BACKGROUND
tau belongs to the family of microtubule-associated proteins , which bind to and/or interact with microtubule.
it has been suggested that tau and other maps serve to promote assembly of microtubule to make them structurally stably, yet dynamic  <cit> .
disruption of the tau gene led to structural abnormalities of microtubule organization in small-calibre axons  <cit> , and behavioural deficits with hyperactivity, impaired motor strength and coordination  <cit> .
in contrast with the initial report  <cit> , there were in vitro phenotypes with delayed development of neuronal polarity and formation of axons in cultured embryonal hippocampal neurons from tau knockout mice  <cit> .
functional redundancy, particularly between tau and map genes likely exists, since map1a protein expression was enhanced in tau knockout mice  <cit> .
indeed tau/map1b double knockout mice displayed brain anomalies with severe defects on axon tract and neuronal layer formation, together with abnormal growth cone morphology and pronounced microtubule disorganization in primary neurons derived from these mice  <cit> .
tau protein synthesis and function is regulated by alternative splicing in a complex species-specific, developmental- and tissue-dependent manner  <cit> .
depending upon the inclusion or exclusion of exon  <dig>  tau mrna isoforms with three  and four  microtubule binding domains are generated, and in total six tau mrna isoforms are produced in the human brain.
in the adult human brain, splicing is balanced with a 1: <dig> expression of 4r- and 3r-tau mrna  <cit> .
this is in contrast to mouse brain in which only 3r-tau mrna is generated at birth and only 4r-tau mrna is synthesized at adulthood  <cit> .
thus equal amounts of 4r- and 3r-tau protein is synthesized in adult human brain, while only 4r-tau protein is produced in adult mouse brain.
interestingly, mutations in exon  <dig> or in adjacent regulatory sequences, can give rise to neurodegenerative disease with accumulation of filamentous inclusions of tau in the human brain.
regulation of alternative splicing of tau is very complex and partly unknown.
in order to investigate the functional role of exon  <dig> in murine tau we ablated the gene on both alleles, and thereby generated e10−/− mice which should in theory only synthesize 3r-tau protein.
moreover, the effect of a 1: <dig> balanced 3r-/4r-tau mrna splicing of human brain was examined by generating mice in which exon  <dig> in tau was deleted on only one allele .
e10+/− mice should in theory synthesize equal amounts of 3r- and 4r-tau protein, as in the human brain .
the effects of fine-tuned regulation of alternative splicing being critical to maintain neuronal functions and viability could thereby be assessed  <cit> .
here we show that e10−/− but not e10+/− mice, display impaired sensorimotor abilities with aging.
RESULTS
generation of mice devoid of exon  <dig> in murine tau
genomic regions  framing exon  <dig> in the murine tau gene were amplified with high-fidelity long-range pcr and subcloned into a pbluescript ii ks-vector that harboured a loxp-neomycin-loxp-cassette .
the gene targeting construct was checked by multiple restriction enzyme digests and sequencing of both introns and the intervening neomycin cassette.
the gene-targeting vector was made linear with sspi and introduced into r <dig> embryonic stem  cell with electroporation.
es cell clones were screened for homologous recombination and in a few clones, integration in the murine tau locus led to deletion of exon  <dig> and adjacent intron sequences .
the clones were screened with pcr-reactions in which one primer was located outside the homologous sequences of the targeting construct.
those experiments resulted in  <dig>  kbp and  <dig>  kbp bands respectively .
one of the positive es cell clones was microinjected into blastocysts and transplanted into pseudopregnant mice.
male chimeric mice were bred with c57bl/6 j and their offspring were examined for evidence of germ line transmission.
these were further bred with transgenic mice expressing phosphoglycerate kinase -cre recombinase to delete the neo cassette.
pcr with genomic dna and primers flanking exon  <dig> provided evidence of an endogenous tau allele  and a null allele  in e10+/− mice, while only a single band was present in wild-type siblings .
lack of exon  <dig> in tau results in 3r-tau protein synthesis in adult mouse brain
next we investigated e10+/− mice for expected deviations in tau mrna expression and protein synthesis.
rt-pcr analyses were performed with primers located within exon  <dig> and exon  <dig> to generate pcr products of 305 bp  and 212 bp  respectively.
as expected, a 1-day-old wild-type mouse  expressed only 3r-tau mrna, while a 9-days-old wild-type mouse  generated equal amounts of 3r- and 4r-tau mrna due to alternative splicing .
an 18-days-old wild-type mouse  produced only 4r-tau mrna.
in contrast, a 2-months-old e10+/− mouse  generated 3r- and 4r-tau mrna in essentially equal proportions, while only 4r-tau mrna was produced in an age-matched wild-type mouse .
protein synthesis was examined with rd <dig> antibody, which is selective for 3r-tau, by western blot and immunohistochemistry.
on western blot, all three isoforms of 3r-tau were found in e10+/−  mouse brain, but the isoform devoid of exon  <dig> and  <dig> was the predominant .
tau- <dig>  an antibody which recognizes both 3r- and 4r-tau, generated extra protein bands in e10+/−  mice as compared to wild-type  mice .
the results were consistent with 3r-tau protein synthesis in e10+/−  mouse brain.
immunohistochemistry with rd <dig> antibody resulted in strong staining in the stratum radiatum in ca1-ca <dig> areas of the hippocampus and faint staining in the soma of pyramidal neurons in adult e10+/− mice , but not in wild-type mice who did not express 3r-tau protein .
in contrast, the non-selective tau- <dig> antibody showed a staining pattern consistent with tau being mainly confined to the axonal compartment  <cit>  in both adult e10+/−  and in wild-type mice .
there was no immunostaining in the absence of a primary antibody .
absence of exon <dig> in tau results in age-dependent sensorimotor dysfunctions
subsequently, we aimed to investigate the physiological and functional role of exon  <dig> in tau.
in previous reports, tau knockout mice developed sensorimotor dysfunctions due to loss of tau protein synthesis  <cit> .
cerebellum, which is implicated in balance and motor learning, develops at postnatal stages when 4r-tau is normally produced.
we therefore examined whether partial or complete absence of exon  <dig> in tau led to sensorimotor anomalies in adult  and middle-aged  gene-manipulated mice.
rotarod and grip strength meter were used to assess sensorimotor dysfunctions.
genotype-dependent motor coordination dysfunction was not detected among adult mice  =  <dig> ; p =  <dig> ), while differences in rotarod performance were observed among middle-aged mice  =  <dig> ; p <  <dig> ).
post-hoc analyses showed that e10−/− mice were impaired compared to e10+/+  and e10+/− .
similar results were found when the highest speed at which the animals remained on the rotarod was used as outcome measure  =  <dig> ; p <  <dig> ).
post-hoc analyses revealed that the muscular strength of adult e10+/− mice was superior to that of e10+/+ mice .
in contrast, middle-aged e10−/− mice were weaker than e10+/− , but not e10+/+ mice  =  <dig> ; figure 3d).
e10−/− mice are less active than e10+/− mice in intellicage
the effect of partial and complete deletion of tau exon  <dig> on general behaviour and cognitive function was assessed in intellicages, a new non-invasive system for behavioural phenotyping  <cit> .
since the behaviours of e10+/− and wild-type mice  in sensorimotor tests were essentially equal, we chose to only examine two experimental groups, 9–10 months-old e10+/− and e10−/− mice.
moreover we wanted to increase group size and statistical power.
first, tendency to explore and habituate to novel environment was examined.
during the first 2 hours of the study, the leds were turned on in two of the four corners.
the e10−/− mice  spent more time in the illuminated corners than e10+/− mice  = − <dig> , p <  <dig> ).
during the first 24 hours, e10+/− mice  tended to perform more visits to the corners than e10−/− mice .
as previously observed, there was a marked diurnal activity pattern, i.e.
more activity during the dark phases compared to light phases.
there was a significant time-dependent decrease in the number of corner visits  =  <dig> , p <  <dig> ) indicating that e10+/− and e10−/− habituated to the new environment .
interestingly, during the habituation, e10+/− mice were significantly more active than e10−/− mice  =  <dig> , p <  <dig> ; figure 4a).
the amount of time spent drinking, measured as lick duration, increased over days for both groups of mice  =  <dig> , p <  <dig> , data not shown), but the length of lick duration did not differ between genotypes.
despite a habituation effect, three e10−/− mice performed few corner visits and drank little, and therefore had to be excluded.
they were replaced by four new e10−/− mice, which were allowed to habituate to the intellicages for additional 7 days.
the inserted mice were initially very active, with high numbers of corner visits.
in the following days, the number of corner visits drastically decreased.
their activity was not significantly different from the rest of the mice during the last three days of the habituation.
place learning or associative learning do not depend on the presence of exon  <dig> in tau
the intellicages are equipped with four corners, each with two doors leading to their respective water bottle.
in the place learning protocol, an error was defined as a nosepoke on any of the seven doors where sucrose water  was not available, i.e.
the closed side of each corner  and the accessible side in the three corners where only water was available .
any visit to a door where reward was not provided was defined as an erroneous behaviour.
the percentage of errors made by e10−/− mice decreased from the first day of testing to the last day .
likewise, the percentage of errors decreased for e10+/− mice from  <dig>  ±  <dig> % to  <dig>  ±  <dig> % .
however, spatial learning ability did not depend on genotype .
during reversal learning, when reward was provided behind the diagonally opposite door, e10−/− mice made more errors on first  <dig>  ±  <dig> % than on the last day of testing  <dig>  ±  <dig> % , but this was not observed for e10+/− mice .
again, reversal learning did not depend on genotype.
when mice again could access water behind all eight doors , e10+/− and e10−/− mice preferred the previously rewarded location  =  <dig> , p <  <dig>  i.e.
above chance level  <dig> %; figure 4d), i.e.
they still preferred the location where reward had been provided during the reversal learning.
to assess cognitive function, e10+/− and e10−/− mice were also exposed to a passive avoidance test with a memory probe trial  <cit> .
at baseline, before being exposed to an aversive stimulus, neither of the gene-manipulated mice preferred any of the four corners .
during training, the mice avoided the corner in which they were exposed to an aversive stimuli , but there was no genotype-dependent difference  =  <dig> , p =  <dig> ; probe trial t =  <dig> , p =  <dig> ; chance level is 25%; figure 4e; middle).
in the probe trial, both e10+/− and e10−/− mice avoided the corner where they had previously been exposed to an aversive stimuli  =  <dig> , p <  <dig> ; probe trial t =  <dig> , p <  <dig> ; chance level is 25%; figure 4e; right).
these findings were indicative of learning and memory retention abilities in both groups of gene-manipulated mice.
no genotype-dependent effects on anxiety-like or exploratory behaviours
since, e10−/− mice were less active than e10+/− mice in the intellicages we wanted to further examine locomotion, emotionality and explorative behaviours with traditional behavioural tests.
behaviour in elevated plus maze is thought to reflect anxiety and risk aversion, while open field measures activity and willingness to explore novel environment.
there were no genotype-dependent differences in the open field test.
the different mice did not differ in terms of location preference  =  <dig> , p =  <dig> ); internal f =  <dig> , p =  <dig> ); periphery f =  <dig> , p =  <dig> ; figure 5a), distance moved  or rearing activity  =  <dig> , p =  <dig> ; figure 5b).
similarly in the elevated plus maze, gene-manipulated mice were equally unwilling to spend time in the open arms as wild-type  mice  =  <dig> , p =  <dig> ); closed arms f =  <dig> , p =  <dig> ); open arms f =  <dig> , p =  <dig> ; figure 5c).
deletion of exon  <dig> does not result in gross morphological abnormalities or tauopathy
brains of 12 months-old wild-type , e10+/− and e10−/− mice were examined for major structural changes.
sections were stained with hematoxylin and luxol fast blue dyes, but we did not find any genotype-dependent differences in the forebrain, cerebellum or spinal cord .
the astroglial marker, glial fibrillary acidic-protein  was used as a rather unspecific sensor of abnormalities in brain tissue.
however, we did not find any differences between the mice .
similarly, there were no genotype-dependent differences in gfap-staining in cerebellar tissue  and no macroscopic differences of whole brain .
we were unable to find any evidence of tau aggregation in the brains of e10+/− or e10−/− mice .
discussion
the cytoskeleton helps cells maintain a dynamic cellular morphology in response to the demands of the surrounding environment.
microtubule-binding proteins likely play a major role in cytoskeletal integrity which is highly critical to neuronal functions and viability, since it enables bidirectional axonal transport of vesicles and organelles along extended processes.
in the adult human brain, splicing of tau protein gives rise to a 1: <dig> balanced expression of 3r- and 4r-tau mrna, while in rodents only 4r-tau mrna is expressed in adult animals.
interestingly, in mouse brain exon  <dig> is not utilized for protein synthesis until postnatal stages  <cit> , when the genome might be less able to activate compensatory mechanisms and mask phenotypes.
this is the first study in which the splicing pattern of tau, a microtubule-binding protein, has been altered in the genome with the purpose of studying physiological functions of exon  <dig> in vivo.
gene targeting is typically associated with the creation of knockout mice, often generated by ablating the basal promoter and exon  <dig>  there are a few reports in which the technique has been used to solely eliminate a single exon  <cit> .
in the current study, equal amounts of 3r- and 4r-tau protein were synthesized in 2 months-old e10+/− mice while wild-type littermates only expressed 4r-tau protein.
moreover, immunostaining with a 3r-tau selective antibody confirmed axonal localization of tau in genetically manipulated mice .
altogether, gene targeting technique led us to the successful generation of gene-manipulated mice expressing 3r:4r-tau  and 3r-tau  mrna and protein with expected subcellular localization.
functional studies revealed an age-dependent decline of sensorimotor skills only in middle-aged e10−/− mice.
adult and middle-aged e10+/− mice had similar motor skills in the rotarod test as e10+/+ mice, consistent with haploinsufficiency seldom giving phenotypes in knockout studies.
interestingly, adult  e10+/− mice were actually stronger than e10+/+ mice, which could be explained by activation of compensatory mechanisms.
it has been suggested that other microtubule-associated proteins, map1a and map1b, are involved in functional compensation.
ablation of tau resulted in ~2-fold increased map1a levels in young mice   <cit> , while levels of map1b and other maps remained unchanged.
compensatory mechanisms seem more active during embryogenesis and early postnatal development, since map1a was unchanged in 12 months-old tau knockout mice  <cit> .
we speculate that functional compensation in adult e10−/− mice was overridden by the effects of aging, resulting in sensorimotor deficits.
cognitive and emotional functions in the gene-manipulated mice were examined, partly because tau anomalies are related to dementia disorders.
we decided to restrict intellicage analyses to two groups of mice; e10+/− and e10−/− because we wanted to attain a reasonable statistical power.
the decision was also due to availability of intellicages and female mice well matched for age.
moreover e10+/− and e10+/+ mice had behaved essentially the same in the sensorimotor tests.
these observations were consistent with many other knockout studies, in which haploinsufficiency seldom results in phenotypes.
e10−/− mice were less active than e10+/− mice in the intellicages, observations which were not reflected in differential anxiety-like behaviours or diminished exploratory behaviour when animals were tested in open field or elevated plus maze.
in intellicages, mice are housed for days undisturbed in social groups in an environment to which they are allowed to habituate.
the procedures of open field and elevated maze testing are markedly different, and this can of course have an impact on the outcome.
both groups habituated well to the new environment and did not show overt cognitive dysfunctions in intellicages.
we conclude that deletion of exon  <dig> in tau does not result in cognitive or emotional phenotypes, although it remains to be investigated if this occurs with aging.
taken together phenotypes in mice genetically manipulated around exon  <dig> in tau were limited to sensorimotor defects and partly similar to those found in tau knockout mice  <cit> .
impaired performance might relate to cerebellar dysfunctions, since this brain region to a large extent develops at postnatal stages when splicing of tau-mrna is abnormal in e10+/− and e10−/− mice  <cit> .
we were unable to find gross morphological changes, but there could be subtle differences in neural tree or synaptodendritic connections leading to network dysfunctions.
electrophysiological and high-resolution quantitative morphology would be needed to investigate such changes, studies which we consider to be outside the scope of an initial characterization of the models.
there is evidence to suggest that in tauopathies, the pathological aggregation of tau can be primary pathogenic e.g.
in frontotemporal lobe dementia  or secondary pathogenic e.g.
to aβ-aggregation in ad  <cit> .
however, it remains unclear why human aβ accumulation by itself does not generate neurofibrillary tangles in aβpp transgenic mice.
perhaps structural differences make murine tau unable to form fibrils but in vitro experiments contradict this theory  <cit> .
murine tau can take part in tau aggregation in vivo but so far seems unable to initiate tau pathology by itself  <cit> .
an unknown factor or a trigger, such as misfolded or aggregated human tau or perhaps aβ seems to be critical.
the differences in splicing of tau in mouse and human brain, i.e.
the 1: <dig> balanced 3r-/4r-tau mrna splicing pattern, could directly impact on the stability of the microtubule structure and on aggregation of tau.
it remains to be investigated whether the microtubule system of e10+/− and e10−/− mice is more vulnerable to a trigger of tau-misfolding e.g.
an aggregate of human aβ or human tau than wild-type mice harbouring intact murine tau .
CONCLUSIONS
this is the first functional study of exon  <dig> in tau, which is linked to neurodegenerative disease.
by deleting exon  <dig> it was possible to alter alternative splicing of tau such that 3r-tau protein was synthesized instead of 4r-tau protein.
the anatomic and subcellular localization of tau protein synthesis was maintained in the gene-manipulated animals.
middle-aged mice lacking exon  <dig> in tau , expressed only 3r-tau protein and displayed mild sensorimotor deficits with reduced grip strength and motor coordination.
haploinsufficient mice , expressing both 3r- and 4r-tau protein, behaved nearly always equal to wild-type mice  which expressed only 4r-tau protein.
there were no genotype-dependent effects on cognition, emotionality or on gross brain structure and tissue morphology.
thus, sensorimotor functions depend on tau exon  <dig>  while perhaps additional functions are masked by related microtubule-binding proteins.

 tau belongs to the family of microtubule-associated proteins, which bind to and/or interact with microtubule. it has been suggested that tau and other maps serve to promote assembly of microtubule to make them structurally stably, yet dynamic. disruption of the tau gene led to structural abnormalities of microtubule organization in small-calibre axons, and behavioural deficits with hyperactivity, impaired motor strength and coordination. in contrast with the initial report, there were in vitro phenotypes with delayed development of neuronal polarity and formation of axons in cultured embryonal hippocampal neurons from tau knockout mice. functional redundancy, particularly between tau and map genes likely exists, since map1a protein expression was enhanced in tau knockout mice. indeed tau/map1b double knockout mice displayed brain anomalies with severe defects on axon tract and neuronal layer formation, together with abnormal growth cone morphology and pronounced microtubule disorganization in primary neurons derived from these mice. tau protein synthesis and function is regulated by alternative splicing in a complex species-specific, developmental- and tissue-dependent manner. depending upon the inclusion or exclusion of exon tau mrna isoforms with three and four microtubule binding domains are generated, and in total six tau mrna isoforms are produced in the human brain. in the adult human brain, splicing is balanced with a 1: expression of 4r- and 3r-tau mrna. this is in contrast to mouse brain in which only 3r-tau mrna is generated at birth and only 4r-tau mrna is synthesized at adulthood. thus equal amounts of 4r- and 3r-tau protein is synthesized in adult human brain, while only 4r-tau protein is produced in adult mouse brain. interestingly, mutations in exon or in adjacent regulatory sequences, can give rise to neurodegenerative disease with accumulation of filamentous inclusions of tau in the human brain. regulation of alternative splicing of tau is very complex and partly unknown. in order to investigate the functional role of exon in murine tau we ablated the gene on both alleles, and thereby generated e10−/− mice which should in theory only synthesize 3r-tau protein. moreover, the effect of a 1: balanced 3r-/4r-tau mrna splicing of human brain was examined by generating mice in which exon in tau was deleted on only one allele. e10+/− mice should in theory synthesize equal amounts of 3r- and 4r-tau protein, as in the human brain. the effects of fine-tuned regulation of alternative splicing being critical to maintain neuronal functions and viability could thereby be assessed. here we show that e10−/− but not e10+/− mice, display impaired sensorimotor abilities with aging.  generation of mice devoid of exon in murine tau genomic regions framing exon in the murine tau gene were amplified with high-fidelity long-range pcr and subcloned into a pbluescript ii ks-vector that harboured a loxp-neomycin-loxp-cassette. the gene targeting construct was checked by multiple restriction enzyme digests and sequencing of both introns and the intervening neomycin cassette. the gene-targeting vector was made linear with sspi and introduced into r embryonic stem cell with electroporation. es cell clones were screened for homologous recombination and in a few clones, integration in the murine tau locus led to deletion of exon and adjacent intron sequences. the clones were screened with pcr-reactions in which one primer was located outside the homologous sequences of the targeting construct. those experiments resulted in kbp and kbp bands respectively. one of the positive es cell clones was microinjected into blastocysts and transplanted into pseudopregnant mice. male chimeric mice were bred with c57bl/6 j and their offspring were examined for evidence of germ line transmission. these were further bred with transgenic mice expressing phosphoglycerate kinase -cre recombinase to delete the neo cassette. pcr with genomic dna and primers flanking exon provided evidence of an endogenous tau allele and a null allele in e10+/− mice, while only a single band was present in wild-type siblings. lack of exon in tau results in 3r-tau protein synthesis in adult mouse brain next we investigated e10+/− mice for expected deviations in tau mrna expression and protein synthesis. rt-pcr analyses were performed with primers located within exon and exon to generate pcr products of 305 bp and 212 bp respectively. as expected, a 1-day-old wild-type mouse expressed only 3r-tau mrna, while a 9-days-old wild-type mouse generated equal amounts of 3r- and 4r-tau mrna due to alternative splicing. an 18-days-old wild-type mouse produced only 4r-tau mrna. in contrast, a 2-months-old e10+/− mouse generated 3r- and 4r-tau mrna in essentially equal proportions, while only 4r-tau mrna was produced in an age-matched wild-type mouse. protein synthesis was examined with rd antibody, which is selective for 3r-tau, by western blot and immunohistochemistry. on western blot, all three isoforms of 3r-tau were found in e10+/− mouse brain, but the isoform devoid of exon and was the predominant. tau- an antibody which recognizes both 3r- and 4r-tau, generated extra protein bands in e10+/− mice as compared to wild-type mice. the results were consistent with 3r-tau protein synthesis in e10+/− mouse brain. immunohistochemistry with rd antibody resulted in strong staining in the stratum radiatum in ca1-ca areas of the hippocampus and faint staining in the soma of pyramidal neurons in adult e10+/− mice, but not in wild-type mice who did not express 3r-tau protein. in contrast, the non-selective tau- antibody showed a staining pattern consistent with tau being mainly confined to the axonal compartment in both adult e10+/− and in wild-type mice. there was no immunostaining in the absence of a primary antibody. absence of exon in tau results in age-dependent sensorimotor dysfunctions subsequently, we aimed to investigate the physiological and functional role of exon in tau. in previous reports, tau knockout mice developed sensorimotor dysfunctions due to loss of tau protein synthesis. cerebellum, which is implicated in balance and motor learning, develops at postnatal stages when 4r-tau is normally produced. we therefore examined whether partial or complete absence of exon in tau led to sensorimotor anomalies in adult and middle-aged gene-manipulated mice. rotarod and grip strength meter were used to assess sensorimotor dysfunctions. genotype-dependent motor coordination dysfunction was not detected among adult mice  = ; p =  ), while differences in rotarod performance were observed among middle-aged mice  = ; p <  ). post-hoc analyses showed that e10−/− mice were impaired compared to e10+/+ and e10+/−. similar results were found when the highest speed at which the animals remained on the rotarod was used as outcome measure  = ; p <  ). post-hoc analyses revealed that the muscular strength of adult e10+/− mice was superior to that of e10+/+ mice. in contrast, middle-aged e10−/− mice were weaker than e10+/−, but not e10+/+ mice  = ; figure 3d). e10−/− mice are less active than e10+/− mice in intellicage the effect of partial and complete deletion of tau exon on general behaviour and cognitive function was assessed in intellicages, a new non-invasive system for behavioural phenotyping. since the behaviours of e10+/− and wild-type mice in sensorimotor tests were essentially equal, we chose to only examine two experimental groups, 9–10 months-old e10+/− and e10−/− mice. moreover we wanted to increase group size and statistical power. first, tendency to explore and habituate to novel environment was examined. during the first 2 hours of the study, the leds were turned on in two of the four corners. the e10−/− mice spent more time in the illuminated corners than e10+/− mice  = −, p <  ). during the first 24 hours, e10+/− mice tended to perform more visits to the corners than e10−/− mice. as previously observed, there was a marked diurnal activity pattern, i.e. more activity during the dark phases compared to light phases. there was a significant time-dependent decrease in the number of corner visits  = , p <  ) indicating that e10+/− and e10−/− habituated to the new environment. interestingly, during the habituation, e10+/− mice were significantly more active than e10−/− mice  = , p < ; figure 4a). the amount of time spent drinking, measured as lick duration, increased over days for both groups of mice  = , p < , data not shown), but the length of lick duration did not differ between genotypes. despite a habituation effect, three e10−/− mice performed few corner visits and drank little, and therefore had to be excluded. they were replaced by four new e10−/− mice, which were allowed to habituate to the intellicages for additional 7 days. the inserted mice were initially very active, with high numbers of corner visits. in the following days, the number of corner visits drastically decreased. their activity was not significantly different from the rest of the mice during the last three days of the habituation. place learning or associative learning do not depend on the presence of exon in tau the intellicages are equipped with four corners, each with two doors leading to their respective water bottle. in the place learning protocol, an error was defined as a nosepoke on any of the seven doors where sucrose water was not available, i.e. the closed side of each corner and the accessible side in the three corners where only water was available. any visit to a door where reward was not provided was defined as an erroneous behaviour. the percentage of errors made by e10−/− mice decreased from the first day of testing to the last day. likewise, the percentage of errors decreased for e10+/− mice from  ±  % to  ±  %. however, spatial learning ability did not depend on genotype. during reversal learning, when reward was provided behind the diagonally opposite door, e10−/− mice made more errors on first  ±  % than on the last day of testing  ±  %, but this was not observed for e10+/− mice. again, reversal learning did not depend on genotype. when mice again could access water behind all eight doors, e10+/− and e10−/− mice preferred the previously rewarded location  = , p <  i.e. above chance level %; figure 4d), i.e. they still preferred the location where reward had been provided during the reversal learning. to assess cognitive function, e10+/− and e10−/− mice were also exposed to a passive avoidance test with a memory probe trial. at baseline, before being exposed to an aversive stimulus, neither of the gene-manipulated mice preferred any of the four corners. during training, the mice avoided the corner in which they were exposed to an aversive stimuli, but there was no genotype-dependent difference  = , p = ; probe trial t = , p = ; chance level is 25%; figure 4e; middle). in the probe trial, both e10+/− and e10−/− mice avoided the corner where they had previously been exposed to an aversive stimuli  = , p < ; probe trial t = , p < ; chance level is 25%; figure 4e; right). these findings were indicative of learning and memory retention abilities in both groups of gene-manipulated mice. no genotype-dependent effects on anxiety-like or exploratory behaviours since, e10−/− mice were less active than e10+/− mice in the intellicages we wanted to further examine locomotion, emotionality and explorative behaviours with traditional behavioural tests. behaviour in elevated plus maze is thought to reflect anxiety and risk aversion, while open field measures activity and willingness to explore novel environment. there were no genotype-dependent differences in the open field test. the different mice did not differ in terms of location preference  = , p =  ); internal f = , p =  ); periphery f = , p = ; figure 5a), distance moved or rearing activity  = , p = ; figure 5b). similarly in the elevated plus maze, gene-manipulated mice were equally unwilling to spend time in the open arms as wild-type mice  = , p =  ); closed arms f = , p =  ); open arms f = , p = ; figure 5c). deletion of exon does not result in gross morphological abnormalities or tauopathy brains of 12 months-old wild-type, e10+/− and e10−/− mice were examined for major structural changes. sections were stained with hematoxylin and luxol fast blue dyes, but we did not find any genotype-dependent differences in the forebrain, cerebellum or spinal cord. the astroglial marker, glial fibrillary acidic-protein was used as a rather unspecific sensor of abnormalities in brain tissue. however, we did not find any differences between the mice. similarly, there were no genotype-dependent differences in gfap-staining in cerebellar tissue and no macroscopic differences of whole brain. we were unable to find any evidence of tau aggregation in the brains of e10+/− or e10−/− mice. discussion the cytoskeleton helps cells maintain a dynamic cellular morphology in response to the demands of the surrounding environment. microtubule-binding proteins likely play a major role in cytoskeletal integrity which is highly critical to neuronal functions and viability, since it enables bidirectional axonal transport of vesicles and organelles along extended processes. in the adult human brain, splicing of tau protein gives rise to a 1: balanced expression of 3r- and 4r-tau mrna, while in rodents only 4r-tau mrna is expressed in adult animals. interestingly, in mouse brain exon is not utilized for protein synthesis until postnatal stages, when the genome might be less able to activate compensatory mechanisms and mask phenotypes. this is the first study in which the splicing pattern of tau, a microtubule-binding protein, has been altered in the genome with the purpose of studying physiological functions of exon in vivo. gene targeting is typically associated with the creation of knockout mice, often generated by ablating the basal promoter and exon there are a few reports in which the technique has been used to solely eliminate a single exon. in the current study, equal amounts of 3r- and 4r-tau protein were synthesized in 2 months-old e10+/− mice while wild-type littermates only expressed 4r-tau protein. moreover, immunostaining with a 3r-tau selective antibody confirmed axonal localization of tau in genetically manipulated mice. altogether, gene targeting technique led us to the successful generation of gene-manipulated mice expressing 3r:4r-tau and 3r-tau mrna and protein with expected subcellular localization. functional studies revealed an age-dependent decline of sensorimotor skills only in middle-aged e10−/− mice. adult and middle-aged e10+/− mice had similar motor skills in the rotarod test as e10+/+ mice, consistent with haploinsufficiency seldom giving phenotypes in knockout studies. interestingly, adult e10+/− mice were actually stronger than e10+/+ mice, which could be explained by activation of compensatory mechanisms. it has been suggested that other microtubule-associated proteins, map1a and map1b, are involved in functional compensation. ablation of tau resulted in ~2-fold increased map1a levels in young mice, while levels of map1b and other maps remained unchanged. compensatory mechanisms seem more active during embryogenesis and early postnatal development, since map1a was unchanged in 12 months-old tau knockout mice. we speculate that functional compensation in adult e10−/− mice was overridden by the effects of aging, resulting in sensorimotor deficits. cognitive and emotional functions in the gene-manipulated mice were examined, partly because tau anomalies are related to dementia disorders. we decided to restrict intellicage analyses to two groups of mice; e10+/− and e10−/− because we wanted to attain a reasonable statistical power. the decision was also due to availability of intellicages and female mice well matched for age. moreover e10+/− and e10+/+ mice had behaved essentially the same in the sensorimotor tests. these observations were consistent with many other knockout studies, in which haploinsufficiency seldom results in phenotypes. e10−/− mice were less active than e10+/− mice in the intellicages, observations which were not reflected in differential anxiety-like behaviours or diminished exploratory behaviour when animals were tested in open field or elevated plus maze. in intellicages, mice are housed for days undisturbed in social groups in an environment to which they are allowed to habituate. the procedures of open field and elevated maze testing are markedly different, and this can of course have an impact on the outcome. both groups habituated well to the new environment and did not show overt cognitive dysfunctions in intellicages. we conclude that deletion of exon in tau does not result in cognitive or emotional phenotypes, although it remains to be investigated if this occurs with aging. taken together phenotypes in mice genetically manipulated around exon in tau were limited to sensorimotor defects and partly similar to those found in tau knockout mice. impaired performance might relate to cerebellar dysfunctions, since this brain region to a large extent develops at postnatal stages when splicing of tau-mrna is abnormal in e10+/− and e10−/− mice. we were unable to find gross morphological changes, but there could be subtle differences in neural tree or synaptodendritic connections leading to network dysfunctions. electrophysiological and high-resolution quantitative morphology would be needed to investigate such changes, studies which we consider to be outside the scope of an initial characterization of the models. there is evidence to suggest that in tauopathies, the pathological aggregation of tau can be primary pathogenic e.g. in frontotemporal lobe dementia or secondary pathogenic e.g. to aβ-aggregation in ad. however, it remains unclear why human aβ accumulation by itself does not generate neurofibrillary tangles in aβpp transgenic mice. perhaps structural differences make murine tau unable to form fibrils but in vitro experiments contradict this theory. murine tau can take part in tau aggregation in vivo but so far seems unable to initiate tau pathology by itself. an unknown factor or a trigger, such as misfolded or aggregated human tau or perhaps aβ seems to be critical. the differences in splicing of tau in mouse and human brain, i.e. the 1: balanced 3r-/4r-tau mrna splicing pattern, could directly impact on the stability of the microtubule structure and on aggregation of tau. it remains to be investigated whether the microtubule system of e10+/− and e10−/− mice is more vulnerable to a trigger of tau-misfolding e.g. an aggregate of human aβ or human tau than wild-type mice harbouring intact murine tau.  this is the first functional study of exon in tau, which is linked to neurodegenerative disease. by deleting exon it was possible to alter alternative splicing of tau such that 3r-tau protein was synthesized instead of 4r-tau protein. the anatomic and subcellular localization of tau protein synthesis was maintained in the gene-manipulated animals. middle-aged mice lacking exon in tau, expressed only 3r-tau protein and displayed mild sensorimotor deficits with reduced grip strength and motor coordination. haploinsufficient mice, expressing both 3r- and 4r-tau protein, behaved nearly always equal to wild-type mice which expressed only 4r-tau protein. there were no genotype-dependent effects on cognition, emotionality or on gross brain structure and tissue morphology. thus, sensorimotor functions depend on tau exon while perhaps additional functions are masked by related microtubule-binding proteins. 
middle-aged e10−/− mice displayed sensorimotor disturbances in the rotarod when compared to age-matched e10+/− and wild-type mice, and their muscular grip strength was less than that of e10+/− mice. the performance of e10+/− mice and wild-type mice was similar in sensorimotor tests. complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. ablation of exon in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. gene-targeting was used to delete exon in murine tau on both alleles to study its functional role. moreover, mice devoid of exon on one allele were generated to investigate the effects of 1: balanced expression of 4r-/3r-tau protein, since equal amounts of 4r-/3r-tau protein are synthesized in human brain. exon has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease. the functional role of alternative splicing of tau e.g.
 complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. the functional role of alternative splicing of tau e.g. exon has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease.  gene-targeting was used to delete exon in murine tau on both alleles to study its functional role. moreover, mice devoid of exon on one allele were generated to investigate the effects of 1: balanced expression of 4r-/3r-tau protein, since equal amounts of 4r-/3r-tau protein are synthesized in human brain. middle-aged e10−/− mice displayed sensorimotor disturbances in the rotarod when compared to age-matched e10+/− and wild-type mice, and their muscular grip strength was less than that of e10+/− mice. the performance of e10+/− mice and wild-type mice was similar in sensorimotor tests. cognitive abilities or anxiety-like behaviours did not depend on exon in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.  ablation of exon in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. presumably related microtubule-stabilizing genes rescue other functions. alzheimer’s diseasetauknockout micealternative splicingmicrotubule 