Presynaptic PTPσ regulates postsynaptic NMDA receptor function through direct adhesion-independent mechanisms

Acceptance summary:

As nicely summarized by one of the reviewers, the authors investigated the role of the presynaptic protein PTPσ, a LAR-type receptor phosphotyrosine-phosphatase, as a transsynpatic regulator of postsynaptic NMDA receptors. They developed a conditional KO mouse (PTPσ-cKO) and provide evidence that deletion of PTPσ reduces postsynaptic NMDAR-mediated transmission and NMDAR-mediated plasticity (LTP and LTD) in the hippocampus that PTPσ-dependent regulation of NMDARs likely involves interactions of PTPσ with cytoplasmic proteins that presumably link PTPσ to phosphotyrosine substrates; and that deletion of PTPσ is associated with impairments in social and reward-related novelty recognition. The findings support the notion that a presynaptic adhesion protein can control postsynaptic function in a non-canonical way that does not involve trans-synaptic protein-protein interactions.

Decision letter after peer review:

Thank you for submitting your article "Presynaptic PTPσ Regulates Postsynaptic NMDA Receptor Function through Cytoplasmic Domains" for consideration by eLife. Your article has been reviewed by Gary Westbrook as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Pablo E Castillo (Reviewer #1); Johannes W Hell (Reviewer #2); Matthijs Verhage (Reviewer #3). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

All three reviewers were interested in the topic and supportive of the work. However, they raised several issues, most of which we think can be dealt with by adjustments in the text and Discussion section. These are enumerated in the comments of the reviewers below.

Reviewer #1:

In this thorough study, the authors investigated the role of the presynaptic protein PTPσ, a LAR-type receptor phosphotyrosine-phosphatase, as a trans-synpatic regulator of postsynaptic NMDA receptors. To this end, they developed a conditional KO mouse (PTPσ-cKO) and used multiple approaches, including synaptic electrophysiology in hippocampal slices, proteomics and behavioral tests. The authors provide experimental evidence in support of the following three main findings. First, deletion of PTPσ reduces postsynaptic NMDAR-mediated transmission and NMDAR-mediated forms of plasticity (LTP and LTD) in the hippocampus. Second, PTPσ-dependent regulation of NMDARs likely involves interactions of PTPσ with cytoplasmic proteins that presumably link PTPσ to phosphotyrosine substrates. Third, deletion of PTPσ is associated with significant impairments in social and reward-related novelty recognition. The findings are exciting and support the notion that a presynaptic adhesion protein can control postsynaptic function in a non-canonical way that does not involve trans-synaptic protein-protein interactions. The study includes a large number of well-designed experiments. The results are clean and support most of the claims that are made. The authors' findings will be of interest to a large audience of neuroscientists and cellular biologists.

Essential revisions:

The authors claim that "PTPσ trans-synaptically regulates the postsynaptic localization and function of NMDARs in the hippocampus". However, their study provides rather indirect evidence that PTPσ changes the localization of NMDARs. The results presented in Figure 1J suggest a global reduction in GluN2B subunits. The authors may want to tone down their claim and provide some explanation for this global change.

Figure 6A: The starting point (familiar) seems to be significantly different between WT and PTPσ-cKO mice. Why? How would this difference affect the interpretation of the results?

The authors indicate that PTPσ is expressed in the cerebellum but the images suggest otherwise (Figure 1—figure supplement 1G).

What is the authors' explanation for a reduced LTD in PTPσ-cKOs?

Did D-cycloserine rescued NMDAR-mediated transmission (e.g. NMDAR-EPSCs) in PTP-cKO mice?

Discussion section. Indicate whether or not previous studies have specifically linked postsynaptic hippocampal LTP/LTD to novel recognition tasks.

Validating the knockdown in cultures is okay but not ideal. The authors may want to show the efficacy of the knockdown in tissue.

"Evoked EPSCs at Schaffer collateral-CA1 pyramidal (SC-CA1) synapses…", add the word "cell" after pyramidal.

Reviewer #2:

PTPs may contribute to adhesion between pre-and postsynaptic sites but numerous aspects of the roles of PTPs in synapse formation and function remain unclear. In this manuscript Eunjoon Kim and his co-workers create conditional KO mice in which PTPσ was deleted in excitatory neurons in cortex and hippocampus. In these mice basal synaptic transmission at the CA3-CA1 synapse was normal including mEPSC amplitude and frequency, evoked transmission and PPR. These findings contrast earlier work in global PTPs KO mice. However, the authors find that LTP as well as LTD were impaired in these cKO. Loss of LTP was rescued by boosting NMDAR activity with D-cycloserine. These data suggest that in these cKO mice postsynaptic NMDAR activity is reduced. Consistent with these functional studies, the content of the NMDAR GluN2B subunit was decreased in PSD fractions.

To define whether pre-or postsynaptic PTPσ expression is relevant here, the authors deleted PTPσ by viral injection in either CA3 or CA1 neurons. Only presynaptic (CA3) but not postsynaptic (CA1) deletion impaired LTP. Using the cKO mice in which PTPσ was deleted in excitatory forebrain neurons the authors further demonstrate that re-expression of WT PTPσ in presynaptic CA3 neurons rescued the LTP. This was also true for expression of several different PTPσ constructs with point and deletion mutations that impaired interactions with different postsynaptic binding partners including HSPG/CSPG and Slitrk/TrcC. However, expression of PTPσ with a point mutation that impaired phosphatase activity and two deletion of intracellular regions that impaired binding to caskin were not able to rescue LTP.

To gain further molecular insight the authors performed an unbiased proteomic approach to define changes in tyrosine phosphorylation in the cKO mice in which PTPσ was deleted in excitatory forebrain neurons. Accordingly, ~1500 proteins showed changes and many of those are synaptic proteins. Presynaptic proteins showed more increases in pTyr as can be expected upon loss of a phosphatase whereas postsynaptic proteins showed more decreases in pTyr. The latter includes several tyrosine residues in GluN2B, which could potentially explain the reduction of GluN2B content of the PSD and thereby reduction in NMDAR activity, as described above.

Finally, the authors performed a number of learning and memory tasks. In mice in which PTPσ was deleted in excitatory forebrain neurons most initial learning was normal when novelty recognition was impaired in several tests (novel object, novel social mouse, novel reward arm in Y maze, and reversal learning in the Morris Water Maze). Remarkably, preference for novel mouse in the social assay was rescued by systemic administration of D-cycloserine, which augments NMDAR activity. Furthermore, deletion of PTPσ in CA3 but not CA1 neurons impaired the novel mouse preference in the social interaction test as well as novel arm preference in the Y maze. Knockout of GluN1 in the postsynaptic CA1 neurons phenocopied these two effects. Collectively these results indicate that loss of presynaptic PTPσ affects postsynaptic NMDAR function and thereby novelty learning.

The above findings are of very high functional significance. This is a very rigorous, comprehensive, and thorough study with functional effects on multiple levels including NMDAR activity in synaptic transmission and novelty learning.

In Figure 6—figure supplement Figure 1 data suggest decreased baseline anxiety including a tendency to increased open field time and a significant increase in open arm time in the elevated plus maze-that should be more detailed in the Results section and discussed.

Reviewer #3:

This study describes how conditional deletion of PTPσ alters synaptic plasticity and posttranslational modification of proteins on either side of the synapse and attempts to connect these alterations to altered cognitive bahaviors. The manuscript presents a massive amount of data, an important new mouse model and important new conclusions on PTPσ function and the trans-synaptic regulation of synaptic strength. These conclusions are different from previous conclusions obtained using classical PTPσ-null mice and challenge the existing concept of how PTPs regulate synapse differentiation and plasticity. The novel concept, postulating that PTP acts through presynaptic interactions with other (trans-synaptic) molecules to alter postsynaptic receptor function and balance, is new, important and convincing. The conclusions are built on an impressive series of in vivo rescue experiments and state of the art phospho-proteomics and analysis. The SynGO analysis of altered pTyr levels presents a spectacular difference between pre- and postsynaptic pTyr proteins (with presynaptic proteins all having increased pTyr levels and postsynaptic proteins decreased pTyr levels). The altered pTyr proteins include relevant new substrates on either side of the synapse.

Essential revisions:

1) PTPσ mutant constructs carrying mutations/deletions in cytoplasmic domains D1-2 do not rescue LTP, which is a central element in the argumentation, but it is unknown if these constructs are targeted correctly. Some evidence for correct targeting is required. Most convincing and more positive evidence would be to show that targeting of the cytoplasmic domains (e.g. using GAP43) is sufficient for normal LTP, but this is probably beyond the scope of the current study.

2) While it seems easy to explain why presynaptic proteins have almost exclusively higher pTyr levels, it is quite intriguing why postsynaptic proteins almost exclusively have lower pTyr levels. Given the proposed indirectness of these effects and the fact that so many postsynaptic substrates are altered, it is quite remarkable that the postsynaptic effects are so one-directional. This should be discussed.

3) Behavioral analyses are extensive and interesting but the connection to cellular/slice data remains loose. Although the behavioral abnormalities are certainly consistent with altered hippocampal NMDA function, the behavioral effects are most likely the net result of many (unknown) cellular and network effects of Emx-expressing neurons, their targets and the targets of these targets etc. A more balanced discussion on this topic would be preferable.