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Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition Background: The present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions. Methods: We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions. Results: In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. Conclusion: These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition. Background low molecular weight PTP and 4) the Asp-based PTPs Tyrosine phosphorylation plays an important role in sev- (Tyr/Ser phosphatase activity) [3]. eral signaling pathways regulating cell growth, differenti- ation, cell cycle, apoptosis and neuronal functions [1,2]. Classical PTPs have been reported to play a key role in The phosphorylation/dephosphorylation balance is con- neural functions, from development to cognitive func- trolled by protein tyrosine kinases and phosphatases. tion. For example, RPTPs such as PTPδ, PTPσ, LAR, and PTPs can be distinguished into four classes: 1) classical especially PTPRO, are important players in axonal growth PTPs that can be subdivided into transmembrane, recep- and guidance during development [4]. Studies on PTPσ- tor-like enzymes, and the intracellular, nonreceptor PTPs, KO (RPTP) mice have shown involvement of this PTP in 2) dual-specificity PTPs (Ser and Tyr phosphatases), 3) the regulation of the developing hypothalamo-pituitary Page 1 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 axis [5,6] and in the development of the CNS architecture experiment was in accordance with Directive 86/609/EEC, [7]. PTPBL-KO (non receptor like PTP-NRPTP) mice dis- enforced by the Italian D.L. No. 116 of January 27, 1992. play impaired motor nerve repair in a model of sciatic Physical facilities and equipment for accommodation and nerve crush lesion [8] and PTPMEG (NRPTP) interacts care of animals were in accordance with the provisions of with key intracellular players leading to the stimulation of EEC Council Directive 86/609. Tail snips from mice were the channel activity of NMDA receptors [9]. collected and genotyped as previously reported [22]. In the present study we focused our attention on a NRPTP, PTPH1 KO design PTPH1, and on its possible role on neural functions. PTPH1-KO mice were generated using the Velocigene Indeed PTPH1 has been shown to be expressed in the CNS technology [23], as described in details elsewhere [22]. [10] but little is currently known on its potential impact Briefly a mouse BAC containing the PTPH1 gene was on CNS functions. PTPH1 (also called PTPN3) belongs to modified: an in-frame LacZ reporter sequence and a neo- a sub-family of non receptor cytosolic PTPs characterized mycin-selectable marker replaced exons 15 to 22 encod- by the presence of a FERM domain (band 4.1, ezrin, ing for the PDZ and the catalytic domain of PTPH1. BAC radixin, moesin) at its N-terminus, responsible for the electroporation into embryonic stem cells was performed. interaction with transmembrane proteins and/or phos- F1 heterozygous mice were bred to generate F2 PTPH1-KO pholipids in the cell membrane [11-13]. In addition mice. Line breeding and animal care were performed in PTPH1 has a PDZ domain in the central part responsible Charles River Italy and France. for the interaction with other proteins, whereas the single catalytic domain is located at the C-terminus. LacZ staining procedure and immunohistochemistry PTPH1-KO and WT mice, 12 months old, n = 2, male and PTPH1 activity has been involved in a variety of cellular females, were sacrificed by ip overdose of thiopental functions including TCR-signaling [14-16], cell cycle reg- (5%), perfused with paraformaldehyde 4%, then washed ulation [11,16,17], endoplasmic reticulum assembly [18], in PBS and incubated overnight at 37°C in the solution cardiac sodium channel modulation [19] and TNFα con- containing the substrate for beta-galactosidase (beta-gal, verting enzyme inhibition [20]. encoded by the LacZ cassette) coupled to a NBT salt. The organs and the tissues in the sections display a green/blue Recently, our group has demonstrated that PTPH1 staining where PTPH1 gene is normally expressed. After dephosphorylates GHR in vitro and in cellular assays [21] rinsing into PBS, organs were postfixed in PFA 4% for 1 and results in an increase of body weight in the functional hour, then incubated in 50% glycerol overnight at 4°C PTPH1-knockout (KO) mice via modulation of IGF1 and finally maintained in 70% glycerol at room tempera- secretion [22] thus demonstrating its in vivo relevance. ture. LacZ staining was observed through a low magnifica- tion microscope and described by an operator blind to the PTPH1 has been shown in the rat to be highly expressed genotypes. in thalamic nuclei as well as various cortical areas [10]. However, no information is currently available on its LacZ staining was also performed on CNS sections. Mice impact on CNS functions. To address this question we (n = 3, 12 months old) were sacrificed by ip injection of have further characterized our PTPH1-KO mice line an overdose of thiopental (5%), perfused with PBS and through behavioral and anatomical approaches. PTPH1 PFA 4%. Brains were removed and postfixed overnight at expression and localization was evaluated by LacZ stain- 4°C in PFA 4%, then placed overnight at 4°C in 15% and ing in the brain and a behavioral test battery evaluated finally in 30% sucrose buffer. The brains were then PTPH1 loss on CNS functions such as locomotor activity included in O.C.T. (Tissue-Tek) and sections were cut on (open field), anxiety-like behavior (open field and ele- slides with a cryostat at 20 μm thickness. The slides were vated plus maze), motor ability, coordination and learn- incubated in LacZ staining solution (see above) overnight ing (accelerating rotarod), spatial working memory (Y at 37°C, washed thrice in PBS (5 min each) and either maze) and nociceptive sensitivity (hot plate). counterstained with H&E (Merck KGaA) [23] or co- expressed with NeuN immunostaining. Briefly, sections were incubated for 3 hours at room temperature in block- Methods Animals ing solution (Vectastain Kit), washed in TBS (Tris-buffered PTPH1-KO and wild type littermates (F2 generation, saline), incubated overnight at 4°C with a solution con- 87.5% C57Bl/6 – 12.5% 129S6SvEv) aged 3–4 months taining the primary antibody mouse anti-mouse Neuro- were used for behavioral phenotyping. Mice were individ- nal Nuclei (Chemicon MAB377, 1/1000). The staining ually caged and maintained in a 12:12 hours light: dark was revealed by ABC kit secondary antibody (mouse cycle (lights on at 7 am) at 21 ± 1°C with food and water Vectastain Kit), and DAB (Sigma). After dehydration, sec- available ad libitum. Protection of animals used in the tions were transferred onto coverslips. LacZ staining and Page 2 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 co-expression with NeuN-immunoreactivity (NeuN-ir) Elevated plus maze was observed by microscopy and described by an operator After one hour of adaptation in the testing room, anxiety- blind to the genotypes. like behavior was tested for each mouse by EPM within one session. The apparatus consists of four arms (29.5 cm Semiquantitative RT-PCR for beta galactosidase gene long and 5 cm wide each). Two arms are open whereas the Semiquantitative RT-PCR for PTPH1 and beta-gal gene 2 others are limited by 2 black walls (20 cm high). The expression was performed in different brain areas of number of entries of each mouse in the open and closed PTPH1-KO and WT mice in order to confirm the presence arms was recorded by a video camera during a period of 5 of beta-gal expression in the KO tissues, replacing PTPH1 minutes and analyzed by the SMART Video-Tracking Soft- PDZ and catalytic domain. Brains from KO and WT mice ware (ViewPoint Life Sci. Inc.). The total number of (n = 5, 6 months old) were freshly removed and rinsed in entries into the arms is an index of locomotion, whereas HBSS. Hippocampus, cerebellum, cortex, striatum, mid- the percentage of time spent and percentage of entries in brain and olfactory bulbs were dissected. Total RNA was the closed arms is an index of anxiety-like behaviors [25]. extracted using Trizol Reagent (Invitrogen) and cleaned- Accelerated rotarod up by RNAeasy columns from Qiagen. 5 μg of total RNA were used to perform the RT-PCR reaction (SuperScript II Motor ability, coordination and learning were evaluated RT kit, Invitrogen). The primer sequences for LacZ ampli- by using an Accelerated Rotarod apparatus for mice (Cat. fication were the following: LacZ – forward 5'-GAT GTA # 7650 by Jones and Roberts, distr. by Basile Instr., Italy). CGT GCC CTG GAA CT/reverse 5'-GGT CCC ACA CTT The apparatus was placed within the animal colony room CAG CAT TT. In order to load equally the reaction mixes, and was cleaned after each trial. Mice were tested for their a 300 bp fragment of Histone 2A was amplified as a house abilities to maintain a balance on a rotating bar, which keeping gene with the following primers: H2Az forward – accelerated from 4 to 40 rpm/min in a 5 min trial. Latency 5' CGT ATT CAT CGA CAC CTG AAA; H2Az reverse – 5' to fall off was measured within one session and all mice CTG TTG TCC TTT CTT CCC GAT. underwent four trials (one every 30 min) [26-28]. The dif- ferences at the rotarod performances in WT and KO were Behavioral phenotyping test battery assessed by a single set of trials [27,28]. This set-up allows Neurological functions of PTPH1-WT and KO mice a major focus on the early phases of motor learning, (males and females, 11 weeks-old, n = 10 per gender per involving a strong activation of prefrontal cortex and of genotype) were assessed through a behavioral test battery. the associative areas of basal ganglia and cerebellum [29,30]. The sequence of the test battery was chosen from the least invasive to the most ones. The schedule of the testing ses- Y-maze alternation sions included one week of recovery from one test to the After one hour of adaptation in the testing room, mice next, as reported in Table 1. were tested on a Y maze apparatus (40 cm long/8 cm wide arms with transparent walls) to investigate spatial work- Open field ing memory [31]. The number and the sequence of the After one hour of adaptation in the testing room, each arm entries for each mouse were recorded during 5 min- mouse was placed in an open field chamber (50 cm wide utes. The locomotion index was calculated as the overall with white floor and walls) (ViewPoint Life Sci. Inc.) to number of arm entries, whereas the working memory test locomotor activity and anxiety-like behaviors. Loco- index was calculated as following: number of exact alter- motion was recorded for one hour by a video camera and nations (entries into three different arms consecutively)/ analyzed automatically by VideoTRACK software (View- possible alternations (i.e. the number of arms entered Point Life Sci. Inc.). Locomotor activity was evaluated by minus 2) × 100. calculating the total path length traveled, whereas the rel- ative time spent in the center was taken as indicative of Hot plate anxiety-like behavior [24]. The tests were performed in Thermal sensitivity was assessed by a hot plate apparatus two sessions with equivalent group representation. for mice (Cat. # 7280 by Biol. Research Apparatus, distr. by Basile Instr., Italy) and lasted a maximum of 45 sec- onds, time at which damages could occur [32]. The appa- Table 1: Schedule of the behavioral test battery. Age (wks) 8 9 10 11 12 13 14 15 10M+10F arrival quarantine adaptation Open Field EPM Rotarod Y-maze Hot plate Page 3 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 ratus was placed in the animal colony room and all the (Figures. 3a, 3b 3c, 4a and 4b) and indusium griseum mice were tested within one session. Animals were placed (Figures 3a, 3b). In the cerebellum, in spite of a strong on a surface heated at 52.5°C and the latency (seconds) to staining in the whole mount (Figure 2), only a faint LacZ shake or lick the paw was recorded by the operator. signal was observed in sections (Figures 5a, 5b) in partic- ular in the granule cells, close to the nuclei. The RT-PCR Statistics on cortical and cerebellar extracts confirmed the presence Statistical comparisons were performed by unpaired two- of LacZ expression in these brain areas (Figure 6). tailed T-test (p < 0.05) and two-way ANOVA (p < 0.05) followed by post-hoc test as necessary. In the accelerated In subcortical regions, LacZ was detected in the anterior rotarod, two-way ANOVA with repeated measures fol- ventral, mediodorsal, ventrolateral, anteromedial and lowed by T-test was used. Results are expressed as mean ± central lateral thalamic nuclear groups (Figure 7a). In SEM. more caudal thalamic areas, LacZ was again detectable in the posterior thalamic nuclear group (Po), and to a lesser Results extent in posteromedial, in posterolateral and in reticular LacZ staining in whole mount thalamic nuclei and also in the dorsal lateral geniculate In PTPH1-KO adult animals, LacZ staining in the brain nuclei (Figures 7b, 7c, 7d and 7e). In the tenia tecta, LacZ was observed in the cerebellum, hippocampus and in the staining visible in the whole mount preparation was con- thalamic nuclei. In addition a strong staining was firmed (Figures 2, 8a, 8b). The RT-PCR on substriatal observed in the cerebral cortex, tenia tecta and septum regions including the thalamus, the midbrain and the (Figures 1a, 1b). pontine areas confirmed the presence of LacZ expression in some of these brain areas (Figure 6). To exclude any LacZ staining in sections and RT-PCR results potential impact of LacZ blood signal contamination in No gross cytoarchitectural brain differences were observed brain areas, RT-PCR on 5 to 20 μl of whole blood was car- by simple visual observation at the microscope in the cor- ried out and did not reveal any significant signal [Addi- tex, hippocampus and thalamus in PTPH1-KO mice com- tional files 2, 3]. pared to WT littermates. In the hippocampus, LacZ expression was observed in the LacZ staining was performed on frozen brain sections to cytoplasm of a few pyramidal cells and through the fibers confirm and to describe the expression of PTPH1 at the of the oriens and radiatum layer in a rostral caudal spread brain structural level (Table 2). (Figure 9a). In rostral sections, LacZ was expressed in the septohippocampal nuclei (Figure 8a). In more caudal sec- In cortical regions, LacZ was expressed in the external tions LacZ was present in the CA1 and CA3, and in a lesser pyramidal (III) and internal granular layer (IVA) of the extent in the CA2 (Figures 9b, 9c and 9d). In the CA3 LacZ cerebral cortex (Figures 2a, 2b), in the retrosplenial cortex was strongly expressed in the oriens and pyramidal cell Co Rc Co Cb Th Cb Tt ab PTPH1-KO adult mouse brain Figure 1 PTPH1-KO adult mouse brain. a: whole brain, dorsal view, staining in cerebellum (Cb) and cortex (Co); b: LacZ staining on brain, sagittal view: detection in the tenia tecta (Tt), cortex (Co), thalamus (Th), hippocampus (H), retrosplenial cortex (Rc), septum (S) and in the granule cell layer of cerebellum. Page 4 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 Table 2: Qualitative estimation of LacZ staining intensity in the different brain areas. Brain Area Intensity of LacZ staining Brain Area Intensity of LacZ staining Cerebral cortex + Dorsal Tenia Tecta ++ Retrosplenial cortex ++ Septohippocampal nu + CA1 oriens layer +++ VPL + CA1 radiatum layer +++ MDL ++ CA1 pyramidal cell layer + AV ++ CA2 oriens layer - VPM ++ CA2 radiatum layer - VL + CA2 pyramidal layer + VM ++ CA3 oriens layer + Po ++ CA3 radiatum layer +/- LD + CA3 pyramidal layer + Rt + DG granular cell layer - DLG ++ DG molecular layer +++ VPPC ++ DG hilus - PF - Fascicola cinereum ++ cerebellum + Indisium griseum + -: absent; +/-: faint; +: present; ++: intense; +++: very intense staining. Cornu Ammonis (CA), Dentate Gyrus (DG), mediodorsal lateral (MDL), anteroventral (AV), ventromedial (VM) ventral posteromedial (VPM), ventrolateral (VL), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt), ventral posteromedial parvicel (VPPC) thalamic nuclei, lateral geniculate nucleus (DLG), parafascicular thalamic nuclei (PF), nuclei (nu). layer (Figure 9d), but its intensity was reduced in the body weight has been detected in PTPH1-KO mice com- radiatum and oriens compared to CA1 (Figure 9b). No pared to WT littermates, more pronounced in male mice staining was detected in the lacunosum-molecular layer in and probably due to an enhanced GHR sensitivity, that CA1, CA2 and CA3 (Figures 9b, 9c and 9d). The dentate leads to increased IGF-1 mRNA and protein expression in gyrus showed a strong positive LacZ signal in the molecu- liver and plasma, respectively [22]. lar layer, but not in the hilus (Figure 9e). In EPM, open field test and hot plate tests (anxiety-related Behavioral phenotyping behavior and thermal pain sensitivity), PTPH1-KO male As previously demonstrated, PTPH1-KO mice were and female mice did not show any significant differences healthy, reproduced normally and did not show any phe- in comparison with their WT littermates (data not notypic traits distinguishing them from their WT litterma- shown). tes by simple visual observations [22,33]. An increased Rc Rc ig II III IVa ig PTPH Figure 3 1-KO cerebral cortex PTPH1-KO cerebral cortex. a: LacZ detection in retrosple- a Figure 2 : PTPH1-KO cerebral cortex (10×, scale bar: 220 μm) nial cortex (Rc) and indusium griseum (ig) staining (4×, scale a: PTPH1-KO cerebral cortex (10×, scale bar: 220 μm). b: bar: 80 μm). b: detail of the Rc and ig (40×, scale bar: 20 μm); positive cytoplasmatic and perinuclear LacZ staining (blue c: positive cytoplasmatic staining of the neurons of Rc (63× dots) in the external pyramidal (III) and internal granular scale bar: 10 μm); the interneural LacZ signals are due to the layer (IVA) (63×, scale bar: 10 μm). presence of trans-sectioned axons and dendrites. Page 5 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 a b PTPH1-KO cerebral cortex Figure 4 PTPH1-KO cerebral cortex. a: colocalization of NeuN-ir and LacZ staining signal in the Rc (100×, scale bar: 4.5 μm).; b: detail of the cytoplasmatic signal of LacZ in neurons. In the accelerated rotarod and Y-maze test, significant dif- Considering this gender effect, the follow up analysis was ferences were observed between PTPH1-KOs and WTs carried out in males or females assessing genotype effects based on gender and genotype factors. In the accelerating on activity. PTPH1-KO female mice performed signifi- rotarod test PTPH1-WT mice did not show any gender dif- cantly worse compared to their matched WT littermates, ferences (P = 0.5824 (WT gender vs WT activity); P starting from the second trial and onwards (P = 0.171; P 2WAY AUC 0 30 = 0.3218 (PTPH1-WT male vs female)) (Figure 10a). = 0.002; P = 0.028; P = 0.025) (Figure 10d). No signif- 60 90 PTPH1-KO male mice displayed an overall significant bet- icant differences were observed in PTPH1-KO male mice ter performance compared to their matched female litter- compared to their matched WT littermates (P = 0.92; P 0 30 mates (P = 0.007 (KO gender vs KO activity) (Figure = 0.363; P = 0.222; P = 0.135) (Figure 10e). 2WAY 60 90 10b). Post-hoc T-test analyses showed that the difference was significant at the second trial of the test (P = 0.109; In the Y-maze test, no differences were detected between P = 0.015; P = 0.067; P = 0.835), and the area under PTPH1-KO and WT female mice either in working mem- 30 60 90 the curve for PTPH1-KO male mice was significantly ory (P = 0.972) or in locomotion indices (P = female female higher (by 50%) compared to the matched values of the 0.73; Figures 11a, 11b). On the other hand, PTPH1 KO female littermates (P = 0.0194) (Figure 10c). male mice displayed a significantly higher working mem- ab PTPH1- Figure 5 KO cerebellar cortex PTPH1-KO cerebellar cortex. a: faint LacZ staining in the granule cell layer (20×, scale bar: 16.5 μm); b: perinuclear staining in the granule cell layer of the cerebellum (63×; scale bar: 5 μm). Page 6 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 adults. We replicated these data in PTPH1-KO mice at WT WT K KO O Es14 and Es16 embryonic stages. PTPH1 is expressed in LacZ LacZ CEREB CEREBE ELL LLUM UM the hypothalamic area and but also in the dorsal root gan- H2 H2A A glia of the spinal cord, excluding the spinal cord itself [Additional file 1] [38]. Moreover, at postnatal P1, PTPH1 WT WT K KO O expression is also present in peripheral organs such as LacZ LacZ CORTEX CORTEX muscles and intestines as in the adults [22]. On the other H2 H2A A hand, the CNS expression at P1 appears weaker than in the adults suggesting a pattern of PTPH1 expression corre- WT WT K KO O sponding to specific developmental stages of the CNS as LacZ LacZ HI HIPPO PPOCA CAM MP PU US S well as peripheral organs (data not shown). These changes in expression may play a role in various developmental H2 H2A A functions that need to be further understood. KO KO WT WT MI MID DB BR RA AI IN N SUBSTRIATAL LacZ LacZ In PTPH1-KO adults, LacZ is expressed in different CNS REGIONS H2 H2A A areas such as cerebral and retrosplenial cortices (Figures 1, 2, 3 and 4), hippocampus (Figure 9), thalamus (preferen- tially ventral thalamus) (Figure 7), cerebellum (Figure 5) RT-PC Figure 6 R for beta-galactosidase expression in brain extracts and in the region of the tenia tecta (Figures 1, 8). This data RT-PCR for beta-galactosidase expression in brain extracts. confirms previously observed expression patterns in the Beta-gal mRNA is expressed in PTPH1-KO cerebellum, cor- rat brain by Sahin et al. [10] and extends the observation tex, hippocampus and substriatal regions (midbrain, thalamic to other brain regions. We, furthermore, demonstrate that nuclei, pontine region); no beta-gal expression detected in WT brain extracts (first lane of each block); histone H2A PTPH1 is expressed within the cytoplasm and close to the gene was used as positive control (second lane). cell membrane of neurons in most of the brain area inves- tigated (Figures 4a, 4b). It is known that the FERM domain is indeed necessary for PTPH1 localization close to the plasma membrane in Jurkat T cells [14] and it could ory index (percentage of exact alteration; P = 0.041) be responsible for the punctate expression pattern of male but similar locomotion activity (total arm entries) (P = PTPH1 in the cytosol of the neurons (Figure 4b) [39]. This male 0.348) compared to their matched WT littermates (Figures supports the concept that PTPH1 may be involved in 11a, 11b). cytoskeleton-membrane interaction within extended neu- ronal population in the CNS, potentially playing a role in various neuronal functions. Discussion PTPs are key factors in multiple signaling pathways, lead- ing to modulated functional activities in various cell types Indeed the neural expression of PTPH1 in CA1, CA3 and [34,35]. Among all PTP forms, PTPH1 has been shown in DG of the hippocampus (Figures 9a, 9b, 9c, 9d and 9e), in vitro to modulate cardiac sodium channel Na 1.5 [19], the retrosplenial cortex (Figures 3, 4) and in a series of tha- that it is also known to be expressed in the axons of cere- lamic nuclei (Figures 7a, 7b, 7c, 7d and 7e) suggests an bral cortex, cerebellum, thalamus and brain stem [36]. involvement of PTPH1 in the modulation of the memory Moreover, PTPH1 contains a domain with high sequence circuit. Both hippocampus and retrosplenial cortex are homology with the members of the band 4.1 superfamily key regions in the spatial working memory functions [40- protein, FERM. This domain mediates the linkage of actin 46]. Moreover, several thalamic nuclei have also been filaments to the plasma membrane [37], and therefore shown to be important in the memory process [47,48]. may be involved in cytoskeleton-membrane interactions, For example, a strong loss of dorsomedial and ventral pos- crucial for axon functionality. To further understand the terior thalamic neurons is associated with severe cognitive potential role of PTPH1 in neural functions in vivo, we first and memory disabilities in patients affected by traumatic investigated its expression pattern in embryonic and adult brain injury [49]. Lesions in the lateral thalamus may lead PTPH1-KO mice CNS by LacZ staining, and second its role to important working memory defects in rodents [50]. in CNS functions by behavioral phenotype characteriza- The anterior thalamic nuclei project via the retrosplenial tion. cortices to the hippocampus [51,52], thus underlying the importance of both these circuits and of PTPH1 in the In rat embryonic stage Es19, PTPH1 expression through mnemonic process. FISH analyses has already been shown in the dorsal tha- lamic nuclei, which give rise to the thalamo-cortical con- Another interesting PTPH1-positive area is the indusium nections in adulthood [10]. Thus, it has been suggested to griseum (Figures 3a, 3b) whose role in the adult brain is play a role in the maintenance of these connections in not clear. It is thought to be part of the limbic system, Page 7 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 MHb LP LD DLG CL Po MD AV Po fr VL VPM AM VPM VPL PF VPL Rt VM VPPC VPM VL b c VM VPL VPM VPL d e PTPH1-KO thalamus [76] Figure 7 PTPH1-KO thalamus [76]. a: LacZ expression detected in several thalamic nuclei (4×; scale bar: 165 μm): mediodorsal (MD), central lateral (CL), anteroventral (AV), anteromedial (AM), ventromedial (VM) ventral posteromedial (VPM) and ventrolateral (VL) thalamic nuclear groups. MHb: medial habendular nuclei. b: LacZ expression detected in the ventral posteromedial tha- lamic nuclei (VPM) and it is present also in ventrolateral (VL), ventromedial (VM), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt) thalamic nuclei (2.5×, scale bar: 130 μm). c: LacZ is expressed in the dorsal lateral genic- ulate nucleus (DLG) and in the lateroposteral thalamic nuclear group. In this caudal section LacZ staining is more intense in the posterior nucleus, but present also in VPM, VPL and VPPC (ventral posteromedial parvicel) thalamic nuclei (2.5×, scale bar: 13 μm). d: Detail of beta-gal expression in neural cell body of VPL and VPM at 40× (scale bar: 20 μm) and e: at 63× (scale bar: 10 μm). receiving afferents from the entorhinal and pyriform cor- The cerebellar cortex is also positive for PTPH1 expres- tex and projecting to the septohippocampal nuclei, olfac- sion, in particular in the cytoplasm of granule cells (Figure tory tubercle (presumably the tenia tecta) and the medial 5b). The cerebellum is known to be the main structure for frontal cortex [53,54]. The expression of PTPH1 in these motor learning functions. In particular, the cerebellar cor- specific regions suggests a potential role in the processing/ tex seems to be involved in the early learning phases of integration of memory and sensory information to the motor activities [59,60] that include also a strong activa- SHi and likely the cortex. tion of other areas such as prefrontal cortex and basal gan- glia [29,30]. PTPH1 expression in the granule cells seems Indeed PTPH1 expression is also detectable in the pyram- to indicate a potential involvement in the processing of idal neurons in layer III and IVA of the cerebral cortex of afferent information to the purkinje cells, since it is the mouse (Figures 2a, 2b), in agreement with Sahin's known that afferents fibers to the cerebellar cortex will findings in the rat brain. The middle layers (III and IV) of project in part through the granule cell layer. the cerebral cortex are key sites for thalamic inputs [55,56] especially for VPM and VPL, primary thalamic nuclei for PTPH1 expression pattern observed in our analysis points somato-sensory information integration [57]. Further- out a potential involvement of this phosphatase in more a strong cortico-cortical communication has been numerous CNS processing functions such as locomotion, assessed between these two layers [58], thus suggesting a sensorial integration, learning and memory. In this study, role for PTPH1 as key regulator in the transmission of the the behavioral phenotyping of the PTPH1-KO mice thalamo-cortical and cortico-cortical information. allowed us to test these hypotheses in vivo. Indeed, as Page 8 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 learning and coordination capacities in PTPH1-KO female mice are significantly impaired (Figures 10b, 10c). The low rotarod performance on the early trials, compen- Dtt sated by the last trial, is suggestive of a delay in learning acquisition (Figures 10b, 10d). Dtt As reported in Pilecka et al., our PTPH1-KO mice express the non-catalytic part of PTPH1 in frame with the enzy- matically active part of LacZ gene. LacZ is widely used as a reporter for promoter activity in KO mice and all those SHi mice express a modified protein, whose full function is not known. So far it was never reported a function of LacZ alone in cognition and we consider quite unlikely that this SHi ab is the case in our mice. Thus, it is very likely that the behavioral phenotype we detect in our mice is linked to PTPH1-KO adult mouse brain Figure 8 the deletion of the catalytic domain of PTPH1. PTPH1-KO adult mouse brain. a: beta-gal expression detected in the dorsal tenia tecta (Dtt) and in the septohip- pocampal nuclei (SHi) (4×; scale bar: 165 μm). b: Detail of The impairment in learning and coordination of PTPH1- cytoplasmatic LacZ staining in the Dtt and SHi (10×; scale KO female mice may be resulting from the involvement of bar: 70 μm). PTPH1 in the GH signaling pathway [21]. Indeed our group has already shown that PTPH1-KO mice display higher GHR response in vivo and consequently a higher already demonstrated by our group [22] and also by oth- expression of its down-stream effector hormone, the IGF1 ers [33], PTPH1-KO mice are healthy and do not display in liver and plasma [22]. GHR is highly expressed in most any phenotype, distinguishing them from their matched areas of the CNS, in particular in the choroid plexus, hip- WT littermates, detectable by simple visual observation. pocampus, putamen, thalamus and hypothalamus. Simi- Therefore PTPH1-WT and KO mice underwent a battery larly IGF1 and IGF1-receptors are localized composed by five behavioral tests, from the least to the predominantly in hippocampus, but also in amygdala, most invasive (Table 1), with the tolerable limitation of cerebellum and cortex [61]. Although IGF1 is considered the handling bias. a neuroprotective hormone, it can be produced in the CNS, it is primarily synthesized in the liver and can cross Behavioral testing revealing locomotor dysfunctions, such the blood-brain barrier [62-65]. The GH-IGF1 axis is also as open-field, EPM and Y-maze did not highlight differ- known to influence cognitive functions due to several ences between the two genotypes (Figure 11b), suggesting neuroprotective effects on the hippocampus [66]. Further- that PTPH1 does not play a critical role in the integration more it has been recently pointed out that old conditional of locomotor information. liver-IGF1-KO mice display impaired spatial learning and memory [67]. The presence of PTPH1 in key CNS regions, Anxiety-like behaviors measured by open-field (as path in as well as the consequent deregulation of the GH-IGF1 the center) and EPM (as time spent in the open arms), axis in KO mice, strengthens the concept that the PTPH1 exploiting rodents natural aversion to open space, did not network (CNS and downstream peripheral effectors) may show any differences between the two genotypes (data not be involved in cognitive functions. shown), leading to the conclusion that PTPH1 may not be involved in the integration of thalamo-limbic informa- The behavioral tests assessing working memory and spe- tion, key paths for anxiety behavior processing. Similar cifically learning revealed not only a genotype effect but conclusions can be drawn from the lack of difference also a gender effect, as mentioned above. Sex hormones between the genotypes regarding integration of nocicep- are known to modulate the somatotropic system [68,69]. tive information, based on hot plate test. In humans, testosterone has an important effect on GH axis, in part by its aromatization to estradiol. Administra- In the behavioral test, that partly depends on working tion of estrogens, or aromatized androgen, modulates GH memory performances (Y maze), PTPH1-KO male mice axis neuroregulation [69,70]. In particular, chronic E2 showed a slightly better short-term memory than their WT administration has been shown to reduce GH-induced littermates (Figure 11a). Thus, PTPH1 may be involved in IGF1 increased expression in liver and plasma via a nega- the integration of memory information. This was further tive feedback mechanism, while acute E2 administration strengthened by results obtained with a test assessing leads to the expected GH-induced IGF1 release [71]. Fur- learning and coordination, the rotarod. Contrary to other thermore, it has been reported that estrogens play not behaviors where little differences have been observed, only regulatory functions on neuroendocrine systems but Page 9 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 lac/mol CA1 CA1 CA2 rad FC CA3 oriens DG pyr CA2 oriens lac/mol rad DG hilus oriens CA3 pyr lac/mol rad pyr lac/mol mol gr d e PTPH1-KO hippoca Figure 9 mpus [76] PTPH1-KO hippocampus [76]. a: Hippocampus at 4×; b: CA1 area of hippocampus shows very intense LacZ staining in both oriens and radiatum layers and to a less extent in the pyramidal cell layer (20×; scale bar: 90 μm). c: CA2 area of hippocampus displays LacZ-positive staining in the pyramidal cell layer. d: CA3 area shows an intense beta-gal expression in the oriens and pyramidal cell layer, and in a less extent in the radiatum (20×). e: The dentate gyrus (DG) displays a strong LacZ staining in the molecular layer and not in the hilus (20×) (scale bar: 20 μm). pyr: pyramidal cell layer; oriens: oriens layer; rad: radiatum layer; mol: molecular layer: gr: granule cell layer; lac/mol: lacunosum-molecular layer. can also have stimulatory or inhibitory impacts on the List of abbreviations inter-connectivity of the hippocampal structure depend- PTPH1: protein tyrosine phosphatase H1; KO: knock-out; ing on the gender [72-75], meaning that the same stimu- WT: wild type; CNS: central nervous system; PTKs: protein lus can have opposite effects in male vs female mice. Thus, tyrosine kinases; PTPs: protein tyrosine phosphatases; the cognitive behavioral differences observed in our KO RPTPs: receptor-like protein tyrosine phosphatases; mice are underlying the potential impact of the PTPH1 NRTPTs: nonreceptor PTPs; FERM: 4.1, Ezrin, Radixin, network on neuroendocrine regulation as well as on cellu- Moesin; TACE: TNFα converting enzyme;GH: growth hor- lar architecture within specific brain regions. mone; GHR: growth hormone receptor; IGF1: insulin-like growth factor 1; BAC: bacterial artificial chromosome; ip: intraperitoneal; PBS: phosphate buffered saline; PFA: Conclusion In conclusion, we have demonstrated that PTPH1 is paraformaldehyde; NBT: nitrobluetetrazolium; beta-gal/ expressed in neural populations present in adult brain LacZ: beta-galactosidase; NeuN: Neuronal Nuclei; HBSS: areas mainly involved in locomotor and cognitive func- Hank's balanced salts solution; H2A: Histone 2A; EPM: tions. The behavioral assessments have allowed us to Elevated plus maze; AUC: area under the curve; Es: embry- reveal PTPH1 functionality especially within cognitive onic stage; E2: estradiol; CA: Cornu Ammonis; DG: Den- domains. Better understanding the interplay between var- tate Gyrus; MDL: mediodorsal lateral thalamic nuclei; AV: ious phosphatases regulating CNS functions, which now anteroventral thalamic nuclei; VM: ventromedial thalamic includes PTPH1, will be key in the future to unravel some nuclei; VPM: ventral posteromedial thalamic nuclei; VL: of the complexity of CNS signaling pathways necessary for ventrolateral thalamic nuclei; VPL: ventral posterolateral information processing. thalamic nuclei; LD: laterodorsal thalamic nuclei; Po: pos- terior thalamic nuclei; Rt: reticular thalamic nuclei; VPPC: Page 10 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 250 150 PTPH1-WT male PTPH1-KO male PTPH1-WT female PTPH1-KO female 0 30 60 90 0 30 60 90 time post-trial (min) a b time post-trial (min) 400 PTPH1-WT female PTPH1-KO male * PTPH1-KO female PTPH1-KO female ** 0 0 KO male KO female 0 30 60 90 c d time post-trial (min) PTPH1-WT male PTPH1-KO male 0 30 60 90 time post-trial (min) Rotarod test on PTPH1-W Figure 10 T and KO mice (n = 10) males and females Rotarod test on PTPH1-WT and KO mice (n = 10) males and females. a: WT males and WT females do not display any signif- icant different performance at the rod (P = 0.5824) b: KO males and KO females display a significant different perform- 2WAY ance (P = 0.007) (post-hoc T-test: P = 0.109; P = 0.015; P = 0.067; P = 0.835). c: 50% difference in the area under the 2WAY 0 30 60 90 curve represented in figure 10a (unpaired T-test, P=0.0194). d: Female KO mice display a worse performance at the rod com- pared to WT females (P = 0.171; P = 0.002; P = 0.028; P = 0.025) e: No significant difference in the performance on the 0 30 60 90 rod between male KO and WT mice. a, b, d, e: All the data were analyzed by Two-way Anova followed by T-test; *: p < 0.05; **: p < 0.01. ventral posteromedial parvicel thalamic nuclei; DLG: lat- Authors' contributions eral geniculate nucleus; PF: parafascicular thalamic nuclei; The study was devised by CP and MCM and carried out by nu: nuclei; SHi: septohippocampal muclei; Tt: tenia tecta; CP. PT was responsible for the genotyping of all the adult Ig: indusium griseum. animals that have been used in this study. MA performed the LacZ staining experiment on adult mice. VM and BG have been deeply involved in the first editing of the man- Competing interests The present work is part of CP’s PhD program at the Uni- uscript and all the authors contributed to modifications in versity of Eastern Piedmont, in close collaboration with subsequent drafts. PFZ has been involved in critically MerckSerono International S.A.. MCM, PT, VM, BG, PFZ revising the manuscript and has given the final approval are employed by MerckSerono International S.A., which is of the version to be published. All the authors read and involved in the discovery and the commercialization of approved the final version of the manuscript. therapeutics for the prevention and treatment of human diseases. Page 11 of 14 (page number not for citation purposes) time on the rod (sec) AUC (mm ) time on the rod (sec) time on the rod (sec) time on the rod (sec) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 Additional file 3 Additional methods. This Loco document motio provides t n inde hx e methods and the refer- Working memory index ences that have been used to perform the experiments represented in Addi- 30 tional file 2 Click here for file 25 [http://www.biomedcentral.com/content/supplementary/1744- * 9081-4-36-S3.doc] 70 20 WT WT KO KO Females Males All Females Males All ab Y-maze behaviora Figure 11 l test on PTPH1-WT and KO mice (n = 10) males and females Y-maze behavioral test on PTPH1-WT and KO mice (n = 10) males and females. a: Male KO mice display higher working memory index compared to WT male littermates (P = 0.041); no differences recorded in the female mice. b: No significant male differences recorded in the locomotion index, represented by the total arm entries between PTPH1-WT and KO males and females. T-test, *: p < 0.05. Additional material Acknowledgements This work was fully supported by MerckSerono International S.A.. We would like to thank Rob Hooft van Huijsduijnen, Andrea Graziani and Linda Additional file 1 Chaabane for their scientific support and for kindly reviewing the manu- LacZ staining on PTPH1-WT and KO embryos. PTPH1-WT embryos do script. A special thank to Sonia Carboni and Gabriele Dati for their helpful not show any staining either at embryological stage 14 (Es14) or at Es16. comments and advices on immunohistochemistry and to Niels Adams for PTPH1-KO embryos display a positive LacZ staining in the hypothalamic his support on LacZ staining data. area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself. References Click here for file 1. Stoker AW: Protein tyrosine phosphatases and signalling. J [http://www.biomedcentral.com/content/supplementary/1744- Endocrinol 2005, 185:19-33. 9081-4-36-S1.ppt] 2. 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Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

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Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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1744-9081
DOI
10.1186/1744-9081-4-36
pmid
18700002
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Abstract

Background: The present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions. Methods: We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions. Results: In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. Conclusion: These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition. Background low molecular weight PTP and 4) the Asp-based PTPs Tyrosine phosphorylation plays an important role in sev- (Tyr/Ser phosphatase activity) [3]. eral signaling pathways regulating cell growth, differenti- ation, cell cycle, apoptosis and neuronal functions [1,2]. Classical PTPs have been reported to play a key role in The phosphorylation/dephosphorylation balance is con- neural functions, from development to cognitive func- trolled by protein tyrosine kinases and phosphatases. tion. For example, RPTPs such as PTPδ, PTPσ, LAR, and PTPs can be distinguished into four classes: 1) classical especially PTPRO, are important players in axonal growth PTPs that can be subdivided into transmembrane, recep- and guidance during development [4]. Studies on PTPσ- tor-like enzymes, and the intracellular, nonreceptor PTPs, KO (RPTP) mice have shown involvement of this PTP in 2) dual-specificity PTPs (Ser and Tyr phosphatases), 3) the regulation of the developing hypothalamo-pituitary Page 1 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 axis [5,6] and in the development of the CNS architecture experiment was in accordance with Directive 86/609/EEC, [7]. PTPBL-KO (non receptor like PTP-NRPTP) mice dis- enforced by the Italian D.L. No. 116 of January 27, 1992. play impaired motor nerve repair in a model of sciatic Physical facilities and equipment for accommodation and nerve crush lesion [8] and PTPMEG (NRPTP) interacts care of animals were in accordance with the provisions of with key intracellular players leading to the stimulation of EEC Council Directive 86/609. Tail snips from mice were the channel activity of NMDA receptors [9]. collected and genotyped as previously reported [22]. In the present study we focused our attention on a NRPTP, PTPH1 KO design PTPH1, and on its possible role on neural functions. PTPH1-KO mice were generated using the Velocigene Indeed PTPH1 has been shown to be expressed in the CNS technology [23], as described in details elsewhere [22]. [10] but little is currently known on its potential impact Briefly a mouse BAC containing the PTPH1 gene was on CNS functions. PTPH1 (also called PTPN3) belongs to modified: an in-frame LacZ reporter sequence and a neo- a sub-family of non receptor cytosolic PTPs characterized mycin-selectable marker replaced exons 15 to 22 encod- by the presence of a FERM domain (band 4.1, ezrin, ing for the PDZ and the catalytic domain of PTPH1. BAC radixin, moesin) at its N-terminus, responsible for the electroporation into embryonic stem cells was performed. interaction with transmembrane proteins and/or phos- F1 heterozygous mice were bred to generate F2 PTPH1-KO pholipids in the cell membrane [11-13]. In addition mice. Line breeding and animal care were performed in PTPH1 has a PDZ domain in the central part responsible Charles River Italy and France. for the interaction with other proteins, whereas the single catalytic domain is located at the C-terminus. LacZ staining procedure and immunohistochemistry PTPH1-KO and WT mice, 12 months old, n = 2, male and PTPH1 activity has been involved in a variety of cellular females, were sacrificed by ip overdose of thiopental functions including TCR-signaling [14-16], cell cycle reg- (5%), perfused with paraformaldehyde 4%, then washed ulation [11,16,17], endoplasmic reticulum assembly [18], in PBS and incubated overnight at 37°C in the solution cardiac sodium channel modulation [19] and TNFα con- containing the substrate for beta-galactosidase (beta-gal, verting enzyme inhibition [20]. encoded by the LacZ cassette) coupled to a NBT salt. The organs and the tissues in the sections display a green/blue Recently, our group has demonstrated that PTPH1 staining where PTPH1 gene is normally expressed. After dephosphorylates GHR in vitro and in cellular assays [21] rinsing into PBS, organs were postfixed in PFA 4% for 1 and results in an increase of body weight in the functional hour, then incubated in 50% glycerol overnight at 4°C PTPH1-knockout (KO) mice via modulation of IGF1 and finally maintained in 70% glycerol at room tempera- secretion [22] thus demonstrating its in vivo relevance. ture. LacZ staining was observed through a low magnifica- tion microscope and described by an operator blind to the PTPH1 has been shown in the rat to be highly expressed genotypes. in thalamic nuclei as well as various cortical areas [10]. However, no information is currently available on its LacZ staining was also performed on CNS sections. Mice impact on CNS functions. To address this question we (n = 3, 12 months old) were sacrificed by ip injection of have further characterized our PTPH1-KO mice line an overdose of thiopental (5%), perfused with PBS and through behavioral and anatomical approaches. PTPH1 PFA 4%. Brains were removed and postfixed overnight at expression and localization was evaluated by LacZ stain- 4°C in PFA 4%, then placed overnight at 4°C in 15% and ing in the brain and a behavioral test battery evaluated finally in 30% sucrose buffer. The brains were then PTPH1 loss on CNS functions such as locomotor activity included in O.C.T. (Tissue-Tek) and sections were cut on (open field), anxiety-like behavior (open field and ele- slides with a cryostat at 20 μm thickness. The slides were vated plus maze), motor ability, coordination and learn- incubated in LacZ staining solution (see above) overnight ing (accelerating rotarod), spatial working memory (Y at 37°C, washed thrice in PBS (5 min each) and either maze) and nociceptive sensitivity (hot plate). counterstained with H&E (Merck KGaA) [23] or co- expressed with NeuN immunostaining. Briefly, sections were incubated for 3 hours at room temperature in block- Methods Animals ing solution (Vectastain Kit), washed in TBS (Tris-buffered PTPH1-KO and wild type littermates (F2 generation, saline), incubated overnight at 4°C with a solution con- 87.5% C57Bl/6 – 12.5% 129S6SvEv) aged 3–4 months taining the primary antibody mouse anti-mouse Neuro- were used for behavioral phenotyping. Mice were individ- nal Nuclei (Chemicon MAB377, 1/1000). The staining ually caged and maintained in a 12:12 hours light: dark was revealed by ABC kit secondary antibody (mouse cycle (lights on at 7 am) at 21 ± 1°C with food and water Vectastain Kit), and DAB (Sigma). After dehydration, sec- available ad libitum. Protection of animals used in the tions were transferred onto coverslips. LacZ staining and Page 2 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 co-expression with NeuN-immunoreactivity (NeuN-ir) Elevated plus maze was observed by microscopy and described by an operator After one hour of adaptation in the testing room, anxiety- blind to the genotypes. like behavior was tested for each mouse by EPM within one session. The apparatus consists of four arms (29.5 cm Semiquantitative RT-PCR for beta galactosidase gene long and 5 cm wide each). Two arms are open whereas the Semiquantitative RT-PCR for PTPH1 and beta-gal gene 2 others are limited by 2 black walls (20 cm high). The expression was performed in different brain areas of number of entries of each mouse in the open and closed PTPH1-KO and WT mice in order to confirm the presence arms was recorded by a video camera during a period of 5 of beta-gal expression in the KO tissues, replacing PTPH1 minutes and analyzed by the SMART Video-Tracking Soft- PDZ and catalytic domain. Brains from KO and WT mice ware (ViewPoint Life Sci. Inc.). The total number of (n = 5, 6 months old) were freshly removed and rinsed in entries into the arms is an index of locomotion, whereas HBSS. Hippocampus, cerebellum, cortex, striatum, mid- the percentage of time spent and percentage of entries in brain and olfactory bulbs were dissected. Total RNA was the closed arms is an index of anxiety-like behaviors [25]. extracted using Trizol Reagent (Invitrogen) and cleaned- Accelerated rotarod up by RNAeasy columns from Qiagen. 5 μg of total RNA were used to perform the RT-PCR reaction (SuperScript II Motor ability, coordination and learning were evaluated RT kit, Invitrogen). The primer sequences for LacZ ampli- by using an Accelerated Rotarod apparatus for mice (Cat. fication were the following: LacZ – forward 5'-GAT GTA # 7650 by Jones and Roberts, distr. by Basile Instr., Italy). CGT GCC CTG GAA CT/reverse 5'-GGT CCC ACA CTT The apparatus was placed within the animal colony room CAG CAT TT. In order to load equally the reaction mixes, and was cleaned after each trial. Mice were tested for their a 300 bp fragment of Histone 2A was amplified as a house abilities to maintain a balance on a rotating bar, which keeping gene with the following primers: H2Az forward – accelerated from 4 to 40 rpm/min in a 5 min trial. Latency 5' CGT ATT CAT CGA CAC CTG AAA; H2Az reverse – 5' to fall off was measured within one session and all mice CTG TTG TCC TTT CTT CCC GAT. underwent four trials (one every 30 min) [26-28]. The dif- ferences at the rotarod performances in WT and KO were Behavioral phenotyping test battery assessed by a single set of trials [27,28]. This set-up allows Neurological functions of PTPH1-WT and KO mice a major focus on the early phases of motor learning, (males and females, 11 weeks-old, n = 10 per gender per involving a strong activation of prefrontal cortex and of genotype) were assessed through a behavioral test battery. the associative areas of basal ganglia and cerebellum [29,30]. The sequence of the test battery was chosen from the least invasive to the most ones. The schedule of the testing ses- Y-maze alternation sions included one week of recovery from one test to the After one hour of adaptation in the testing room, mice next, as reported in Table 1. were tested on a Y maze apparatus (40 cm long/8 cm wide arms with transparent walls) to investigate spatial work- Open field ing memory [31]. The number and the sequence of the After one hour of adaptation in the testing room, each arm entries for each mouse were recorded during 5 min- mouse was placed in an open field chamber (50 cm wide utes. The locomotion index was calculated as the overall with white floor and walls) (ViewPoint Life Sci. Inc.) to number of arm entries, whereas the working memory test locomotor activity and anxiety-like behaviors. Loco- index was calculated as following: number of exact alter- motion was recorded for one hour by a video camera and nations (entries into three different arms consecutively)/ analyzed automatically by VideoTRACK software (View- possible alternations (i.e. the number of arms entered Point Life Sci. Inc.). Locomotor activity was evaluated by minus 2) × 100. calculating the total path length traveled, whereas the rel- ative time spent in the center was taken as indicative of Hot plate anxiety-like behavior [24]. The tests were performed in Thermal sensitivity was assessed by a hot plate apparatus two sessions with equivalent group representation. for mice (Cat. # 7280 by Biol. Research Apparatus, distr. by Basile Instr., Italy) and lasted a maximum of 45 sec- onds, time at which damages could occur [32]. The appa- Table 1: Schedule of the behavioral test battery. Age (wks) 8 9 10 11 12 13 14 15 10M+10F arrival quarantine adaptation Open Field EPM Rotarod Y-maze Hot plate Page 3 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 ratus was placed in the animal colony room and all the (Figures. 3a, 3b 3c, 4a and 4b) and indusium griseum mice were tested within one session. Animals were placed (Figures 3a, 3b). In the cerebellum, in spite of a strong on a surface heated at 52.5°C and the latency (seconds) to staining in the whole mount (Figure 2), only a faint LacZ shake or lick the paw was recorded by the operator. signal was observed in sections (Figures 5a, 5b) in partic- ular in the granule cells, close to the nuclei. The RT-PCR Statistics on cortical and cerebellar extracts confirmed the presence Statistical comparisons were performed by unpaired two- of LacZ expression in these brain areas (Figure 6). tailed T-test (p < 0.05) and two-way ANOVA (p < 0.05) followed by post-hoc test as necessary. In the accelerated In subcortical regions, LacZ was detected in the anterior rotarod, two-way ANOVA with repeated measures fol- ventral, mediodorsal, ventrolateral, anteromedial and lowed by T-test was used. Results are expressed as mean ± central lateral thalamic nuclear groups (Figure 7a). In SEM. more caudal thalamic areas, LacZ was again detectable in the posterior thalamic nuclear group (Po), and to a lesser Results extent in posteromedial, in posterolateral and in reticular LacZ staining in whole mount thalamic nuclei and also in the dorsal lateral geniculate In PTPH1-KO adult animals, LacZ staining in the brain nuclei (Figures 7b, 7c, 7d and 7e). In the tenia tecta, LacZ was observed in the cerebellum, hippocampus and in the staining visible in the whole mount preparation was con- thalamic nuclei. In addition a strong staining was firmed (Figures 2, 8a, 8b). The RT-PCR on substriatal observed in the cerebral cortex, tenia tecta and septum regions including the thalamus, the midbrain and the (Figures 1a, 1b). pontine areas confirmed the presence of LacZ expression in some of these brain areas (Figure 6). To exclude any LacZ staining in sections and RT-PCR results potential impact of LacZ blood signal contamination in No gross cytoarchitectural brain differences were observed brain areas, RT-PCR on 5 to 20 μl of whole blood was car- by simple visual observation at the microscope in the cor- ried out and did not reveal any significant signal [Addi- tex, hippocampus and thalamus in PTPH1-KO mice com- tional files 2, 3]. pared to WT littermates. In the hippocampus, LacZ expression was observed in the LacZ staining was performed on frozen brain sections to cytoplasm of a few pyramidal cells and through the fibers confirm and to describe the expression of PTPH1 at the of the oriens and radiatum layer in a rostral caudal spread brain structural level (Table 2). (Figure 9a). In rostral sections, LacZ was expressed in the septohippocampal nuclei (Figure 8a). In more caudal sec- In cortical regions, LacZ was expressed in the external tions LacZ was present in the CA1 and CA3, and in a lesser pyramidal (III) and internal granular layer (IVA) of the extent in the CA2 (Figures 9b, 9c and 9d). In the CA3 LacZ cerebral cortex (Figures 2a, 2b), in the retrosplenial cortex was strongly expressed in the oriens and pyramidal cell Co Rc Co Cb Th Cb Tt ab PTPH1-KO adult mouse brain Figure 1 PTPH1-KO adult mouse brain. a: whole brain, dorsal view, staining in cerebellum (Cb) and cortex (Co); b: LacZ staining on brain, sagittal view: detection in the tenia tecta (Tt), cortex (Co), thalamus (Th), hippocampus (H), retrosplenial cortex (Rc), septum (S) and in the granule cell layer of cerebellum. Page 4 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 Table 2: Qualitative estimation of LacZ staining intensity in the different brain areas. Brain Area Intensity of LacZ staining Brain Area Intensity of LacZ staining Cerebral cortex + Dorsal Tenia Tecta ++ Retrosplenial cortex ++ Septohippocampal nu + CA1 oriens layer +++ VPL + CA1 radiatum layer +++ MDL ++ CA1 pyramidal cell layer + AV ++ CA2 oriens layer - VPM ++ CA2 radiatum layer - VL + CA2 pyramidal layer + VM ++ CA3 oriens layer + Po ++ CA3 radiatum layer +/- LD + CA3 pyramidal layer + Rt + DG granular cell layer - DLG ++ DG molecular layer +++ VPPC ++ DG hilus - PF - Fascicola cinereum ++ cerebellum + Indisium griseum + -: absent; +/-: faint; +: present; ++: intense; +++: very intense staining. Cornu Ammonis (CA), Dentate Gyrus (DG), mediodorsal lateral (MDL), anteroventral (AV), ventromedial (VM) ventral posteromedial (VPM), ventrolateral (VL), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt), ventral posteromedial parvicel (VPPC) thalamic nuclei, lateral geniculate nucleus (DLG), parafascicular thalamic nuclei (PF), nuclei (nu). layer (Figure 9d), but its intensity was reduced in the body weight has been detected in PTPH1-KO mice com- radiatum and oriens compared to CA1 (Figure 9b). No pared to WT littermates, more pronounced in male mice staining was detected in the lacunosum-molecular layer in and probably due to an enhanced GHR sensitivity, that CA1, CA2 and CA3 (Figures 9b, 9c and 9d). The dentate leads to increased IGF-1 mRNA and protein expression in gyrus showed a strong positive LacZ signal in the molecu- liver and plasma, respectively [22]. lar layer, but not in the hilus (Figure 9e). In EPM, open field test and hot plate tests (anxiety-related Behavioral phenotyping behavior and thermal pain sensitivity), PTPH1-KO male As previously demonstrated, PTPH1-KO mice were and female mice did not show any significant differences healthy, reproduced normally and did not show any phe- in comparison with their WT littermates (data not notypic traits distinguishing them from their WT litterma- shown). tes by simple visual observations [22,33]. An increased Rc Rc ig II III IVa ig PTPH Figure 3 1-KO cerebral cortex PTPH1-KO cerebral cortex. a: LacZ detection in retrosple- a Figure 2 : PTPH1-KO cerebral cortex (10×, scale bar: 220 μm) nial cortex (Rc) and indusium griseum (ig) staining (4×, scale a: PTPH1-KO cerebral cortex (10×, scale bar: 220 μm). b: bar: 80 μm). b: detail of the Rc and ig (40×, scale bar: 20 μm); positive cytoplasmatic and perinuclear LacZ staining (blue c: positive cytoplasmatic staining of the neurons of Rc (63× dots) in the external pyramidal (III) and internal granular scale bar: 10 μm); the interneural LacZ signals are due to the layer (IVA) (63×, scale bar: 10 μm). presence of trans-sectioned axons and dendrites. Page 5 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 a b PTPH1-KO cerebral cortex Figure 4 PTPH1-KO cerebral cortex. a: colocalization of NeuN-ir and LacZ staining signal in the Rc (100×, scale bar: 4.5 μm).; b: detail of the cytoplasmatic signal of LacZ in neurons. In the accelerated rotarod and Y-maze test, significant dif- Considering this gender effect, the follow up analysis was ferences were observed between PTPH1-KOs and WTs carried out in males or females assessing genotype effects based on gender and genotype factors. In the accelerating on activity. PTPH1-KO female mice performed signifi- rotarod test PTPH1-WT mice did not show any gender dif- cantly worse compared to their matched WT littermates, ferences (P = 0.5824 (WT gender vs WT activity); P starting from the second trial and onwards (P = 0.171; P 2WAY AUC 0 30 = 0.3218 (PTPH1-WT male vs female)) (Figure 10a). = 0.002; P = 0.028; P = 0.025) (Figure 10d). No signif- 60 90 PTPH1-KO male mice displayed an overall significant bet- icant differences were observed in PTPH1-KO male mice ter performance compared to their matched female litter- compared to their matched WT littermates (P = 0.92; P 0 30 mates (P = 0.007 (KO gender vs KO activity) (Figure = 0.363; P = 0.222; P = 0.135) (Figure 10e). 2WAY 60 90 10b). Post-hoc T-test analyses showed that the difference was significant at the second trial of the test (P = 0.109; In the Y-maze test, no differences were detected between P = 0.015; P = 0.067; P = 0.835), and the area under PTPH1-KO and WT female mice either in working mem- 30 60 90 the curve for PTPH1-KO male mice was significantly ory (P = 0.972) or in locomotion indices (P = female female higher (by 50%) compared to the matched values of the 0.73; Figures 11a, 11b). On the other hand, PTPH1 KO female littermates (P = 0.0194) (Figure 10c). male mice displayed a significantly higher working mem- ab PTPH1- Figure 5 KO cerebellar cortex PTPH1-KO cerebellar cortex. a: faint LacZ staining in the granule cell layer (20×, scale bar: 16.5 μm); b: perinuclear staining in the granule cell layer of the cerebellum (63×; scale bar: 5 μm). Page 6 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 adults. We replicated these data in PTPH1-KO mice at WT WT K KO O Es14 and Es16 embryonic stages. PTPH1 is expressed in LacZ LacZ CEREB CEREBE ELL LLUM UM the hypothalamic area and but also in the dorsal root gan- H2 H2A A glia of the spinal cord, excluding the spinal cord itself [Additional file 1] [38]. Moreover, at postnatal P1, PTPH1 WT WT K KO O expression is also present in peripheral organs such as LacZ LacZ CORTEX CORTEX muscles and intestines as in the adults [22]. On the other H2 H2A A hand, the CNS expression at P1 appears weaker than in the adults suggesting a pattern of PTPH1 expression corre- WT WT K KO O sponding to specific developmental stages of the CNS as LacZ LacZ HI HIPPO PPOCA CAM MP PU US S well as peripheral organs (data not shown). These changes in expression may play a role in various developmental H2 H2A A functions that need to be further understood. KO KO WT WT MI MID DB BR RA AI IN N SUBSTRIATAL LacZ LacZ In PTPH1-KO adults, LacZ is expressed in different CNS REGIONS H2 H2A A areas such as cerebral and retrosplenial cortices (Figures 1, 2, 3 and 4), hippocampus (Figure 9), thalamus (preferen- tially ventral thalamus) (Figure 7), cerebellum (Figure 5) RT-PC Figure 6 R for beta-galactosidase expression in brain extracts and in the region of the tenia tecta (Figures 1, 8). This data RT-PCR for beta-galactosidase expression in brain extracts. confirms previously observed expression patterns in the Beta-gal mRNA is expressed in PTPH1-KO cerebellum, cor- rat brain by Sahin et al. [10] and extends the observation tex, hippocampus and substriatal regions (midbrain, thalamic to other brain regions. We, furthermore, demonstrate that nuclei, pontine region); no beta-gal expression detected in WT brain extracts (first lane of each block); histone H2A PTPH1 is expressed within the cytoplasm and close to the gene was used as positive control (second lane). cell membrane of neurons in most of the brain area inves- tigated (Figures 4a, 4b). It is known that the FERM domain is indeed necessary for PTPH1 localization close to the plasma membrane in Jurkat T cells [14] and it could ory index (percentage of exact alteration; P = 0.041) be responsible for the punctate expression pattern of male but similar locomotion activity (total arm entries) (P = PTPH1 in the cytosol of the neurons (Figure 4b) [39]. This male 0.348) compared to their matched WT littermates (Figures supports the concept that PTPH1 may be involved in 11a, 11b). cytoskeleton-membrane interaction within extended neu- ronal population in the CNS, potentially playing a role in various neuronal functions. Discussion PTPs are key factors in multiple signaling pathways, lead- ing to modulated functional activities in various cell types Indeed the neural expression of PTPH1 in CA1, CA3 and [34,35]. Among all PTP forms, PTPH1 has been shown in DG of the hippocampus (Figures 9a, 9b, 9c, 9d and 9e), in vitro to modulate cardiac sodium channel Na 1.5 [19], the retrosplenial cortex (Figures 3, 4) and in a series of tha- that it is also known to be expressed in the axons of cere- lamic nuclei (Figures 7a, 7b, 7c, 7d and 7e) suggests an bral cortex, cerebellum, thalamus and brain stem [36]. involvement of PTPH1 in the modulation of the memory Moreover, PTPH1 contains a domain with high sequence circuit. Both hippocampus and retrosplenial cortex are homology with the members of the band 4.1 superfamily key regions in the spatial working memory functions [40- protein, FERM. This domain mediates the linkage of actin 46]. Moreover, several thalamic nuclei have also been filaments to the plasma membrane [37], and therefore shown to be important in the memory process [47,48]. may be involved in cytoskeleton-membrane interactions, For example, a strong loss of dorsomedial and ventral pos- crucial for axon functionality. To further understand the terior thalamic neurons is associated with severe cognitive potential role of PTPH1 in neural functions in vivo, we first and memory disabilities in patients affected by traumatic investigated its expression pattern in embryonic and adult brain injury [49]. Lesions in the lateral thalamus may lead PTPH1-KO mice CNS by LacZ staining, and second its role to important working memory defects in rodents [50]. in CNS functions by behavioral phenotype characteriza- The anterior thalamic nuclei project via the retrosplenial tion. cortices to the hippocampus [51,52], thus underlying the importance of both these circuits and of PTPH1 in the In rat embryonic stage Es19, PTPH1 expression through mnemonic process. FISH analyses has already been shown in the dorsal tha- lamic nuclei, which give rise to the thalamo-cortical con- Another interesting PTPH1-positive area is the indusium nections in adulthood [10]. Thus, it has been suggested to griseum (Figures 3a, 3b) whose role in the adult brain is play a role in the maintenance of these connections in not clear. It is thought to be part of the limbic system, Page 7 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 MHb LP LD DLG CL Po MD AV Po fr VL VPM AM VPM VPL PF VPL Rt VM VPPC VPM VL b c VM VPL VPM VPL d e PTPH1-KO thalamus [76] Figure 7 PTPH1-KO thalamus [76]. a: LacZ expression detected in several thalamic nuclei (4×; scale bar: 165 μm): mediodorsal (MD), central lateral (CL), anteroventral (AV), anteromedial (AM), ventromedial (VM) ventral posteromedial (VPM) and ventrolateral (VL) thalamic nuclear groups. MHb: medial habendular nuclei. b: LacZ expression detected in the ventral posteromedial tha- lamic nuclei (VPM) and it is present also in ventrolateral (VL), ventromedial (VM), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt) thalamic nuclei (2.5×, scale bar: 130 μm). c: LacZ is expressed in the dorsal lateral genic- ulate nucleus (DLG) and in the lateroposteral thalamic nuclear group. In this caudal section LacZ staining is more intense in the posterior nucleus, but present also in VPM, VPL and VPPC (ventral posteromedial parvicel) thalamic nuclei (2.5×, scale bar: 13 μm). d: Detail of beta-gal expression in neural cell body of VPL and VPM at 40× (scale bar: 20 μm) and e: at 63× (scale bar: 10 μm). receiving afferents from the entorhinal and pyriform cor- The cerebellar cortex is also positive for PTPH1 expres- tex and projecting to the septohippocampal nuclei, olfac- sion, in particular in the cytoplasm of granule cells (Figure tory tubercle (presumably the tenia tecta) and the medial 5b). The cerebellum is known to be the main structure for frontal cortex [53,54]. The expression of PTPH1 in these motor learning functions. In particular, the cerebellar cor- specific regions suggests a potential role in the processing/ tex seems to be involved in the early learning phases of integration of memory and sensory information to the motor activities [59,60] that include also a strong activa- SHi and likely the cortex. tion of other areas such as prefrontal cortex and basal gan- glia [29,30]. PTPH1 expression in the granule cells seems Indeed PTPH1 expression is also detectable in the pyram- to indicate a potential involvement in the processing of idal neurons in layer III and IVA of the cerebral cortex of afferent information to the purkinje cells, since it is the mouse (Figures 2a, 2b), in agreement with Sahin's known that afferents fibers to the cerebellar cortex will findings in the rat brain. The middle layers (III and IV) of project in part through the granule cell layer. the cerebral cortex are key sites for thalamic inputs [55,56] especially for VPM and VPL, primary thalamic nuclei for PTPH1 expression pattern observed in our analysis points somato-sensory information integration [57]. Further- out a potential involvement of this phosphatase in more a strong cortico-cortical communication has been numerous CNS processing functions such as locomotion, assessed between these two layers [58], thus suggesting a sensorial integration, learning and memory. In this study, role for PTPH1 as key regulator in the transmission of the the behavioral phenotyping of the PTPH1-KO mice thalamo-cortical and cortico-cortical information. allowed us to test these hypotheses in vivo. Indeed, as Page 8 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 learning and coordination capacities in PTPH1-KO female mice are significantly impaired (Figures 10b, 10c). The low rotarod performance on the early trials, compen- Dtt sated by the last trial, is suggestive of a delay in learning acquisition (Figures 10b, 10d). Dtt As reported in Pilecka et al., our PTPH1-KO mice express the non-catalytic part of PTPH1 in frame with the enzy- matically active part of LacZ gene. LacZ is widely used as a reporter for promoter activity in KO mice and all those SHi mice express a modified protein, whose full function is not known. So far it was never reported a function of LacZ alone in cognition and we consider quite unlikely that this SHi ab is the case in our mice. Thus, it is very likely that the behavioral phenotype we detect in our mice is linked to PTPH1-KO adult mouse brain Figure 8 the deletion of the catalytic domain of PTPH1. PTPH1-KO adult mouse brain. a: beta-gal expression detected in the dorsal tenia tecta (Dtt) and in the septohip- pocampal nuclei (SHi) (4×; scale bar: 165 μm). b: Detail of The impairment in learning and coordination of PTPH1- cytoplasmatic LacZ staining in the Dtt and SHi (10×; scale KO female mice may be resulting from the involvement of bar: 70 μm). PTPH1 in the GH signaling pathway [21]. Indeed our group has already shown that PTPH1-KO mice display higher GHR response in vivo and consequently a higher already demonstrated by our group [22] and also by oth- expression of its down-stream effector hormone, the IGF1 ers [33], PTPH1-KO mice are healthy and do not display in liver and plasma [22]. GHR is highly expressed in most any phenotype, distinguishing them from their matched areas of the CNS, in particular in the choroid plexus, hip- WT littermates, detectable by simple visual observation. pocampus, putamen, thalamus and hypothalamus. Simi- Therefore PTPH1-WT and KO mice underwent a battery larly IGF1 and IGF1-receptors are localized composed by five behavioral tests, from the least to the predominantly in hippocampus, but also in amygdala, most invasive (Table 1), with the tolerable limitation of cerebellum and cortex [61]. Although IGF1 is considered the handling bias. a neuroprotective hormone, it can be produced in the CNS, it is primarily synthesized in the liver and can cross Behavioral testing revealing locomotor dysfunctions, such the blood-brain barrier [62-65]. The GH-IGF1 axis is also as open-field, EPM and Y-maze did not highlight differ- known to influence cognitive functions due to several ences between the two genotypes (Figure 11b), suggesting neuroprotective effects on the hippocampus [66]. Further- that PTPH1 does not play a critical role in the integration more it has been recently pointed out that old conditional of locomotor information. liver-IGF1-KO mice display impaired spatial learning and memory [67]. The presence of PTPH1 in key CNS regions, Anxiety-like behaviors measured by open-field (as path in as well as the consequent deregulation of the GH-IGF1 the center) and EPM (as time spent in the open arms), axis in KO mice, strengthens the concept that the PTPH1 exploiting rodents natural aversion to open space, did not network (CNS and downstream peripheral effectors) may show any differences between the two genotypes (data not be involved in cognitive functions. shown), leading to the conclusion that PTPH1 may not be involved in the integration of thalamo-limbic informa- The behavioral tests assessing working memory and spe- tion, key paths for anxiety behavior processing. Similar cifically learning revealed not only a genotype effect but conclusions can be drawn from the lack of difference also a gender effect, as mentioned above. Sex hormones between the genotypes regarding integration of nocicep- are known to modulate the somatotropic system [68,69]. tive information, based on hot plate test. In humans, testosterone has an important effect on GH axis, in part by its aromatization to estradiol. Administra- In the behavioral test, that partly depends on working tion of estrogens, or aromatized androgen, modulates GH memory performances (Y maze), PTPH1-KO male mice axis neuroregulation [69,70]. In particular, chronic E2 showed a slightly better short-term memory than their WT administration has been shown to reduce GH-induced littermates (Figure 11a). Thus, PTPH1 may be involved in IGF1 increased expression in liver and plasma via a nega- the integration of memory information. This was further tive feedback mechanism, while acute E2 administration strengthened by results obtained with a test assessing leads to the expected GH-induced IGF1 release [71]. Fur- learning and coordination, the rotarod. Contrary to other thermore, it has been reported that estrogens play not behaviors where little differences have been observed, only regulatory functions on neuroendocrine systems but Page 9 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 lac/mol CA1 CA1 CA2 rad FC CA3 oriens DG pyr CA2 oriens lac/mol rad DG hilus oriens CA3 pyr lac/mol rad pyr lac/mol mol gr d e PTPH1-KO hippoca Figure 9 mpus [76] PTPH1-KO hippocampus [76]. a: Hippocampus at 4×; b: CA1 area of hippocampus shows very intense LacZ staining in both oriens and radiatum layers and to a less extent in the pyramidal cell layer (20×; scale bar: 90 μm). c: CA2 area of hippocampus displays LacZ-positive staining in the pyramidal cell layer. d: CA3 area shows an intense beta-gal expression in the oriens and pyramidal cell layer, and in a less extent in the radiatum (20×). e: The dentate gyrus (DG) displays a strong LacZ staining in the molecular layer and not in the hilus (20×) (scale bar: 20 μm). pyr: pyramidal cell layer; oriens: oriens layer; rad: radiatum layer; mol: molecular layer: gr: granule cell layer; lac/mol: lacunosum-molecular layer. can also have stimulatory or inhibitory impacts on the List of abbreviations inter-connectivity of the hippocampal structure depend- PTPH1: protein tyrosine phosphatase H1; KO: knock-out; ing on the gender [72-75], meaning that the same stimu- WT: wild type; CNS: central nervous system; PTKs: protein lus can have opposite effects in male vs female mice. Thus, tyrosine kinases; PTPs: protein tyrosine phosphatases; the cognitive behavioral differences observed in our KO RPTPs: receptor-like protein tyrosine phosphatases; mice are underlying the potential impact of the PTPH1 NRTPTs: nonreceptor PTPs; FERM: 4.1, Ezrin, Radixin, network on neuroendocrine regulation as well as on cellu- Moesin; TACE: TNFα converting enzyme;GH: growth hor- lar architecture within specific brain regions. mone; GHR: growth hormone receptor; IGF1: insulin-like growth factor 1; BAC: bacterial artificial chromosome; ip: intraperitoneal; PBS: phosphate buffered saline; PFA: Conclusion In conclusion, we have demonstrated that PTPH1 is paraformaldehyde; NBT: nitrobluetetrazolium; beta-gal/ expressed in neural populations present in adult brain LacZ: beta-galactosidase; NeuN: Neuronal Nuclei; HBSS: areas mainly involved in locomotor and cognitive func- Hank's balanced salts solution; H2A: Histone 2A; EPM: tions. The behavioral assessments have allowed us to Elevated plus maze; AUC: area under the curve; Es: embry- reveal PTPH1 functionality especially within cognitive onic stage; E2: estradiol; CA: Cornu Ammonis; DG: Den- domains. Better understanding the interplay between var- tate Gyrus; MDL: mediodorsal lateral thalamic nuclei; AV: ious phosphatases regulating CNS functions, which now anteroventral thalamic nuclei; VM: ventromedial thalamic includes PTPH1, will be key in the future to unravel some nuclei; VPM: ventral posteromedial thalamic nuclei; VL: of the complexity of CNS signaling pathways necessary for ventrolateral thalamic nuclei; VPL: ventral posterolateral information processing. thalamic nuclei; LD: laterodorsal thalamic nuclei; Po: pos- terior thalamic nuclei; Rt: reticular thalamic nuclei; VPPC: Page 10 of 14 (page number not for citation purposes) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 250 150 PTPH1-WT male PTPH1-KO male PTPH1-WT female PTPH1-KO female 0 30 60 90 0 30 60 90 time post-trial (min) a b time post-trial (min) 400 PTPH1-WT female PTPH1-KO male * PTPH1-KO female PTPH1-KO female ** 0 0 KO male KO female 0 30 60 90 c d time post-trial (min) PTPH1-WT male PTPH1-KO male 0 30 60 90 time post-trial (min) Rotarod test on PTPH1-W Figure 10 T and KO mice (n = 10) males and females Rotarod test on PTPH1-WT and KO mice (n = 10) males and females. a: WT males and WT females do not display any signif- icant different performance at the rod (P = 0.5824) b: KO males and KO females display a significant different perform- 2WAY ance (P = 0.007) (post-hoc T-test: P = 0.109; P = 0.015; P = 0.067; P = 0.835). c: 50% difference in the area under the 2WAY 0 30 60 90 curve represented in figure 10a (unpaired T-test, P=0.0194). d: Female KO mice display a worse performance at the rod com- pared to WT females (P = 0.171; P = 0.002; P = 0.028; P = 0.025) e: No significant difference in the performance on the 0 30 60 90 rod between male KO and WT mice. a, b, d, e: All the data were analyzed by Two-way Anova followed by T-test; *: p < 0.05; **: p < 0.01. ventral posteromedial parvicel thalamic nuclei; DLG: lat- Authors' contributions eral geniculate nucleus; PF: parafascicular thalamic nuclei; The study was devised by CP and MCM and carried out by nu: nuclei; SHi: septohippocampal muclei; Tt: tenia tecta; CP. PT was responsible for the genotyping of all the adult Ig: indusium griseum. animals that have been used in this study. MA performed the LacZ staining experiment on adult mice. VM and BG have been deeply involved in the first editing of the man- Competing interests The present work is part of CP’s PhD program at the Uni- uscript and all the authors contributed to modifications in versity of Eastern Piedmont, in close collaboration with subsequent drafts. PFZ has been involved in critically MerckSerono International S.A.. MCM, PT, VM, BG, PFZ revising the manuscript and has given the final approval are employed by MerckSerono International S.A., which is of the version to be published. All the authors read and involved in the discovery and the commercialization of approved the final version of the manuscript. therapeutics for the prevention and treatment of human diseases. Page 11 of 14 (page number not for citation purposes) time on the rod (sec) AUC (mm ) time on the rod (sec) time on the rod (sec) time on the rod (sec) Behavioral and Brain Functions 2008, 4:36 http://www.behavioralandbrainfunctions.com/content/4/1/36 Additional file 3 Additional methods. This Loco document motio provides t n inde hx e methods and the refer- Working memory index ences that have been used to perform the experiments represented in Addi- 30 tional file 2 Click here for file 25 [http://www.biomedcentral.com/content/supplementary/1744- * 9081-4-36-S3.doc] 70 20 WT WT KO KO Females Males All Females Males All ab Y-maze behaviora Figure 11 l test on PTPH1-WT and KO mice (n = 10) males and females Y-maze behavioral test on PTPH1-WT and KO mice (n = 10) males and females. a: Male KO mice display higher working memory index compared to WT male littermates (P = 0.041); no differences recorded in the female mice. b: No significant male differences recorded in the locomotion index, represented by the total arm entries between PTPH1-WT and KO males and females. T-test, *: p < 0.05. Additional material Acknowledgements This work was fully supported by MerckSerono International S.A.. We would like to thank Rob Hooft van Huijsduijnen, Andrea Graziani and Linda Additional file 1 Chaabane for their scientific support and for kindly reviewing the manu- LacZ staining on PTPH1-WT and KO embryos. PTPH1-WT embryos do script. A special thank to Sonia Carboni and Gabriele Dati for their helpful not show any staining either at embryological stage 14 (Es14) or at Es16. comments and advices on immunohistochemistry and to Niels Adams for PTPH1-KO embryos display a positive LacZ staining in the hypothalamic his support on LacZ staining data. area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself. References Click here for file 1. Stoker AW: Protein tyrosine phosphatases and signalling. J [http://www.biomedcentral.com/content/supplementary/1744- Endocrinol 2005, 185:19-33. 9081-4-36-S1.ppt] 2. 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