APP Causes Hyperexcitability in Fragile X Mice

Amyloid-beta protein precursor (APP) and metabolite levels are altered in fragile X syndrome (FXS) patients and in the mouse model of the disorder, Fmr1KO mice. Normalization of APP levels in Fmr1KO mice (Fmr1KO/APPHET mice) rescues many disease phenotypes. Thus, APP is a potential biomarker as well as therapeutic target for FXS. Hyperexcitability is a key phenotype of FXS. Herein, we determine the effects of APP levels on hyperexcitability in Fmr1KO brain slices. Fmr1KO/APPHET slices exhibit complete rescue of UP states in a neocortical hyperexcitability model and reduced duration of ictal discharges in a CA3 hippocampal model. These data demonstrate that APP plays a pivotal role in maintaining an appropriate balance of excitation and inhibition (E/I) in neural circuits. A model is proposed whereby APP acts as a rheostat in a molecular circuit that modulates hyperexcitability through mGluR5 and FMRP. Both over- and under-expression of APP in the context of the Fmr1KO increases seizure propensity suggesting that an APP rheostat maintains appropriate E/I levels but is overloaded by mGluR5-mediated excitation in the absence of FMRP. These findings are discussed in relation to novel treatment approaches to restore APP homeostasis in FXS.


INTRODUCTION
Amyloid-beta protein precursor (APP) levels are dysregulated in numerous neurological disorders that are comorbid with a seizure phenotype including fragile X syndrome (FXS) (Westmark, 2013). FXS is a trinucleotide repeat disorder caused by a CGG repeat expansion at the 5 ′ -end of the FMR1 gene. Hypermethylation of the repeat expansion results in transcriptional silencing of the FMR1 gene and loss of expression of fragile X mental retardation protein (FMRP) (Jin and Warren, 2000). FMRP is an RNA binding protein (RBP) that plays a pivotal role in synaptic function. It is one of numerous RBP that interact with amyloid precursor protein (App) mRNA to regulate post-transcriptional and/or translational events involved in the synthesis of APP (Westmark and Malter, 2012). Specifically, FMRP binds to a guanine-rich region in the coding region of App mRNA and regulates APP translation through a metabotropic glutamate receptor 5 (mGluR 5 )-dependent pathway (Westmark and Malter, 2007). We hypothesize that altered expression of APP in FXS contributes to disease severity. In support of this hypothesis, genetic knockout of one App allele in Fmr1 KO mice (Fmr1 KO /APP HET mice) reduces APP expression in the Fmr1 KO to wild type (WT) levels and rescues audiogenic-induced seizures (AGS), the percentage of mature spines, open field and marble burying behavioral phenotypes, and mGluR-LTD (Westmark et al., 2011). APP and metabolite levels are altered in Fmr1 KO mice and FXS patients (Sokol et al., 2006;Westmark et al., 2011;Erickson et al., 2014;Pasciuto et al., 2015;Ray et al., 2016). Thus, APP is a potential therapeutic target as well as bloodbased biomarker for FXS (Berry-Kravis et al., 2013;Westmark et al., 2016), and it is of interest to determine the effect(s) of APP levels on additional disease phenotypes. Herein, we ascertain the effects of App knockdown on hyperexcitability in the Fmr1 KO mouse.

GENETIC REDUCTION OF APP RESCUES HYPEREXCITABILITY IN Fmr1 KO MICE
The psychiatric phenotype of FXS includes hyperexcitability traits such as tactile defensiveness, attention deficits, hyperactivity, and hyperarousal to sensory stimulation (Tranfaglia, 2011). There is high comorbidity of epilepsy in FXS with electroencephalogram (EEG) patterns most often consisting of a centrotemporal spike pattern resembling Benign Focal Epilepsy of Childhood (BFEC) (Berry-Kravis, 2002). Hyperexcitability can be modeled in the Fmr1 KO mice both in vivo and in vitro (brain slices). In vivo, the Fmr1 KO mice are susceptible to AGS (Chen and Toth, 2001). In the AGS model, mice are exposed to 110 dB siren, which elicits out-of-control (wild) running and jumping followed by convulsive seizures and often death. There is substantial evidence that dysregulated APP expression alters seizure propensity. AGS are exacerbated by overexpression of APP in the Fmr1 KO mouse (FRAXAD mice) and partially rescued by reduced expression of APP in Fmr1 KO /APP HET mice (Westmark et al., 2010(Westmark et al., , 2011. Alzheimer's disease (Tg2576) and Down syndrome (Ts65Dn) mice, which overexpress human and mouse APP respectively, are highly susceptible to AGS (Westmark et al., 2010). Numerous mouse models that express altered APP or metabolite levels exhibit elevated rates of spontaneous or provoked seizures (Moechars et al., 1996;Steinbach et al., 1998;Del Vecchio et al., 2004;Lalonde et al., 2005;Palop et al., 2007;Kobayashi et al., 2008;Westmark et al., 2008;Minkeviciene et al., 2009;Ziyatdinova et al., 2011;Sanchez et al., 2012) while suppression of transgenic APP in Alzheimer's disease mice during postnatal development delays the onset of EEG abnormalities (Born et al., 2014). In brain slices, hyperexcitability can be measured by recording UP states and epileptiform discharges. UP states are short periods of local network activity that generate a steady-state level of depolarization and synchronous firing among groups of neighboring neurons (Gibson et al., 2008). Fmr1 KO mice exhibit an increased duration of the UP state, consistent with network hyperexcitability (Gibson et al., 2008;Goncalves et al., 2013). Specifically, spontaneously occurring UP states are 38-67% longer in Fmr1 KO than in WT slices (Hays et al., 2011). Deletion of Fmr1 selectively in excitatory neurons mimics the prolonged UP states whereas knockdown of mGluR 5 rescues the hyperexcitability in the Fmr1 KO with no effect in WT (Hays et al., 2011). To determine if hyperexcitability was rescued in Fmr1 KO mice by knockdown of App, we recorded UP states in Fmr1 KO /App HET mice and littermate controls per previously described methods (Gibson et al., 2008). Briefly, Fmr1 HET /App HET females were bred with App HET males to generate WT, Fmr1 KO , App HET and Fmr1 KO /App HET male littermates. Thalamocortical slices (400 µm) from postnatal day 24-28 (P24-P28) males were transected parallel to the pia mater to remove the thalamus and midbrain, and spontaneously generated UP states were recorded in layer 4 of the somatosensory cortex. The increased duration of the UP states observed in the Fmr1 KO was completely rescued in Fmr1 KO /APP HET mice (Figures 1A,B) where UP state duration decreased from 931 ± 55 milliseconds (ms) in Fmr1 KO to 597 ± 30 ms in Fmr1 KO /APP HET , (p < 0.001). UP state duration was not significantly different between APP HET and WT slices suggesting that rescue was not a consequence of a general reduction in excitability due to lower APP levels. histogram depicting a significant increase in UP state activity in Fmr1 KO slices compared to WT, which was completely rescued in the Fmr1 KO /App HET . Error bars represent SEM. Horizontal bars denote statistically different levels by one-way ANOVA and Bonferroni's multiple comparison test (P < 0.0001). DHPG-induced prolonged epileptiform discharges were assessed in hippocampal slices from Fmr1 KO and Fmr1 KO /App HET male mice (n = 6 mice per cohort). The recordings were continuous for 3 or more hours in a single slice per animal. (C) Summary frequency histogram of synchronized epileptiform discharges from Fmr1 KO and Fmr1 KO /App HET slices in the presence of DHPG (60 min) and after DHPG washout at the indicated times up to 2 h. The mean durations of epileptiform discharges in Fmr1 KO /App HET slices at 30, 60, 90, and 120 min after DHPG washout are significantly shorter than those in Fmr1 KO for all times tested (P < 0.001).
Hyperexcitability can also be evaluated in slices of the CA3 region of the hippocampus in Fmr1 KO mice. Prolonged epileptic bursts can be induced by group 1 mGluR agonists in both WT and Fmr1 KO mice and with a GABAergic antagonist only in Fmr1 KO (Chuang et al., 2005;Zhong et al., 2009). In WT slices, DHPG elicits short (∼500 ms) synchronized discharges that gradually extend to reach an average duration of 4.4 ± 0.14 s at 60 min; and in untreated Fmr1 KO slices, bicuculline elicits short <1 ms synchronized discharges that progressively increase in duration over 60 min (average duration 2.3 ïĆś 0.13 s) (Osterweil et al., 2013). These prolonged epileptiform discharges resemble the ictal discharges observed in the CA3 region in epilepsy (Merlin and Wong, 1997;Wong et al., 2004). The number and duration of ictal-like discharges were assessed by intracellular CA3 recordings in juvenile Fmr1 KO and Fmr1 KO /App HET slices in the presence of DHPG (60 min) and after DHPG washout for up to 2 h as previously described (Chuang et al., 2005) ( Figure 1C, Supplementary Figure 1). In the presence of DHPG, a distinct population of ictal-like discharges (burst duration > 1500 ms) occurred in both Fmr1 KO and Fmr1 KO /App HET slices. After DHPG washout, the ictal-like discharges remained distinct for the duration of the recording (up to 2 h post-DHPG washout) in the Fmr1 KO , but not in the Fmr1 KO /App HET slices. Thus, a major difference between Fmr1 KO and Fmr1 KO /App HET slices is that while ictal-like discharges were transiently expressed in both genotypes, they were not maintained in the Fmr1 KO /App HET upon termination of receptor stimulation. The seizure activity modeled in the hippocampal slice paradigm is congruent with the AGS phenotype observed in Fmr1 KO /App HET mice where wild running and seizures are attenuated but not completely rescued to WT levels (Westmark et al., 2011). The two critical components of plasticity include the initiating factors required for induction of the modification and the downstream effectors that maintain expression of the enhanced response (Bianchi et al., 2012). Our data suggest that genetic reduction of App in the Fmr1 KO background does not prevent the induction of seizure activity, but can attenuate progression; thus, APP appears to be a downstream effector that maintains hyperexcitability in the context of the Fmr1 KO .
The complete rescue of hyperexcitability in the neocortex compared to the partial rescue in the hippocampus in the Fmr1 KO /App HET mice is in accord with studies in immature mice demonstrating that the hippocampus has a lower seizure threshold compared to neocortex (Abdelmalik et al., 2005). This could be due differential expression and/or activity of group 1 mGluRs (mGluR 1 and mGluR 5 ) in the respective neurons under study. In fast spiking inhibitory neurons (neocortical slice model), mGluR 1 is more highly expressed than mGluR 5 (Sun et al., 2009); however reduced expression of mGluR 5 or APP in the Fmr1 KO completely rescues neocortical hyperexcitability whereas UP states are still longer in the Fmr1 KO after treatment with the mGluR 1 inhibitor LY367385 (Hays et al., 2011). These data suggest that mGluR 5 is the critical group 1 mGluR that modulates Fmr1-dependent hyperexcitability in the neocortex. Alternatively, in CA3 hippocampal neurons, both group 1 mGluR subtypes are involved in the induction and maintenance of mGluR-mediated bursts, but mGluR 5 plays a greater role in the induction and mGluR 1 in the maintenance of the prolonged epileptic bursts (Merlin, 2002). As burst duration but not induction are rescued in the Fmr1 KO /APP HET , these data suggest that the hyperexcitability elicited by elevated APP expression in the Fmr1 KO CA3 region is dependent on mGluR 1 .
Synaptic dysfunction occurs when the appropriate balance of excitation and inhibition (E/I) in neural circuits is not maintained (Gatto and Broadie, 2010). The absence of FMRP during postnatal development results in an E/I imbalance dominated by excitation. Our results demonstrate that E/I balance is predominantly restored when APP expression is reduced to WT levels in the Fmr1 KO . Thus, APP plays a critical role in modulating excitability. The other half of E/I balance is the inhibitory feedback on circuits. FMRP normally binds to multiple GABA A R mRNAs, and their expression is decreased in juvenile Fmr1 KO (Braat et al., 2015) resulting in delay of the developmental GABA switch in Fmr1 KO (He et al., 2014). Selective deletion of Fmr1 in inhibitory neurons has no effect on prolonged UP states suggesting that impaired GABA A R signaling in FXS does not account for increased hyperexcitability in the neocortex (Hays et al., 2011). Conversely, a competitive antagonist of GABA A R, bicuculline, elicits epileptiform discharges in the CA3 region of the hippocampus (Osterweil et al., 2013). These findings suggest that inhibitory feedback is differentially regulated in the neocortex and hippocampus in Fmr1 KO . Overall, the neocortical hyperexcitability and hippocampal epileptiform discharge slice models share the features of prolonged activity states and dependence on mGluR 5 , FMRP, and APP, but differ in induction mode (neocortical slices exhibit baseline excitation vs. hippocampal slices require pharmacological stimulation), inhibitory feedback (hippocampal slices are dependent of GABA A R), and protein synthesis requirements (CA3 bursts require extracellular signal-regulated kinase (ERK)1/2 activation and new protein synthesis) (Zhao et al., 2004;Chuang et al., 2005;Hays et al., 2011).

A MODEL FOR AN APP-INDUCED SHORT CIRCUIT IN FRAGILE X
Regarding possible mechanisms for APP-mediated hyperexcitability, (Westmark, 2013) APP or a metabolite could interfere with cell surface receptor activation. For example, Aβ oligomers cause redistribution of mGluR 5 to synapses (Renner et al., 2010) and trigger multiple distinct signaling events through mGluR 5 /prion protein complexes (Um et al., 2013;Hu et al., 2014;Haas and Strittmatter, 2016). In neurons that overexpress APP, Aβ depresses excitatory synaptic transmission (Kamenetz et al., 2003). In Fmr1 KO mice, Aβ levels are elevated in older mice but reduced in juvenile mice compared to WT controls (Westmark et al., 2011;Pasciuto et al., 2015). Thus, increased α-secretase and/or decreased BACE1 processing during postnatal development could result in decreased Aβ levels and increased synaptic transmission (Jin and Warren, 2000) Altered APP expression could affect scaffolding protein interactions at the postsynaptic density. For example, APP co-immunoprecipitates with Homer2 and Homer3 (Parisiadou et al., 2008). These scaffolding proteins inhibit APP processing, reduce cell surface APP expression, and prevent maturation of BACE1 (Parisiadou et al., 2008). Uncoupled Homer1-mGluR 5 interactions underlie Fmr1 KO phenotypes, and genetic deletion of Homer1a rescues prolonged UP states in Fmr1 KO mice similar to the complete rescue observed herein in the Fmr1 KO /APP HET mice (Ronesi et al., 2012). APP does not co-immunoprecipitate with Homer1 (Parisiadou et al., 2008); however, Aβ induces disassembly of Homer1b and Shank1 clusters (Roselli et al., 2009). (Westmark and Malter, 2012) APP or metabolites could alter the activity of intracellular signaling pathways such as ERK and mTOR (Young et al., 2009;Ma et al., 2010;Caccamo et al., 2011;Chasseigneaux et al., 2011;Pasciuto et al., 2015). Both of these pathways play pivotal roles in FXS pathology (Osterweil et al., 2010;Hoeffer et al., 2012). And Westmark and Malter (2007) APP metabolites could function in feedback loops to regulate the aforementioned pathways or even the transcription of the APP and APP processing enzymes. Aβ binds to the promoter regions of the APP and BACE1 genes and may function as a transcription factor to regulate its own production and/or processing (Bailey et al., 2011). Thus, there are numerous molecular junctures where altered expression of APP or metabolites could interfere with synaptic function and lead to a hyperexcitable circuit.
Overall, these data suggest a model whereby mGluR 5 inhibitors act as a circuit breaker, FMRP as an automatic transfer switch and APP as a rheostat in a circuit that controls hyperexcitability (Figure 2). The mGluR 5 circuit breaker: Genetic reduction of mGluR 5 in the Fmr1 KO mouse rescues plasticity (ocular dominance plasticity, neocortical hyperexcitability), dendritic spines (density on cortical pyramidal neurons), protein synthesis, behavior (inhibitory avoidance extinction), and AGS (Dolen et al., 2007;Hays et al., 2011). Pharmaceutical inhibition of mGluR 5 likewise rescues numerous Fmr1 KO phenotypes (Michalon et al., 2012(Michalon et al., , 2014. Thus, inhibiting mGluR 5 appears to break a circuit that mediates hyperexcitability in the Fmr1 KO mouse. The FMRP automatic transfer switch: mGluR 5 activation causes a rapid dephosphorylation of FMRP, which permits protein synthesis (Ceman et al., 2003;Narayanan et al., 2007), as well as a biphasic change in FMRP levels (initial decrease followed by increase) (Zhao et al., 2011). Thus, FMRP appears to function as an automatic transfer switch downstream of mGluR 5 to control protein synthesis in response to receptor activation. In FXS models, loss of the FMRP switch that modulates mGluR 5 signaling permits a constitutively-on circuit. The APP rheostat: Born and colleagues demonstrated that juvenile overexpression of APP contributes to sharp wave EEG discharges in APP transgenic mice, and proposed that APP expression functions as a rheostat that regulates synaptic balance in the brain (Born et al., 2014). We have observed that both over-and underexpression of APP increases seizure propensity in juvenile Fmr1 KO mice suggesting that tight regulation of this protein may be necessary to mitigate hyperexcitability in FXS (Westmark et al., 2010(Westmark et al., , 2011. Genetic reduction of APP in Fmr1 KO mice rescues plasticity (mGluR-LTD, neocortical hyperexcitability, epileptiform discharge duration but not induction), dendritic spines (percent mature spines but not dendritic spine length), protein synthesis, behavior (open field, marble burying), and AGS (Westmark et al., 2011;Pasciuto et al., 2015). The partial rescue of dendritic spines and epileptiform discharges in the Fmr1 KO /APP HET suggests that APP is necessary but not sufficient to maintain synaptic homeostasis. Thus, in the context of WT mice, an APP variable resistor is capable of maintaining an appropriate E/I balance, but in Fmr1 KO and some APP transgenic mice, excess APP appears to cause a short circuit through overload of the APP rheostat resulting in hyperexcitability. Likewise, complete loss of APP would bypass the APP rheostat. Fmr1 KO /App KO mice exhibit an extremely strong AGS phenotype (97%, n = 36 mice) (Westmark et al., 2013a), which is not observed in App KO mice (11%, n = 36 mice). These data suggest that exacerbated hyperexcitability is a result of the combined loss of both FMRP and APP.
APP and metabolites play key roles in regulating synaptic activity with both Aβ and sAPPα implicated in positive feedback loops that facilitate mGluR 5 signaling (Casley et al., 2009;Renner et al., 2010;Ferreira and Klein, 2011;Westmark et al., 2011Westmark et al., , 2013bPasciuto et al., 2015). Thus, the APP rheostat may provide a graded response to mGluR 5 activation through feedback loops involving amyloidogenic and non-amyloidogenic secretase processing. We found that AGS are attenuated in Fmr1 KO mice with BACE1 inhibitor treatment (Supplementary Figure 2). Prox and colleagues found that seizures are increased in the ADAM10 conditional knockout mouse (loss of αsecretase processing) (Prox et al., 2013). The effect of sAPPα overexpression on hyperexcitability, which could be studied in TgAPPα (Bailey et al., 2013) and TgAPPα/Fmr1 KO mice, remains to be determined. Thus, multiple APP fragments may play roles in hyperexcitability and seizure susceptibility. A caveat to this model is that over-expression of APP alone is not sufficient to increase seizure propensity in either WT or Fmr1 KO mice. We tested seizures in two alternative Alzheimer's disease mouse models, R1.40 and J20, which exhibit elevated APP expression (Lamb et al., 1997;Mucke et al., 2000). Neither strain exhibited a strong AGS phenotype (Supplementary Figures 3, 4) in contrast to Tg2576 and Ts65Dn (Westmark et al., 2010). A genetic cross of J20 with Fmr1 KO mice that produced mice over-expressing human APP in the context of the Fmr1 KO background did not result in exacerbated AGS rates in comparison to Fmr1 KO unlike the FRAXAD mice (cross of Tg2576 with Fmr1 KO ) (Westmark et al., 2010). The inclusion of flanking sequences in the transgenic constructs used for the R1.40 and J20 mice are expected to affect posttranscriptional regulation of the APP gene, which could alter the temporal and spatial expression of APP and metabolites and thus their contribution to seizure threshold. Of note, Fmr1 HET /J20 female mice exhibited a 50% wild running rate, which was significantly higher than WT, Fmr1 HET and J20 controls, supporting the assertion that APP works in synergy with FMRP to regulate hyperexcitability (Supplementary Figure 4). This synergistic effect is also observed in mGluR-LTD studies where loss of FMRP and APP modulate synaptic transmission in opposite directions (Westmark et al., 2011). The Fmr1 KO /APP HET mice used herein were a constitutive App knockdown. It remains to be determined how conditional knockdown of App during development affects Fmr1 phenotypes. FIGURE 2 | Model for an APP-induced short circuit in FXS. APP acts as a rheostat (i.e., variable resistor, dimmer switch) in a circuit where mGluR 5 inhibitors are a circuit breaker and FMRP is an automatic transfer switch that regulate neuronal excitability. The FMRP switch is dependent on a rapid dephosphorylation reaction in response to mGluR 5 activation. In the absence of FMRP, the circuit is constitutively on. In the presence of mGluR 5 inhibitors, the circuit is shut down. The downstream APP circuitry appears to be wired differently dependent on brain region. In the neocortex, knockdown of individual proteins including mGluR 5 , Homer and APP completely rescues excitability levels suggesting that these components are arranged in a parallel circuit whereby there is more than one continuous signaling pathway between mGluR 5 activation and excitability output. Rescue of any one of the parallel components is sufficient to restore synaptic homeostasis. In the hippocampus, ictal burst duration, but not induction, is rescued in Fmr1 KO /App HET slices in response to DHPG treatment. This incomplete rescue suggests that APP and FMRP are wired in series downstream of mGluR 5 , and that the APP rheostat is overloaded in the absence of FMRP resulting in a short circuit. This model has important implications for therapeutic development and suggests that APP should be considered as a drug target for FXS as part of a multi-drug therapeutic strategy.

RELEVANCE TO THERAPEUTIC DEVELOPMENT
All major Fmr1 KO phenotypes can be corrected by inhibition or knockdown of mGluR 5 in mice; however, neural circuitry is likely more complicated in humans and it may be necessary to employ pharmaceutical cocktails for disease treatment. Drugs under study for FXS such as acamprosate, AFQ056, donepezil, ganaxolone, lithium, lovastatin, memantine, minocycline and sertraline exhibit on-and/or off-site effects that are expected to modulate APP, Aβ, BACE1, and/or ADAM10 (Westmark et al., 2013b). Targeting APP and metabolites in FXS may allow fine tuning of excitability levels as part of a multi-drug therapeutic approach. Both amyloidogenic and non-amyloidogenic therapies have been proposed to treat FXS (Westmark et al., 2013b;Pasciuto et al., 2015). Both amyloidogenic (Aβ 1−42 ) and non-amyloidogenic (sAPPα) metabolites of APP stimulate phosphorylation of ERK and modulate synthesis of multiple synaptic proteins predicted to be regulated through mGluR 5 /FMRP and to contribute to altered synaptic plasticity (Westmark et al., 2011;Pasciuto et al., 2015). Thus, it may be necessary to simultaneously modulate both α-and β-secretase processing to attain homeostatic levels of APP metabolites and rescue hyperexcitability in FXS.

ACKNOWLEDGMENTS
The authors thank the Research Animal Resources Centers (RARC) staffs at the University of Wisconsin, the State University of New York Downstate Medical Center and the University of Texas Southwestern for expert animal care and assistance with transport between facilities. All mouse procedures were performed in accordance with NIH guidelines and approved University of Wisconsin-Madison, State University of New York Downstate Medical Center and University of Texas Southwestern Medical Center animal care protocols administered through their RARC.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnmol. 2016.00147/full#supplementary-material