PTPs are key factors in multiple signaling pathways, leading to modulated functional activities in various cell types [34
]. Among all PTP forms, PTPH1 has been shown in vitro
to modulate cardiac sodium channel Nav
], that it is also known to be expressed in the axons of cerebral cortex, cerebellum, thalamus and brain stem [36
]. Moreover, PTPH1 contains a domain with high sequence homology with the members of the band 4.1 superfamily protein, FERM. This domain mediates the linkage of actin filaments to the plasma membrane [37
], and therefore may be involved in cytoskeleton-membrane interactions, crucial for axon functionality. To further understand the potential role of PTPH1 in neural functions in vivo
, we first investigated its expression pattern in embryonic and adult PTPH1-KO mice CNS by LacZ staining, and second its role in CNS functions by behavioral phenotype characterization.
In rat embryonic stage Es19, PTPH1 expression through FISH analyses has already been shown in the dorsal thalamic nuclei, which give rise to the thalamo-cortical connections in adulthood [10
]. Thus, it has been suggested to play a role in the maintenance of these connections in adults. We replicated these data in PTPH1-KO mice at Es14 and Es16 embryonic stages. PTPH1 is expressed in the hypothalamic area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself [Additional file 1
]. Moreover, at postnatal P1, PTPH1 expression is also present in peripheral organs such as muscles and intestines as in the adults [22
]. On the other hand, the CNS expression at P1 appears weaker than in the adults suggesting a pattern of PTPH1 expression corresponding to specific developmental stages of the CNS as well as peripheral organs (data not shown). These changes in expression may play a role in various developmental functions that need to be further understood.
In PTPH1-KO adults, LacZ is expressed in different CNS areas such as cerebral and retrosplenial cortices (Figures , , and ), hippocampus (Figure ), thalamus (preferentially ventral thalamus) (Figure ), cerebellum (Figure ) and in the region of the tenia tecta (Figures , ). This data confirms previously observed expression patterns in the rat brain by Sahin et al. [10
] and extends the observation to other brain regions. We, furthermore, demonstrate that PTPH1 is expressed within the cytoplasm and close to the cell membrane of neurons in most of the brain area investigated (Figures ). 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 be responsible for the punctate expression pattern of PTPH1 in the cytosol of the neurons (Figure ) [39
]. This supports the concept that PTPH1 may be involved in cytoskeleton-membrane interaction within extended neuronal population in the CNS, potentially playing a role in various neuronal functions.
Indeed the neural expression of PTPH1 in CA1, CA3 and DG of the hippocampus (Figures and ), in the retrosplenial cortex (Figures , ) and in a series of thalamic nuclei (Figures and ) suggests an involvement of PTPH1 in the modulation of the memory circuit. Both hippocampus and retrosplenial cortex are key regions in the spatial working memory functions [40
]. Moreover, several thalamic nuclei have also been shown to be important in the memory process [47
]. For example, a strong loss of dorsomedial and ventral posterior thalamic neurons is associated with severe cognitive and memory disabilities in patients affected by traumatic brain injury [49
]. Lesions in the lateral thalamus may lead to important working memory defects in rodents [50
]. The anterior thalamic nuclei project via the retrosplenial cortices to the hippocampus [51
], thus underlying the importance of both these circuits and of PTPH1 in the mnemonic process.
Another interesting PTPH1-positive area is the indusium griseum (Figures ) whose role in the adult brain is not clear. It is thought to be part of the limbic system, receiving afferents from the entorhinal and pyriform cortex and projecting to the septohippocampal nuclei, olfactory tubercle (presumably the tenia tecta) and the medial frontal cortex [53
]. The expression of PTPH1 in these specific regions suggests a potential role in the processing/integration of memory and sensory information to the SHi and likely the cortex.
Indeed PTPH1 expression is also detectable in the pyramidal neurons in layer III and IVA of the cerebral cortex of the mouse (Figures ), in agreement with Sahin's findings in the rat brain. The middle layers (III and IV) of the cerebral cortex are key sites for thalamic inputs [55
] especially for VPM and VPL, primary thalamic nuclei for somato-sensory information integration [57
]. Furthermore a strong cortico-cortical communication has been assessed between these two layers [58
], thus suggesting a role for PTPH1 as key regulator in the transmission of the thalamo-cortical and cortico-cortical information.
The cerebellar cortex is also positive for PTPH1 expression, in particular in the cytoplasm of granule cells (Figure ). The cerebellum is known to be the main structure for motor learning functions. In particular, the cerebellar cortex seems to be involved in the early learning phases of motor activities [59
] that include also a strong activation of other areas such as prefrontal cortex and basal ganglia [29
]. PTPH1 expression in the granule cells seems to indicate a potential involvement in the processing of afferent information to the purkinje cells, since it is known that afferents fibers to the cerebellar cortex will project in part through the granule cell layer.
PTPH1 expression pattern observed in our analysis points out a potential involvement of this phosphatase in numerous CNS processing functions such as locomotion, sensorial integration, learning and memory. In this study, the behavioral phenotyping of the PTPH1-KO mice allowed us to test these hypotheses in vivo
. Indeed, as already demonstrated by our group [22
] and also by others [33
], PTPH1-KO mice are healthy and do not display any phenotype, distinguishing them from their matched WT littermates, detectable by simple visual observation. Therefore PTPH1-WT and KO mice underwent a battery composed by five behavioral tests, from the least to the most invasive (Table ), with the tolerable limitation of the handling bias.
Behavioral testing revealing locomotor dysfunctions, such as open-field, EPM and Y-maze did not highlight differences between the two genotypes (Figure ), suggesting that PTPH1 does not play a critical role in the integration of locomotor information.
Anxiety-like behaviors measured by open-field (as path in the center) and EPM (as time spent in the open arms), exploiting rodents natural aversion to open space, did not show any differences between the two genotypes (data not shown), leading to the conclusion that PTPH1 may not be involved in the integration of thalamo-limbic information, key paths for anxiety behavior processing. Similar conclusions can be drawn from the lack of difference between the genotypes regarding integration of nociceptive information, based on hot plate test.
In the behavioral test, that partly depends on working memory performances (Y maze), PTPH1-KO male mice showed a slightly better short-term memory than their WT littermates (Figure ). Thus, PTPH1 may be involved in the integration of memory information. This was further strengthened by results obtained with a test assessing learning and coordination, the rotarod. Contrary to other behaviors where little differences have been observed, learning and coordination capacities in PTPH1-KO female mice are significantly impaired (Figures ). The low rotarod performance on the early trials, compensated by the last trial, is suggestive of a delay in learning acquisition (Figures ).
As reported in Pilecka et al., our PTPH1-KO mice express the non-catalytic part of PTPH1 in frame with the enzymatically active part of LacZ gene. LacZ is widely used as a reporter for promoter activity in KO mice and all those 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 is the case in our mice. Thus, it is very likely that the behavioral phenotype we detect in our mice is linked to the deletion of the catalytic domain of PTPH1.
The impairment in learning and coordination of PTPH1-KO female mice may be resulting from the involvement of 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 expression of its down-stream effector hormone, the IGF1 in liver and plasma [22
]. GHR is highly expressed in most areas of the CNS, in particular in the choroid plexus, hippocampus, putamen, thalamus and hypothalamus. Similarly IGF1 and IGF1-receptors are localized predominantly in hippocampus, but also in amygdala, cerebellum and cortex [61
]. Although IGF1 is considered a neuroprotective hormone, it can be produced in the CNS, it is primarily synthesized in the liver and can cross the blood-brain barrier [62
]. The GH-IGF1 axis is also known to influence cognitive functions due to several neuroprotective effects on the hippocampus [66
]. Furthermore it has been recently pointed out that old conditional liver-IGF1-KO mice display impaired spatial learning and memory [67
]. The presence of PTPH1 in key CNS regions, as well as the consequent deregulation of the GH-IGF1 axis in KO mice, strengthens the concept that the PTPH1 network (CNS and downstream peripheral effectors) may be involved in cognitive functions.
The behavioral tests assessing working memory and specifically learning revealed not only a genotype effect but also a gender effect, as mentioned above. Sex hormones are known to modulate the somatotropic system [68
]. In humans, testosterone has an important effect on GH axis, in part by its aromatization to estradiol. Administration of estrogens, or aromatized androgen, modulates GH axis neuroregulation [69
]. In particular, chronic E2 administration has been shown to reduce GH-induced IGF1 increased expression in liver and plasma via a negative feedback mechanism, while acute E2 administration leads to the expected GH-induced IGF1 release [71
]. Furthermore, it has been reported that estrogens play not only regulatory functions on neuroendocrine systems but can also have stimulatory or inhibitory impacts on the inter-connectivity of the hippocampal structure depending on the gender [72
], meaning that the same stimulus can have opposite effects in male vs
female mice. Thus, the cognitive behavioral differences observed in our KO mice are underlying the potential impact of the PTPH1 network on neuroendocrine regulation as well as on cellular architecture within specific brain regions.