clathrin - mediated endocytosis ( cme ) is an evolutionarily conserved process that cells use to internalize specific components of the plasma membrane [ 1 , 2 ] . in neurons ,
cme plays a particularly important role , functioning both on the presynaptic and postsynaptic sides of neuronal synapses [ 3 , 4 ] .
numerous studies have revealed that cme regulates the recycling of synaptic vesicles ( svs ) at presynaptic membranes [ 4 , 5 ] , mediates the internalization of neurotransmitter receptors , and contributes to synaptic plasticity by controlling postsynaptic excitability at postsynaptic membranes [ 58 ] .
for instance , endophilin1 is expressed only in the brain , endophilin2 exists in multiple tissues , and endophilin3 is found mainly in the brain and testis [ 1214 ] .
endophilin dysfunction has been linked to both cancer and neurodegenerative diseases [ 9 , 1517 ] .
microinjection of endophilin antibodies into giant axons causes a stimulus - dependent depletion of sv in lamprey .
mutant drosophila larvae lacking endophilin fail to take up fm1 - 43 dye , indicating an inability to retrieve synaptic membranes .
endophilin1 assists in downregulating epidermal growth factor receptor ( egfr ) and other growth factor receptors .
similarly , overexpression of endophilin2 in 293 t cells increases egf - induced endocytosis of egfrs .
the cos-7 cells transfected with endophilin1 do not affect transferrin uptake efficiency , whereas transfection with full - length endophilin3 strongly reduces it . at the subcellular level ,
all three endophilin isoforms are concentrated at synaptic terminals and in the cytosol of neurons .
electron microscopy of immunogold - labeled endophilin shows that endophilin is localized at both presynaptic and postsynaptic membranes .
endophilin functions as a membrane - bending molecule and is delivered to endocytic zones by exocytosis .
endophilin2 and endophilin3 , but not endophilin1 , are involved in arc / arg3.1-mediated ampa receptor endocytosis on postsynaptic membranes , suggesting that isoform specificity can confer particular properties to specific endocytic pathways . at the presynaptic terminal , a series of studies revealed that the functional domains of endophilin affect several stages of cme [ 9 , 10 ] .
an amino acid sequence analysis showed that all three endophilin isoforms consist of an n - terminal bin / amphiphysin / rvs ( bar ) domain , a variable middle region , and a c - terminal src homology-3 ( sh3 ) domain .
the bar domain mainly participates at the base of the membrane invagination , an early step in endocytosis of the membrane .
the sh3 domain is involved in the interaction of endophilin with other endocytic machinery molecules that contain a proline - rich domain ( prd ) , such as synaptojanin and gtpase dynamin [ 1214 ] . disrupting the interactions perturbs the fission and uncoating of synaptic clathrin - coated vesicles .
our previous studies found an unconventional prd embedded within endophilin2 , and the intramolecular interaction between this unconventional prd and the c - terminal sh3 domain is ca - dependent .
further biochemical studies demonstrated that endophilin1 has a similar ca - dependent interaction with voltage - gated ca channels and dynamin as endophilin2 , whereas endophilin3 does not .
these findings imply that these structurally similar endophilin isoforms may have distinct characteristics in interactions with other endocytic machineries .
however , the precise functional differences of each endophilin isoform on presynaptic sv endocytosis remain unknown . here
we found that sv endocytosis was impaired in endophilin1 or endophilin2 knockdown neurons but was unaffected in endophilin3 knockdown neurons .
our results indicate that endophilin1 or endophilin2 , but not endophilin3 , is the main regulatory machinery involved in clathrin - mediated sv endocytosis .
all animal procedures were performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals produced by the national institutes of health .
the protocol was approved by the institutional animal care and use committee at zhongshan school of medicine in sun yat - sen university .
neurons cultured in 24-well culture plates at 1214 days in vitro were used to perform the transfection .
endophilin sirna ( 100  pmol ) was combined with 37  l of 2.5  m cacl2 solution in sterile , deionized water for a final volume of 300  l and then mixed well with 300  l of 2x hepes - buffered saline .
the mixture was vortexed and incubated at 25c for 15  min . in each well , 30  l of the mixture was added dropwise to the cells and allowed to incubate for 40  min .
the full - length endophilin1 , endophilin2 , and endophilin3 cdna fragments were subcloned into pegfp - c1 plasmids ( clontech ) .
a detailed description of the methods used for constructing cdna plasmids is available in our previous studies [ 25 , 26 ] .
the 19-nucleotide sirnas for each endophilin isoform and their related negative controls ( nc , scrambled sequence ) were designed using online software ( http://www.promega.com/sirnadesigner/ ) and were synthesized by shanghai gene - pharma co. , ltd .
these included the si1 - 1- and si1 - 2-targeted sequences 5-gggctaaactcagtatgat-3 and 5-ccgacgcttagactttgat-3 of endophilin1 mrna ( nm_053935 ) , si2 - 1- and si2 - 2-targeted sequences 5-gaggttctattacctttct-3 and 5-gcttcgtcatcatttagat-3 of endophilin2 mrna ( nm_031239 ) , and si3 - 1- and si3 - 2-targeted sequences 5-gccgaaagaagcatgttta-3 and 5-gcttcgtcatcatttagat-3 of endophilin3 mrna ( nm_031238 ) .
human embryonic kidney ( hek ) 293 cell culture was performed as described previously . to determine the efficacy and specificity of sirnas , cotransfection of 100  pmol of sirnas or nc together with 2  g of the corresponding endophilin - pegfpc1 plasmids into hek293 cells
the cells were lysed and separated 48  h after cotransfection using 10% sds - page and electrophoretically transferred to pvdf membranes ( pierce , rockford , il ) .
the membranes were blocked with 5% nonfat milk in tbs and 0.1% tween-20 and then incubated with an isoform - specific endophilin antibody or anti - endophilins13 antibody ( santa cruz ) .
after three to four washes with tbs and 0.1% tween-20 , the membranes were incubated with hrp - conjugated secondary antibodies ( jackson immunoresearch ) .
the protein bands were detected after developing the blots using an ecl kit ( pierce ) .
three days after sirna transfection , the hippocampal neurons were fixed with 4% paraformaldehyde ( sigma ) .
the primary antibodies , anti - endophilin1 , 2 , or 3 , were used at a dilution of 1  :  100 , and donkey anti - goat igg ( h + l ) dylight 549 ( jackson immunoresearch ) was used at a dilution of 1  :  500 . after staining ,
the cells were mounted on glass slides using fluoro gel ii with dapi ( ems ) and imaged with a carl zeiss lsm 710 confocal microscope .
images were acquired with the same optical slice thickness in every channel using a 63x oil objective and a resolution of 1024  1024 pixels . the rna interference efficiency in hippocampal neurons was determined by calculating the percentage of endophilin - positive cells , as previously described .
excitatory postsynaptic currents ( epscs ) were recorded in cultured hippocampal neurons using dual whole - cell recordings by evoking an action potential ( ap ) or a train of aps in transfected neurons .
the aps were evoked in transfected neurons by delivering brief ( 3  ms ) and large ( 300400  pa ) depolarizations in current - clamp mode , and epscs were recorded in voltage - clamp ( 70  mv ) mode in a nearby , nontransfected neuron . for the input - output relationship of aps , aps were evoked using a 400  ms current injection at various intensities ( 0200  pa ) . to measure the depletion of the readily releasable pool ( rrp ) of svs
, the transfected neurons were challenged with high - frequency stimulation ( 100 pulses at 5  hz ) . to compare the size of the frequency - dependent depression of epscs during high - frequency stimulation ,
the 2nd to 100th responses were normalized to the 1st response , and the last 20 responses were averaged and normalized to the 1st response to compare among the groups . to measure the replenishment of the rrp , the transfected neurons were stimulated with consecutive high - frequency stimulation . for consecutive high - frequency stimulation ,
four trains of stimulation ( 5  hz , 100 stimuli ) were applied with a 3  min interval , during which the transfected neuron was stimulated at 0.1  hz . to compare the recoveries of the rrp , the epsc amplitudes recorded at 0.1  hz after the 1st , 2nd , 3rd , and 4th train were normalized to the amplitude recorded before the 1st train .
the experiment was conducted at room temperature using an epc-10 patch - clamp amplifier and patchmaster software ( heka electroniks ) .
the epscs were recorded with a patch electrode ( 35  m tip resistance ) and filtered at 2  khz .
the extracellular bath solution contained ( in mm ) nacl , 128 ; kcl , 5 ; cacl2 , 2 ; mgcl2 , 1 ; glucose , 15 ; and hepes , 20 ( ph 7.3 ) .
the pipette solution contained ( in mm ) : kcl , 147 ; na2-phosphocreatine , 5 ; egta , 2 ; mgatp , 2 ; na2gtp , 0.3 ; and hepes , 10 ( ph 7.2 ) .
the statistical significance of the differences was analyzed using student 's t - test between two groups and one - way anova with newman - keuls post hoc tests for comparisons among more than two groups .
the efficacy of the designed sirnas against the endophilin isoforms was tested in hek293 cells .
at least one interference fragment selectively inhibited the expression of its corresponding endophilin ( figure 1(a ) ) .
the specificity of each endophilin isoform sirna was also determined . as shown in figure 1(b ) , isoform - specific sirnas did not reduce the expression of other endophilin isoforms .
as shown in figure 1(c ) , compared with the nc , the expression of endogenous endophilin1 was significantly knocked down in the endo1 sirna - transfected neurons .
similar results for the sirna effect were also observed with the other two endophilin isoforms ( data not shown ) .
isoform - specific sirna resulted in 7080% knockdown of the corresponding endogenous endophilin ( figure 1(d ) ) .
these results indicate that the designed endophilin isoform - specific sirnas are effective . to examine whether endophilin isoforms affected sv endocytosis , we performed dual - cell patch - clamp recordings in cultured hippocampal neurons .
neurons transfected with sirna or ncs ( figure 2(a ) left , green ) were challenged with various stimulation patterns , and epscs were recorded from neighboring nontransfected cells ( figure 2(a ) right , gray ) .
we first assessed whether the intrinsic electrophysiological features of the transfected neurons were altered by transfection of sirna or nc .
the number of aps induced under the same stimulation parameters in neurons transfected with either sirna ( n = 16 , including five endo1 sirnas , five endo2 sirnas , and six endo3 sirnas ) or nc ( n = 16 ) was consistent with that in nontransfected neurons ( n = 18 ; p > 0.05 ; figure 2(c ) ) , indicating that introducing sirna or nc into cultured neurons did not change the intrinsic electrophysiological features of neurons . when stimulating at a low frequency ( 0.1  hz for 3  min ) , neurons transfected with either sirna or nc released neurotransmitters in response to brief depolarizing pulses ( figure 2(b ) , right ) .
the epsc amplitudes in endophilin isoform knockdown groups were 569.328  89.497  pa for endo1 sirna ( n = 13 ) , 478.991  97.81  pa for endo2 sirna ( n = 11 ) , and 615.125  129.694  pa for endo3 sirna ( n = 10 ) , which were not significantly different from that in the nc group ( p > 0.05 , figure 2(d ) ) .
these results suggest that knocking down endophilin isoforms has no apparent effect on sv exocytosis , consistent with a report that ablation of endophilin in drosophila yields no significant evoked excitatory junctional potential changes . when stimulating at high frequency , a reduction in the synaptic response ( called short - term synaptic depression , std )
was observed as a result of depleting the rrp of the sv ( figure 3(a ) , nc ) . at a stimulating intensity of 5  hz ( 100 stimuli ) ,
synaptic depression was enhanced in neurons transfected with endo1 sirna compared with that in neurons transfected with nc ( figures 3(a ) and 3(b ) ) .
similar results were also observed for endo2 sirna - transfected neurons ( figures 3(a ) and 3(c ) ) .
the marked std in endo1 sirna- and endo2 sirna - transfected neurons suggested that sv endocytosis was impaired .
these results were consistent with previous observations when sv endocytosis was impaired [ 27 , 29 ] .
however , the epsc amplitude in endo3 sirna - transfected neurons was not decreased compared with that in nc - transfected neurons ( figures 3(a ) and 3(d ) ) .
the mean normalized epsc amplitudes for the last 20 responses in neurons transfected with endo1 sirna and endo2 sirna were 0.266  0.029 ( n = 13 ) and 0.281  0.012 ( n = 8) , respectively , significantly lower than that in the nc group ( 0.586  0.034 ,
. however , there was no significant difference in the mean normalized epsc amplitude between the endo3 sirna ( 0.566  0.027 , n = 12 ) and nc groups ( p > 0.05 , figure 3(e ) ) . when sv endocytosis is impaired , replenishment of the rrp is also hindered .
thus , to further confirm that these three endophilin isoforms played distinct roles in sv endocytosis , presynaptic neurons were challenged with consecutive high - frequency stimulation .
four trains of stimulation ( 5  hz , 100 stimuli ) were applied with a 3  min interval , during which time the presynaptic neuron was stimulated at 0.1  hz . when multiple train stimulations were applied , a steady decrease was observed in the averaged epsc amplitude evoked at 0.1  hz during the interval between single train stimulations .
neurons transfected with endo1 sirna exhibited an accelerated decrease in the epsc amplitude from the beginning of the 1st train to the 4th train compared with neurons transfected with nc (
a similar result was also found in the endo2 sirna group ( n = 12 , p < 0.05 after the 1st train , p < 0.005 after the 2nd , 3rd , and 4th trains ; figure 3(g ) ) .
however , no significant difference was detected in the epsc amplitude between the endo3 sirna group and the nc group ( n = 9 , p > 0.05 ; figure 3(h ) ) .
these electrophysiological results suggest that replenishment of the rrp is hindered in endo1 sirna- and endo2 sirna - transfected neurons but not in endo3 sirna - transfected neurons .
to investigate the relationship between endophilin1 and endophilin2 in sv endocytosis , neurons cotransfected with endo1 sirna and endo2 sirna ( endo1 , 2 sirna ) were stimulated using single high - frequency stimulation and consecutive high - frequency stimulation .
the response of the endo1 , 2 sirna knockdown neurons during a single high - frequency stimulation revealed that the epsc amplitude rapidly decreased to reach a significantly lower plateau ( approximately 30% of the control ) compared with that in the nc group ( n = 8 , figure 4(a ) ) .
the mean normalized epsc amplitude for the last 20 responses in the endo1 , 2 sirna group was significantly lower than that in the control group ( 0.240  0.023 versus 0.586  0.034 , n = 8 , p < 0.005 ; figure 4(b ) ) .
however , no significant difference was detected for this measure among the endo1 sirna , endo2 sirna , and endo1 , 2 sirna groups ( figures 3(e ) and 4(b ) ) . when given multiple train stimulations , neurons transfected with endo1 , 2 sirna exhibited an accelerated decrease in the epsc amplitude from the beginning of the 1st train to the 4th train compared with neurons in the nc group ( n = 10 , p < 0.005 ; figure 4(c ) ) .
the normalized epsc amplitudes after the 1st and 2nd trains in the endo1 , 2 sirna cotransfected group were consistent with those in both endo1 sirna and endo2 sirna groups . although the endo1 , 2 sirna group exhibited lower epsc amplitudes than either endo1 sirna or endo2 sirna groups after the 3rd and 4th trains , there was no significant difference among them ( p > 0.05 , figure 4(d ) ) .
these results suggest that endophilin1 and endophilin2 influence sv endocytosis , functioning together , but not independently , to mediate sv endocytosis .
in our previous study , we found that all endophilin isoforms formed endophilin - ca channel complexes in neurons .
endophilin1 and endophilin2 demonstrated clear ca - dependent interactions with the ca channel , whereas endophilin3 did not .
however , it remained unknown whether this characteristic would affect sv endocytosis . to address this issue in the present study , we generated endophilin isoform - specific knockdowns in cultured hippocampal neurons .
each endophilin sirna successfully reduced the expression of its corresponding isoform approximately 7080% ( figure 1 ) .
neither transfected sirna nor nc affected the tested intrinsic electrophysiological properties of the neurons . as shown in figure 2(d ) , the epsc amplitude evoked at low frequency in each sirna group exhibited no significant difference from that in neurons transfected with nc , suggesting that none of the endophilin isoform knockdowns affected sv exocytosis , consistent with results from a study that ablated endophilin in drosophila . by microinjecting anti - endophilin antibodies into the giant axon of the lamprey ,
fly and worm mutants lacking endophilin also demonstrated that endophilin is an important part of the machinery driving sv recycling [ 29 , 30 ] . using cultured cortical neurons derived from endophilin triple knockout ( tko ) mice , milosevic et al . showed that endophilin was implicated in sv endocytosis at mammalian central synapses , particularly in the process of clathrin uncoating . in our study , through selectively knocking down endophilin isoforms in cultured hippocampal neurons , we found that neurons with endophilin1 or endophilin2 knockdowns exhibited synaptic depression , similar to the results in flies and tko mice [ 9 , 29 ] , whereas neurons with the endophilin3 knockdown were not different from control neurons in this regard .
this result demonstrates that sv endocytosis is sustained by endophilin1 or endophilin2 isoforms , but not by endophilin3 .
although all three endophilins contain bar and sh3 domains , which are considered the molecular basis of the endophilin involvement in sv endocytosis [ 13 , 23 ] , they do not exhibit the same functions in sv endocytosis .
this may be related to differences in their variable regions between the bar and sh3 domains .
previous analysis on the secondary structure of the proteins indicated that the variable regions in endophilin1 and endophilin2 carry short -helices , whereas a -turn was detected in the n - terminal region of endophilin3 .
our previous study showed that the variable regions of endophilin1 and endophilin2 also harbor a calcium binding site ( e264 ) , but no calcium binding site was found in endophilin3 .
the calcium binding site enables endophilin to interact with other proteins at resting ca levels and dissociate at high ca levels [ 25 , 27 ] . in
the present study , compared with endophilin3 , the amplitude of the epsc evoked from endophilin1 or endophilin2 isoform - specific knockdown neurons rapidly decreased after stimulation ( figure 3 ) .
in this situation , neurons are depolarized and the ca concentration is considered to be at a high level ( over 1  m ) .
these data suggest that although the bar and sh3 domains are necessary for the role of endophilin in sv endocytosis , the contribution of the calcium binding sites in endophilin1 and endophilin2 may be more important for endophilin function . reducing the endogenous levels of either endophilin1 or endophilin2 through
rna inference impedes sv endocytosis ( figure 3 ) . however , the response of cotransfected endo1 , 2 sirna neurons to single high - frequency or multiple high - frequency stimulation displayed no obvious difference compared with those of neurons transfected with endo1 or endo2 sirna alone .
studies have shown that endophilin1 and endophilin2 are found predominantly as stable dimers through a coiled - coil domain in their conserved nh2-terminal moiety .
this suggests that endophilin1 and endophilin2 may function together , not independently , to mediate sv endocytosis .
dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery . in conclusion
, our study suggests that , in cultured mammalian primary neurons , endocytosis of presynaptic vesicles is sustained by endophilin1 or endophilin2 isoforms , but not by endophilin3 , suggesting that isoform specificity confers particular properties on specific endocytic pathways .
 endophilin isoforms perform distinct characteristics in their interactions with n - type ca2 + channels and dynamin 
 . however , precise functional differences for the endophilin isoforms on synaptic vesicle ( sv ) endocytosis remain unknown . by coupling rna interference and electrophysiological recording techniques in cultured rat hippocampal neurons , we investigated the functional differences of three isoforms of endophilin in sv endocytosis . 
 the results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations . 
 similar results were found using endophilin2 knockdown neurons , whereas endophilin3 sirna exhibited no change compared with control neurons . 
 endophilin1 and endophilin2 affected sv endocytosis , but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone . 
 this result suggested that endophilin1 and endophilin2 functioned together but not independently during sv endocytosis . taken together , our results indicate that sv endocytosis is sustained by endophilin1 and endophilin2 isoforms , but not by endophilin3 , in primary cultured hippocampal neurons . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion