Growing genetic evidence is converging in favor of common pathogenic mechanisms for autism spectrum disorders (ASD), intellectual disability (ID or mental retardation) and schizophrenia (SCZ), three neurodevelopmental disorders affecting cognition and behavior. Copy number variations and deleterious mutations in synaptic organizing proteins including NRXN1 have been associated with these neurodevelopmental disorders, but no such associations have been reported for NRXN2 or NRXN3. From resequencing the three neurexin genes in individuals affected by ASD (n = 142), SCZ (n = 143) or non-syndromic ID (n = 94), we identified a truncating mutation in NRXN2 in a patient with ASD inherited from a father with severe language delay and family history of SCZ. We also identified a de novo truncating mutation in NRXN1 in a patient with SCZ, and other potential pathogenic ASD mutations. These truncating mutations result in proteins that fail to promote synaptic differentiation in neuron coculture and fail to bind either of the established postsynaptic binding partners LRRTM2 or NLGN2 in cell binding assays. Our findings link NRXN2 disruption to the pathogenesis of ASD for the first time and further strengthen the involvement of NRXN1 in SCZ, supporting the notion of a common genetic mechanism in these disorders.

SUMMARY Perturbations of cell surface synapse organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-β) as a novel synapse organizing protein unique in binding and recruiting α-neurexins but not β-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3 shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3 −/− mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.

Selective suppression of inhibitory synapses—with no effect on excitatory synapses—results from interaction of the autism- and schizophrenia-associated proteins MDGA1 and neuroligin-2 through effects on the neuroligin–neurexin pathway.

Brain-derived neurotrophic factor (BDNF) has been reported to exert an acute potentiation of synaptic activity. Here we examined the action of BDNF on synchronous spontaneous Ca 2؉ oscillations in cultured cerebral cortical neurons prepared from postnatal 2-3-dayold rats. The synchronous spontaneous Ca 2؉ oscillations beganatapproximatelyDIV5.Itwasrevealedthatvoltagedependent Ca 2؉ channels and ionotropic glutamate receptors were involved in the synchronous spontaneous oscillatory activity. BDNF potentiated the frequency of these oscillations. The BDNF-potentiated activity reached 207 ؎ 20.1% of basal oscillatory activity. NT-3 and NT-4/5 also induced the potentiation. However, nerve growth factor did not. We examined the correlation between BDNF-induced glutamate release and the BDNF-potentiated oscillatory activity. Both up-regulation of phospholipase C-␥ (PLC-␥) expression and the BDNF-induced glutamate release occurred at approximately DIV 5 when the BDNF-potentiated oscillations appeared. We confirmed that the BDNF-induced glutamate release occurred through a glutamate transporter that was dependent on the PLC-␥/IP 3 /Ca 2؉ pathway. Transporter inhibitors blocked the BDNF-potentiated oscillations, demonstrating that BDNF enhanced the glutamatergic transmissions in the developing cortical network by inducing glutamate release via a glutamate transporter.Neurotrophins play important roles in the survival and differentiation of the peripheral nervous system and CNS 1 neurons. Besides having long term effects, neurotrophins play a fundamental role in neuronal plasticity in the short term (1). In particular, BDNF is essential to neuronal transmissions and activity-dependent neuronal plasticity (2-11). Application of BDNF to cultured hippocampal neurons induced an excitatory synaptic transmission (12), cation influx (13), generation of action potential (14), and Ca 2ϩ mobilization (15). Ca 2ϩ appears to affect processes that are central to the development and plasticity of the CNS (16), and several patterns of Ca 2ϩ dynamics are known. Spontaneous oscillations in the intracellular Ca 2ϩ concentration occur in developing CNS neurons, and their mechanisms are highly distinct. For example, the activation of nicotinic acetylcholine receptors is involved in the oscillatory activity of the retina, whereas a depolarization through the GABA A receptors is required for hippocampal oscillatory activity. Retinal oscillations are spatially restricted to the domains of amacrine and ganglion cells (17,18). By contrast, hippocampal activity consists of transient bursts in the intracellular Ca 2ϩ recurring synchronously over the entire population of pyramidal and interneurons (19). In the cerebral cortex, several patterns of Ca 2ϩ activity have been observed during cortical development, and the metabotropic glutamate receptors (mGluR), GABA A receptors, gap junctions, and ionotropic glutamate receptors are all able to mediate them. The Ca 2ϩ oscillations mediated by mGluR are triggered by mGluR agonists in neonatal or embryonic ...

Changes in synaptic efficacy are considered necessary for learning and memory. Recently, it has been suggested that estrogen controls synaptic function in the central nervous system. However, it is unclear how estrogen regulates synaptic function in central nervous system neurons. We found that estrogen potentiated presynaptic function in cultured hippocampal neurons. Chronic treatment with estradiol (1 or 10 nm) for 24 h significantly increased a high potassium-induced glutamate release. The estrogen-potentiated glutamate release required the activation of both phosphatidylinositol 3-kinase and MAPK. The high potassium-evoked release with or without estradiol pretreatment was blocked by tetanus neurotoxin, which is an inhibitor of exocytosis. In addition, the reduction in intensity of FM1-43 fluorescence, which labeled presynaptic vesicles, was enhanced by estradiol, suggesting that estradiol potentiated the exocytotic mechanism. Furthermore, protein levels of synaptophysin, syntaxin, and synaptotagmin (synaptic proteins, respectively) were up-regulated by estradiol. We confirmed that the up-regulation of synaptophysin was blocked by the MAPK pathway inhibitor, U0126. These results suggested that estrogen enhanced presynaptic function through the up-regulated exocytotic system. In this study, we propose that estrogen reinforced excitatory synaptic transmission via potentiated-glutamate release from presynaptic sites.

Calsyntenin-2 has an evolutionarily conserved role in cognition. In a human genome-wide screen, the CLSTN2 locus was associated with verbal episodic memory, and expression of human calsyntenin-2 rescues the associative learning defect in orthologous Caenorhabditis elegans mutants. Other calsyntenins promote synapse development, calsyntenin-1 selectively of excitatory synapses and calsyntenin-3 of excitatory and inhibitory synapses. We found that targeted deletion of calsyntenin-2 in mice results in a selective reduction in functional inhibitory synapses. Reduced inhibitory transmission was associated with a selective reduction of parvalbumin interneurons in hippocampus and cortex. Clstn2(-/-) mice showed normal behavior in elevated plus maze, forced swim test, and novel object recognition assays. However, Clstn2(-/-) mice were hyperactive in the open field and showed deficits in spatial learning and memory in the Morris water maze and Barnes maze. These results confirm a function for calsyntenin-2 in cognitive performance and indicate an underlying mechanism that involves parvalbumin interneurons and aberrant inhibitory transmission.

We reported previously that BDNF induced glutamate release was dependent on intracellular Ca(2+) but not extracellular Ca(2+) in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca(2+) and Na(+) in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca(2+) level. We found that the Ca(2+) increase induced by BDNF was completely blocked by xestospongin C, an IP(3) receptor antagonist, and U-73122, a PLC-gamma inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca(2+) through the activation of the PLC-gamma/ IP(3) pathway was essential for the glutamate release. We found that BDNF induced a Na(+) influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na(+) influx, suggesting that the Na(+)influx required the BDNF-induced Ca(2+) increase. Next, we examined the possibility that a co-transporter of Na(+) and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK(+))-induced release was blocked by t-PDC. In addition, DL-threo-beta-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na(+)-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-gamma/IP(3)-mediated Ca(2+) signaling.

Brain-derived neurotrophic factor (BDNF), a critical neurotrophin, regulates many neuronal aspects including cell differentiation, cell survival, neurotransmission, and synaptic plasticity in the central nervous system (CNS). Though BDNF has two types of receptors, high affinity tropomyosin-related kinase (Trk)B and low affinity p75 receptors, BDNF positively exerts its biological effects on neurons via activation of TrkB and of resultant intracellular signaling cascades including mitogen-activated protein kinase/extracellular signal-regulated protein kinase, phospholipase Cγ, and phosphoinositide 3-kinase pathways. Notably, it is possible that alteration in the expression and/or function of BDNF in the CNS is involved in the pathophysiology of various brain diseases such as stroke, Parkinson's disease, Alzheimer's disease, and mental disorders. On the other hand, glucocorticoids, stress-induced steroid hormones, also putatively contribute to the pathophysiology of depression. Interestingly, in addition to the reduction in BDNF levels due to increased glucocorticoid exposure, current reports demonstrate possible interactions between glucocorticoids and BDNF-mediated neuronal functions. Other steroid hormones, such as estrogen, are involved in not only sexual differentiation in the brain, but also numerous neuronal events including cell survival and synaptic plasticity. Furthermore, it is well known that estrogen plays a role in the pathophysiology of Parkinson's disease, Alzheimer's disease, and mental illness, while serving to regulate BDNF expression and/or function. Here, we present a broad overview of the current knowledge concerning the association between BDNF expression/function and steroid hormones (glucocorticoids and estrogen).