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Keywords:

  • Agrin;
  • Agrn;
  • Atp1a3;
  • bipolar disorder;
  • circadian rhythm;
  • mania;
  • mutant mouse;
  • Na+;
  • K+-ATPase alpha3

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Myshkin mice heterozygous for an inactivating mutation in the neuron-specific Na+,K+-ATPase α3 isoform show behavior analogous to mania, including an abnormal endogenous circadian period. Agrin is a proteoglycan implicated as a regulator of synapses that has been proposed to inhibit activity of Na+,K+-ATPase α3. We examined whether the mania-related behavior of Myshkin mice could be rescued by a reduction in the expression of agrin through genetic knockout. The suppression of agrin reduced hyperambulation and holeboard exploration, restored anxiety-like behavior (or reduced risk-taking behavior), improved prepulse inhibition and shortened the circadian period. Hence, agrin is important for regulating mania-like behavior and circadian rhythms. In Myshkin mice, the suppression of agrin increased brain Na+,K+-ATPase activity by 11 ± 4%, whereas no effect on Na+,K+-ATPase activity was detected when agrin was suppressed in mice without the Myshkin mutation. These results introduce agrin as a potential therapeutic target for the treatment of mania and other neurological disorders associated with reduced Na+,K+-ATPase activity and neuronal hyperexcitability.

Preliminary human studies have implicated the Na+,K+-ATPase and its regulation in the etiology of bipolar disorder, while mouse and rat models provide support for a role of Na+,K+-ATPase activity in regulating mania and depression-related behavior (Crema et al. 2010; De Vasconcellos et al. 2005; El-Mallakh et al. 2003; Gamaro et al. 2003; Kirshenbaum et al. 2011a,b; Moseley et al. 2007). The Na+,K+-ATPase, composed of a catalytic α subunit and a β subunit, regulates electrochemical gradients in neurons by active exchange of Na+ and K+ and thereby controls neuronal excitability. Three isoforms of the α subunit (α1–3) are expressed in the brain and each has distinct expression patterns (Kaplan 2002). Na+,K+-ATPase α1 is expressed in neurons and glia, Na+,K+-ATPase α2 is primarily expressed in glia and Na+,K+-ATPase α3 is only expressed in neurons (Mcgrail et al. 1991). Post-mortem brains of bipolar individuals show reduced genetic expression of the Na+,K+-ATPase α2 subunit in temporal cortex (Rose et al. 1998) and α3 in the prefrontal cortex (Tochigi et al. 2008), while associations between bipolar disorder and some variants of α1, α2 and α3 have been reported with unknown functional effects (Goldstein et al. 2009; Mynett-Johnson et al. 1998). In addition, endogenous ouabain-like compounds, which selectively bind and inhibit Na+,K+-ATPase α isoforms (Croyle et al. 1997), are present at reduced concentration in serum (El-Mallakh et al. 2010; Grider et al. 1999) and at higher concentration in parietal cortex, and have higher binding affinity in the parietal cortex (Goldstein et al. 2006) in bipolar vs. healthy humans.

Myshkin (Atp1a3Myk/+; Myk/+) mice carry a missense mutation in the neuron-specific α3 isoform of the Na+,K+-ATPase that results in a normally expressed, but inactive enzyme, leading to a 36–42% reduction in total Na+,K+-ATPase activity in the brain (Clapcote et al. 2009; Kirshenbaum et al. 2011a). Myk/+ mice exhibit several behavioral abnormalities that are consistent with mania, including a lengthened circadian period, enhanced sensitivity to d-amphetamine, and response to chronic treatment by the mood stabilizers lithium and valproic acid (Kirshenbaum et al. 2011a). The behavior of Myk/+ mice is comparable to the pharmacological ouabain model of mania in rats, which acts by inhibition of the activity of all Na+,K+-ATPase isoforms in the brain (El-Mallakh et al. 2003). Myk/+ behavior is also comparable to existing mouse genetic models of mania including knockdown of GluR6 (Shaltiel et al. 2008), DAT (Ralph-Williams et al. 2003; Young et al. 2011), inactivation of Clock (Mcclung et al. 2005; Roybal et al. 2007), reduction of ERK (Creson et al. 2009; Engel et al. 2009), overexpression of GSK3β (Prickaerts et al. 2006), phosphorylation resistant GSK3β (Ackermann et al. 2010) and mtDNA polymerase-dependent deletions in mtDNA (Kasahara et al. 2006). These genes may interact in a common pathway in mania-related circuitry.

Transgenic restoration of functional Na+,K+-ATPase α3 in Myk/+ mice increases Na+,K+-ATPase α3 expression and whole brain Na+,K+-ATPase activity and rescues mania-related behavior (Clapcote et al. 2009; Kirshenbaum et al. 2011a). Consequently, therapies aimed at increasing Na+,K+-ATPase activity may prove valuable in the treatment of mania-related behavior. Because it has been suggested that the endogenous protein agrin acts in the brain to regulate neuronal excitability via inhibition of Na+,K+-ATPase α3 (Hilgenberg et al. 2006; Tidow et al. 2010, 2011), interference with this inhibition might be a useful strategy. The interaction of agrin with Na+,K+-ATPase has not been replicated in vitro or showed in vivo, though. To genetically suppress agrin expression in Myk/+ mice, we crossed them with agrin heterozygous knockout mice (Agrn/+) that carry a null allele affecting all isoforms of agrin. Herein, we report that suppression of neuronal agrin by inheritance of the Agrn null allele ameliorates mania-like behavior and rescues circadian period lengthening in Myk/+ mice. Our findings provide evidence that agrin regulates neuronal excitability to influence behavior.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Animals and housing

All procedures were approved by the Toronto Centre for Phenogenomics Animal Care Committee, followed the Province of Ontario Animals for Research Act 1971 and adhered to requirements of the Canadian Council on Animal Care. Myk/+ mice have been previously described and were backcrossed 20 generations on a C57BL/6NCr strain (National Cancer Institute) (Clapcote et al. 2009; Kirshenbaum et al. 2011a). Agrn/+ mice (Agrntm4Jrs) carrying a deletion that ablates expression of both the neural and non-neural isoforms of agrin were generously provided by Dr. Robert Burgess (The Jackson Laboratory) and have been described previously (Lin et al. 2001). All test subjects were bred from six separate breeding cages of one Myk/+ male and two Agrn/+ females to produce wild-type (+/+), agrin heterozygous (Agrn/+), Myshkin heterozygous (Myk/+) and compound heterozygous (Myk/+/Agrn) mice. Pups were genotyped using previously described polymerase chain reaction assays (Clapcote et al. 2009; Lin et al. 2001; Serpinskaya et al. 1999). At 4 weeks of age, pups of mixed genotypes were weaned and housed by sex in groups of three to five. Mice were housed in filtered cages containing corn cob bedding, nesting material and ad libitum sterile food (Harlan Teklad 2918) and water. Housing conditions were maintained at 21 ± 1°C and 50–60% humidity under a 12:12 h light–dark cycle (lights on: 0700–1900 h).

Behavioral studies

Behavioral testing was conducted during the light phase between 0900 and 1500 h with observers being blind to genotype. Behavioral experiments began at 8 weeks of age and tests were separated by 1 week when mice performed more than one test. One cohort of mice was tested in the open field and elevated plus maze (EPM). A second cohort was tested in the light–dark box (LDB) and holeboard exploration tests. A third cohort was tested in prepulse inhibition (PPI) and for circadian rhythms. Males and females were included in all experiments in balanced numbers. As no sex differences were detected, results were pooled. Mice were left undisturbed in a testing environment for 30 min to allow for acclimation before experimentation. A solution of 70% ethanol or Clidox was used to clean surfaces and equipment between subjects. Behavior was scored using Observer 5.0 software (Noldus Information Technology, Wageningen, Netherlands).

Open field

Mice (+/+ n = 18, Agrn/+ n = 17, Myk/+n = 21, Myk/+/Agrn mice n = 15) were placed in the middle of a transparent Plexiglas open field illuminated by 200 lux (41.25 cm × 41.25 cm × 31.25 cm). Locomotor activity was recorded for 30 min by the VersaMax Animal Activity Monitoring System (Columbus, OH, USA).

Elevated plus maze

The EPM was performed as described (Crawley & Goodwin 1980). Experiments were conducted in a dark room. The apparatus was elevated 50 cm from the floor and was made of opaque Plexiglas. There was a center platform (5 × 5 cm) with two opposing open arms (25 × 5 cm, illuminated by 700 lux) and two opposing closed arms enclosed by black Plexiglas walls (25 × 5 × 30 cm). Behavior was scored for 5 min and the experiment began by placing a mouse on the center platform facing a closed arm (+/ + n = 20, Agrn/+ n = 16, Myk/+ n = 25, Myk/+/Agrn n = 12).

Light–dark box

The LDB was performed as described (Crawley & Goodwin 1980), and consisted of transparent Plexiglas (69 cm × 31 cm × 31 cm). One third of the box was separated by a partition with a door (20 cm × 10 cm). The partitioned area was painted black and covered by a roof. The experiments were conducted in a dark room and a bright light (2000 lux) was directed at the unpartitioned area. The darkened partition had very dim light. A mouse was placed in the darkened area, and durations in the dark and light areas were scored for 6 min (+/+ n = 20, Agrn/+ n = 16, Myk/+ n = 25, Myk/+/Agrn n = 12).

Prepulse inhibition

PPI was performed as described (Lalonde & Botez 1985). Mice were placed in a confined chamber within a calibrated sound attenuating acoustic isolation chamber with a load-cell platform (Startle Reflex System, MED Associates, St. Albans, VT, USA; ENV-022s). A sound generator and Med Associates software (MED-ASR-PRO; Startle Reflex package version 5.98) regulated sound pulses from the amplifier. Mice (+/+ n = 20, Agrn/+ n = 16, Myk/+ n = 25, Myk/+/Agrn n = 12) had a 5-min acclimation period to 65 dB background white noise. Mice were then exposed to a series of five startle-pulse-alone (P) trials that were single white noise bursts at 100 dB for 40 milliseconds. Next, mice were given trials consisting of (1) no stimulus, (2) a startle-pulse-alone or (3) one of four prepulse intensities (70, 78, 86 and 90 dB, 20 milliseconds) presented 100 milliseconds before a startle pulse. Trials were presented in 10 blocks and each block contained all 6 trial types (no stimulus, P, 70 dB + P, 78 dB + P, 86 dB + P, 90 dB + P) in pseudorandom order. Lastly, mice were presented with five startle-pulse-alone trials. Intertrial intervals were 12–30 seconds. The peak startle was recorded for each trial. PPI was calculated as %PPI = 100 – (startle response on prepulse trials/startle response on startle-pulse-alone trials) × 100.

Holeboard exploration

The holeboard exploration task was modified from a previous study (Lalonde & Botez 1985). The experiment was performed in an open field made of transparent Plexiglas (41.25 cm × 41.25 cm× 31.25 cm) in dim light on previously handled animals. The floor of the open field had 11 holes and the walls had 2 holes. Mice (+/+ n = 13, Agrn/+ n = 9, Myk/+ n = 13, Myk/+/Agrn n = 11) were placed in the center of the apparatus, and behavior was observed for 6 min per day for 5 days.

Circadian rhythms

Mice (+/+ n = 8, Agrn/+ n = 7, Myk/+ n = 5, Myk/+/Agrn n = 5) were individually housed in cages containing a running wheel (17 cm diameter) with ad libitum access to food and water at a room temperature of 20 ± 2°C. Continuous wheel running activity was recorded by VitalView (Minimitter Co., Bend, OR, USA) and analyzed using Actiview (Minimitter) and Clocklab (Actimetrics Inc, Evanston, IL, USA). Initially, mice were housed under a 14 h:10 h light–dark (L:D) cycle for 14 days. To assess free running period, mice were then housed in constant dark for 7–14 days.

Na+,K+ -ATPase assay of brain homogenates

At 20 weeks of age, mice were sacrificed by cervical dislocation. Brains were immediately removed and frozen in liquid nitrogen. As previously described, Na+,K+-ATPase activity was measured in brain homogenates and related to total protein concentration in the homogenate (Vilsen 1992). Brain samples (whole brain) were homogenized in 85 mm NaCl, 20 mm KCl, 4 mm MgCl2, 0.2 mm ethyleneglycoltetraacetic acid (EGTA), 30 mm histidine (pH 7.2) and 10% sucrose (wt/vol). Tissue was permeabilized further by incubation with 0.65 mg sodium deoxycholate/ml at a total brain protein concentration of 0.3 mg/ml in the presence of 2 mm ethylenediaminetetraacetic acid and 2 mm imidazole at 20°C. The ouabain sensitive Na+ and K+ activated ATP hydrolysis rate was determined at 37°C by a colorimetric assay for liberated Pi (Vilsen 1992). The deoxycholate-treated brain tissue homogenate (25 µl) was added to 500 µl ATPase buffer containing 30 mm histidine (pH 7.5), 140 mm NaCl, 20 mm KCl, 3 mm MgCl2 and 1mM EGTA, and was allowed to react for 5 min with 3 mm ATP in the presence and absence of 3 mm ouabain (+/+ n = 10, Agrn/+ n = 7, Myk/+ n = 8, Myk/+/Agrn n = 7).

Statistical analysis

All statistics were calculated by STATISTICA (StatSoft, Tulsa, OK, USA). All data are presented as the mean ± SEM with significance set at P < 0.05. Biochemical and behavioral results were analyzed using two-way, three-way or repeated-measures analysis of variance (anova) with Agrn genotype, Atp1a3 genotype and sex as the between-subject factors. Within-subject factors were ‘day’ in the holeboard test, ‘prepulse intensity’ in the PPI test and ‘5 minute bin interval’ in the open field test. Fisher's least significant difference (LSD) post hoc comparisons were performed when anova yielded statistically significant main effects or interactions.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Agrin deficiency reduces mania-related behavior in Myk/+ mice

When agrin was suppressed in Myk/+ mice by inheritance of the Agrn null allele, hyperambulation was reduced (main effect of Agrn/+ genotype F1,69 = 3.62, P = 0.03, main effect of Myk/+ genotype F1,69 = 34.43, P < 0.0001, Agrn/+ ×Myk/+ genotype interaction F1,69 = 8.94, P = 0.0007, Fig. 1a). Overall, Myk/+ and Myk/+/Agrn mice showed higher locomotor activity compared to +/+ mice, but Myk/+/Agrn mice had significantly reduced locomotor activity than Myk/+ littermates. This difference was most notable during the initial exploration of the open field (main effect of genotype F3,69 = 30.724, P < 0.0003, main effect of 5-min bin F5,69 = 19.183, P < 0.0001, genotype × 5-min bin interaction F15,69 = 3.211, P < 0.0001, Fig. S1). In a holeboard exploration test, Myk/+ mice showed increased exploration on all 5 testing days compared to +/+ mice (main effect of genotype F3,42 = 37.054, P < 0.0001, main effect of day F4,42 = 25.817, P < 0.0001). In addition, suppression of agrin reduced nosepoke exploration in Myk/+/Agrn mice relative to Myk/+ mice (Fig. 1b).

image

Figure 1. Mania-related behavior is partially restored by a reduction of agrin in Myshkin mice. (a) In an open field for 30 min, Myk/+/Agrn mice travel a shorter distance than Myk/+ mice. (b) In a holeboard exploration test, Myk/+ mice make more nosepokes than Myk/+/Agrn mice on all 5 days of testing. (c) In the EPM, Myk/+/Agrn mice explore the open arm for a shorter duration than Myk/+ mice. (d) In the LDB, Myk/+ mice explore the light side for a longer duration than Myk/+/Agrn mice. (e) Compared to +/+ mice, Myk/+ mice show reduced PPI scores and Myk/+/Agrn mice do not. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to +/+; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to Myk/+.

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Myk/+ and Myk/+/Agrn mice explored the open arm of the EPM for an increased duration compared to +/+ mice (main effect of Agrn/+ genotype F1,69 = 7.26, P = 0.0002, main effect of Myk/+ genotype F1,69 = 50.59, P < 0.0001, Agrn/+ ×Myk/+ genotype interaction F1,69 = 5.15, P = 0.0015) and made more exploratory head dips (main effect of Agrn/+ genotype F1,69 = 7.60, P = 0.0006, main effect of Myk/+ genotype F1,69 = 42.50, P < 0.0001, Agrn/+ ×Myk/+ genotype interaction F1,69 = 5.38, P = 0.0034). However, Myk/+/Agrn mice explored the open arm for a shorter duration and made less frequent head dips compared to Myk/+ mice, indicating an increase in anxiety or a reduction in risk-taking behavior (Figs. 1c and S1). All four genotypes showed similar total locomotor activity, but Myk/+ mice showed increased entries to the open arm compared to +/+ mice (main effect of Myk/+ genotype F1,69 = 18.54, P < 0.0001), whereas Myk/+/Agrn mice did not (Fig. S1). In the LDB, Myk/+ mice spent more time exploring the light side than +/+, Agrn/+ and Myk/+/Agrn mice, which all spent equal time exploring the light side (main effect of Agrn/+ genotype F1,51 = 11.74, P = 0.0016, main effect of Myk/+ genotype F1,51 = 27.82, P < 0.0001 Fig. 1d). Locomotor activity did not differ between the four genotypes in the LDB test (Fig. S1). Overall, compared to Myk/+ mice, Myk/+/Agrn mice showed increased measures of anxiety in the EPM and LDB, comparable to those of +/+ mice.

Similar PPI scores were shown by +/+, Agrn/+ and Myk/+/Agrn mice, but Myk/+ mice had reduced scores compared to +/+ mice at all prepulse intensities (main effect of genotype F3,69 = 7.157, P = 0.0003, main effect of prepulse intensity F3,69 = 9.974, Fig. 1e). The suppression of agrin in Myk/+ mice thus restored measures of PPI.

Agrin deficiency restores circadian period in Myk/+ mice

In constant darkness, Myk/+ mice show an extended endogenous free-running circadian period of 25 h, which was normalized to +/+ levels of 23.5 h (Possidente et al. 1995) by inheritance of the Agrn null allele (main effect of Agrn/+ genotype F1,21 = 28.43, P = 0.0004, main effect of Myk/+ genotype F1,21 = 35.63, P = 0.0001, Agrn/+ ×Myk/+ genotype interaction F1,21 = 7.38, P = 0.05 Fig. 2a,b; summary data are shown in Fig. 2a, and representative actograms in constant dark are shown in Fig. 2b).

image

Figure 2. Free-running circadian period. (a) Myk/+ mice have a lengthened free-running circadian period of about 25 h that is rescued to +/+ duration (23.5 h) by a reduction in agrin. Data are presented as the mean ± SEM. ***P < 0.001 compared to +/+; ##P < 0.01 compared to Myk/+. (b) Representative actograms. Activity is double plotted. Genotype and period are indicated above each panel. Onsets (red dots) were determined using Clocklab. Period was determined by linear regression, indicated by solid lines through onset on two actograms.

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Reductions in whole brain Na+,K+-ATPase activity are related to mania-like behavior

Relative to +/+ mice, Agrn/+ mice show similar whole brain Na+,K+-ATPase activity (main effect of Myk/+ genotype F1,28 = 61.32, P < 0.0001, Agrn/+ ×Myk/+ genotype interaction F1,28 = 10.23, P = 0.001, Fig. 3a). The lack of effect of agrin deficiency on Na+,K+-ATPase activity is somewhat surprising considering that agrin has been proposed to be an inhibitor of the neuronal α3 isoform of Na+,K+-ATPase (Hilgenberg et al. 2006; Tidow et al. 2010, 2011). Myk/+ and Myk/+/Agrn mice show lower whole brain Na+,K+-ATPase activity than +/+ mice. However, the activity of Na+,K+-ATPase is 11 ± 4% higher in Myk/+/Agrn mice than in Myk/+ mice (Na+,K+-ATPase activity is reduced by 36 ± 4% of +/+ levels in Myk/+ mice and by 25 ± 4.6% of +/+ levels in Myk/+/Agrn mice at 20 weeks of age; Fig. 3a). These results suggest that the Agrn null allele modestly increases Na+,K+-ATPase activity in Myk/+ mice, either directly or indirectly.

image

Figure 3. Whole brain Na + ,K + -ATPase activity is increased by a reduction of agrin in Myk/+ mice and related to mania-like behavior. (a) Whole brain Na+,K+-ATPase activity is increased by approximately 11% in Myk/+/Agrn mice compared to Myk/+ mice. (b) Total distance in the open field and (c) duration on the open arm of the EPM are negatively correlated with whole brain Na+,K+-ATPase activity (P < 0.0001) (+/+ n = 10, Agrn/+ n = 7, Myk/+ n = 8, Myk/+/Agrn n = 7).

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Whole brain Na+,K+-ATPase activity showed a significant negative correlation with total distance traveled in the open field in 30 min (Pearson's correlation r = −0.78, P < 0.0001) and the duration on the open arm of the EPM (Pearson's correlation r = −0.76, P < 0.0001; Fig. 3b,c), regardless of Agrn and Atp1a3 genotype, indicating that reductions in Na+,K+-ATPase activity are related to increased mania-like behavior.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Myk/+ mice have a missense mutation I810N in the neuronally expressed Na+,K+-ATPase α3 isoform that leads to a 36 ± 4% reduction in whole brain Na+,K+-ATPase activity and model several symptoms of the manic phase of bipolar disorder (Clapcote et al. 2009; Kirshenbaum et al. 2011a). Manic bipolar individuals show prominent hyperactivity, hyperexploration and novelty seeking (Minassian et al. 2011). Myk/+ mice model these symptoms by exhibiting hyperambulation in a novel open field and increased and sustained exploration of novelty (Kirshenbaum et al. 2011a). Manic individuals show increased risk-taking behavior (Holmes et al. 2009; Young et al. 2011). Myk/+ mice exhibit reduced measures of anxiety in the EPM and LDB (Kirshenbaum et al. 2011a), which can be interpreted as an increase in risk-taking behavior (Einat 2006). Deficits in PPI are found in manic individuals and their unaffected siblings (Giakoumaki et al. 2007; Perry et al. 2001). Myk/+ mice also model this facet of mania (Kirshenbaum et al. 2011a). Finally, endogenous circadian rhythms are altered in the majority of bipolar individuals (Jones 2001). Myk/+ mice exhibit aberrant endogenous circadian rhythms (Kirshenbaum et al. 2011a).

We crossed Myk/+ mice with mice deficient for agrin (Agrn/+ mice) to produce Myk/+/Agrn mice. The deficiency of agrin caused by inheritance of the Agrn null allele reduced mania-related behavior in mice with the Myk mutation, but had little effect on behavior in Agrn/+ mice with an otherwise wild-type genetic background. Similarly, we previously reported that the mood stabilizers lithium and valproic acid had anti-manic effects in Myk/+ mice but did not affect the behavior of their +/+ littermates (Kirshenbaum et al. 2011a). In comparison to Myk/+ mice, Myk/+/Agrn mice showed a reduction in hyperambulation, reduced exploration of novelty, increased anxiety-like behavior (or reduced risk-taking behavior), improved PPI and a shorter circadian period. Notably, sex differences were not detected in our experiments, but may possibly be observed with larger sample sizes. While reduced anxiety measures in the EPM and LDB are indicative of an increase in risk-taking behavior, newer paradigms such as the Iowa Gambling Task for mice directly test risk-taking (Young et al. 2011) and may be applied to future studies to separate anxiety from risk-taking. Taken together, our results suggest that agrin is important for regulating mania-like behavior and circadian rhythms.

Because agrin is a putative inhibitor of Na+,K+-ATPase α3 activity (Hilgenberg et al. 2006; Tidow et al. 2010, 2011), the observed behavioral effects of reducing agrin expression might be a consequence of relief of Na+,K+-ATPase inhibition. Hence, it is possible that agrin–Na+,K+-ATPase α3 interactions modulate neuronal circuits involved in the regulation of behavior, including locomotor activity, exploration, anxiety, sensorimotor gating and endogenous circadian rhythms. Na+,K+-ATPase α3 is expressed in excitatory glutamatergic and inhibitory GABAergic neurons in the mouse brain, with higher GABAergic expression in particular brain regions such as the hippocampus and basal ganglia (Bottger et al. 2011; Chu et al. 2009; Richards et al. 2007). On the other hand, agrin is localized to glutamatergic synapses in the mouse brain (Ksiazek et al. 2007). Therefore, agrin–Na+,K+-ATPase α3 interactions could be predominant at excitatory synapses and synapses between excitatory and inhibitory neurons within circuits that regulate behavior. Studies of conditional Agrn mutant mice with targeted suppression of agrin in specific brain regions known to regulate behavior are required to further characterize the roles of such interactions. Theoretically, an increase in agrin expression or activity could lead to a decrease in Na+,K+-ATPase activity and induction of mania. An activity-dependent model for agrin–Na+,K+-ATPase α3 signaling has been proposed. Upon synaptic activity, the protease neurotrypsin is released from presynaptic terminals (Frischknecht et al. 2008) and cleaves pre- and postsynaptic transmembrane agrin to release agrin fragments into the synapse (Reif et al. 2007). A 22-kDa agrin fragment is thought to diffuse and bind to pre- and postsynaptic Na+,K+-ATPase α3 subunits and inhibit their activity; this may be dependent on N-methyl-d-aspartate receptor activation on the postsynaptic membrane (Matsumoto-Miyai et al. 2009; Tidow et al. 2011). An inactivating mutation in the neurotrypsin gene PRSS12 leads to mental retardation (OMIM #249500; Molinari et al. 2002). Consequently, enhancement of agrin signaling by undiscovered mutations in the genes encoding neurotrypsin, agrin or Na+,K+-ATPase α3 might alter synaptic physiology and lead to mania.

In apparent contradiction with this emerging hypothesis, Agrn/+ mice did not show the predicted increase in Na+,K+-ATPase activity relative to +/+ mice, which was unexpected given that Agrn/+ mice and agrin-deficient neurons show attenuated responses to excitatory stimuli (Hilgenberg et al. 2002). Only in Myk/+ mice did reduction in agrin expression lead to a modest increase of total brain Na+,K+-ATPase activity of 11 ± 4%. These findings introduce the possibility that agrin does not directly inhibit Na+,K+-ATPase activity and suggest that more complex mechanisms are responsible for the behavioral differences between Myk/+/Agrn and Myk/+ mice. For example, agrin can initiate Ca2+ signaling and activation of CaMKII and MAPK in neurons, and may act through these mechanisms (Hilgenberg & Smith 2004). In contrast to Agrn homozygous knockout mice, which completely lack agrin in the brain and show synaptic deficits, Agrn/+ mice still express agrin, show normal synapse development, and perform all behavioral measures at +/+ levels (Ksiazek et al. 2007; Li et al. 1999; Serpinskaya et al. 1999). As the proposed role of agrin in relation to Na+,K+-ATPase is activity-dependent (Tidow et al. 2010), it is possible that Na+,K+-ATPase activity is only higher in Agrn/+ mice compared to +/+ mice during neuronal activity, which would not be observed in our measurements of Na+,K+-ATPase activity in brain homogenates. An alternative possibility is that the Myk mutation leads to a higher binding affinity for agrin in Na+,K+-ATPase α3 subunits, so that the amount of free agrin available to interact with the wild-type α3 protein is limited in the Myk/+ mice, which could account for the larger changes observed in Myk/+ mice than in +/+ mice with a reduction in agrin. It should, however, also be taken into consideration that the 11% increase in total brain Na+,K+-ATPase activity in Myk/+/Agrn mice with reduced agrin level could arise from an increased amount of Na+,K+-ATPase α3 protein or of any of the other two Na+,K+-ATPase isoforms, α1 and α2, present in the brain, as a result of changes in signaling pathways affecting expression levels.

Irrespective of whether the effect of agrin suppression on Na+,K+-ATPase activity is direct or indirect, our results introduce agrin as a potential therapeutic target for the treatment of mania and, possibly, other disorders characterized by reduced Na+,K+-ATPase activity, such as rapid-onset dystonia parkinsonism (Einholm et al. 2010). The anti-mania-like efficacy of reduced agrin expression in Myk/+ mice is similar to that of the mood stabilizers lithium and valproic acid (Kirshenbaum et al. 2011a). Consequently, chemical agents that achieve suppression of agrin in the brain may be at least as therapeutically effective as currently available treatments for mania. The potential applicability of this approach would be further illuminated by determining whether agrin deficiency also has anti-manic effects in other mouse genetic models for mania, such as the Clock mutant (Mcclung et al. 2005; Roybal et al. 2007).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

This work was supported by grants to J.C.R. from the Canadian Institutes of Health Research (MOP 94856) and the Amalgamated Transit Union, Local 113. JCR holds a Canada Research Chair. M.R.R. was supported by the Natural Sciences Research Council of Canada. G.S.K. was supported by a Research Studentship from the Ontario Mental Health Foundation. S.J.C. was supported by a grant from the UK Medical Research Council (G0900625) and a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression. B.V. was supported by grants from the Lundbeck Foundation, the Novo Nordisk Foundation (Fabrikant Vilhelm Pedersen og Hustrus Legat) and the Danish Medical Research Council. The authors declare no conflict of interest.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: Open field, elevated plus maze and light-dark box. (a) In the open field, Myk/+/Agrn mice show reduced locomotor activity compared to Myk/+ mice. (b) In the elevated plus maze, Myk/+ mice show more entries to the open arm than +/+ mice and (c) total entries do not differ between genotypes. (d) Myk/+/Agrn mice show reduced head dips compared to Myk/+ mice (+/+ n = 18, Agrn/+ n = 17, Myk/+ n = 21, Myk/+/Agrn n = 15). (e) In the light–dark, locomotor activity does not differ between genotypes (+/+ n = 16, Agrn/+ n = 12, Myk/+ n = 15, Myk/+/Agrn n = 12). Data are presented as the mean ± SEM. &ast;&ast;&ast;P < 0.001 compared to +/+; &num;&num;&num;P < 0.001 compared to Myk/+.

Table S1: Behavioral phenotypes in genetic mouse models of mania

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