SGLT2 inhibition, plasma proteins, and heart failure: a proteome-wide Mendelian Randomization and colocalization study.

Objective: To investigate the causal contributions of Sodium-glucose cotransporter 2 (SGLT2) inhibition on Heart Failure (HF) and identify the circulating proteins that mediate SGLT2 inhibition's effects on HF. Methods: Applying a two-sample, two-step Mendelian Randomization (MR) analysis, we aimed to estimate: (1) the causal impact of SGLT2 inhibition on HF; (2) the causal correlation of SGLT2 inhibition on 4,907 circulating proteins; (3) the causal association of SGLT2 inhibition-driven plasma proteins on HF. Genetic variants linked to SGLT2 inhibition derived from the previous studies. The 4,907 circulating proteins were derived from the deCODE study. Genetic links to HF were obtained through the Heart Failure Molecular Epidemiology for Therapeutic Targets (HERMES) consortium. Results: SGLT2 inhibition demonstrated a lower risk of HF (odds ratio [OR] = 0.44, 95% CI [0.26, 0.76], P = 0.003). Among 4,907 circulating proteins, we identified leucine rich repeat transmembrane protein 2 (LRRTM2), which was related to both SGLT2 inhibition and HF. Mediation analysis revealed that the impact of SGLT2 inhibition on HF operates indirectly through LRRTM2 [ß = -0.20, 95% CI (-0.39, -0.06), P = 0.02] with a mediation proportion of 24.6%. Colocalization analysis provided support for the connections between LRRTM2 and HF. Conclusion: The study indicated a causative link between SGLT2 inhibition and HF, with plasma LRRTM2 potentially serving as a mediator.

Novel presynaptic assay system revealed that metformin ameliorates exaggerated synaptic release and Munc18-1 accumulation in presynapses of neurons from Fragile X syndrome mouse model.

Fragile X syndrome (FXS) is a developmental disorder characterized by intellectual disability and autistic-like behaviors. These symptoms are supposed to result from dysregulated translation in pre- and postsynapses, resulting in aberrant synaptic plasticity. Although most drug development research on FXS has focused on aberrant postsynaptic functions by excess translation in postsynapses, the effect of drug candidates on FXS in presynaptic release is largely unclear. In this report, we developed a novel assay system using neuron ball culture with beads to induce presynapse formation, allowing for the analysis of presynaptic phenotypes, including presynaptic release. Metformin, which is shown to rescue core phenotypes in FXS mouse model by normalizing dysregulated translation, ameliorated the exaggerated presynaptic release of neurons of FXS model mouse using this assay system. Furthermore, metformin suppressed the excess accumulation of the active zone protein Munc18-1, which is supposed to be locally translated in presynapses. These results suggest that metformin rescues both postsynaptic and presynaptic phenotypes by inhibiting excess translation in FXS neurons.

Hemisynapse Formation Between Target Astrocytes and Cortical Neuron Axons in vitro.

One of the most fundamental organizing principles in the mammalian brain is that neurons do not establish synapses with the other major cell type, the astrocytes. However, induced synapse formation between neurons and astrocytes appears conceivable, because astrocytes are well known to express functional ionotropic glutamate receptors. Here, we attempted to trigger synapse formation between co-cultured neurons and astrocytes by overexpressing the strongly synaptogenic adhesion protein LRRTM2 in astrocytes physically contacted by cortical axons. Interestingly, control experiments with immature cortical astrocytes without any overexpression resulted in the induction of synaptic vesicle clustering in contacting axons (hemisynapse formation). This synaptogenic activity correlated with the endogenous expression of the synaptogenic protein Neuroligin1. Hemisynapse formation was further enhanced upon overexpression of LRRTM2 in cortical astrocytes. In contrast, cerebellar astrocytes required overexpression of LRRTM2 for induction of synaptic vesicle clustering in contacting axons. We further addressed, whether hemisynapse formation was accompanied by the appearance of fully functional glutamatergic synapses. We therefore attempted to record AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in innervated astrocytes using the whole-cell patch-clamp technique. Despite the endogenous expression of the AMPA receptor subunits GluA2 and to a lesser extent GluA1, we did not reliably observe spontaneous AMPA mEPSCs. In conclusion, overexpression of the synaptogenic protein LRRTM2 induced hemisynapse formation between co-cultured neurons and astrocytes. However, the formation of fully functional synapses appeared to require additional factors critical for nano-alignment of presynaptic vesicles and postsynaptic receptors.

Differential Properties of the Synaptogenic Activities of the Neurexin Ligands Neuroligin1 and LRRTM2.

Synaptic cell adhesion molecules are well established to exhibit synaptogenic activity when overexpressed in target cells, indicating that they are involved in formation and functional maturation of synapses. The postsynaptic adhesion proteins Neuroligin1 and LRRTM2 both induce synaptic vesicle clusters in presynaptic axons in vitro by transsynaptically interacting with neurexins. In neurons, this is accompanied by the induction of glutamatergic, but not GABAergic synapses. Although the synaptogenic activity of Neuroligin1 has been well characterized, the properties of the synaptogenic activities of other synaptic adhesion molecules are largely unknown. In this paper, we now compared characteristics of the synaptogenic activities of Neuroligin1 and LRRTM2 upon overexpression in cultured mouse cortical neurons. Individual cortical neurons were transfected with Neuroligin1 and LRRTM2 expression plasmids, respectively, and synaptic vesicle clustering in contacting axons was examined by immunostaining for the vesicle membrane protein VAMP2. In immature neurons at 6-7 days in vitro (DIV) both Neuroligin1 and LRRTM2 exhibited strong synaptogenic activity. However, upon further neuronal differentiation only LRRTM2 retained significant synaptogenic activity at 12-13 DIV. A similar differential developmental maturation of the synaptogenic activities of Neuroligin1 and LRRTM2 was observed for the induction of glutamatergic synapses, which were detected by co-immunostaining for VGLUT1 and Homer1. Most interestingly, the synaptogenic activity of Neuroligin1 was strongly dependent on the expression and function of the synaptic adhesion molecule N-cadherin in immature neurons. In contrast, the synaptogenic activity of LRRTM2 was independent of N-cadherin expression and function in both immature (6-7 DIV) and more mature neurons (14-15 DIV). Taken together, our results with overexpression in cultured cortical neurons revealed striking differences in the properties of the synaptogenic activities of Neuroligin1 and LRRTM2, although both transsynaptically interact with presynaptic neurexins.

Fragile X mental retardation protein regulates accumulation of the active zone protein Munc18-1 in presynapses via local translation in axons during synaptogenesis.

Fragile X mental retardation protein (FMRP), a causative gene (FMR1) product of Fragile X syndrome (FXS), is an RNA-binding protein to regulate local protein synthesis in dendrites for postsynaptic functions. However, involvement of FMRP in local protein synthesis in axons for presynaptic functions remains unclear. Here we investigated role of FMRP in local translation of the active zone protein Munc18-1 during presynapse formation. We found that leucine-rich repeat transmembrane neuronal 2 (LRRTM2)-conjugated beads, which promotes synchronized presynapse formation, induced simultaneous accumulation of FMRP and Munc18-1 in presynapses of axons of mouse cortical neurons in neuronal cell aggregate culture. The LRRTM2-induced accumulation of Munc18-1 in presynapses was observed in axons protein-synthesis-dependently, even physically separated from cell bodies. The accumulation of Munc18-1 was enhanced in Fmr1-knockout (KO) axons as compared to wild type (WT), suggesting FMRP-regulated suppression for local translation of Munc18-1 in axons during presynapse formation. Using naturally formed synapses of dissociated culture, structured illumination microscope revealed that accumulation of Munc18-1 puncta in Fmr1-KO neurons increased significantly at 19 days in vitro, as compared to WT. Our findings lead the possibility that excessive accumulation of Munc18-1 in presynapses at early stage of synaptic development in Fmr1-KO neurons may have a critical role in impaired presynaptic functions in FXS.

Cadherins Interact With Synaptic Organizers to Promote Synaptic Differentiation.

Classical cadherins, a set of ~20 related recognition and signaling molecules, have been implicated in many aspects of neural development, including the formation and remodeling of synapses. Mechanisms underlying some of these steps have been studied by expressing N-cadherin (cdh2), a Type 1 cadherin, in heterologous cells, but analysis is complicated because widely used lines express cdh2 endogenously. We used CRISPR-mediated gene editing to generate a Human embryonic kidney (HEK)293 variant lacking Cdh2, then compared the behavior of rodent cortical and hippocampal neurons co-cultured with parental, cdh2 mutant and cdh2-rescued 293 lines. The comparison demonstrated that Cdh2 promotes neurite branching and that it is required for three synaptic organizers, neurologin1 (NLGL1), leucine-rich repeat transmembrane protein 2 (LRRtm2), and Cell Adhesion Molecule 1 (Cadm1/SynCAM) to stimulate presynaptic differentiation, assayed by clustering of synaptic vesicles at sites of neurite-293 cell contact. Similarly, Cdh2 is required for a presynaptic organizing molecule, Neurexin1ß, to promote postsynaptic differentiation in dendrites. We also show that another Type I cadherin, Cdh4, and a Type II cadherin, Cdh6, can substitute for Cdh2 in these assays. Finally, we provide evidence that the effects of cadherins require homophilic interactions between neurites and the heterologous cells. Together, these results indicate that classical cadherins act together with synaptic organizers to promote synaptic differentiation, perhaps in part by strengthening the intracellular adhesion required for the organizers to act efficiently. We propose that cadherins promote high affinity contacts between appropriate partners, which then enable synaptic differentiation.

5q31 Microdeletions: Definition of a Critical Region and Analysis of LRRTM2, a Candidate Gene for Intellectual Disability.

Microdeletions including 5q31 have been reported in only few patients to date. Apart from intellectual disability/developmental delay (ID/DD) of varying degrees, which is common to all reported patients, the clinical spectrum is wide and includes short stature, failure to thrive, congenital heart defects, encephalopathies, and dysmorphic features. We report a patient with a 0.9-Mb de novo deletion in 5q31.2, the smallest microdeletion in 5q31 reported thus far. His clinical presentation includes mild DD, borderline short stature, postnatal microcephaly, and mild dysmorphic signs including microretrognathia. Together with data from 7 reported overlapping microdeletions, analysis of our patient enabled the tentative delineation of a phenotype map for 5q31 deletions. In contrast to the mild phenotype of small microdeletions affecting only 5q31.2, carriers of larger microdeletions which also include subbands 5q31.1 and/or 5q31.3 seem to be more severely affected with congenital malformations, growth anomalies, and severe encephalopathies. A 240-kb smallest region of overlap in 5q31.2 is delineated which contains only 2 genes, CTNNA1 and LRRTM2. We propose LRRTM2 as the most promising candidate gene for ID/DD due to its expression pattern, function as a key regulator of excitatory development, and interaction with Neurexin 1. However, sequence analysis of LRRTM2 in 330 patients with ID/DD revealed no relevant alterations, excluding point mutations in LRRTM2 as a frequent cause of ID/DD in patients without microdeletions.