the major pathology in the brain of app / ps1 mice .
a plaques were assessed by immunohistochemisty in the cortex and hippocampus ( cornu ammonis 1 [ ca1 ] is illustrated in  figure  2a ) .
l  + t600 treatment reduced the area ( p  <  0.01 ; figure  2b ) and the number ( p  < 0.001 ; figure  2c ) of a plaques compared with vehicle . although t600 decreased the a plaque number ( p  < 0.05 ; figure  2c ) , it did not significantly change the percentage of the a plaque area . to further confirm these results , tissue homogenates from cortex and hippocampus were used to detect a by western blotting ( figures 2d2f ) .
l  + t600 reduced a levels both in cortex ( p  < 0.05 ; figure  2e ) and hippocampus ( p  < 0.01 ; figure  2f ) compared with vehicle .
these results indicate that tat - hafgf14154-loaded cationic liposomes decreased a burden in  the brain of app / ps1 mice and were more effective than tat - hafgf14154 alone.figure  2tat - hafgf14154-loaded cationic liposomes reduced a loading in the brain of app / ps1 mice(a ) representative images of a plaques in coronal section and ca1 area of brain ( scale bar , 80  m ) .
( b and c ) percentage of a plaque area ( b ) and plaque number ( c ) in coronal sections were analyzed by image - pro plus ( n  = 8) .
( d  f ) representative immunoblots ( d ) and quantification of a expression in cortex ( e ) and hippocampus ( f ) ( n  = 6 ) . all values are presented as means   sem ( * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
tat - hafgf14154-loaded cationic liposomes reduced a loading in the brain of app / ps1 mice ( a ) representative images of a plaques in coronal section and ca1 area of brain ( scale bar , 80  m ) .
( b and c ) percentage of a plaque area ( b ) and plaque number ( c ) in coronal sections were analyzed by image - pro plus ( n  = 8) .
( d  f ) representative immunoblots ( d ) and quantification of a expression in cortex ( e ) and hippocampus ( f ) ( n  = 6 ) .
all values are presented as means   sem ( * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
od , relative optical density . to determine whether the reduction of a loading could be due to a  change in app - processing modulation , the expression of app and its processing proteins ( adam10 , bace1 , and ps1 ) in cortex and hippocampus was measured by western blotting ( figure  3a ) .
the expression of adam10 ( p  < 0.01 in cortex , figure  3b ; p  < 0.05 in hippocampus , figure  3f ) , but not full - length app ( fl - app , figures 3c and 3 g ) or bace1 ( figures 3d and 3h ) , was enhanced significantly by l  + t600 both in cortex and hippocampus compared with vehicle .
l  + t600 enhanced the expression of ps1 ( p  < 0.05 ; figure  3i ) in hippocampus , but not in cortex .
t600 had no influence on the expression of the above proteins ( figures 3b3i ) .
enzyme activities of adam10 and bace1 , two major secretases in app processing , were assayed from hippocampus homogenates of app / ps1 mice .
the activity of adam10 ( p  < 0.05 ; figure  3j ) , but not bace1 ( figure  3k ) , was increased by l  + t600 compared with vehicle .
these results revealed that the reduction of a loading in the brain of app / ps1 mice treated with tat - hafgf14154-loaded cationic liposomes could be due to the enhancement of adam10 in expression and activity.figure  3tat - hafgf14154-loaded cationic liposomes enhanced adam10 expression and activity in the brain of app / ps1 mice(a  i ) representative immunoblots ( a ) and quantification expression of app - processing proteins in cortex ( b  e ) and hippocampus ( f  i ) of app / ps1 mice .
app - processing proteins , including fl - app , adam10 , bace1 , and ps1 , were assayed by western blotting .
( j and k ) adam10 activity ( j ) and bace1 activity ( k ) in homogenate of hippocampus were measured by an enzymatic cleavage assays .
all values are presented as means   sem ( n  = 6 ; * p  < 0.05 and * * p  < 0.01 , one - way anova with bonferroni post hoc test ) .
tat - hafgf14154-loaded cationic liposomes enhanced adam10 expression and activity in the brain of app / ps1 mice ( a  i ) representative immunoblots ( a ) and quantification expression of app - processing proteins in cortex ( b  e ) and hippocampus ( f  i ) of app / ps1 mice . app - processing proteins , including fl - app , adam10 , bace1 , and ps1 , were assayed by western blotting .
( j and k ) adam10 activity ( j ) and bace1 activity ( k ) in homogenate of hippocampus were measured by an enzymatic cleavage assays .
all values are presented as means   sem ( n  = 6 ; * p  < 0.05 and * * p  < 0.01 , one - way anova with bonferroni post hoc test ) .
primary cortical neurons at 5  days in  vitro ( div ) were incubated with a142 oligomers for 48  hr at 37c .
a142 ( 1.5  m ) suppressed the viability of primary cortical neurons to 69.7%   1.4% ( p 
< 0.001 ; figure  4a ) , and apoptotic bodies / karyopyknosis affected 34.3%   4.4% of the cells ( p  < 0.001 ; figures 4b and 4c ) , compared to control cells . neurons at 5 div were pre - treated with tat - hafgf14154 ( 60  ng / ml [ t60 ] , 300  ng / ml [ t300 ] , and 1,500  ng / ml [ t1500 ] ) for 2  hr at  37c . 
a was added to the culture medium with continuous tat - hafgf14154 treatment for 48  hr . upon treatment with tat - hafgf14154 , cell viability
was significantly increased to 84.5%   3.0% ( t60  + a ) , 90.4%   3.5% ( t300  + a ) , and 97.4%   1.9% ( t1500  + a ) , compared with untreated cells ( p  < 0.01 for t60  + a , p  < 0.001 for t300  + a , and p  < 0.001 for t1500  + a ; figure  4a ) .
nuclear staining ( hoechst 33342 ) revealed that the prevalence of karyopyknosis or apoptotic body formation induced by a142 was diminished by tat - hafgf14154 in a concentration - dependent manner ( p  < 0.05 for t60  + a , p  < 0.001 for t300  + a , and p  < 0.001 for t1500  + a ; figures 4b and 4c ) .
morphologic structure of neurons was observed by scanning electron microscopy ( figure  4d ) . as showed in micrographs ,
somata were flat and fractured and neurites were fragmented in neurons injured by a. this damaged appearance was reversed by the treatment with tat - hafgf14154 in a concentration - dependent manner.figure  4tat - hafgf14154 protected neurons from damage induced by a142primary cortical neurons were isolated from brains of newborn sd rats .
div5 ( 5  days in  vitro ) neurons were pre - treated with tat - hafgf14154 ( 60 , 300 , and 1,500  ng / ml ) for 2  hr at 37c , and 1.5  m a142 was added into the culture medium with continuous tat - hafgf14154 treatment for 48  hr .
( a ) cell viability was detected by mtt assay ( n  = 6 ) .
( b ) representative immunofluorescence images of hoechst 33342 staining ( scale bar , 20  m ) .
( c ) the percentage of cells exhibiting apoptotic bodies or karyopyknosis ( blue ) was calculated ( n  = 20 ) .
( d ) representative images of cell surface structure were captured by scanning electron microscope ( scale bar , 4  m ) .
the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or shrinking somata .
values are presented as means   sem ( * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
tat - hafgf14154 protected neurons from damage induced by a142 primary cortical neurons were isolated from brains of newborn sd rats .
div5 ( 5  days in  vitro ) neurons were pre - treated with tat - hafgf14154 ( 60 , 300 , and 1,500  ng / ml ) for 2  hr at 37c , and 1.5  m a142 was added into the culture medium with continuous tat - hafgf14154 treatment for 48  hr .
( a ) cell viability was detected by mtt assay ( n  = 6 ) .
( b ) representative immunofluorescence images of hoechst 33342 staining ( scale bar , 20  m ) .
( c ) the percentage of cells exhibiting apoptotic bodies or karyopyknosis ( blue ) was calculated ( n  = 20 ) .
( d ) representative images of cell surface structure were captured by scanning electron microscope ( scale bar , 4  m ) .
the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or shrinking somata .
values are presented as means   sem ( * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) . to further estimate the degree of injury in neurites , two synaptic markers , synaptophysin ( syn ) and growth - associated protein 43 ( gap43 ) , were assessed by immunofluorescence and western blotting in primary cortical neurons ( figure  s4 ) . both assays revealed that levels of syn and gap43 were suppressed by a ( p  < 0.05 or p  <  0.01 ) but that this was successfully rescued by the tat - hafgf14154 treatment ( p  < 0.01 or p  < 0.001 )
. levels of syn and gap43 were also assessed in the brains of app / ps1 mice by immunohistochemistry and western blotting ( figure  s5 ) .
the immunohistochemistry detected syn and gap43 located in different regions of hippocampus in wild - type healthy control mice ( figure  s5a ) .
syn was mainly located in the dentate gyrus ( dg ) area , whereas gap43 was mainly located in ca1 area .
therefore , the effects of treatment on the levels of syn in the dg area ( figures s5b and s5d ) and gap43 in the ca1 area ( figures s5c and s5e ) were assessed .
l  +  t600 increased the levels of syn  in the dg area ( p  < 0.01 ; figure  s5d ) and gap43 in the ca1 area ( p  < 0.001 ; figure  s5e ) compared with vehicle .
western blotting for syn and gap43 ( figures s5f  s5j ) validated these conclusions .
although there were no significant changes of syn and gap43 levels in the cortex ( figures s5 g and s5h ) , l  + t600 enhanced the levels of  syn ( p  < 0.05 ; figure  s5i ) and gap43 ( p  < 0.05 ; figure  s5j ) in the hippocampus compared with vehicle .
t600 did not change the expression levels of syn and gap43 in the cortex and hippocampus ( figures s5d  s5j ) .
the expression of adam10 , both mrna and protein , in primary neurons was significantly enhanced by tat - hafgf14154 ( p  < 0.05 for t300  + a and p  < 0.01 for t1500  + a in mrna level , figure  5a ; p  < 0.01 for t1500  + a in protein level , figure  5c ) .
adam10 activity in primary neurons was also elevated by tat - hafgf14154 ( p  < 0.01 for t300  + a and p  < 0.001 for t1500  + a ; figure  5d ) .
the enhancement of adam10 in expression and enzyme activity both in  vivo ( figure  3 ) and in  vitro indicated that adam10 might be a key target for tat - hafgf14154 to protect neurons from injury in ad models.figure  5tat - hafgf14154 enhanced expression and activity of adam10 in primary neurons injured by a142expressions of adam10 mrna and protein levels were assayed by qrt - pcr and western blotting , respectively .
( b and c ) representative immunoblots ( b ) and quantification ( c ) of adam10 expression .
all values are presented as means   sem ( n  = 6 ; * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
tat - hafgf14154 enhanced expression and activity of adam10 in primary neurons injured by a142 expressions of adam10 mrna and protein levels were assayed by qrt - pcr and western blotting , respectively .
( b and c ) representative immunoblots ( b ) and quantification ( c ) of adam10 expression .
all values are presented as means   sem ( n  = 6 ; * p  < 0.05 , * * p  < 0.01 , and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
od , relative optical density . to validate this possibility , small interfering rna ( sirna ) targeting of adam10 was tested in rat primary cortical neurons at 5 div .
knockdown was confirmed over 2472  hr by qrt - pcr and western blotting , and it was not affected by a treatment for 48  hr ( figure  s6 ) .
normal neurons ( t1500  + a ) or adam10-knockdown ( kd ) neurons ( kd  + t1500  + a ) were incubated with tat - hafgf14154 and a142 as described above .
the sirna knockdown blocked the elevation of adam10 induced by tat - hafgf14154 ( p  < 0.01 for a and p  < 0.001 for kd  + t1500  + a ; figure  6b ) , and it significantly impaired the ability of t1500 to rescue a toxicity ( p  < 0.01 ; figure  6c ) and a damage to soma and neurites ( figure  6d).figure  6knockdown of adam10 by sirna blocks tat - hafgf14154 neuroprotectionneurons were treated with or without a142/tat - hafgf14154/sirna as described above .
( a and b ) expression of adam10 in neurons was detected by western blotting .
the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas .
all values are presented as means   sem ( * * p  < 0.01 and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
knockdown of adam10 by sirna blocks tat - hafgf14154 neuroprotection neurons were treated with or without a142/tat - hafgf14154/sirna as described above .
( a and b ) expression of adam10 in neurons was detected by western blotting .
the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas .
all values are presented as means   sem ( * * p  < 0.01 and * * * p  < 0.001 , one - way anova with bonferroni post hoc test ) .
x - box - binding protein 1 ( xbp1 ) is one of the transcription factors of adam10 , and it is also linked with the unfolded protein response ( upr ) .
spliced xbp1 protein ( xbp1s ) is the activated form of xbp1 , and the ratio of xbp1s and unspliced xbp1 ( xbp1u ) reflects the activity of xbp1 .
after nuclear translocation , xbp1s binds to its binding site on  the adam10 mrna promoter and promotes transcription .
xbp1s / xbp1u was significantly increased by l  + t600 in the cortex ( p  < 0.05 ; figure  s7b ) , by t600 and l  + t600 in the hippocampus ( p  < 0.01 for t600 and p  < 0.05 for l  + t600 ; figure  s7c ) compared to vehicle - treated mice , indicating that xbp1 was activated by tat - hafgf14154 .
primary cortical neurons in  vitro were used to confirm these results and explore the signaling pathway related to the upregulation of adam10 by tat - hafgf14154 .
inositol - requiring enzyme 1 ( ire1 ) , one of the three endoplasmic reticulum ( er ) stress sensors , can active xbp1 by splicing xbp1u to xbp1s .
phosphoinositide 3-kinase ( pi3k ) is one of the major signaling mediators of afgf , and its activation can be blocked by ly294002 .
regulatory subunits p85 of pi3k promote the nuclear translocation of xbp1s.35 , 36 , 37 camp response element - binding protein ( creb ) is activated in ad models by pi3k / akt , and its phosphorylation is neuroprotective.38 , 39 creb is also activated by er stress , and it binds to the promoter of the ire1 gene and regulates its expression .
based on these findings , we hypothesized that tat - hafgf14154 promotes the expression of adam10 through this pi3k - creb - ire1/xbp1 pathway .
levels of xbp1u , xbp1s , p - pi3k ( p - p85 and tyr467 ) , pi3k ( p85 ) , p - creb ( ser133 ) , and creb were assayed by western blotting , and the effects of pi3k inhibitor ( ly294002 ) were assessed ( figures 7a7e ) .
after treatment with tat - hafgf14154 , xbp1s / xbp1u , phosphorylation of pi3k ( p - pi3k / p - p85 and tyr467 ) , and phosphorylation of creb ( p - creb and ser133 ) were significantly increased in neurons injured by a ( p  < 0.01 ) , and they were suppressed by pi3k inhibitor ( p  < 0.001 in figures 7c and 7d and p  < 0.01 in figure  7e ) .
these results fueled our hypothesis that the pi3k - creb - ire1/xbp1 pathway was activated by tat - hafgf14154 .
pi3k inhibition abolished the rise in adam10 expression seen in neurons treated with t1500  + a ( p  < 0.01 for p  + t1500  + a ; figures 7f and 7 g ) as well as thwarted hafgf14154 rescue of cell viability ( p  <  0.001 for p  + t1500  + a ; figure  7h ) and morphological damage ( figure  7i).figure  7tat - hafgf14154 upregulated the expression of adam10 via the pi3k - creb - ire1/xbp1 pathwayprimary neurons were pre - treated for 30  min with pi3k inhibitor ( ly294002 , p ) , followed by incubation with tat - hafgf14154 and a142 as described above .
( a and b ) representative immunoblots of xbp1s , xbp1u , p - pi3k(p - p85 and tyr467 ) , pi3k(p85 ) , p - creb ( ser133 ) , and creb .
( c  e ) quantitative analysis for xbp1s / xbp1u ( c ) , expression of pi3k p85 and p - p85 ( d ) , and p - creb ( ser133 ) and creb ( e ) . ( f and g ) upregulation of adam10 induced by tat - hafgf14154 was blocked by the pi3k inhibitor .
( i ) the pi3k inhibitor subverted the improvement effect of tat - hafgf14154 on cell morphology .
the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas .
all values are presented as means   sem ( n  = 6 ; * p  < 0.05 , * * p  < 0.01 , and * * * p  <  0.001 , one - way anova with bonferroni post hoc test for c , g , and h , two - way anova with bonferroni s post hoc test for d and e ) .
p , pi3k inhibitor ly294002 ; xbp1s , spliced xbp1 protein ; xbp1u , unspliced xbp1protein ; od , relative optical density .
tat - hafgf14154 upregulated the expression of adam10 via the pi3k - creb - ire1/xbp1 pathway primary neurons were pre - treated for 30  min with pi3k inhibitor ( ly294002 , p ) , followed by incubation with tat - hafgf14154 and a142 as described above .
( a and b ) representative immunoblots of xbp1s , xbp1u , p - pi3k(p - p85 and tyr467 ) , pi3k(p85 ) , p - creb ( ser133 ) , and creb .
( c  e ) quantitative analysis for xbp1s / xbp1u ( c ) , expression of pi3k p85 and p - p85 ( d ) , and p - creb ( ser133 ) and creb ( e ) . ( f and g ) upregulation of adam10 induced by tat - hafgf14154 was blocked by the pi3k inhibitor .
( i ) the pi3k inhibitor subverted the improvement effect of tat - hafgf14154 on cell morphology .
the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas .
all values are presented as means   sem ( n  = 6 ; * p  < 0.05 , * * p  < 0.01 , and * * * p  <  0.001 , one - way anova with bonferroni post hoc test for c , g , and h , two - way anova with bonferroni s post hoc test for d and e ) .
p , pi3k inhibitor ly294002 ; xbp1s , spliced xbp1 protein ; xbp1u , unspliced xbp1protein ; od , relative optical density .
ad has become a major public health problem owing to the increasing prevalence , long course of disease , burden on caregivers , and high financial cost of care .
new effective treatments that will prevent , delay , or treat the symptoms of ad are urgently needed .
neuroprotection of afgf has been widely reported over the recent years,15 , 42 , 43 yet its mechanism is still uncertain . in our study ,
tat - hafgf14154-loaded cationic liposomes were delivered to app / ps1 mice via intranasal administration to investigate neuroprotection of tat - hafgf14154 , and the related mechanism was explored in primary cortical neurons of rats injured by a142 .
one of the great challenges for drugs targeting neurodegenerative diseases is that the bbb restricts the access of drugs to the brain by oral and intravenous administration .
intranasal delivery has proven to be a new and noninvasive strategy , which can circumvent the bbb and facilitate direct transport of the drugs to the brain through neuronal and extracellular pathways.44 , 45 our previous studies showed that intranasal administration of tat - hafgf could improve cognition and reduce a deposits more significantly in app / ps1 mice compared with intravenous injection.18 , 21 , 22 , 23 in an animal model of parkinson s disease induced by 6-ohda , liposomes markedly assisted the delivery of basic fibroblast growth factor ( bfgf ) to the striatum and substantia nigra ( sn ) , and they enhanced the neuroprotective effects of bfgf on dopaminergic neurons .
liposomes also enhanced the levels of afgf significantly in the ec , accompanied by amelioration in cognitive and non - cognitive behavioral deficits .
consistent with the behavioral improvement , a burden was reduced in both the cortex and hippocampus of brain by intranasal treatment with tat - hafgf14154-loaded cationic liposomes .
a originates from proteolysis of app by the sequential enzymatic actions of bace1 and ps1 , called app amyloidogenic processing .
conversely , adam10 precludes a generation by cleaving app within the eventual a sequence , called non - amyloidogenic processing .
there was a marked increase of adam10 in both expression and activity in the brains of mice with treatment of tat - hafgf14154-loaded cationic liposomes .
adam10-mediated non--amyloidosis processing via enhancing expression and activity of adam10 may be involved in the amelioration of behavioral deficits and the  reduction of a burden in app / ps1 mice treated with tat - hafgf14154 .
recent reports have provided several candidate drugs or therapeutic methods to attenuate pathology in ad ; most of them show its mechanism of modulating app processing so as to alter app toward non - amyloidogenic processing.30 , 47 , 48 , 49 , 50 overexpression of adam10 impressively reduced a plaque load and soluble a , and it additionally restored learning deficits in double - transgenic alzheimer model mice .
adam10 is a metalloprotease with multiple functions in the brain . besides the -secretase in app processing , adam10 also plays a critical role in regulating functional membrane proteins at the synapse
. postnatal disruption of the adam10 in mouse brain caused epileptic seizures , learning deficits , altered spine morphology , and defective synaptic functions . with the upregulation of adam10 , tat - hafgf14154 dose - dependently enhanced cell viability , and it reversed damage in soma and neurites of neurons damaged by  a.  tat - hafgf14154 increased synaptic proteins syn and gap43 significantly both in  vitro and in  vivo , indicating that tat - hafgf14154 promoted nerve growth and possessed the potential of modulating synaptic plasticity .
after sirna knockdown of adam10 , damage in soma and neurites induced by a could not be reversed by the tat - hafgf14154 treatment with a decrease in cell viability , which suggested that adam10 is a key target for tat - hafgf14154 to protect neurons from injury in the ad model .
pischedda and piccoli also reported that pharmacological inhibition of adam10 results in impairment of neurite outgrowth , and this modulation is involved in the fgfr2-signaling pathway .
recent progress in understanding the substrates and function as well as the regulation and cell biology of adam10 in the cns highlights the value of adam10 as a drug target in brain diseases . according to our results that tat - hafgf14154 promoted mrna expression of adam10
xbp1 efficiently upregulated adam10 expression ; a reduced activity or presence of xbp1 could potentially be associated with an increased plaque burden and progression of ad .
pc12 cells overexpressing mxbp1s were fully protected at lethal doses of a oligomers ( 18  mg / ml ) and partially protected at higher concentrations of a. endogenous xbp1 plays a critical role in preventing a neurotoxicity , as shown by the exacerbated cell death induced by a in ad models when xbp1 was eliminated .
ire1 cleaves xbp1 pre - mrna encoding for xbp1u in the cytoplasm , and this cytoplasmic splicing results in a new protein with transcriptional activity called xbp1s .
mammalian ire1 initiates diverse downstream signaling of the upr either through splicing of  xbp1 pre - mrna to maintain cell survival or through posttranscriptional modifications via regulated ire1-dependent decay ( ridd ) of  multiple substrates , which triggers cell apoptosis.55 , 56 tat - hafgf14154 enhanced the ratio of xbp1s / xbp1u in the brain of app / ps1 mice and primary neurons injured by a , indicating that splicing xbp1 was activated by tat - hafgf14154 .
nuclear translocation of active xbp1s could be improved by the overexpression of pi3k regulatory subunits p85 , and deletion of p85 showed a reduced accumulation of xbp1s in the nucleus , revealing a link between pi3k and the ire1/xbp1 pathway .
the pi3k pathway is one of the most important intracellular signaling cascades induced by afgf too .
phosphorylation of pi3k was upregulated by tat - hafgf14154 in primary neurons damaged by a , but no change of pi3k expression was found .
it meant that tat - hafgf14154 activated the pi3k pathway but did nt influence the translocation of xbp1s .
creb is critically involved in the regulation of synaptic plasticity , intrinsic excitability , and long - term memory formation .
its activation exhibits neuroprotection via the pi3k / akt / creb pathway in ad models.38 , 39 creb is also activated by er stress : a chromatin immunoprecipitation assay indicated that creb binds to the promoter region of ire1 genes and regulates its expression .
expression of ire1 was increased concomitantly with creb phosphorylation in human glioma cells treated with nitric oxide ( no ) .
phosphorylation of creb was upregulated by tat - hafgf14154 , and no change of creb expression was found in primary neurons injured by a. the activation of the creb pathway and the splicing of xbp1 induced by tat - hafgf14154 were blocked by pi3k inhibitor , resulting in a disappearing rescue of cell viability and morphological damage .
these results suggest that the activation of the pi3k pathway is related to the activation of creb and the downstream splicing of xbp1 induced by ire1. in conclusion , tat - hafgf14154 treatment significantly ameliorated the behavioral deficits of app / ps1 mice , relieved a burden , and increased the expression and activity of adam10 in the brain .
tat - hafgf14154 antagonized a142-induced cell death and structure damage in primary neurons in an adam10-dependent manner following the activation of xbp1 splicing and the pi3k - creb pathway .
as shown in figure  8 , tat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the xbp1s mrna generated is translated to active xbp1s protein , a transcription factor for adam10 .
these results highlight the important role of adam10 as a key target in neuroprotection of tat - hafgf14154 , suggesting that tat - hafgf14154 has the potential to be an alternative therapy to attenuate the ad pathological process.figure  8signaling pathway for tat - hafgf14154 neuroprotectiontat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the xbp1s mrna generated is translated to active xbp1s protein , a transcription factor for adam10 .
the nuclear translocation of active xbp1s can be promoted by the pi3k regulatory subunit p85,35 , 36 , 37 but tat - hafgf14154 does not alter the level of p85. ly294002 inhibits the phosphorylation of pi3k and the splicing of xbp1 , inhibiting the expression of adam10 . signaling pathway for tat - hafgf14154 neuroprotection tat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the xbp1s mrna generated is translated to active xbp1s protein , a transcription factor for adam10 .
the nuclear translocation of active xbp1s can be promoted by the pi3k regulatory subunit p85,35 , 36 , 37 but tat - hafgf14154 does not alter the level of p85. ly294002 inhibits the phosphorylation of pi3k and the splicing of xbp1 , inhibiting the expression of adam10 .
recombinant fusion protein tat - hafgf14154 was expressed and purified from our lab as reported previously.14 , 18 liposomes were produced by the method of ph gradient .
tat - hafgf14154 was incubated with liposomes in different ratios ( 1:100 , 1:50 , 1:10 , 1:5 , 1:2 , and 4:5 ) for 30  min at 25c or 30c .
encapsulation efficiency of cationic liposomes was calculated by a formula : encapsulation efficiency ( q ) %  = ( ctotal  cfree)/ctotal  100 .
free tat - hafgf14154 ( cfree ) and total tat - hafgf14154 ( ctotal ) were separated by microcolumn centrifugation method .
the content of tat - hafgf14154-loaded cationic liposomes was assayed by human fgf acidic elisa kit  ( r&d systems ) , according to the manufacturer s instructions .
coomassie blue staining was applied after electrophoresis with 12%  sds - page to detect sds - resistant polymerization of tat - hafgf14154 after 0 , 0.5 , or 1  hr incubation with cationic liposomes .
appswe / ps1d9 double - transgenic mice ( app / ps1 ) and wild - type background - matched mice ( 7  months old ) were purchased from beijing huafukang biotechnology .
mice were kept under conditions of controlled temperature ( 24c   1c ) , humidity ( 50%60% ) , and a  light / dark cycle of 12/12  hr ( light on at 8:00 a.m. ) .
aliquots of 0.9% saline ( vehicle ) , 600  g / kg tat - hafgf14154 ( t600 ) , and 600  g / kg tat - hafgf14154-loaded cationic liposomes ( l  + t600 ) were intranasally administered as a single dose to wild - type mice , which were then sacrificed in after 30  min .
app / ps1 mice were treated with the same doses of the same agents by intranasal delivery every 2  days for 6  weeks according to the modified method of capsoni .
all experiments were conducted according to the guidelines for animal care and use of china , and they were approved by the animal ethics committee of the chinese academy of medical science .
after treatment for 5  weeks , learning and memory abilities of mice were assayed by mwm .
the maze consisted of a steel pool ( 120-cm diameter ) filled with opaque water at 22c   1c and a steel platform ( 10  cm ) .
an overhead video camera connected to ethovsion xt ( noldus information technology ) was used to track the movement of mice and record all trials . in the place navigation test ,
the hidden platform ( 1  cm under water level ) was kept constant in the middle of one quadrant throughout training .
the training consisted of 6-day trials ( one trial per quadrant and four quadrants per day , with a 20-min interval between each trial ) .
each trial lasted until the mouse climbed onto the hidden platform target within 60 s , and the escape latency onto the platform and the path length were recorded .
the spatial probe test without the platform was conducted 24  hr after completing the last training trial ( day 5 ) .
each mouse was put into the pool for 60  s from the most distant to the target quadrant , and the percentage of time spent on each quadrant and times of crossing the removed - platform area were measured .
mice were transferred to individual cages with bedding but no  environmental enrichment items 1  hr before the dark phase commenced .
the mouse was given access to a piece of cotton gauze ( 5-cm squares , weighing 3 g ) in each cage .
the next morning the nesting score of each mouse was calculated according to a standard five - point nest - rating scale .
the chamber was a brightly and evenly illuminated square area ( 50   50   25  cm ) made of white glacial polyvinyl chloride and illuminated with four 60-w lamps ( mounted 1.5  m above ) .
mice were placed individually in the center of the of , left to explore for 5  min , and videotaped under white illumination .
time spent in the  center , number of crossings through the central area , and total distance movement were recorded and analyzed by ethovision xt ( noldus information technology ) .
the right hemisphere of app / ps1 mice was fixed in 4% paraformaldehyde ( guoyao ) and embedded with paraffin .
coronal paraffin sections ( 6-m thick ) were dewaxed , rehydrated , and treated with 3% h2o2/methanol solution for 10  min .
then , sections were boiled for 15  min in 0.01  m citrate buffer ( ph 6.0 ) .
after the citrate buffer returned to room temperature , sections were washed with pbs and incubated in blocking solution ( 5% bsa ) for 1  hr at 37c , followed by being incubated overnight with primary antibodies at 4c in a humidified chamber .
primary antibodies included anti - synaptophysin ( 1:250 , abcam ) and anti - gap43 ( 1:250 , abcam ) rabbit antibodies and mouse monoclonal anti - a antibody ( 6e10 , 1:500 , covance ) .
after being washed with pbs , the sections were incubated with anti - mouse or rabbit igg - horseradish peroxidase ( hrp ) secondary antibody for 1 
hr at 37c , followed by diaminobenzidine ( dab ) ( boster biotech ) and hematoxylin staining .
slides were assessed by light microscope ( ix71 , olympus ) and micrographs were analyzed by image - pro plus 6.0 software .
tissue homogenates and cultured cells were lysed in radioimmunoprecipitation assay ( ripa ) lysis buffer ( beyotime ) with pmsf ( sigma - aldrich ) and protease inhibitor cocktail ( roche ) on ice .
lysates in supernatant were separated by centrifuging at 13,000  rpm at 4c for 15  min .
the concentration of total protein was measured by  bca protein assay kit ( thermo fisher scientific ) .
samples were electrophoresed in nupage 4%12% bis - tris gels ( thermo fisher scientific ) with nupage mes sds running buffer ( thermo fisher scientific ) , and they were electrophoretically transferred to polyvinylidene difluoride ( pvdf ) membranes ( millipore ) . after being dipped in 0.2% glutaraldehyde ( sigma - aldrich ) for 30  min , membranes were blocked for 12  hr at room temperature and then incubated at 4c overnight with the following primary antibodies : mouse afgf monoclonal antibody ( 1:1,000 , sigma ) , rabbit anti - synaptopysin antibody ( 1:1,000 , abcam ) , rabbit anti - gap43 antibody ( 1:1,000 , abcam ) , 6e10 mouse monoclonal antibody ( 1:2,000 , covance ) , adam10 rabbit monoclonal antibody , bace1 rabbit monoclonal antibody , ps1  rabbit monoclonal antibody ( 1:1,000 , abcam ) , anti - app(c - terminal ) rabbit monoclonal antibody ( 1:4,000 , sigma ) , phospho - pi3k p85 / p55 ( tyr 467/tyr 199 ) rabbit antibody , pi3 kinase p85 / p55 rabbit monoclonal antibody ( 1:1,000 , bioworld ) , -actin rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) , xbp-1 rabbit polyclonal antibody ( 1:1,000 , abcam ) , creb rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) , phospho - creb ( ser 133 ) rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) , and gapdh rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) .
after incubation with secondary antibody ( peroxidase - conjugated affinipure goat anti - rabbit / mouse igg , 1:2,000 , proteintech ) for 2  hr at room temperature , the blots were detected with supersignal west pico chemiluminescent substrate ( pierce biotechnology ) .
activities of adam10 and bace1 were measured by sensolyte 520 adam10 activity assay kit ( anaspec ) and -secretase ( bace1 ) activity detection kit ( sigma - aldrich ) , according to the manufacturers instructions .
primary cortical neurons were separated from brains of newborn sprague - dawley ( sd ) rats .
tissues were cut rapidly into small pieces in d - hank s buffer , then digested with 0.25% trypsin for 10  min at 37c .
dmem ( high glucose , thermo fisher scientific ) containing 10% fetal bovine serum ( fbs ) ( thermo fisher scientific ) was then added .
the cell suspension was filtered , then centrifuged at 250   g for 5  min .
cells were cultured in neurobasal - a medium ( thermo fisher scientific ) supplemented with 2% b-27 supplement ( 50 , serum free , thermo fisher scientific ) at 37c .
small tissue mass and dead cells were removed by changing culture medium 46  hr after seeding .
neurons ( 5 div ) were pre - treated with tat - hafgf14154 ( 60 , 300 , and 1,500  ng / ml ) for 2  hr at 37c , and 1.5  m a142 ( millipore ) was added into the culture medium with continuous tat - hafgf14154 treatment for 48  hr .
a142 was dissolved in 0.1% dmso and incubated in 37c for 24  hr . in some cases ,
cells were treated with 50  nm pi3k inhibitor ( ly294002 , cell signaling technology ) 30  min before tat - hafgf14154 treatment . for viability analysis , cells were seeded in a 96-well culture plate at a density of 1   10 cells / well .
after treatments , cells were incubated with 10  ml 5  mg / ml methyl thiazolyl tetrazolium ( mtt ) ( sigma ) for 4  hr at 37c .
the supernatants were aspirated , and the formazan precipitates were solubilized by the addition of 100  l dmso per well .
absorbance at 570  nm was monitored by a multiskan go spectrophotometer ( thermo scientific ) . for apoptosis analysis
, cells were seeded in a six - well culture plate at a density of 1.5   10 cells / well .
after treatments , cells were washed with pbs and fixed with 4% paraformaldehyde for 10  min at room temperature , then exposed to hoechst 33342 ( 5  g / ml )
images were recorded by interactive laser cytometer ( lsm 710 meta , carl zeiss ) .
cells were seeded on 15-mm circular glass coverslips ( nest ) in a 24-well culture plate .
after treatments , samples were fixed with glutaraldehyde ( sigma - aldrich ) overnight at 4c , followed by being rinsed and dehydrated in an alcohol gradient ( 30% , 50% , 70% , and 90% for 3  min each and 100% for 15  min ) .
cells on the coverslips were incubated with isoamyl acetate ( dongzheng ) for 5  min twice , dried by a critical point dryer ( emitech k850x , quorum ) , followed by gold - palladium sputtering .
the coverslips were fixed on specimen mount with conductive adhesive , and the cell surfaces were examined by scanning electron microscope ( carl zeiss ) . to detect the location and expression of synaptic proteins ,
primary cortical neurons were seeded in 35-mm confocal dishes ( corning ) at a density of 1.5   10 cells / well .
after treatments , cells were fixed with 4% paraformaldehyde ( pfa ) for 15  min and permeabilized with 0.1% triton x-100 ( sigma ) for 20  min at room temperature , followed by incubation with blocking buffer ( beyotime ) for 1  hr at room temperature .
then , samples were incubated with primary antibodies overnight at 4c , including rabbit anti - gap43 antibody ( 1:250 , abcam ) and rabbit anti - synaptopysin antibody ( 1:250 , abcam ) .
after  being washed with pbs , cells were incubated with anti - rabbit igg - h&l secondary antibody ( dylight 488 , 1:200 , abcam ) for 1  hr at  37c and kept in the dark , followed by dapi nuclear staining ( 0.5  g / ml , beyotime ) for 15  min .
images of cells were recorded by  interactive laser cytometer ( lsm 710 meta , carl zeiss ) , and micrographs were analyzed using image - pro plus 6.0 software .
total rna of cultured cells was extracted with hipure total rna mini kit ( magen ) and quantified by nanodrop 2000 ( thermo fisher scientific ) .
the iscript cdna synthesis kit ( bio - rad ) was used to  synthesize the first - strand cdna .
the qpcr was performed with  ssoadvanced universal supermixes ( bio - rad ) by cfx96 connect real - time pcr detection system ( bio - rad ) .
-actin was used as an internal control and its primers were cccgcgagtacaaccttcttg ( forward ) and tcatccatggcgaactggtgg ( reverse ) .
the sirna duplexes against adam10 were designed by us and synthesized by genepharma with the following sequences : ccagcagagagauauauuatt ( sense ) and uaauauaucucucugcuggtt ( anti - sense ) .
scrambled - sirna sequences were uucuccgaacgugucacgutt ( sense ) and acgugacacguucggagaatt ( anti - sense ) .
primary cortical neurons were seeded on six - well  plates at a density of 1.5   10 cells / well .
cells ( 5 div ) were transfected with 20  nm scrambled - sirna or targeted - sirna using lipofectamine rnaimax reagent ( invitrogen ) .
the knockdown efficiency was validated by qrt - pcr and western blot after sirnas were transfected for 48 or 72  hr .
one - way anova followed by bonferroni post hoc test or two - way  anova followed by bonferroni multiple comparison tests was used for data analyses with statistics software ( spss 19.0 ) .
all experimental data represent means   sem , and p  < 0.05 was considered statistically significant .
 acid fibroblast growth factor ( afgf ) has shown neuroprotection in alzheimer s disease ( ad ) models in previous studies , yet its mechanism is still uncertain . here 
 we report that the efficacy of tat - hafgf14154 is markedly increased when loaded cationic liposomes for intranasal delivery are intranasally administered to app / ps1 mice . 
 our results demonstrated that liposomal tat - hafgf14154 treatment significantly ameliorated behavioral deficits , relieved brain a burden , and increased the expression and activity of disintegrin and metalloproteinase domain - containing protein 10 ( adam10 ) in the brain . 
 tat - hafgf14154 antagonized a142-induced cell death and structural damage in rat primary neurons in an adam10-dependent manner , which , in turn , was promoted by the activation of xbp1 splicing and modulated by the pi3k - creb pathway . 
 both knockdown of adam10 and inhibition of  pi3k ( ly294002 ) negated tat - hafgf14154 rescue . 
 thus , tat - hafgf14154 activates the ire1/xbp1 pathway of the unfolded protein response ( upr ) against the endoplasmic reticulum ( er ) stress induced by a , and , subsequently , the nuclear translocation of spliced xbp1 ( xbp1s ) promotes transcription of adam10 . 
 these results highlight the important role of adam10 and its activation through the pi3k - creb - ire1/xbp1 pathway as a key factor in the mechanism of neuroprotection for tat - hafgf14154 . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest