Distinct but overlapping roles of LRRTM1 and LRRTM2 in developing and mature hippocampal circuits.

LRRTMs are postsynaptic cell adhesion proteins that have region-restricted expression in the brain. To determine their role in the molecular organization of synapses in vivo, we studied synapse development and plasticity in hippocampal neuronal circuits in mice lacking both Lrrtm1 and Lrrtm2. We found that LRRTM1 and LRRTM2 regulate the density and morphological integrity of excitatory synapses on CA1 pyramidal neurons in the developing brain but are not essential for these roles in the mature circuit. Further, they are required for long-term-potentiation in the CA3-CA1 pathway and the dentate gyrus, and for enduring fear memory in both the developing and mature brain. Our data show that LRRTM1 and LRRTM2 regulate synapse development and function in a cell-type and developmental-stage-specific manner, and thereby contribute to the fine-tuning of hippocampal circuit connectivity and plasticity.

NBI-921352, a first-in-class, NaV1.6 selective, sodium channel inhibitor that prevents seizures in Scn8a gain-of-function mice, and wild-type mice and rats.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

An LRRTM4-HSPG complex mediates excitatory synapse development on dentate gyrus granule cells.

Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.

The transcription factor MEF2 directs developmental visually driven functional and structural metaplasticity.

Natural sensory input shapes both structure and function of developing neurons, but how early experience-driven morphological and physiological plasticity are interrelated remains unclear. Using rapid time-lapse two-photon calcium imaging of network activity and single-neuron growth within the unanesthetized developing brain, we demonstrate that visual stimulation induces coordinated changes to neuronal responses and dendritogenesis. Further, we identify the transcription factor MEF2A/2D as a major regulator of neuronal response to plasticity-inducing stimuli directing both structural and functional changes. Unpatterned sensory stimuli that change plasticity thresholds induce rapid degradation of MEF2A/2D through a classical apoptotic pathway requiring NMDA receptors and caspases-9 and -3/7. Knockdown of MEF2A/2D alone is sufficient to induce a metaplastic shift in threshold of both functional and morphological plasticity. These findings demonstrate how sensory experience acting through altered levels of the transcription factor MEF2 fine-tunes the plasticity thresholds of brain neurons during neural circuit formation.

Neurexin-neuroligin cell adhesion complexes contribute to synaptotropic dendritogenesis via growth stabilization mechanisms in vivo.

Cell adhesion molecules are well characterized for mediating synapse initiation, specification, differentiation, and maturation, yet their contribution to directing dendritic arborization during early brain circuit formation remains unclear. Using two-photon time-lapse imaging of growing neurons within intact and awake embryonic Xenopus brain, we examine roles of ß-neurexin (NRX) and neuroligin-1 (NLG1) in dendritic arbor development. Using methods of dynamic morphometrics for comprehensive 3D quantification of rapid dendritogenesis, we find initial trans-synaptic NRX-NLG1 adhesions confer transient morphologic stabilization independent of NMDA receptor activity, whereas persistent stabilization requires NMDA receptor-dependent synapse maturation. Disrupting NRX-NLG1 function destabilizes filopodia while reducing synaptic density and AMPA receptor mEPSC frequency. Altered dynamic growth culminates in reduced dendritic arbor complexity as neurons mature over days. These results expand the synaptotropic model of dendritogenesis to incorporate cell adhesion molecule-mediated morphological stabilization necessary for directing normal dendritic arborization, providing a potential morphological substrate for developmental cognitive impairment associated with cell adhesion molecule mutations.

PKM zeta restricts dendritic arbor growth by filopodial and branch stabilization within the intact and awake developing brain.

The molecular mechanisms underlying activity-dependent neural circuit growth and plasticity during early brain development remain poorly understood. Protein kinase Mzeta (PKMz), an endogenous constitutively active kinase associated with late-phase long-term synaptic potentiation and memory in the mature brain, is expressed in the embryonic Xenopus retinotectal system with heightened levels during peak periods of dendrite growth and synaptogenesis. In vivo rapid time-lapse imaging of actively growing tectal neurons and comprehensive three-dimensional tracking of dynamic dendritic growth behavior finds that altered PKMz activity affects morphologic stabilization. Exogenous expression of PKMz within single neurons stabilizes dendritic filopodia by increasing dendritic filopodial lifetimes and decreasing filopodial additions, eliminations, and motility, whereas long-term in vivo imaging demonstrates restricted expansion of the dendritic arbor. Alternatively, blocking endogenous PKMz activity in individual growing tectal neurons with an inhibitory peptide (zeta-inhibitory peptide) destabilizes dendritic filopodia and over long periods promotes excessive arbor expansion. Furthermore, inhibiting endogenous PKMz throughout the tectum decreases colocalization of immunostained presynaptic and postsynaptic markers, SNAP-25 and PSD-95, respectively, suggesting impaired synapse maintenance. Together, these results implicate PKMz activity in restricting dendritic arborization during embryonic brain circuit development through synaptotropic stabilization of dynamic processes.