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Impaired motor functions in mice lacking the RNA-binding protein Hzf
Neuroscience Research 58 (2007) 183–189 www.elsevier.com/locate/neures
Impaired motor functions in mice lacking the RNA-binding protein Hzf Takatoshi Iijima a, Hiroo Ogura c, Kanako Takatsuki d, Shigenori Kawahara d, Kenichiro Wakabayashi a,b, Daisuke Nakayama a, Masato Fujioka a,b, Yuki Kimura e, Alan Bernstein e, Hirotaka James Okano a, Yutaka Kirino d, Hideyuki Okano a,* a
b
Department of Physiology, School of Medicine, Keio University, Shinjyuku, Tokyo 160-8582, Japan Department of Otolatoryngology, Head and Neck Surgery, School of Medicine, Keio University, Shinjyuku, Tokyo 160-8582, Japan c Tsukuba Research laboratories, Eisai Co., Ltd., Tsukuba, Ibaraki 300-2635, Japan d Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan e Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada Received 14 December 2006; accepted 21 February 2007 Available online 28 February 2007
Abstract Local protein synthesis in dendrites plays an important role in some aspects of neuronal development and synaptic plasticity. Neuronal RNAbinding proteins regulate the transport and/or translation of the localized mRNAs. Previously, we reported that hematopoietic zinc finger (Hzf) is one of the neuronal RNA-binding proteins that regulate these processes. The Hzf protein is highly expressed in neuronal cells including hippocampal pyramidal neurons and cerebellar Purkinje cells, and plays essential roles in the dendritic mRNA localization and translation. In the present study we demonstrated that mice lacking Hzf (Hzf / mice) exhibited severe impairments of motor coordination and cerebellumdependent motor learning. These findings raise the possibility that the post-transcriptional regulation by Hzf may contribute to some aspects of synaptic plasticity and motor learning in the cerebellum. # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Hzf; Neuronal RNA-binding protein; Purkinje cell; mRNA localization; Motor coordination; Motor learning
1. Introduction In neuronal cells, various mRNAs are localized in the somatodendritic region, and are locally translated in response to neuronal activity (Steward and Schuman, 2001). Local protein synthesis plays a crucial role in neuronal development and synaptic plasticity. Indeed, protein synthesis in the postsynapse has been shown to be required for induction of chemically induced long-term potentiation (LTP) and longterm depression (LTD) in the hippocampus (Kang and
Abbreviations: IP3R1, type1 inositol 1,4,5-trisphosphate receptor; 30 UTR, 3 untranslated region; FMRP, fragile X mental retardation protein; EMG, electromyograms; LTD, long-term depression; LTP, long-term potentiation; US, unconditioned stimulus; CS, conditioned stimulus; CR, conditioned response; CNS, central nervous system * Corresponding author at: Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjyuku, Tokyo 160-8582, Japan. Tel.: +81 3 5363 3747; fax: +81 3 3357 5445. E-mail address: [email protected] (H. Okano). 0
Schuman, 1996; Huber et al., 2000). Importantly, neuronal RNA-binding proteins are known to regulate a series of the processes including stabilization, storage, trafficking and translation of the localized mRNAs via binding to their 30 UTRs. Examples of RNA-binding proteins which are involved in dendritically localized mRNAs include Staufen (Kiebler et al., 1999; Kohrmann et al., 1999) and ZBP1 (Ross et al., 1997; Zhang et al., 2001; Tiruchinapalli et al., 2003). Genetically disrupting some neuronal RNA-binding proteins leads to impairment of synaptic plasticity, and subsequently extends to cover behavioral and cognitive abnormalities. Among them, the fragile X mental retardation protein (FMRP) was one of the most characterized RNA-binding proteins. FMRP could regulate the expression of multiple mRNAs in dendrites (Brown et al., 2001; Miyashiro et al., 2003). Loss of FMRP decreases cortical LTP (Li et al., 2002) and enhances hippocampal and cerebellar LTD (Huber et al., 2002; Koekkoek et al., 2005). In addition, knockout of the fmr1 gene results in consistent neurobehavioral abnormalities, analogous to the clinical and pathological symptoms observed in fragile X
0168-0102/$ – see front matter # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2007.02.013
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patients (Kooy, 2003). In our previous study, we identified hematopoietic zinc finger (Hzf) as a new RNA-binding protein which regulates the dendritic localization of mRNA(s) in neuronal cells. The Hzf protein was highly expressed in cerebellar Purkinje cells, and was associated with type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) mRNA through binding to the 30 UTR (Iijima et al., 2005). Here, in this study, we report on behavioral studies, including locomotor activity, muscle strength, cerebellar motor learning and hippocampus-dependent spatial and non-spatial memory, using mice lacking Hzf (Hzf / mice). Hzf / mice exhibited severe impairments in motor coordination and motor learning related to cerebellar functions. These findings suggest that the post-transcriptional regulation by Hzf may be involved in synaptic plasticity and motor learning in the cerebellum. 2. Materials and methods 2.1. Hzf/ mice Hzf / mice were generated by homologous recombination as previously described (Kimura et al., 2002). Largely, male mice generated on a CD1 background were used as described previously (Houchi et al., 2005; Mikics et al., 2006). Briefly, heterozygous mice were bred for five generations on a CD1 background, and 11–13 weeks old male mice were used in this study. A battery of behavioral tests to assess motor function was performed as described previously (Ogura et al., 2001a,b).
2.2. Locomotor activity Briefly, locomotor activity was measured by placing an animal in a clear Plexiglas box (30 cm 20 cm 13 cm) that was then positioned in a frame through which infrared beams were passed (Scanet SV-10, Toyo Industry Co. Ltd. Japan). Beam interruptions were summed in 10-min bins over a period of 60 min.
2.3. Rotating rod test Motor coordination was assessed with a rotating rod apparatus (KN-75, Natsume Seisakujo Co. Ltd., Tokyo, Japan), which consisted of a plastic rod (3 cm diameter, 8 cm long) with a gritted surface flanked by two large discs (40 cm diameter). Latency until a fall occurred was recorded in four trials. The mouse was lifted by the tail and allowed to grasp the hold bar with its forepaws. The experimenter slowly pulled the mouse back by the tail and the maximum tension in the cable was recorded.
2.6. Elevated plus-maze test The elevated plus maze is a cross-shaped structure of gray plastic consisting of two arms that are open to the environment (open arms, 30.5 cm 5.5 cm) and two arms that are enclosed by side and end walls (closed arms, 30.5 cm 5.5 cm 15 cm) (Lister, 1987), and the arms are connected by a central area (5.5 cm 5.5 cm). The maze is elevated from the floor (46.5 cm). Behavioral testing began by placing an animal in the central area of the maze facing an open arm. Exploratory behavior was recorded with a video camera, and a remote monitor was located in an adjacent room. The number of open- and closed-arm entries and the time spent in each type of arm was measured. An entry was defined as all four paws crossing into one arm.
2.7. Delay eyeblink conditioning test The delay eyeblink conditioning test was performed as described previously (Kishimoto et al., 2001a,b). Mice were kept on a 12 h light/12 h dark cycle with ad libitum access to food and water. We used male and female mice weighing 24–45 g at the time of surgery. (Care of the animals throughout the experiments conformed to the guidelines published in the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.) Under anesthesia with ketamine (80 mg/kg, i.p., Sankyo, Tokyo, Japan) and xylazine (20 mg/kg, i.p., Bayer, Tokyo, Japan), four Teflon-coated stainless steel wires (No. 7910, AM Systems Inc., Carlsborg, WA, USA) were implanted in the left upper eyelid, two to record eyelid electromyograms (EMG) and the other two to deliver the unconditioned stimulus (US). Three days after the surgery, spontaneous eyeblink frequency was measured. The conditioning began the next day. Daily conditioning consisted of 90 conditioned stimulus (CS)–US paired trials and 10 CS-alone trials on every 10th trial, with a pseudorandomized inter-trial interval of 20–40 s. In the paired trials, a 352-ms tone CS (1 kHz, 85 dB) was followed by a 100-ms periorbital shock US (100 Hz square pulses), eliciting an eyeblink/head-turn response. In the present study, we used the delay paradigm in which the CS preceded and terminated simultaneously with the US. The conditioned response (CR) was monitored through eyelid EMG activity. The average + S.D. of the amplitudes of the EMG activities for 300 ms before CS onset in 100 trials was defined as the threshold and was used in the analyses described below. In each trial, average values of EMG amplitude above the threshold were calculated for a period of 300 ms before CS onset (prevalue), 30 ms after CS onset (startle-value), and of 20 ms before US onset (CRvalue). If the pre-values and startle-values were less than 10% and 30% of threshold, respectively, the trial was regarded as a ‘‘valid’’ trial. Among the valid trials, a trial was regarded to contain the CR if the CR value was larger than 1% of the threshold value and was more than twice the pre-value. In CS-alone trials, the period for CR-value calculation was extended to the CS termination. To evaluate the effect on the startle response, we calculated the frequency of trials in which the startle-value exceeded 20% of threshold. The frequency of CRs in the valid trials (CR%) was expressed as mean S.E.M.
2.8. Spontaneous Y-maze alternation task 2.4. Wire-hanging test The apparatus was a length of stainless-steel bar (50 cm; 2-mm diameter) resting on two vertical supports and elevated 37 cm above a flat surface. A mouse was placed on the bar at a point midway between the supports and observed for 30 s in three trials. The amount of time spent hanging was recorded and scored according to the following system: 0, fell off; 1, hung onto the bar with two forepaws; 2, in addition to 1, attempted to climb onto the bar; 3, hung onto the bar with two forepaws and one or both hind paws; 4, hung onto the bar with all four paws with tail wrapped around the bar; 5, escaped to one of the supports.
A mouse was placed in one arm (10 cm in length, 4 cm in width and 6 cm in height) of a Y-maze and allowed to explore the maze for 10 min. Interruptions of the infrared beams in each arm were recorded automatically through a digital I/O interface (Muromachi Kikai Co. Ltd., Tokyo Japan) at 0.1 s intervals by a computer outside the testing room. Any three consecutive choices of three different arms were counted as an alternation and the percentage of alternation was calculated by dividing the total alternations by the total arm choices minus 2.
2.9. Object recognition task 2.5. Traction test The grip strength of a mouse was measured with a traction apparatus (FU-1, Muromachi Kikai Co. Ltd., Tokyo, Japan). Mice were made to grasp the attached bar (2 mm diameter) with the forepaws and were slowly pulled back by the tail. The maximum tension before release was recorded.
For object recognition, mice were given a 1 h exposure in their home cage to the first object in an object pair. Following a 3 h retention interval, mice were reexposed to this familiar object along with a novel object, and the time spent exploring each object recorded for 2 min. Objects were thoroughly washed between presentations to remove odor cues. Exploration scoring included
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orientation toward the object with the mouse’s nose within 1–2 cm of the object and touching or chewing the object, but did not include standing or sitting on the object, propping up on the object to look out of the cage, or incidental touching of the object while digging through the bedding. Objects were plastic and were matched approximately for size, weight, and innate interest level based on preliminary tests with control mice. For each retention interval, training and testing were done twice, using different pairs of objects, and the scores were averaged.
2.10. Statistical analysis A repeated-measures analysis of variance (ANOVA) with genotypes as a between subjects factor and time or trial as a within-subject factor was used in the statistical analysis of spontaneous locomotor activity, rotating rod test and delayed eyeblink test. When the genotype factor was significant, a post hoc individual comparison was carried out. The statistical significance of differences between genotypes was analyzed by Student’s t-test, except that the nonparametric Mann– Whitney U-test was applied to the results of the wire hanging and elevated plus maze test. A p-value of < 0.05 was considered significant.
3. Results In general, Hzf / mice exhibited such ataxia-like phenotypes as tremor, ataxic gate, and tilted head, and could be easily distinguished from the wild-type or Hzf +/ mice. Here, we applied the following behavioral tests to characterize the behavioral phenotypes more in detail. 3.1. Motor activity and coordination We first investigated motor function and motor learning in the animals. Motor coordination was assessed by the spontaneous motor activity and the rotating rod test, Hzf / mice showed a significant difference from the wild-type in spontaneous motor activity (F 1,24 = 5.510, p < 0.05, ANOVA) (Fig. 1). Surprisingly, the motor coordination imposed by the rotating rod test was severely impaired in Hzf / mice. Most of the mutant mice were unable to stand on the bar under even when stationary (0 rpm) for more than 10 s (F 1,20 = 48.665, p < 0.001, ANOVA) (Fig. 2). 3.2. Muscular strength The above motor abnormalities could be caused by either impairment of motor coordination controlled by the supraspinal
Fig. 2. Rotating rod test. The test was performed to assess the motor coordination, equilibrium, learning capacity in Hzf/ mice (n = 10) and the littermate wild type (n = 12). Four trials were performed at intervals of at least 30 min per day. Filled circle, wild type; open box, Hzf/ mice. Values are mean S.E.M. (***) p < 0.001.
central nervous system (CNS) or by the muscle weakness or other defects in the lower motor systems. Next, high grip strength was measured by the wire-hanging test or the traction test. In the wirehanging test, neither the hanging time nor the score was of significant difference between wild-type and Hzf / mice (Fig. 3A and B). In contrast, the grip strength of Hzf / mice was lower than that of wild-type mice in the traction test. (t = 2.269, p < 0.01, Student’s t-test) (Fig. 3C). The difference in the traction test might be explained by differences in body weight among genotypes, since body weights in animals strongly reflect the total amount of skeletal muscle. Hzf / mice have been reported to exhibit growth retardation (Kimura et al., 2002). Indeed, body weights of Hzf / mice used in this study were significantly lower than those of wild-type mice (wild type, 34.3g 0.8; Hzf /, 30.1g 0.6) (t = 3.663, p < 0.02, Student’s t-test). Therefore, the significant difference in the hanging test might be affected by growth retardation rather than an intrinsic muscular deficit. In addition, gross anatomical and histological abnormalities of the muscle and skeleton of the Hzf / mice have not been observed in our study (data not shown). Thus, the results indicated that the motor dysfunction observed in Hzf / mice might be mainly due to impairment of motor coordination controlled by the CNS rather than to muscle weakness or other defects in the lower motor systems. 3.3. Learning and memory
Fig. 1. Spontaneous motor activity. Hzf/ mice and their littermates were assessed for their horizontal activity in a novel environment (n = 12 per genotype). Filled circle, wild type; open box, Hzf/ mice. Values are mean S.E.M. (*) p < 0.05.
Hzf protein is highly expressed in the cerebellum and the thalamus. In particular, the cerebellar Purkinje cell is one of the most prominent expression sites. The cerebellum is a center for motor functions including motor coordination and motor learning. Additionally, Hzf protein has been suggested to be required for posttranscriptional regulation of IP3R1 (Iijima et al., 2005), which is necessary for cerebellar synaptic plasticity and motor coordination (Inoue et al., 1998; Ogura et al., 2001b). Taken together, we hypothesized that the observed motor discoordinations may be mainly due to cerebellar dysfunction. Here, we assessed the learning ability of Hzf / mice by classical delay eyeblink conditioning, a form of motor learning that largely depends on the cerebellum (Thompson et al., 1997).
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Fig. 3. Traction and wire-hanging tests. Neuromuscular strength was evaluated by these tests. The wire-hanging tests were calculated on the basis of both mean scores and mean retention time in every third trials. (A) Score in the wire-hanging test in Hzf/ mice (n = 10) and the littermate wild type (n = 12). (B) Retention time in the wire-hanging test. In A and B, the left panels indicate the mean score or the retention time in three successive trials and the right panels indicate the change in values in each trial. (C) Traction test (n = 10 per genotype). Values are mean S.E.M. (**) p < 0.01.
Interestingly, Hzf / mice showed a distinct disruption in the delay paradigm (F 1,14 = 11.859, p < 0.004, ANOVA) (Fig. 4A and B). In addition, there were no significant differences in startle responses to a tone between the wild-type and Hzf / mice (Fig. 4C), indicating that the sensory input to CS were almost comparable between wild-type and Hzf / mice. These findings
indicated that the loss of Hzf had profound effects on the cerebellum-dependent motor learning. Hzf is also expressed in the cerebral cortex and the hippocampus. In addition, the protein is localized in dendrites of CA1-2 neurons in the hippocampus. The Morris water maze task is one of the most general procedures for assessing
Fig. 4. Delay eyeblink conditioning test. Primary cerebellar motor learning was examined by a classical conditioning task. (A) Temporal relationship between the CS and US in delay eyeblink conditioning. There is temporal overlap of the US with the preceding CS. (B) Development of CR during delay eyeblink conditioning (n = 8 per genotype). Spontaneous eyeblink frequency (Sp) was measured for two days prior to the acquisition session. In the acquisition session of day 1–day 7, daily session consisted of 10 blocks of trials and a block consisted of nine CS US paired trials and one CS-only trial. In the following 3-day extinction session, 100 CS-only presentations were given daily. (C) The frequency of the startle response to the CS (tone) in Hzf/ mice (n = 8 per genotype). Values are mean S.E.M. (**) p < 0.01.
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(Fig. 5). These findings suggested that the loss of Hzf does not have a profound effect on the hippocampus-dependent spatial and non-spatial memory despite its high expression in the hippocampus. 3.4. Other tests Hzf / mice and wild-type littermates were indistinguishable from each other in other behavioral tests, including elevated-plus maze tests (Fig. 6) and cliff avoidance (data not shown). These results suggested that the loss of the Hzf gene has little effect on the visual system and anxiety-related behavior. 4. Discussion
Fig. 5. Spontaneous alternation in the Y-maze task and the object recognition task. Learning and memory was evaluated by two tasks. (A) Spontaneous alternation in the Y-maze task in Hzf/ mice (n = 10) and the littermate wild type (n = 12). (B) Object recognition task (n = 10 per genotype). Values are mean S.E.M.
hippocampal-dependent spatial learning and memory. Unfortunately, we could not obtain sufficient data in the task, because of the poor swimming performance of Hzf / mice. Instead, we assessed the spontaneous alternation in the Y-maze, one of the spatial memory tasks which are largely dependent on the hippocampus, and the object recognition task, which is a hippocampus-dependent nonspatial memory task (Clark et al., 2000). However, the mutant mice were not significantly different from the wild-type in either the spontaneous alternation in the Y-maze or the object recognition task
In this study, we represented a comprehensive behavioral analysis of Hzf / mice summarized in Table 1. We will first discuss the behavioral characteristics of Hzf / mice and then the link with biological roles of the Hzf protein in the neuronal system. 4.1. Disruption of motor functions in Hzf/ mice Hzf gene deficiency severely disrupted motor functions (Figs. 1 and 2), whereas distinct abnormalities were not observed in the lower motor system (e.g. motor strength, Fig. 3 and Table 1). Notably, cerebellar motor learning during the delay eyeblink conditioning was impaired in Hzf / mice (Fig. 4). On the other hand, trace eyeblink conditioning was not performed in this study, since it depends on the hippocampus (Moyer et al., 1990; Weiss et al., 1991) as well as on the cerebellum (WoodruffPak et al., 1985; Takehara et al., 2003). However, hippocampusselective memory tasks such as spontaneous alternation in the Ymaze and object recognition task were not significantly affected (Fig. 5), indicating that the hippocampal functions are not severely impaired in Hzf / mice. Taken together, the motor impairment is likely to be mainly caused by deficits of some cerebellar neuronal functions of the Hzf protein. Table 1 The summary for behavioral analyses using Hzf/ mice
Fig. 6. Elevated-plus maze test. Anxiety-related behavior was evaluated in the test. (A) The number of open- and closed-arm entries in Hzf/ mice (n = 10) and the littermate wild type (n = 12). (B) The time spent in each type of arm. Values are mean S.E.M.
Tests
Results
Motor activity and coordination Locomotor activity Rota rod test
Impaired Impaired (drastic)
Muscle strength Wire-hanging test Traction test
Normal Impaired (slightly)
Anxiety Elevated plus maze
Normal
Learning and memory Eyeblink conditoning test (delay) Morris water maze test Spontaneous Y-maze test Object recognition test
Impaired N.I. Normal Normal
N.I., no informative.
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Nevertheless, we cannot completely exclude the possibility that the loss of Hzf in other regions or systems might have induced the behavioral phenotypes observed in the present study. A previous study has reported that Hzf is expressed in megakaryocytes, a precursor of platelets, and that internal hemorrhaging is observed in the brains and gastrointestinal tracts in Hzf / neonates (Hidaka et al., 2000; Kimura et al., 2002). Brain hemorrhaging could cause motor dysfunction in the null mice. In that respect, at least, we did not observe marked internal hemorrhaging in animals used in the eyeblink conditioning task (data not shown). Based on these points, it is unlikely that these severe motor dysfunctions in Hzf / mice can be simply explained by internal hemorrhaging. It has been suggested that one of the mechanisms underlying eyeblink conditioning is cerebellar plasticity at parallel fiberPurkinje cell synapses, since cerebellar LTD-deficient mice exhibit severe impairments of eyeblink conditioning (Aiba et al., 1994; Shibuki et al., 1996; Kishimoto et al., 2001a,b; Koekkoek et al., 2003). The cerebellum is crucial for motor coordination and learning in animals. Since Hzf is highly expressed in Purkinje cells within the cerebellum, we guessed that some portion of the observed motor abnormalities might be due to cerebellar dysfunction. 4.2. The link between posttranscriptional regulations by Hzf and motor functions It is known that an abnormality of climbing fiber translocation and innervation of Purkinje cells often causes ataxia-phenotypes in animals. However, no such morphological defects were observed in the Hzf / mice (unpublished data). By what mechanism was a series of motor impairments caused in Hzf / mice? Disruption of dendritic mRNA localization and activity-dependent mRNA translation impair the long-phase synaptic plasticity and memory consolidation in the hippocampus (Miller et al., 2002; Kelleher et al., 2004). This could also be the case in the cerebellum. A particular form of cerebellar LTD depends on postsynaptic protein synthesis in cultured Purkinje cells (Linden, 1996). The group 1 mGluRdependent LTD is enhanced by deletion of the FMR1 gene in both the hippocampus and cerebellum (Huber et al., 2002; Koekkoek et al., 2005). Furthermore, overall and Purkinje cellspecific deletions of FMR1 in mice and fragile X patients distinctly attenuate cerebellar eyeblink conditioning (Koekkoek et al., 2005). These findings suggest that newly synthesized proteins controlled by specific RNA-binding proteins play an important role in the maintenance of particular forms of cerebellar synaptic plasticity. Like FMRP, Hzf could be a component of a neuronal RNA granule, which transports mRNAs into dendrites (Kanai et al., 2004). Interestingly, our previous study suggests that Hzf functions as a regulator of dendritic localization and their translation of IP3R1 mRNA in cerebellar Purkinje cells (Iijima et al., 2005). Hzf could potentially target multiple mRNAs. Nevertheless, IP3R1 mRNA would be one of the intriguing targets for Hzf, since it is the intracellular Ca2+ channel (Furuichi et al., 1989), and
required for cerebellar LTD (Inoue et al., 1998), the basis for cerebellar motor learning (Ito, 1989). Mice lacking IP3R1 exhibit low birth rate and postnatally began to show severe cerebellar ataxia and truncal torsions (Matsumoto et al., 1996). Although the phenotypes of Hzf / mice largely differed from those of IP3R1/ mice at the point where such severe phenotypes are not observed, there are somewhat similar to IP3R1+/ mice, which exhibit motor dysfunctions (Ogura et al., 2001b). Therefore, the observed motor dysfunctions in Hzf / mice, at least, might partially reflect the impairment of posttranscriptional regulations of the target mRNAs (including IP3R1 mRNA) for Hzf protein in the cerebellum (particularly in Purkinje cells), although the physiological relevance of posttranscriptional regulation controlled by Hzf should be elucidated in future studies. In addition, as mentioned above, the behavioral phenotypes in animals may reflect the interplay of multiple regions and systems. To demonstrate the involvement of cerebellar systems in the behavioral impairments of Hzf / mice which were observed in the present study, we are currently planning the rescue experiments of these behavioral phenotypes by a cerebellar Purkinje cells-selective Hzf transgene. Thus, the genetic manipulation within a specific brain region or cell type will reveal the biological significance of Hzf in motor functions in animal more clearly in our future study. Acknowledgements We appreciate the invariable assistance of all members of the Okano laboratory. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.O.), a Grant-in-aid for 21st Century COE program to Keio University. References Aiba, A., Kano, M., Chen, C., Stanton, M.E., Fox, G.D., Herrup, K., Zwingman, T.A., Tonegawa, S., 1994. Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79, 377–388. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wilkinson, K.D., Keene, J.D., Darnell, R.B., Warren, S.T., 2001. Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107, 477– 487. Clark, R.E., Zola, S.M., Squire, L.R., 2000. Impaired recognition memory in rats after damage to the hippocampus. J. Neurosci. 20, 8853–8860 similar results. Furuichi, T., Yosikawa, S., Miyawaki, A., Wada, K., Maeda, N., Mikoshiba, K., 1989. Primary structure and functional expression of the inositol 1,4,5trisphosphate-binding protein P400. Nature 342, 32–38. Hidaka, M., Caruana, G., Stanford, W.L., Sam, M., Correll, P.H., Bernstein, A., 2000. Gene trapping of two novel genes, Hzf and Hhl, expressed in hematopoietic cells. Mech. Dev. 90, 3–15. Houchi, H., Babovic, D., Pierrefiche, O., Ledent, C., Daoust, M., Naassila, M., 2005. CB1 receptor knockout mice display reduced ethanol-induced conditioned place preference and increased striatal dopamine D2 receptors. Neuropsychopharmacology 30, 339–349. Huber, K.M., Kayser, M.S., Bear, M.F., 2000. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 288, 1254–1257.
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Impaired motor functions in mice lacking the RNA-binding protein Hzf Takatoshi Iijima a, Hiroo Ogura c, Kanako Takatsuki d, Shigenori Kawahara d, Kenichiro Wakabayashi a,b, Daisuke Nakayama a, Masato Fujioka a,b, Yuki Kimura e, Alan Bernstein e, Hirotaka James Okano a, Yutaka Kirino d, Hideyuki Okano a,* a
b
Department of Physiology, School of Medicine, Keio University, Shinjyuku, Tokyo 160-8582, Japan Department of Otolatoryngology, Head and Neck Surgery, School of Medicine, Keio University, Shinjyuku, Tokyo 160-8582, Japan c Tsukuba Research laboratories, Eisai Co., Ltd., Tsukuba, Ibaraki 300-2635, Japan d Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan e Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada Received 14 December 2006; accepted 21 February 2007 Available online 28 February 2007
Abstract Local protein synthesis in dendrites plays an important role in some aspects of neuronal development and synaptic plasticity. Neuronal RNAbinding proteins regulate the transport and/or translation of the localized mRNAs. Previously, we reported that hematopoietic zinc finger (Hzf) is one of the neuronal RNA-binding proteins that regulate these processes. The Hzf protein is highly expressed in neuronal cells including hippocampal pyramidal neurons and cerebellar Purkinje cells, and plays essential roles in the dendritic mRNA localization and translation. In the present study we demonstrated that mice lacking Hzf (Hzf / mice) exhibited severe impairments of motor coordination and cerebellumdependent motor learning. These findings raise the possibility that the post-transcriptional regulation by Hzf may contribute to some aspects of synaptic plasticity and motor learning in the cerebellum. # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Hzf; Neuronal RNA-binding protein; Purkinje cell; mRNA localization; Motor coordination; Motor learning
1. Introduction In neuronal cells, various mRNAs are localized in the somatodendritic region, and are locally translated in response to neuronal activity (Steward and Schuman, 2001). Local protein synthesis plays a crucial role in neuronal development and synaptic plasticity. Indeed, protein synthesis in the postsynapse has been shown to be required for induction of chemically induced long-term potentiation (LTP) and longterm depression (LTD) in the hippocampus (Kang and
Abbreviations: IP3R1, type1 inositol 1,4,5-trisphosphate receptor; 30 UTR, 3 untranslated region; FMRP, fragile X mental retardation protein; EMG, electromyograms; LTD, long-term depression; LTP, long-term potentiation; US, unconditioned stimulus; CS, conditioned stimulus; CR, conditioned response; CNS, central nervous system * Corresponding author at: Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjyuku, Tokyo 160-8582, Japan. Tel.: +81 3 5363 3747; fax: +81 3 3357 5445. E-mail address: [email protected] (H. Okano). 0
Schuman, 1996; Huber et al., 2000). Importantly, neuronal RNA-binding proteins are known to regulate a series of the processes including stabilization, storage, trafficking and translation of the localized mRNAs via binding to their 30 UTRs. Examples of RNA-binding proteins which are involved in dendritically localized mRNAs include Staufen (Kiebler et al., 1999; Kohrmann et al., 1999) and ZBP1 (Ross et al., 1997; Zhang et al., 2001; Tiruchinapalli et al., 2003). Genetically disrupting some neuronal RNA-binding proteins leads to impairment of synaptic plasticity, and subsequently extends to cover behavioral and cognitive abnormalities. Among them, the fragile X mental retardation protein (FMRP) was one of the most characterized RNA-binding proteins. FMRP could regulate the expression of multiple mRNAs in dendrites (Brown et al., 2001; Miyashiro et al., 2003). Loss of FMRP decreases cortical LTP (Li et al., 2002) and enhances hippocampal and cerebellar LTD (Huber et al., 2002; Koekkoek et al., 2005). In addition, knockout of the fmr1 gene results in consistent neurobehavioral abnormalities, analogous to the clinical and pathological symptoms observed in fragile X
0168-0102/$ – see front matter # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2007.02.013
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patients (Kooy, 2003). In our previous study, we identified hematopoietic zinc finger (Hzf) as a new RNA-binding protein which regulates the dendritic localization of mRNA(s) in neuronal cells. The Hzf protein was highly expressed in cerebellar Purkinje cells, and was associated with type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) mRNA through binding to the 30 UTR (Iijima et al., 2005). Here, in this study, we report on behavioral studies, including locomotor activity, muscle strength, cerebellar motor learning and hippocampus-dependent spatial and non-spatial memory, using mice lacking Hzf (Hzf / mice). Hzf / mice exhibited severe impairments in motor coordination and motor learning related to cerebellar functions. These findings suggest that the post-transcriptional regulation by Hzf may be involved in synaptic plasticity and motor learning in the cerebellum. 2. Materials and methods 2.1. Hzf/ mice Hzf / mice were generated by homologous recombination as previously described (Kimura et al., 2002). Largely, male mice generated on a CD1 background were used as described previously (Houchi et al., 2005; Mikics et al., 2006). Briefly, heterozygous mice were bred for five generations on a CD1 background, and 11–13 weeks old male mice were used in this study. A battery of behavioral tests to assess motor function was performed as described previously (Ogura et al., 2001a,b).
2.2. Locomotor activity Briefly, locomotor activity was measured by placing an animal in a clear Plexiglas box (30 cm 20 cm 13 cm) that was then positioned in a frame through which infrared beams were passed (Scanet SV-10, Toyo Industry Co. Ltd. Japan). Beam interruptions were summed in 10-min bins over a period of 60 min.
2.3. Rotating rod test Motor coordination was assessed with a rotating rod apparatus (KN-75, Natsume Seisakujo Co. Ltd., Tokyo, Japan), which consisted of a plastic rod (3 cm diameter, 8 cm long) with a gritted surface flanked by two large discs (40 cm diameter). Latency until a fall occurred was recorded in four trials. The mouse was lifted by the tail and allowed to grasp the hold bar with its forepaws. The experimenter slowly pulled the mouse back by the tail and the maximum tension in the cable was recorded.
2.6. Elevated plus-maze test The elevated plus maze is a cross-shaped structure of gray plastic consisting of two arms that are open to the environment (open arms, 30.5 cm 5.5 cm) and two arms that are enclosed by side and end walls (closed arms, 30.5 cm 5.5 cm 15 cm) (Lister, 1987), and the arms are connected by a central area (5.5 cm 5.5 cm). The maze is elevated from the floor (46.5 cm). Behavioral testing began by placing an animal in the central area of the maze facing an open arm. Exploratory behavior was recorded with a video camera, and a remote monitor was located in an adjacent room. The number of open- and closed-arm entries and the time spent in each type of arm was measured. An entry was defined as all four paws crossing into one arm.
2.7. Delay eyeblink conditioning test The delay eyeblink conditioning test was performed as described previously (Kishimoto et al., 2001a,b). Mice were kept on a 12 h light/12 h dark cycle with ad libitum access to food and water. We used male and female mice weighing 24–45 g at the time of surgery. (Care of the animals throughout the experiments conformed to the guidelines published in the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.) Under anesthesia with ketamine (80 mg/kg, i.p., Sankyo, Tokyo, Japan) and xylazine (20 mg/kg, i.p., Bayer, Tokyo, Japan), four Teflon-coated stainless steel wires (No. 7910, AM Systems Inc., Carlsborg, WA, USA) were implanted in the left upper eyelid, two to record eyelid electromyograms (EMG) and the other two to deliver the unconditioned stimulus (US). Three days after the surgery, spontaneous eyeblink frequency was measured. The conditioning began the next day. Daily conditioning consisted of 90 conditioned stimulus (CS)–US paired trials and 10 CS-alone trials on every 10th trial, with a pseudorandomized inter-trial interval of 20–40 s. In the paired trials, a 352-ms tone CS (1 kHz, 85 dB) was followed by a 100-ms periorbital shock US (100 Hz square pulses), eliciting an eyeblink/head-turn response. In the present study, we used the delay paradigm in which the CS preceded and terminated simultaneously with the US. The conditioned response (CR) was monitored through eyelid EMG activity. The average + S.D. of the amplitudes of the EMG activities for 300 ms before CS onset in 100 trials was defined as the threshold and was used in the analyses described below. In each trial, average values of EMG amplitude above the threshold were calculated for a period of 300 ms before CS onset (prevalue), 30 ms after CS onset (startle-value), and of 20 ms before US onset (CRvalue). If the pre-values and startle-values were less than 10% and 30% of threshold, respectively, the trial was regarded as a ‘‘valid’’ trial. Among the valid trials, a trial was regarded to contain the CR if the CR value was larger than 1% of the threshold value and was more than twice the pre-value. In CS-alone trials, the period for CR-value calculation was extended to the CS termination. To evaluate the effect on the startle response, we calculated the frequency of trials in which the startle-value exceeded 20% of threshold. The frequency of CRs in the valid trials (CR%) was expressed as mean S.E.M.
2.8. Spontaneous Y-maze alternation task 2.4. Wire-hanging test The apparatus was a length of stainless-steel bar (50 cm; 2-mm diameter) resting on two vertical supports and elevated 37 cm above a flat surface. A mouse was placed on the bar at a point midway between the supports and observed for 30 s in three trials. The amount of time spent hanging was recorded and scored according to the following system: 0, fell off; 1, hung onto the bar with two forepaws; 2, in addition to 1, attempted to climb onto the bar; 3, hung onto the bar with two forepaws and one or both hind paws; 4, hung onto the bar with all four paws with tail wrapped around the bar; 5, escaped to one of the supports.
A mouse was placed in one arm (10 cm in length, 4 cm in width and 6 cm in height) of a Y-maze and allowed to explore the maze for 10 min. Interruptions of the infrared beams in each arm were recorded automatically through a digital I/O interface (Muromachi Kikai Co. Ltd., Tokyo Japan) at 0.1 s intervals by a computer outside the testing room. Any three consecutive choices of three different arms were counted as an alternation and the percentage of alternation was calculated by dividing the total alternations by the total arm choices minus 2.
2.9. Object recognition task 2.5. Traction test The grip strength of a mouse was measured with a traction apparatus (FU-1, Muromachi Kikai Co. Ltd., Tokyo, Japan). Mice were made to grasp the attached bar (2 mm diameter) with the forepaws and were slowly pulled back by the tail. The maximum tension before release was recorded.
For object recognition, mice were given a 1 h exposure in their home cage to the first object in an object pair. Following a 3 h retention interval, mice were reexposed to this familiar object along with a novel object, and the time spent exploring each object recorded for 2 min. Objects were thoroughly washed between presentations to remove odor cues. Exploration scoring included
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orientation toward the object with the mouse’s nose within 1–2 cm of the object and touching or chewing the object, but did not include standing or sitting on the object, propping up on the object to look out of the cage, or incidental touching of the object while digging through the bedding. Objects were plastic and were matched approximately for size, weight, and innate interest level based on preliminary tests with control mice. For each retention interval, training and testing were done twice, using different pairs of objects, and the scores were averaged.
2.10. Statistical analysis A repeated-measures analysis of variance (ANOVA) with genotypes as a between subjects factor and time or trial as a within-subject factor was used in the statistical analysis of spontaneous locomotor activity, rotating rod test and delayed eyeblink test. When the genotype factor was significant, a post hoc individual comparison was carried out. The statistical significance of differences between genotypes was analyzed by Student’s t-test, except that the nonparametric Mann– Whitney U-test was applied to the results of the wire hanging and elevated plus maze test. A p-value of < 0.05 was considered significant.
3. Results In general, Hzf / mice exhibited such ataxia-like phenotypes as tremor, ataxic gate, and tilted head, and could be easily distinguished from the wild-type or Hzf +/ mice. Here, we applied the following behavioral tests to characterize the behavioral phenotypes more in detail. 3.1. Motor activity and coordination We first investigated motor function and motor learning in the animals. Motor coordination was assessed by the spontaneous motor activity and the rotating rod test, Hzf / mice showed a significant difference from the wild-type in spontaneous motor activity (F 1,24 = 5.510, p < 0.05, ANOVA) (Fig. 1). Surprisingly, the motor coordination imposed by the rotating rod test was severely impaired in Hzf / mice. Most of the mutant mice were unable to stand on the bar under even when stationary (0 rpm) for more than 10 s (F 1,20 = 48.665, p < 0.001, ANOVA) (Fig. 2). 3.2. Muscular strength The above motor abnormalities could be caused by either impairment of motor coordination controlled by the supraspinal
Fig. 2. Rotating rod test. The test was performed to assess the motor coordination, equilibrium, learning capacity in Hzf/ mice (n = 10) and the littermate wild type (n = 12). Four trials were performed at intervals of at least 30 min per day. Filled circle, wild type; open box, Hzf/ mice. Values are mean S.E.M. (***) p < 0.001.
central nervous system (CNS) or by the muscle weakness or other defects in the lower motor systems. Next, high grip strength was measured by the wire-hanging test or the traction test. In the wirehanging test, neither the hanging time nor the score was of significant difference between wild-type and Hzf / mice (Fig. 3A and B). In contrast, the grip strength of Hzf / mice was lower than that of wild-type mice in the traction test. (t = 2.269, p < 0.01, Student’s t-test) (Fig. 3C). The difference in the traction test might be explained by differences in body weight among genotypes, since body weights in animals strongly reflect the total amount of skeletal muscle. Hzf / mice have been reported to exhibit growth retardation (Kimura et al., 2002). Indeed, body weights of Hzf / mice used in this study were significantly lower than those of wild-type mice (wild type, 34.3g 0.8; Hzf /, 30.1g 0.6) (t = 3.663, p < 0.02, Student’s t-test). Therefore, the significant difference in the hanging test might be affected by growth retardation rather than an intrinsic muscular deficit. In addition, gross anatomical and histological abnormalities of the muscle and skeleton of the Hzf / mice have not been observed in our study (data not shown). Thus, the results indicated that the motor dysfunction observed in Hzf / mice might be mainly due to impairment of motor coordination controlled by the CNS rather than to muscle weakness or other defects in the lower motor systems. 3.3. Learning and memory
Fig. 1. Spontaneous motor activity. Hzf/ mice and their littermates were assessed for their horizontal activity in a novel environment (n = 12 per genotype). Filled circle, wild type; open box, Hzf/ mice. Values are mean S.E.M. (*) p < 0.05.
Hzf protein is highly expressed in the cerebellum and the thalamus. In particular, the cerebellar Purkinje cell is one of the most prominent expression sites. The cerebellum is a center for motor functions including motor coordination and motor learning. Additionally, Hzf protein has been suggested to be required for posttranscriptional regulation of IP3R1 (Iijima et al., 2005), which is necessary for cerebellar synaptic plasticity and motor coordination (Inoue et al., 1998; Ogura et al., 2001b). Taken together, we hypothesized that the observed motor discoordinations may be mainly due to cerebellar dysfunction. Here, we assessed the learning ability of Hzf / mice by classical delay eyeblink conditioning, a form of motor learning that largely depends on the cerebellum (Thompson et al., 1997).
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Fig. 3. Traction and wire-hanging tests. Neuromuscular strength was evaluated by these tests. The wire-hanging tests were calculated on the basis of both mean scores and mean retention time in every third trials. (A) Score in the wire-hanging test in Hzf/ mice (n = 10) and the littermate wild type (n = 12). (B) Retention time in the wire-hanging test. In A and B, the left panels indicate the mean score or the retention time in three successive trials and the right panels indicate the change in values in each trial. (C) Traction test (n = 10 per genotype). Values are mean S.E.M. (**) p < 0.01.
Interestingly, Hzf / mice showed a distinct disruption in the delay paradigm (F 1,14 = 11.859, p < 0.004, ANOVA) (Fig. 4A and B). In addition, there were no significant differences in startle responses to a tone between the wild-type and Hzf / mice (Fig. 4C), indicating that the sensory input to CS were almost comparable between wild-type and Hzf / mice. These findings
indicated that the loss of Hzf had profound effects on the cerebellum-dependent motor learning. Hzf is also expressed in the cerebral cortex and the hippocampus. In addition, the protein is localized in dendrites of CA1-2 neurons in the hippocampus. The Morris water maze task is one of the most general procedures for assessing
Fig. 4. Delay eyeblink conditioning test. Primary cerebellar motor learning was examined by a classical conditioning task. (A) Temporal relationship between the CS and US in delay eyeblink conditioning. There is temporal overlap of the US with the preceding CS. (B) Development of CR during delay eyeblink conditioning (n = 8 per genotype). Spontaneous eyeblink frequency (Sp) was measured for two days prior to the acquisition session. In the acquisition session of day 1–day 7, daily session consisted of 10 blocks of trials and a block consisted of nine CS US paired trials and one CS-only trial. In the following 3-day extinction session, 100 CS-only presentations were given daily. (C) The frequency of the startle response to the CS (tone) in Hzf/ mice (n = 8 per genotype). Values are mean S.E.M. (**) p < 0.01.
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(Fig. 5). These findings suggested that the loss of Hzf does not have a profound effect on the hippocampus-dependent spatial and non-spatial memory despite its high expression in the hippocampus. 3.4. Other tests Hzf / mice and wild-type littermates were indistinguishable from each other in other behavioral tests, including elevated-plus maze tests (Fig. 6) and cliff avoidance (data not shown). These results suggested that the loss of the Hzf gene has little effect on the visual system and anxiety-related behavior. 4. Discussion
Fig. 5. Spontaneous alternation in the Y-maze task and the object recognition task. Learning and memory was evaluated by two tasks. (A) Spontaneous alternation in the Y-maze task in Hzf/ mice (n = 10) and the littermate wild type (n = 12). (B) Object recognition task (n = 10 per genotype). Values are mean S.E.M.
hippocampal-dependent spatial learning and memory. Unfortunately, we could not obtain sufficient data in the task, because of the poor swimming performance of Hzf / mice. Instead, we assessed the spontaneous alternation in the Y-maze, one of the spatial memory tasks which are largely dependent on the hippocampus, and the object recognition task, which is a hippocampus-dependent nonspatial memory task (Clark et al., 2000). However, the mutant mice were not significantly different from the wild-type in either the spontaneous alternation in the Y-maze or the object recognition task
In this study, we represented a comprehensive behavioral analysis of Hzf / mice summarized in Table 1. We will first discuss the behavioral characteristics of Hzf / mice and then the link with biological roles of the Hzf protein in the neuronal system. 4.1. Disruption of motor functions in Hzf/ mice Hzf gene deficiency severely disrupted motor functions (Figs. 1 and 2), whereas distinct abnormalities were not observed in the lower motor system (e.g. motor strength, Fig. 3 and Table 1). Notably, cerebellar motor learning during the delay eyeblink conditioning was impaired in Hzf / mice (Fig. 4). On the other hand, trace eyeblink conditioning was not performed in this study, since it depends on the hippocampus (Moyer et al., 1990; Weiss et al., 1991) as well as on the cerebellum (WoodruffPak et al., 1985; Takehara et al., 2003). However, hippocampusselective memory tasks such as spontaneous alternation in the Ymaze and object recognition task were not significantly affected (Fig. 5), indicating that the hippocampal functions are not severely impaired in Hzf / mice. Taken together, the motor impairment is likely to be mainly caused by deficits of some cerebellar neuronal functions of the Hzf protein. Table 1 The summary for behavioral analyses using Hzf/ mice
Fig. 6. Elevated-plus maze test. Anxiety-related behavior was evaluated in the test. (A) The number of open- and closed-arm entries in Hzf/ mice (n = 10) and the littermate wild type (n = 12). (B) The time spent in each type of arm. Values are mean S.E.M.
Tests
Results
Motor activity and coordination Locomotor activity Rota rod test
Impaired Impaired (drastic)
Muscle strength Wire-hanging test Traction test
Normal Impaired (slightly)
Anxiety Elevated plus maze
Normal
Learning and memory Eyeblink conditoning test (delay) Morris water maze test Spontaneous Y-maze test Object recognition test
Impaired N.I. Normal Normal
N.I., no informative.
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Nevertheless, we cannot completely exclude the possibility that the loss of Hzf in other regions or systems might have induced the behavioral phenotypes observed in the present study. A previous study has reported that Hzf is expressed in megakaryocytes, a precursor of platelets, and that internal hemorrhaging is observed in the brains and gastrointestinal tracts in Hzf / neonates (Hidaka et al., 2000; Kimura et al., 2002). Brain hemorrhaging could cause motor dysfunction in the null mice. In that respect, at least, we did not observe marked internal hemorrhaging in animals used in the eyeblink conditioning task (data not shown). Based on these points, it is unlikely that these severe motor dysfunctions in Hzf / mice can be simply explained by internal hemorrhaging. It has been suggested that one of the mechanisms underlying eyeblink conditioning is cerebellar plasticity at parallel fiberPurkinje cell synapses, since cerebellar LTD-deficient mice exhibit severe impairments of eyeblink conditioning (Aiba et al., 1994; Shibuki et al., 1996; Kishimoto et al., 2001a,b; Koekkoek et al., 2003). The cerebellum is crucial for motor coordination and learning in animals. Since Hzf is highly expressed in Purkinje cells within the cerebellum, we guessed that some portion of the observed motor abnormalities might be due to cerebellar dysfunction. 4.2. The link between posttranscriptional regulations by Hzf and motor functions It is known that an abnormality of climbing fiber translocation and innervation of Purkinje cells often causes ataxia-phenotypes in animals. However, no such morphological defects were observed in the Hzf / mice (unpublished data). By what mechanism was a series of motor impairments caused in Hzf / mice? Disruption of dendritic mRNA localization and activity-dependent mRNA translation impair the long-phase synaptic plasticity and memory consolidation in the hippocampus (Miller et al., 2002; Kelleher et al., 2004). This could also be the case in the cerebellum. A particular form of cerebellar LTD depends on postsynaptic protein synthesis in cultured Purkinje cells (Linden, 1996). The group 1 mGluRdependent LTD is enhanced by deletion of the FMR1 gene in both the hippocampus and cerebellum (Huber et al., 2002; Koekkoek et al., 2005). Furthermore, overall and Purkinje cellspecific deletions of FMR1 in mice and fragile X patients distinctly attenuate cerebellar eyeblink conditioning (Koekkoek et al., 2005). These findings suggest that newly synthesized proteins controlled by specific RNA-binding proteins play an important role in the maintenance of particular forms of cerebellar synaptic plasticity. Like FMRP, Hzf could be a component of a neuronal RNA granule, which transports mRNAs into dendrites (Kanai et al., 2004). Interestingly, our previous study suggests that Hzf functions as a regulator of dendritic localization and their translation of IP3R1 mRNA in cerebellar Purkinje cells (Iijima et al., 2005). Hzf could potentially target multiple mRNAs. Nevertheless, IP3R1 mRNA would be one of the intriguing targets for Hzf, since it is the intracellular Ca2+ channel (Furuichi et al., 1989), and
required for cerebellar LTD (Inoue et al., 1998), the basis for cerebellar motor learning (Ito, 1989). Mice lacking IP3R1 exhibit low birth rate and postnatally began to show severe cerebellar ataxia and truncal torsions (Matsumoto et al., 1996). Although the phenotypes of Hzf / mice largely differed from those of IP3R1/ mice at the point where such severe phenotypes are not observed, there are somewhat similar to IP3R1+/ mice, which exhibit motor dysfunctions (Ogura et al., 2001b). Therefore, the observed motor dysfunctions in Hzf / mice, at least, might partially reflect the impairment of posttranscriptional regulations of the target mRNAs (including IP3R1 mRNA) for Hzf protein in the cerebellum (particularly in Purkinje cells), although the physiological relevance of posttranscriptional regulation controlled by Hzf should be elucidated in future studies. In addition, as mentioned above, the behavioral phenotypes in animals may reflect the interplay of multiple regions and systems. To demonstrate the involvement of cerebellar systems in the behavioral impairments of Hzf / mice which were observed in the present study, we are currently planning the rescue experiments of these behavioral phenotypes by a cerebellar Purkinje cells-selective Hzf transgene. Thus, the genetic manipulation within a specific brain region or cell type will reveal the biological significance of Hzf in motor functions in animal more clearly in our future study. Acknowledgements We appreciate the invariable assistance of all members of the Okano laboratory. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.O.), a Grant-in-aid for 21st Century COE program to Keio University. References Aiba, A., Kano, M., Chen, C., Stanton, M.E., Fox, G.D., Herrup, K., Zwingman, T.A., Tonegawa, S., 1994. Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79, 377–388. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wilkinson, K.D., Keene, J.D., Darnell, R.B., Warren, S.T., 2001. Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107, 477– 487. Clark, R.E., Zola, S.M., Squire, L.R., 2000. Impaired recognition memory in rats after damage to the hippocampus. J. Neurosci. 20, 8853–8860 similar results. Furuichi, T., Yosikawa, S., Miyawaki, A., Wada, K., Maeda, N., Mikoshiba, K., 1989. Primary structure and functional expression of the inositol 1,4,5trisphosphate-binding protein P400. Nature 342, 32–38. Hidaka, M., Caruana, G., Stanford, W.L., Sam, M., Correll, P.H., Bernstein, A., 2000. Gene trapping of two novel genes, Hzf and Hhl, expressed in hematopoietic cells. Mech. Dev. 90, 3–15. Houchi, H., Babovic, D., Pierrefiche, O., Ledent, C., Daoust, M., Naassila, M., 2005. CB1 receptor knockout mice display reduced ethanol-induced conditioned place preference and increased striatal dopamine D2 receptors. Neuropsychopharmacology 30, 339–349. Huber, K.M., Kayser, M.S., Bear, M.F., 2000. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 288, 1254–1257.
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