Functional characterization of the AMPA receptor interacting proteins Shisa6 and
Shisa7
Schmitz, L.J.M.
2018
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citation for published version (APA)
Schmitz, L. J. M. (2018). Functional characterization of the AMPA receptor interacting proteins Shisa6 and
Shisa7.
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Chapter
7
7
Summary and scope of the discussion
AMPA receptor (AMPAR) complexes mostly consist of the pore-forming subunits decorated with various associated proteins151. Studies of transmembrane-type AMPAR interactors, the so-called auxiliary subunits, revealed that these associated proteins can modulate AMPAR cell surface expression, intracellular traffi cking, and channel
properties152,261. Th e Shisa protein family forms a distinct group of transmembrane
AMPAR proteins177,179, of which Shisa9/CKAMP44 was the fi rst one intensively
studied165,170,180. Th e aim of this thesis was to elucidate the function of two uncharacterized
Shisa family members, Shisa6 and -7, by making use of newly generated knockout (KO) mice.
In chapter 2 and 3, we identifi ed Shisa6 and -7 as components of native hippocampal AMPAR complexes and we explored the expression and interactome of these proteins. For functional characterization, the eff ects of Shisa6 and -7 on AMPAR kinetics and transmission were studied in HEK293 cells and in the neuronal context of hippocampal pyramidal cells in acute brain slices. In addition to basal AMPAR function, eff ects of Shisa proteins on short-term and long-term hippocampal plasticity were measured in hippocampal slices. Subsequently, the functional implication of Shisa6 and -7 was studied at the behavioral level in chapter 4. To this end Shisa6, -7 and -6/7 (d)KO mice were studied using a series of cognitive tests in a behavioral test battery focused on hippocampus-dependent learning and memory. In chapter 5, we studied the eff ects of Shisa deletion on dendritic development and synapse formation in vitro to evaluate neuronal phenotypes. Finally, in chapter 6, we aimed to explore changes in the hippocampal synaptic proteome of Shisa6, -7 and -6/7 (d)KO mice, using FASP/SWATH mass spectrometry analysis, and we focused on the validation of protein regulation detected by this novel proteomics approach.
In this chapter, I will discuss how Shisa6 and -7 might aff ect AMPAR gating, plasticity and behavior in light of the established characteristics of Shisa9 and other auxiliary subunits. Th e comparison of these mutant phenotypes at the physiological and behavioral level stresses the unique role of Shisa6 and -7. Finally, I will discuss possibilities for future research that might further the understanding of the shared and diff erential functions of the Shisa proteins.
Shisa proteins modulate AMPAR kine� c proper� es
One of the key features of AMPAR auxiliary subunits is their ability to modify the
biophysical properties of the AMPAR152,349. In chapter 2 and 3 we showed that the
7
up and Shisa6 slows desensitization and only Shisa6 modulates deactivation rates. Ex vivo, in a brain slice preparation, the deactivation time of miniature excitatory postsynaptic currents (mEPCS) is shorter in both the Shisa6 and -7 KO. Th ese fi ndings suggest that modulation of AMPAR kinetics occurs via direct interaction of the AMPAR with Shisa6, not with Shisa7. Th e modulatory role of Shisa7 on the deactivation time in the hippocampus likely depends on the neuronal, synaptic context of the AMPARs. AMPAR modulation might be mediated by other auxiliary subunits after Shisa7 deletion. Th ere is currently no experimental evidence to support this view. Furthermore, as deactivationkinetics is also determined by AMPAR subunit composition26,184, a shift towards more
GluA2/3-containing receptors in the hippocampus might explain the reduction in the deactivation time of receptors in the Shisa7 KO. Th is shift could be specifi c for certain synaptic subdomains, and current techniques used would not reveal this.
In contrast with Shisa6, the eff ect of Shisa7 on AMPAR kinetics in HEK293 cells was only investigated in GluA1 monomers. Whether deactivation and desensitization rate of GluA1/2 heteromers are aff ected by Shisa7 remains to be determined. In a recent HEK293 cell study, no eff ect of Shisa7 on GluA1 and GluA2 monomer kinetics was
found179. In contrast with our data, the recovery of desensitization was not modulated
TARP Shisa9 Shisa9 Neurons HEK293/oocyte Shisa7 Shisa9 Shisa9 Recovery of desensitization Shisa7 Shisa6 Shisa9 Shisa9 Shisa7 Desensitization Shisa6 TARPTARP TARPTARPTARPTARPTARPTARPTARP TARP TARPTARP Deactivation Shisa6 Shisa6 Slower Faster
7
by Shisa7 expression, suggesting that in this study the expression of Shisa7 associated with the AMPAR at the cell surface might have been insuffi cient to modulate AMPAR function. Alternatively, the diff erences observed might be ascribed to the use of diff erent alternatively spliced forms of Shisa7. Of note is that in all our studies (in chapters 2 and 3), the long exon3/4-containing Shisa forms were used, in contrast to use of the short
forms in the study by Farrow et al.179.
In addition to a change in AMPAR composition, the AMPAR-associated proteins might modulate GluA1/2 and GluA2/3 receptors diff erentially, as previously suggested
for CNIH2/3 in hippocampal neurons167. To dissect whether potential subunit-specifi c
interactions in the hippocampal AMPAR complexes in the Shisa7 KO mouse induce faster deactivation, fi rst the ratio of GluA1/2 vs. GluA2/3 complexes should be determined. In chapter 2 and chapter 3 we have identifi ed GluA1–3, non-discriminatively, as interactors of both Shisa proteins and we revealed that general synaptic expression of GluA1 and GluA2 and the mEPSC amplitude in the hippocampus was normal in the Shisa6 and -7 KO mice. On the other hand, in chapter 6 a minor, non-signifi cant, increase in synaptic GluA3 expression was detected in the Shisa6 KO, suggesting there might be subtle changes in the composition of the receptors that mediate synaptic signaling.
Shisa6 and Shisa7 do not control basal AMPAR expression and
transmission
Besides direct modulation of the AMPAR biophysical properties, several AMPAR auxiliary subunits have been implicated in synaptic targeting of the AMPAR by modulating
traffi cking, surface mobility and stabilization of receptors at the postsynapse173. In the
hippocampus, deletion of TARP γ-8 reduces surface expression of AMPARs, in which
it mainly aff ects the extrasynaptic pool of receptors158,172. Synaptically, the interaction
of TARP with the postsynaptic scaff olding protein PSD-95 induces trapping of the
AMPAR via interaction with the PDZ domain92,350.
In this thesis, we studied the eff ect of Shisa6 and -7 on synaptic expression of the AMPAR by measuring CA1 pyramidal cell mEPSC amplitudes and protein expression in biochemically isolated synaptic membrane fractions and cultured hippocampal neurons. Together, these data suggest that under basal conditions these two Shisa family members do not regulate synaptic targeting of the receptor. In contrast, Shisa9 deletion and overexpression in dentate gyrus granule cells shows that Shisa9 positively regulates
synaptic AMPAR expression170, thereby enhancing mEPSC peak amplitudes.
AMPAR-mediated signaling controls growth and arborization of dendrites138,147,307
and spines316. In rat hippocampal organotypic cultures, it has been shown that inhibition
of AMPAR signaling induces a reduction in dendrite growth and complexity148. In
7
dentate gyrus stimulates development of dendrites and spines170, whereas Shisa9 deletion
reduces spine density. Th is suggests that by facilitation of synaptic AMPAR signaling, Shisa9 contributes to development of synapses and dendritic tree complexity. Similarly, mEPSC recordings and spine density analysis after overexpression and knockdown of TARP γ-8 revealed that its presence increases synaptic AMPAR transmission and
the number of excitatory synapses170. Interestingly, although there is no evidence
for reduction in AMPAR expression and mEPSC amplitude current in absence of Shisa6 and -7, dendritic outgrowth is reduced in Shisa6 KO, and enhanced in Shisa7 KO, dissociated hippocampal neuronal cultures. On the other hand, gross dendritic morphology in the Shisa KO mice is not aff ected, suggesting that activity-independent mechanisms in the hippocampus may compensate for the disturbed dendritic growth process. Such mechanism might involve intact brain structure, extracellular molecular
signals, and glia, which guide neuronal development133,323,351. In addition, fi ne dendritic
morphology has not been examined in vivo in Shisa6 and -7 KO mice, and might match the altered morphology of KO dissociated hippocampal cultures. Th e Shisa7 KO mice display a slight reduction in basal and apical spines in the CA1 pyramidal cells. Strikingly, mEPSC frequencies are not aff ected in the Shisa7 KO, suggesting that the number of AMPAR containing synapses is not diff erent. A possible explanation could be that deletion of Shisa7 specifi cally reduces the number of silent synapses, which lack
surface expression of the AMPAR352. In order to test whether there is a shift in the
proportion of AMPAR-containing synapses versus total number of synapses, AMPAR and NMDAR EPSCs should be measured.
Although our data suggest that Shisa6 and -7 do not modulate AMPAR surface expression under basal conditions, interestingly, diff usion of the AMPAR is regulated
by Shisa6178. Using quantum dot labeling of specifi c antibodies directed against GluA2
we observed that the membrane diff usion rate of the receptor is reduced by Shisa6
at synaptic and extrasynaptic sites178. Shisa6 switches AMPAR between a mobile and
immobile state, being more immobile in the postsynaptic nanodomains204,353. As the
reduction in mobility of the AMPAR by Shisa6 is mediated by the C-terminal PDZ domain, Shisa6 probably stimulates trapping in nanodomains by interaction with PSD-95. Remarkably, extrasynaptic diff usion of the receptor is also modulated by Shisa6. Th is eff ect might be caused by interactions with extrasynaptic scaff olds, possibly as a result of artifi cially high levels of Shisa6 due to overexpression. Whether Shisa7 also modulates diff usion of the receptor is hitherto unknown and remains to be investigated.
Shisa proteins modulate synap� c plas� city
Short-term plasti city
7
kinetics aff ects short-term synaptic plasticity. Th e HEK293 cell data on recovery of desensitization suggest that in KO animals receptors return faster to a resting state, allowing them to continue fi ring upon repetitive stimulation. However, high-frequent repetitive stimulation of the Schaff er collaterals induced synaptic depression in the Shisa6 KO, but not in WT animals. Th erefore, it is likely that by slowing the desensitization rate the presence of Shisa6 in the synaptic AMPAR complex is able to maintain fi ring rates in WT neurons. Corresponding to the absence of a direct eff ect of Shisa7 on desensitization, short-term plasticity is not modulated by Shisa7. Th e eff ect of Shisa9 on short-term synaptic plasticity has been studied by overexpression in the CA1 region, in which its endogenous expression is non-detectable. In the CA1 Shisa9 reduces the
paired-pulse ratio of AMPARs165. Slowed recovery of desensitization is the putative
mechanism for this reduced paired-pulse facilitation of Shisa9. Similar to Shisa9, both Shisa6 and -7 slow recovery of desensitization in HEK293 cells. However, paired-pulse facilitation is normal in these KO mice. Because endogenous Shisa6 and -7 expression levels are high in the CA1 region, the eff ect of overexpression of Shisa9 and deletion of Shisa6 or -7 might not be comparable.
Short-term plasticity is not only modulated by the gating kinetics of AMPARs, but also by their mobility. In previous studies, mobility of receptors has been manipulated by digestion of the extracellular matrix to increase mobility, or by increasing local
calcium levels and cross-linking of AMPARs to reduce mobility62,354. Without aff ecting
sensitization or desensitization kinetics, these interventions alter paired-pulse ratios, suggesting that exchange of receptors prevents synaptic depression. Furthermore,
desensitized receptors are more mobile than resting receptors63. For TARP γ-2 it has
been suggested that when glutamate binds to the AMPAR and receptors desensitize,
TARP-AMPAR interactions become weaker and the receptors become more mobile63.
Shisa6 reduces the desensitization rate of the AMPAR. However, when the receptor desensitizes, it can be replaced with resting AMPARs. Such replacement would overcome the eff ect of reduced recovery from desensitization induced by Shisa6 to maintain fi ring rates
LTP inducti on
Despite the minor eff ect of Shisa7 on basal AMPAR function and short-term plasticity, in chapter 3 we concluded that long-term synaptic plasticity is severely disturbed in the absence of Shisa7. A paired theta-burst stimulation protocol was applied to induce LTP in the Schaff er collaterals, demonstrating a reduction in EPSC slope and amplitude both in the initial and maintenance phase of LTP.
LTP induction depends on activation of kinases, including Ca2+
/calmodulin-dependent protein kinase II (CaMKII) and calcium/phospholipid-/calmodulin-dependent protein kinase (PKC), that phosphorylate the AMPAR and induce potentiation
7
Furthermore, CaMKII activation induces translocation of the kinase and binding to the NMDAR. As such, CaMKII is positioned in close proximity to the AMPAR, PSD-95 and TARP. During LTP, AMPARs move towards the PSD via lateral diff usion and get captured at the postsynaptic site. TARP γ-2 is involved in trapping the AMPAR, whichis mediated by CaMKII-induced phosphorylation of TARP γ-283. TARP γ-8, which is
more abundant in the hippocampus, also plays a crucial role in synaptic plasticity172,226,
as TARP γ-8 gene deletion attenuates LTP induction and maintenance172. Remarkably,
TARP γ-8∆4/∆4 knock-in mice lacking the C-terminal PDZ ligand have unaltered LTP,
indicating that LTP is not dependent on protein-protein interactions of the C-terminal
PDZ domain173. Rather, a knock-in approach deleting the CaMKII sites of TARP γ-8
revealed that CaMKII phosphorylation of TARP γ-8 is required for enhanced
AMPAR-mediated transmission during LTP226.
Th ere are several possible mechanisms that might explain an initial defi cit in synaptic potentiation, as observed in Shisa7 KO mice. First, surface mobility of the AMPAR might be controlled by Shisa7, although this has not been experimentally tested. An overexpression study is required to explore whether Shisa7 reduces AMPAR surface diff usion via the C-terminal PDZ domain, as was observed for Shisa6. Such eff ect on AMPAR mobility, suggests that AMPAR diff usion in the PSD of the Shisa7 KO is enhanced and receptor capturing is reduced.
Second, the signal transduction cascade required for AMPAR trapping, likely activated by postsynaptic depolarization and a local rise in calcium, might be disturbed. As Shisa7 interacts with postsynaptic scaff olds and the AMPAR, it might act as scaff old protein, contributing to proper positioning of the AMPAR in nanodomains consisting of PSD-95 and the NMDAR. Identifi cation of such nanodomain residing proteins in Shisa7 complexes might further support this scaff olding role of Shisa7.
Th ird, Shisa proteins might be involved in phosphorylation-induced trapping mediated by postsynaptic kinases, similar to the phosphorylation of TARP γ-8 by
CaMKII226. To investigate this hypothesis the putative phosphorylation sites at
Shisa7 should be identifi ed to characterize and manipulate phosphorylation after LTP induction. Although basal AMPAR transmission is normal in the Shisa7 KO mouse, a defi cit in AMPAR trapping might be more pronounced during potentiation of the synapse.
Finally, as Shisa7 directly interacts with the AMPAR, it might modulate AMPAR
phosphorylation, e.g. at Ser-83179,80, critical for the threshold of LTP induction357.
7
LTP maintenance
After dissecting the role of Shisa7 in early LTP induction processes, the question remains whether reduced LTP in the Shisa7 KO is exclusively dependent on disturbed initiation of the molecular cascade, or also relies on reduced recruitment of AMPARs to the synapse during the LTP maintenance phase. In this phase, AMPARs remain phosphorylated at
the Ser-831 residue up to 1 h after induction of LTP101,358. In addition, by exocytosis
of AMPAR- containing vesicles extrasynaptic receptor pools are replenished which allows diff usion and trapping of newly recruited AMPARs in synapses to maintain the
potentiated state93–95.
Th e consensus that C-terminal interactions of the GluA1-subunit specifi cally
mediate AMPAR traffi cking and capturing in synapses during LTP359,360, has been
challenged by a molecular replacement study361. Th e authors demonstrated that LTP
can still be induced when AMPARs are either replaced with a construct lacking the C-terminal GluA1 tail, or fully replaced with kainate receptors (KARs). Although AMPARs and regulating proteins like TARP and postsynaptic kinases might control LTP, these experiments suggest that independent machinery can account for increased synaptic transmission. Th erefore, this study supports the crucial role of structural
remodeling of the actin cytoskeleton and molecular reorganization in LTP113,362.
Interestingly, actin-associated proteins can regulate AMPAR traffi cking by facilitating exocytosis and endocytosis of the receptor required for LTP maintenance
(for review see 363). Although synaptic proteome changes detected by SWATH analysis
in chapter 6 require careful interpretation due to the lack of independent validation, regulation of actin-associated proteins was observed in Shisa6, -7 and -6/7 (d)KO mice. Th e Shisa6/7 dKO showed downregulation of actin-associated proteins, e.g. the
Arp2/3 complex subunits. In addition, the F-actin stabilizing protein Drebrin345, which
modulates synaptic plasticity and memory364, was diff erentially regulated between Shisa6
and -7 KO mice. Th ese basal changes in the synaptic proteome might contribute to disturbed stimulus-induced potentiation of synapses, by aff ecting AMPAR traffi cking.
Monitoring of AMPAR traffi cking could provide new insights in the role of Shisa7 in LTP maintenance. In previous studies, to visualize AMPAR traffi cking and to discriminate between intracellular and surface receptors, GluA1 tagged with a
pH-sensitive form of eGFP, (GluA1-SEP) was expressed in organotypic cultures93,95.
Expression of GluA1-SEP in Shisa7 KO cultures might demonstrate whether basal and/or stimulus-induced AMPAR exocytosis and lateral diff usion are aff ected. Since Shisa7 is strongly enriched in biochemically isolated PSD fractions, the association with the AMPAR probably occurs postsynaptically. If Shisa7 modulates stimulus-induced exocytosis and traffi cking, the question lingers, which intermediate signaling cascade is controlled by Shisa7. Possibly, Shisa7 interferes with early activation of kinases, such as PKA and CaMKII, which modulate AMPAR exocytosis. Phosphorylation of the PKA
7
on the other hand, activates small GTPases and can thereby also control AMPARexocytosis71,96.
Enhanced LTP in the Shisa6 KO
Shisa6 reduces mobility of the AMPAR, in which it probably facilitates diff usional trapping of the AMPAR initially after LTP induction. Unlike the eff ect of Shisa7
deletion on long-term synaptic plasticity, Shisa6 KO mice display enhanced LTP262.
Given the role of Shisa6 in AMPAR mobility, the question arises how in absence of Shisa6, AMPAR-mediated transmission increases in stimulated synapses.
GSG1L is an auxiliary subunit that, similar to Shisa6, negatively regulates LTP,
but in contrast with Shisa6, also aff ects basal AMPAR transmission168. Extrasynaptic
AMPAR-mediated currents are enhanced in the GSG1L KO, suggesting that enlarged extrasynaptic reserve pools might facilitate synaptic recruitment of AMPARs and
stimulate LTP168. Furthermore, immunolabeling experiments in dissociated cultures
indicated that endocytosis of surface AMPARs is reduced in GSG1L KO neurons, which
might underlie increased expression of AMPARs during LTP168. As in chapter 2 and 5
we demonstrated that Shisa6 does not modulate basal synaptic and somatic
AMPAR-currents178 and synaptic AMPAR expression, it is not likely that the reserve pool of
receptors is altered in the Shisa6 KO. Since Shisa6 deletion, in contrast with GSG1L, only enhances stimulus-induced, and not basal synaptic AMPAR expression, activity-dependent AMPAR endocytosis might be aff ected by Shisa6. Th is form of AMPAR endocytosis is required for long-term depression (LTD), and involves an active process of rapid AMPAR removal, mediated via clathrin-coated pits, endocytic proteins including
dynamin and specifi c AMPAR-interacting proteins27,367–369. Initiation of
NMDAR-dependent LTD is characterized by phosphatase activity370 and dephosphorylation
of the Ser-845 PKA phosphorylation site the GluA1 subunit358,370,371. In addition to
AMPAR phosphorylation also TARP phosphorylation controls endocytosis of the AMPAR in hippocampal neurons, as dephosphorylation of TARP γ-2 is required for
NMDAR-dependent LTD91. Dephosphorylated TARP γ-2 forms a complex with the
adaptor protein AP-2, which regulates clathrin-mediated endocytosis372. Matsuda et
al.372 propose that dephosphorylation of TARP γ-2, via NMDAR-mediated activation
of calcineurin, facilitates interaction with AP-2 to induce endocytosis of the AMPAR. Several AMPAR-interacting proteins involved in AMPAR traffi cking and
endocytosis, including Protein interacting with C kinase-1 (PICK1) and GRIP1373,374,
have been characterized as Shisa family interactors with use of yeast two-hybrid (YTH)
screenings375. PICK1 is an AMPAR binding protein that facilitates endocytosis of
the receptor, thereby modulating both LTP and LTD376,377. Another Shisa6 and -7
interactor, the E3 ligase Nedd4375, is responsible for AMPAR ubiquitination, which
facilitates endocytosis and degradation of the receptor378,379. Nedd4 heterozygous mice
7
that Nedd4 plays a role in long-term synaptic plasticity via AMPAR ubiquitination and
degradation380. In chapter 6, we did not confi dently quantify PICK1 or Nedd4 with the
SWATH proteomics screening in Shisa6 and -7 KO mice. Still, loss of Shisa6 and -7 might alter the interaction of these proteins with the AMPAR and hence might lead to disturbed internalization of AMPARs during plasticity. Th e question remains whether interaction of the Shisa proteins with PICK1 and Nedd4 contributes to AMPAR endocytosis.
LTD measurements in mice carrying a Shisa6 or -7 deletion might demonstrate whether these proteins aff ect AMPAR recycling. Possibly, a unique balance between stimulus-induced AMPAR exocytosis and synaptic recruitment as well as endocytosis might underlie the distinct LTP phenotypes of Shisa KO mice. In order to dissect whether altered AMPAR endocytosis or degradation aff ect synaptic plasticity in the KO mouse, the rate of AMPAR endocytosis could be determined by labeling surface receptors and monitoring internalization and targeting of receptors to late endosomes
after application of an AMPAR agonist378,381,382. Alternatively, AMPAR expression in
biochemically isolated membrane and soluble fractions, as well as surface expression might be determined in basal and stimulated synapses.
Th e distinct eff ects of Shisa6 or -7 deletion on synaptic plasticity might be explained by a diff erential modulation of the LTP induction threshold, which could be tested by comparison of multiple stimulation protocols. Similarly, it has been shown that modulation of the phosphorylation state of the GluA1 receptors at Ser831 and
Ser845 residues changes the threshold of LTP induction357. A shift in threshold for LTP
and LTD induction has also been observed in mutant mice with a deletion of either PSD-95 or PSD-9388. Knowledge about the full profi le of LTP (and LTD) induction
and maintenance induced with diff erent stimulation protocols could reveal whether lack of Shisa proteins shifts the threshold for LTP and LTD.
Shisa proteins modulate learning & memory
Long-term adaptations of AMPAR-mediated transmission take place in the hippocampus
during consolidation of memory101,104,256. Disruption of hippocampal synaptic plasticity
has been associated with disturbed memory performance80,383. Interestingly, enhanced
hippocampal synaptic plasticity relates to enhanced learning and memory in some
mutant mice101,251,384, but to impaired memory in other385,386. As Shisa6 deletion
7
strong reduction in freezing during retrieval of short-term and long-term contextual fear memory and impaired working memory. Strikingly, whereas Shisa7 mice showed intact spatial retention memory in the Morris water maze probe session, Shisa6 KO memory performance was impaired. Together, these results suggest that although Shisa6 KO mice display enhanced LTP, this is not benefi cial for cognitive function in general. On the other hand, Shisa6 KO mice show increased performance during reversal learning in the CognitionWall, suggesting that loss of Shisa6 leads to increased cognitive fl exibility. Long-term depression (LTD) has been suggested as mechanism for depotentiation of synapses to allow storage of new memories and has been associatedwith cognitive fl exibility303,387,388. Whether Shisa6 is involved in LTD remains unknown,
but a hypothetical increase in depotentiation could mediate the enhanced fl exibility in the CognitionWall reversal phase.
Th e GSG1L KO has enhanced LTP, similar to the Shisa6 KO, but it does show
normal Morris water maze learning. It should be noted that the eff ect of GSG1L on spatial memory has been studied in KO rats. Subjecting mice to the Morris water maze induces a rise in corticosterone levels, which might have a negative impact on cognitive
function285,389. As Shisa6 mice display increased anxiety-like behavior in an open fi eld
paradigm, they might be more sensitive for stress induced by novelty and handling, which could underlie the spatial reference memory defi cits. In line with these results, the Shisa6 mice do not show impaired learning in an automated home-cage, when
experimenter-induced stress is reduced to a minimum272.
Th e TARP γ-8 KO mouse has reduced LTP172, comparable with the Shisa7
KO. Pharmacological disruption of the AMPAR-TARP γ-8 binding does only mildly aff ect the rate of acquisition in the Morris water maze, and modulates working memory, resembling the Shisa7 KO phenotype, although it is not reported whether the pharmacological intervention also reduces synaptic plasticity. Mice with mutated CaMKII phosphorylation sites in TARP γ-8 have reduced hippocampal LTP and
reduced contextual and auditory fear memory226.
Th e remarkable distinct phenotypic profi le of Shisa6 and -7 in hippocampus-dependent memory tests (Fig. 3) might be explained by the diff erence in expression
profi le. At the transcript and protein level178,252, the expression of Shisa6 and -7 overlaps
in the hippocampus. Although contextual fear conditioning and Morris water maze learning rely on the hippocampus, these memory tests require integrity of a network of
brain regions275. Spatial learning is facilitated by parallel signaling between the medial
entorhinal cortex and lateral entorhinal cortex and hippocampus275,390. Contextual
fear memories strongly depend on a neural circuit comprising of the hippocampus, striatum, medial prefrontal cortex the basolateral amygdala (BLA), and central
amygdala (CEA) (reviewed by Maren et al.270). Since Shisa7 is expressed in cortical
7
is intact, implying that mice can associate the aversive foot shock with a conditioned stimulus and in specifi c, only contextual memory is aff ected. A rescue experiment to reintroduce Shisa7 in the hippocampus of KO mice might support the putative causal role of disrupted hippocampal plasticity in impaired contextual fear memory.
Th e opposing learning and memory phenotypes in Shisa6 and -7 KO mice might either suggest a true specifi c role of each Shisa protein in the process of learning and memory or anxiety. Alternatively, it might hint to compensation, such that Shisa6 compensates for the lack of Shisa7 in tests where there is no phenotype present in Shisa7 KO mice, and vice versa. Th e fi rst hypothesis suggests that each memory defi cit observed in single KO mice resembles the memory impairment in double Shisa6/7 dKO mice. Th e second option implies that since functional compensation would not be possible in Shisa6/7 dKO mice, phenotypes would be more exaggerated compared with the single mutants. In our behavioral study, described in chapter 4, fear conditioning data is missing, and only a small sample size of the Shisa6/7 dKO group was tested. Yet, the memory defi cits observed in the Morris water maze and the novelty-induced locomotor behavior and anxiety-like behavior in the dark-light box and open fi eld indicate an additive eff ect of the double Shisa deletion. Other defi cits, including CognitionWall discrimination learning, T-maze spontaneous alternation, and rotarod learning, are a mere resemblance of the single mutant. Th us, although some behavioral tests support compensatory mechanisms, specifi c learning and memory and motor function phenotypes can be assigned to individual Shisa proteins.
Dis� nct phenotypes for Shisa6 and Shisa7 - Future direc� ons
7
AMPAR milliseconds Shisa6 KO Shisa7 KO Short-term plasticity Long-term plasticity 0 Amplitude EPSPs minutes/hours stimulationIncreased depression No effect
Enhanced LTP Reduced LTP PLASTICITY AMPAR Desensitized AMPAR AMPAR Desensitized AMPAR
Figure 2. Summary model of short-term and long-term plas� city phenotypes in the Shisa6 and -7 KO.
Figure 3. Summary overview of hippocampus-dependent memory phenotypes in Shisa6 and -7 KO mice.
Spatial memory
Morris water maze
MEMORY WT KO WT KO Impaired memory WT KO WT KO Impaired memory WT KO WT KO No effect
Contextual fear memory
Short-term & Long-term
7
vice versa, as discussed above for behavioral defi cits.
Th e increase and decrease in dendritic complexity observed in Chapter 5 in Shisa6 KO cultures, respectively, suggests that Shisa6 might be crucial for dendritic growth. If absence of Shisa6 inhibits dendritic growth, absence of Shisa7 might allow Shisa6 to functionally dominate and increase dendritic growth. To test this hypothesis, the shRNA-mediated knockdown, explored in chapter 5, could provide more acute manipulation of Shisa expression to reduce development of compensatory mechanisms. Moreover, Shisa6/7 dKO cultures might be used, in combination with re-expression of either Shisa6, or -7. Likewise, induction of LTP in Shisa6/7 dKO mice might demonstrate whether compensation between Shisa6 and -7 underlies the observed reverse LTP eff ects in KO mice.
Besides Shisa6 and -7, other AMPAR-associated proteins that are part of the AMPAR-Shisa complex might dominate AMPAR basal fi ring and plasticity changes induced by Shisa deletion. Th is type of interplay between AMPAR-associated proteins
MORPHOLOGY Shisa6 KO Shisa7 KO
Hippocampal primary culture Reduced dendritic length Increaseddendritic length
Increased synapse density No effect
Biocytin labeled CA1 pyramidal cells
No effect Reduced spine density
Figure 4. Summary overview of morphology phenotypes in Shisa6 and -7 KO hippocampal cultured neurons and CA1 pyramidal cells.
TARP TARP TARP TARP TARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARP TARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARP
TARPTARP TARP TARPTARP TARPTARP TARPTARP TARP TARPTARP TARPTARPTARP TARP TARP TARPTARP TARP TARPTARP TARP Shisa9 Shisa7
Shisa6 Shisa6 TARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARP TARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARPTARP Shisa7 Shisa6 Shisa7 Shisa9 TARP
7
has been observed for several AMPAR auxiliary subunits. Both Shisa9 and CNIH2 can actsynergistically with TARP to modulate AMPAR function167,170,232,391,392. Th e modulatory
role of CNIH2 on TARP function has been illustrated by recording resensitization after
prolonged glutamate exposure392. TARP γ-8 induces resensitization when co-expressed
with GluA1 and GluA2 in HEK293 cells or when overexpressed in hippocampal neurons, although resensitization does not take place in CA1 pyramidal cells. Only when co-expressing CNIH2, TARP γ-8 and the AMPAR, resensitization is blocked, similar to the physiology of endogenous hippocampal receptors. TARP γ-8 and Shisa9 also collectively modulate hippocampal AMPAR transmission. Deletion of both proteins reduces mEPSC amplitude and frequency in dentate gyrus granule cells, but double deletion induces a stronger, cumulative eff ect. Modulation of Shisa6 and -7 expression might therefore similarly enhance or suppress the function of other associated proteins in the AMPAR complex.
Shisa9170 and -6180 are part of distinct hippocampal AMPAR complexes that
