These results define the major function of the gene mutated to cause human ADLTE and uncover a key molecular agent regulating postnatal glutamatergic synapse development. LGI1 regulated the functional maturation and structural pruning of glutamatergic synapses during postnatal development (Supplementary Fig. 12
). LGI4, another family member, plays a similar postnatal role in developmental myelination of peripheral nerves35
. ADLTE mutant LGI1 arrested the development of hippocampal MPP-GC excitatory synapses, causing a drastic increase of excitatory transmission. As the dentate gyrus gates seizure propagation through hippocampus36,37
, the findings could have direct relevance to human seizure dissemination. Indeed mice carrying mLGI1 were prone to kindling epileptogenesis.
Our results suggest LGI1 acts at the presynaptic terminal to overcome Kvβ1-mediated Kv1.1 channel inhibition in vivo
during postnatal development. Kv1 channel blocker DTX eliminated the differences caused by the LGI1
transgenes on presynaptic release probability (), suggesting LGI1 reduces presynaptic release probability by up-regulating presynaptic Kv1 potassium channels in vivo
. The result is consistent with in vitro
studies showing LGI1 antagonizes Kvβ1-promoted Kv1.1 channel inactivation14
. Significantly, Kv1.1 is already expressed at the adult level during the third postnatal week38
, and Kvβ1 is developmentally up-regulated during this same time period (Anderson, unpublished data). Directly measuring Kv1.1 channels function at the minute MPP synaptic terminus is currently not technically feasible, but future studies aimed at larger synapses that express LGI1 will be important.
Postsynaptic NMDA receptor trafficking is of particular importance to synapse maturation and plasticity39
. Our results indicate that Src kinase activity plays a critical role in maintaining the high level of NR2B-containing NMDA receptor function present at MPP-GC synapses during early postnatal development (). Interestingly, both LGI1 and integrin5
seem to coordinate the developmental maturation of pre- and postsynaptic function of glutamatergic synapses. Furthermore, both LGI1 and integrin bind the same disintegrin domain of the ADAM family proteins13,40
and regulate NR2 subunit composition via
tyrosine kinase signaling ()5
. In future studies, it will be important to determine whether LGI1 and integrin interact via
ADAM family members to promote glutamatergic synapse maturation and pruning.
In contrast to our results, exogenous application of LGI protein to hippocampal brain slices failed to affect release probability at Schaffer collateral-CA1 pyramidal cell synapses in p24–p29 rodents13
. Our results indicate release probability is already fully down-regulated by the high levels of native LGI1 present by p24–p29. Furthermore, ADAM22 is only weakly expressed in the Schaffer collateral (www.neuromab.org/datasheet/N46_30.pdf
). Exogenous LGI1 was also reported to produce a small increase of the AMPA/NMDA ratio, mEPSC amplitude, and mEPSC frequency13
. By contrast, our transgenic mice with excess LGI1 displayed a decreased mEPSC frequency and no change in mEPSC amplitude. Differences in the amount, distribution, or target of exogenous vs. endogenous LGI1 may explain these differing results.
Our results indicate LGI1 acts to remodel the hippocampal dentate glutamatergic circuitry. Dendritic arbor pruning may remove excess excitatory connections formed during early development. Although the detailed cellular mechanisms remain unknown, interactions between LGI1 and the postsynaptic cellular scaffolding molecule PSD95 might be important41
LGI1 did not regulate GABAergic synaptic functions measured in dentate gyrus, but inhibitory exceeded excitatory transmission by approximately 20-fold (). This observation suggests the strong inhibition might mask the increased excitatory transmission caused by mLGI142
. Future studies should evaluate whether LGI1 regulates glutamatergic synapses onto GABAergic neurons as these could also partially compensate for the pro-epileptic effects of mLGI1.
The opposing effects of mLGI1 and excess full-length LGI1 indicate mLGI1 acts as a dominant negative inhibitor to cause epilepsy14
, rather than through haploid insufficiency7,11
. The dominant negative mechanism remains undefined, but could result from hetero-multimer formation between mLGI1 and wild-type LGI1 via
cysteine residues within the N-terminal leucine-rich repeat domain13
. Since the dominant negative effects of mLGI1 occurred in the critical period of postnatal development, we suggest ADLTE mLGI1 may disrupt normal brain development in early childhood to promote epilepsy throughout adulthood.