Inhibitory CCK+ basket synapse defects in mouse models of dystroglycanopathy

Reviewer #1 (Public Review):

This important study from Jahncke et al. demonstrates inhibitory synaptic defects and elevated seizure susceptibility in multiple models of dystroglycanopathy. A strength of the paper is the use of a wide range of genetic models to disrupt different aspects of dystroglycan protein or glycosylation in forebrain neurons. The authors use a combination of immunohistochemistry and electrophysiology to identify cellular migration, lamination, axonal targeting, synapse formation/function, and seizure phenotypes in forebrain neurons. This is an elegant study with extensive data supporting the conclusions. The role of dystroglycan and the dystrophin glycoprotein complex (DGC) in cellular migration and synapse formation are of broad interest.

A strength of this paper is the use of several transgenic mouse lines with mutations in genes involved in glycosylation of dystroglycan. Knockout of POMT2 abolishes the majority of dystroglycan glycosylation, while point mutations in B4GAT and FKRP presumably produce more minor changes in glycosylation. This is a powerful approach to investigate the role of glycosylation in dystroglycan function. However, the authors do not address how mutations in these genes may affect glycosylation or expression of proteins other than dystroglycan. It is possible, even likely, that some of the phenotypes observed are due to changing glycosylation in any number of other proteins. The paper would be strengthened by addressing this possibility more directly.
It would be helpful to have a more clear description of how dystroglycan glycosylation is altered in B4GAT1M155T or FKRPP448L mice. For example, Figure 1 makes it appear that the distal sugar moieties are missing, however, the IIH6 antibody, which binds to terminal matriglycan repeats on the glycan chain, recognizes dystroglycan in these mutants.

In Figure 1, the authors use the IIH6 antibody, which recognizes the terminal portion of the dystroglycan glycan chain, to label dystroglycan in the hippocampus. As expected, Emx1Cre,POMT2cKO mice, which lack glycosylation of dystroglycan, do not show any labelling. However, this experiment does not reveal anything about dystroglycan expression, only that the IIH6 antibody no longer recognizes dystroglycan. It would be very helpful in interpreting the later results to know whether the level and pattern of dystroglycan expression is normal or absent in the POMT2cKO mice, perhaps using another antibody that does not target the glycosylated region. For example, figure 3 shows reduced axon targeting to the cell body layer in POMT2cKO, however, it is unclear whether this is due to absence/mislocalization of dystroglycan at the cell surface, or if dystroglycan expression is normal, but glycosylation is directly required for axon targeting.

In Figures 3 and 5, the authors use CB1R labelling to measure axon targeting and synapses formation. However, it is not clear how the authors measure axon targeting and synapses number separately using the same CB1R antibody. In addition, figure 3 shows reduced CB1R labelling in Dag1cyto pyramidal cell layer, but Figure 5 shows no change in CB1R labelling in the same mice. These results would appear to be contradictory.

The authors measure spontaneous IPSCs (sIPSC) in CA1 pyramidal neurons to measure inhibitory synaptic function. This measure assesses inhibitory synaptic input from all sources, but dystroglycan mutations primarily impairs synapses arising from CCK+/CB1R interneurons, leaving synapses arising from PV or other interneurons relatively unchanged. To assess changes in CCK+/CB1R interneurons the authors apply the cholinergic receptor agonist Carbachol (which selectively activates CCK+/CB1R interneurons) and measure the change in sIPSC amplitude and frequency. While this is an interesting and reasonable experiment, the observed effects could be due to altered carbachol sensitivity in the transgenic mice. Control experiments showing that the effect of Carbachol on excitability of CCK+/CB1R interneurons is similar across mouse lines is missing.

Earlier work has shown that selective deletion of dystroglycan from pyramidal neurons produces near complete loss of CCK+/CB1R interneurons and synapse formation, a more severe deficit than observed here using a more widespread Cre-driver. This finding is surprising, as generally more wide-spread gene deletion results in more severe, not less severe, phenotypes. The authors make the reasonable claim that more wide-spread gene deletion better mimics human pathologies. However, possible speculation on why this is the case for dystroglycan could provide insight into the nature of CNS deficits in different forms of dystroglycanopathies.

Reviewer #2 (Public Review):

The manuscript by Jahncke and colleagues is centered on the CCK+ synaptic defects that are a consequence of Dystroglycanopathy and/or impaired dystroglycan-related protein function. The authors use conditional mouse models for Dag1 and Pomt2 to ablate their function in mouse forebrain neurons and demonstrate significant impairment of CCK+/CB1R+ interneuron (IN) development in addition to being prone to seizures. Mice lacking the intracellular domain of Dystroglycan have milder defects, but impaired CCK+/CB1R+ IN axon targeting. The authors conclude that the milder dystroglycanopathy is due to the partially reduced glycosylation that occurs in the milder mouse models as opposed to the more severe Pomt2 models. Additionally, the authors postulate that inhibitory synaptic defects and elevated seizure susceptibility are hallmarks of severe dystroglycanopathy and are required for the organization of functional inhibitory synapse assembly.

The manuscript is overall, fairly well-written and the description of the phenotypic impact of disruption of Dystroglycan forebrain neurons (and similar glycosyltransferase pathway proteins) demonstrate impairment in axon targeting and organization. There are some questions with regards to interpretation of some of the results from these conditional mouse models. The study is mostly descriptive, and some validation of subunits of the dystroglycan-glycoprotein complex and laminin interactions would go towards defining the impact of disruption of dystroglycan's function in the brain. The statistics and basic analysis of the manuscript appear to be appropriate and within parameters for a study of this nature. Some clarification between the discrepancies between the Walker Warburg Syndrome (WWS) patient phenotypes and those observed in these conditional mouse models is warranted. This manuscript has the potential to be impactful in the Dystroglycanopathy and general neurobiology fields.

Reviewer #3 (Public Review):

The study presents a systematic analysis of how a range of dystroglycan mutations alter CCK/CB1 axonal targeting and inhibition in hippocampal CA1 and impact seizure susceptibility. The study follows up on prior literature identifying a role for dystroglycan in CCK/CB1 synapse formation. The careful assay includes comparison of 5 distinct dystroglycan mutation types known to be associated with varying degrees of muscular dystrophy phenotypes: a forebrain specific Dag1 knockout in excitatory neurons at 10.5, a forebrain specific knockout of the glycosyltransferase enzyme in excitatory neurons, mice with deletion of the intracellular domain of beta-Dag1 and 2 lines with missense mutations with milder phenotypes. They show that forebrain glutamatergic deletion of Dag1 or glycosyltransferase alters cortical lamination while lamination is preserved in mice with deletion of the intracellular domain or missense mutation. The study extends prior works by identifying that forebrain deletion of Dag1 or glycosyltransferase in excitatory neurons impairs CCK/CB1 and not PV axonal targeting and CB1 basket formation around CA1 pyramidal cells. Mice with deletion of the intracellular domain or missense mutation show limited reductions in CCK/CB1 fibers in CA1. Carbachol enhancement of CA1 IPSCs was reduced both in forebrain knockouts. Interestingly, carbachol enhancement of CA1 IPSCs was reduced when the intracellular domain of beta-Dag1was deleted, but not I the missense mutations, suggesting a role of the intracellular domain in synapse maintenance. All lines except the missense mutations , showed increased susceptibility to chemically induced behavioral seizures. Together, the study, is carefully designed, well controlled and systematic. The results advance prior findings of the role for dystroglycans in CCK/CB1 innervations of PCs by demonstrating effects of more selective cellular deletions and site specific mutations in extracellular and intracellular domains. The interesting finding that deletion of intracellular domain reduces both CB1 terminals in CA1 and carbachol modulation of IPSCs warrants further analysis. Lack of EEG evaluation of seizure latency is a limitation.

Specific comments
1. Whether CCK/CB1 cell numbers in the CA1 are differentially affected in the transgenic mice is not clarified.
2. Whether basal synaptic inhibition is altered by the changes in CCK innervation is not examined.