Cavß3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalitis.

BACKGROUND: Tight control of cytoplasmic Ca2+ in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavß3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. METHODS: We investigated the function of Cavß3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavß3-/- (Cav ß3-deficient) mice as controls. RESULTS: We identified Cavß3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavß3. After induction of experimental autoimmune encephalomyelitis, Cavß3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavß3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavß3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavß3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavß3 with inositol 1,4,5-trisphosphate receptor proteins. CONCLUSIONS: Independent of its function as a subunit of Cav channels, Cavß3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavß3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

Glutamate transporter contribution to retinal ganglion cell vulnerability in a rat model of multiple sclerosis.

Glial glutamate transporters actively participate in neurotransmission and have a fundamental role in determining the ambient glutamate concentration in the extracellular space. Their expression is dynamically regulated in many diseases, including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In EAE, a downregulation has been reported which may render neurons more susceptible to glutamate excitotoxicity. In this study, we have investigated the expression of GLAST (EAAT1) and GLT-1 (EAAT2) in the retina of Brown Norway rats following induction of myelin oligodendrocyte glycoprotein (MOG)-EAE, which results in retinal ganglion cell (RGC) degeneration and dysfunction. In addition, we tested whether AAV-mediated overexpression of GLAST in the retina can protect RGCs from degeneration. To address the impact of glutamate transporter modulation on RGCs, we performed whole-cell recordings and measured tonic NMDA receptor-mediated currents in the absence and presence of a glutamate-uptake blocker. We report that αOFF-RGCs show larger tonic glutamate-induced currents than αON-RGCs, in line with their greater vulnerability under neuroinflammatory conditions. We further show that increased AAV-mediated expression of GLAST in the retina does indeed protect RGCs from degeneration during the inflammatory disease. Collectively, our study highlights the neuroprotective role of glutamate transporters in the EAE retina and provides a characterization of tonic glutamate-currents of αRGCs. The larger effects of increased extracellular glutamate concentration on the αOFF-subtype may underlie its enhanced vulnerability to degeneration.

Co-modulation of TNFR1 and TNFR2 in an animal model of multiple sclerosis.

BACKGROUND: Tumour necrosis factor (TNF) is a pleiotropic cytokine and master regulator of the immune system. It acts through two receptors resulting in often opposing biological effects, which may explain the lack of therapeutic potential obtained so far in multiple sclerosis (MS) with non-receptor-specific anti-TNF therapeutics. Under neuroinflammatory conditions, such as MS, TNF receptor-1 (TNFR1) is believed to mediate the pro-inflammatory activities associated with TNF, whereas TNF receptor-2 (TNFR2) may instead induce anti-inflammatory effects as well as promote remyelination and neuroprotection. In this study, we have investigated the therapeutic potential of blocking TNFR1 whilst simultaneously stimulating TNFR2 in a mouse model of MS. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG35-55) in humanized TNFR1 knock-in mice. These were treated with a human-specific TNFR1-selective antagonistic antibody (H398) and a mouse-specific TNFR2 agonist (EHD2-sc-mTNFR2), both in combination and individually. Histopathological analysis of spinal cords was performed to investigate demyelination and inflammatory infiltration, as well as axonal and neuronal degeneration. Retinas were examined for any protective effects on retinal ganglion cell (RGC) degeneration and neuroprotective signalling pathways analysed by Western blotting. RESULTS: TNFR modulation successfully ameliorated symptoms of EAE and reduced demyelination, inflammatory infiltration and axonal degeneration. Furthermore, the combinatorial approach of blocking TNFR1 and stimulating TNFR2 signalling increased RGC survival and promoted the phosphorylation of Akt and NF-κB, both known to mediate neuroprotection. CONCLUSION: These results further support the potential of regulating the balance of TNFR signalling, through the co-modulation of TNFR1 and TNFR2 activity, as a novel therapeutic approach in treating inflammatory demyelinating disease.

Alterations in Lymphocytic Metabolism-An Emerging Hallmark of MS Pathophysiology?

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterised by acute inflammation and subsequent neuro-axonal degeneration resulting in progressive neurological impairment. Aberrant immune system activation in the periphery and subsequent lymphocyte migration to the CNS contribute to the pathophysiology. Recent research has identified metabolic dysfunction as an additional feature of MS. It is already well known that energy deficiency in neurons caused by impaired mitochondrial oxidative phosphorylation results in ionic imbalances that trigger degenerative pathways contributing to white and grey matter atrophy. However, metabolic dysfunction in MS appears to be more widespread than the CNS. This review focuses on recent research assessing the metabolism and mitochondrial function in peripheral immune cells of MS patients and lymphocytes isolated from murine models of MS. Emerging evidence suggests that pharmacological modulation of lymphocytic metabolism may regulate their subtype differentiation and rebalance pro- and anti-inflammatory functions. As such, further understanding of MS immunometabolism may aid the identification of novel treatments to specifically target proinflammatory immune responses.

Influence of retinal NMDA receptor activity during autoimmune optic neuritis.

Autoimmune optic neuritis (AON), a model of multiple sclerosis-associated optic neuritis, is accompanied by degeneration of retinal ganglion cells (RGCs) and optic nerve demyelination and axonal loss. In order to investigate the role of N-methyl-d-aspartate (NMDA) receptors in mediating RGC degeneration, upstream changes in the optic nerve actin cytoskeleton and associated deterioration in visual function, we induced AON in Brown Norway rats by immunization with myelin oligodendrocyte glycoprotein. Subsequently, visual acuity was assessed by recording visual evoked potentials and electroretinograms prior to extraction of optic nerves for western blot analysis and retinas for quantification of RGCs. As previously reported, in Brown Norway rats RGC degeneration is observed prior to onset of immune cell infiltration and demyelination of the optic nerves. However, within the optic nerve, destabilization of the actin cytoskeleton could be seen as indicated by an increase in the globular to filamentous actin ratio. Interestingly, these changes could be mimicked by intravitreal injection of glutamate, and similarly blocked by application of the NMDA receptor blocker MK-801, leading us to propose that prior to optic nerve lesion formation, NMDA receptor activation within the retina leads to retinal calcium accumulation, actin destabilization within the optic nerve as well as a deterioration of visual acuity during AON.

Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis.

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB-crystallin is a heat-shock protein induced in cells undergoing cellular stress and has been reported to be up-regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB-crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)-immunized animals induced an increase in αB-crystallin expression, as did passive transfer of sera from MOG-immunized animals to unimmunized recipients, we propose that the partially permeable blood-brain barrier of the optic nerve head may present an opportunity for blood-borne components such as anti-MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development.

Calcium-Binding Proteins as Determinants of Central Nervous System Neuronal Vulnerability to Disease.

Neuronal subpopulations display differential vulnerabilities to disease, but the factors that determine their susceptibility are poorly understood. Toxic increases in intracellular calcium are a key factor in several neurodegenerative processes, with calcium-binding proteins providing an important first line of defense through their ability to buffer incoming calcium, allowing the neuron to quickly achieve homeostasis. Since neurons expressing different calcium-binding proteins have been reported to be differentially susceptible to degeneration, it can be hypothesized that rather than just serving as markers of different neuronal subpopulations, they might actually be a key determinant of survival. In this review, we will summarize some of the evidence that expression of the EF-hand calcium-binding proteins, calbindin, calretinin and parvalbumin, may influence the susceptibility of distinct neuronal subpopulations to disease processes.

Dysfunction of neuronal calcium signalling in neuroinflammation and neurodegeneration.

Neurodegeneration has been increasingly recognised as the leading structural correlate of disability progression in autoimmune diseases such as multiple sclerosis. Since calcium signalling is known to regulate the development of degenerative processes in many cell types, it is believed to play significant roles in mediating neurodegeneration. Because of its function as a major juncture linking various insults and injuries associated with inflammatory attack on neuronal cell bodies and axons, it provides potential for the development of neuroprotective strategies. This is of great significance because of the lack of neuroprotective agents presently available to supplement the current array of immunomodulatory treatments. In this review, we summarise the role that various calcium channels and pumps have been shown to play in the development of neurodegeneration under inflammatory autoimmune conditions. The identification of suitable targets might also provide insights into applications in non-inflammatory neurodegenerative diseases.

Neurological deficits caused by tissue hypoxia in neuroinflammatory disease.

OBJECTIVE: To explore the presence and consequences of tissue hypoxia in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: EAE was induced in Dark Agouti rats by immunization with recombinant myelin oligodendrocyte glycoprotein and adjuvant. Tissue hypoxia was assessed in vivo using 2 independent methods: an immunohistochemical probe administered intravenously, and insertion of a physical, oxygen-sensitive probe into the spinal cord. Indirect markers of tissue hypoxia (eg, expression of hypoxia-inducible factor-1α [HIF-1α], vessel diameter, and number of vessels) were also assessed. The effects of brief (1 hour) and continued (7 days) normobaric oxygen treatment on function were evaluated in conjunction with other treatments, namely administration of a mitochondrially targeted antioxidant (MitoQ) and inhibition of inducible nitric oxide synthase (1400W). RESULTS: Observed neurological deficits were quantitatively, temporally, and spatially correlated with spinal white and gray matter hypoxia. The tissue expression of HIF-1α also correlated with loss of function. Spinal microvessels became enlarged during the hypoxic period, and their number increased at relapse. Notably, oxygen administration significantly restored function within 1 hour, with improvement persisting at least 1 week with continuous oxygen treatment. MitoQ and 1400W also caused a small but significant improvement. INTERPRETATION: We present chemical, physical, immunohistochemical, and therapeutic evidence that functional deficits caused by neuroinflammation can arise from tissue hypoxia, consistent with an energy crisis in inflamed central nervous system tissue. The neurological deficit was closely correlated with spinal white and gray matter hypoxia. This realization may indicate new avenues for therapy of neuroinflammatory diseases such as MS.

Protocol for generating and analyzing organ-on-chip using human and mouse intestinal organoids.

Organ-on-chip technology is a powerful tool for in vitro modeling. Combining it with organoids overcomes lumen inaccessibility while preserving cellular diversity and function of the intestinal epithelium. Here, we present a protocol for generating and analyzing organ-on-chips using human and mouse intestinal organoids. This protocol covers organoid line establishment, single-cell dissociation, chip preparation, and seeding. It outlines procedures for permeability assays, RNA isolation, staining, and imaging. Additionally, we describe independent stimulation and sampling of the apical and basal side.

Preclinical retinal neurodegeneration in a model of multiple sclerosis.

Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.

Neuroprotective effects of the cellular prion protein in autoimmune optic neuritis.

Although the pathologic role of the prion protein in transmissible spongiform encephalopathic diseases has been widely investigated, the physiologic role of the cellular prion protein (PrP(C)) is not known. Among the many functions attributed to PrP(C), there is increasing evidence that it is involved in cell survival and mediates neuroprotection. A potential role in the immune response has also been suggested. However, how these two functions interplay in autoimmune disease is unclear. To address this, autoimmune optic neuritis, a model of multiple sclerosis, was induced in C57Bl/6 mice, and up-regulation of PrP(C) was observed throughout the disease course. In addition, compared with wild-type mice, in PrP(C)-deficient mice and mice overexpressing PrP(C), histopathologic analysis demonstrated that optic neuritis was exacerbated, as indicated by axonal degeneration, inflammatory infiltration, and demyelination. However, significant neuroprotection of retinal ganglion cells, the axons of which form the optic nerve, was observed in mice that overexpressed PrP(C). Conversely, mice lacking PrP(C) demonstrated significantly more neurodegeneration. This suggests that PrP(C) may have a neuroprotective function independent of its role in regulating the immune response.

Retinal Glutamate Neurotransmission: From Physiology to Pathophysiological Mechanisms of Retinal Ganglion Cell Degeneration.

Glutamate neurotransmission and metabolism are finely modulated by the retinal network, where the efficient processing of visual information is shaped by the differential distribution and composition of glutamate receptors and transporters. However, disturbances in glutamate homeostasis can result in glutamate excitotoxicity, a major initiating factor of common neurodegenerative diseases. Within the retina, glutamate excitotoxicity can impair visual transmission by initiating degeneration of neuronal populations, including retinal ganglion cells (RGCs). The vulnerability of RGCs is observed not just as a result of retinal diseases but has also been ascribed to other common neurodegenerative and peripheral diseases. In this review, we describe the vulnerability of RGCs to glutamate excitotoxicity and the contribution of different glutamate receptors and transporters to this. In particular, we focus on the N-methyl-d-aspartate (NMDA) receptor as the major effector of glutamate-induced mechanisms of neurodegeneration, including impairment of calcium homeostasis, changes in gene expression and signalling, and mitochondrial dysfunction, as well as the role of endoplasmic reticular stress. Due to recent developments in the search for modulators of NMDA receptor signalling, novel neuroprotective strategies may be on the horizon.

Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components.

Neurexins are cell-surface molecules that bind neuroligins to form a heterophilic, Ca(2+)-dependent complex at central synapses. This transsynaptic complex is required for efficient neurotransmission and is involved in the formation of synaptic contacts. In addition, both molecules have been identified as candidate genes for autism. Here we performed mutagenesis experiments to probe for essential components of the neurexin/neuroligin binding interface at the single-amino acid level. We found that in neurexins the contact area is sharply delineated and consists of hydrophobic residues of the LNS domain that surround a Ca(2+) binding pocket. Point mutations that changed electrostatic and shape properties leave Ca(2+) coordination intact but completely inhibit neuroligin binding, whereas alternative splicing in alpha- and beta-neurexins and in neuroligins has a weaker effect on complex formation. In neuroligins, the contact area appears less distinct because exchange of a more distant aspartate completely abolished binding to neurexin but many mutations of predicted interface residues had no strong effect on binding. Together with calculations of energy terms for presumed interface hot spots that complement and extend our mutagenesis and recent crystal structure data, this study presents a comprehensive structural basis for the complex formation of neurexins and neuroligins and their transsynaptic signaling between neurons.

Pathophysiological Ionotropic Glutamate Signalling in Neuroinflammatory Disease as a Therapeutic Target.

Glutamate signalling is an essential aspect of neuronal communication involving many different glutamate receptors, and underlies the processes of memory, learning and synaptic plasticity. Despite neuroinflammatory diseases covering a range of maladies with very different biological causes and pathophysiologies, a central role for dysfunctional glutamate signalling is becoming apparent. This is not just restricted to the well-described role of glutamate in mediating neurodegeneration, but also includes a myriad of other influences that glutamate can exert on the vasculature, as well as immune cell and glial regulation, reflecting the ability of neurons to communicate with these compartments in order to couple their activity with neuronal requirements. Here, we discuss the role of pathophysiological glutamate signalling in neuroinflammatory disease, using both multiple sclerosis and Alzheimer's disease as examples, and how current steps are being made to harness our growing understanding of these processes in the development of neuroprotective strategies. This review focuses in particular on N-methyl-D-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methylisooxazol-4-yl) propionate (AMPA) type ionotropic glutamate receptors, although metabotropic, G-protein-coupled glutamate receptors may also contribute to neuroinflammatory processes. Given the indispensable roles of glutamate-gated ion channels in synaptic communication, means of pharmacologically distinguishing between physiological and pathophysiological actions of glutamate will be discussed that allow deleterious signalling to be inhibited whilst minimising the disturbance of essential neuronal function.

The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation.

Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses via TNFR2 intact, to induce therapeutic effects. Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.

Role of n-type voltage-dependent calcium channels in autoimmune optic neuritis.

OBJECTIVE: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis. METHODS: Calcium ion (Ca(2+)) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using omega-conotoxin GVIA, an N-type specific blocker. RESULTS: We observed that pathological Ca(2+) influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of alpha(1B), the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with omega-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. INTERPRETATION: We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca(2+) influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage. Ann Neurol 2009;66:81-93.

VEGFD Protects Retinal Ganglion Cells and, consequently, Capillaries against Excitotoxic Injury.

In the central nervous system, neurons and the vasculature influence each other. While it is well described that a functional vascular system is trophic to neurons and that vascular damage contributes to neurodegeneration, the opposite scenario in which neural damage might impact the microvasculature is less defined. In this study, using an in vivo excitotoxic approach in adult mice as a tool to cause specific damage to retinal ganglion cells, we detected subsequent damage to endothelial cells in retinal capillaries. Furthermore, we detected decreased expression of vascular endothelial growth factor D (VEGFD) in retinal ganglion cells. In vivo VEGFD supplementation via neuronal-specific viral-mediated expression or acute intravitreal delivery of the mature protein preserved the structural and functional integrity of retinal ganglion cells against excitotoxicity and, additionally, spared endothelial cells from degeneration. Viral-mediated suppression of expression of the VEGFD-binding receptor VEGFR3 in retinal ganglion cells revealed that VEGFD exerts its protective capacity directly on retinal ganglion cells, while protection of endothelial cells is the result of upheld neuronal integrity. These findings suggest that VEGFD supplementation might be a novel, clinically applicable approach for neuronal and vascular protection.

ERG Responses in Mice with Deletion of the Synaptic Ribbon Component RIBEYE.

Purpose: To determine the influence of RIBEYE deletion and the resulting absence of synaptic ribbons on retinal light signaling by electroretinography. Methods: Full-field flash electroretinograms (ERGs) were recorded in RIBEYE knock-out (KO) and wild-type (WT) littermate mice under photopic and scotopic conditions, with oscillatory potentials (OPs) extracted by digital filtering. Flicker ERGs and ERGs following intravitreal injection of pharmacological agents were also obtained under scotopic conditions. Results: The a-wave amplitudes were unchanged between RIBEYE KO and WT mice; however, the b-wave amplitudes were reduced in KOs under scotopic, but not photopic, conditions. Increasing stimulation frequency led to a greater reduction in RIBEYE KO b-wave amplitudes compared with WTs. Furthermore, we observed prominent, supernormal OPs in RIBEYE KO mice in comparison with WT mice. Following intravitreal injections with l-2 amino-4-phosphonobutyric acid and cis-2,3 piperidine dicarboxylic acid to block ON and OFF responses at photoreceptor synapses, OPs were completely abolished in both mice types, indicating a synaptic origin of the prominent OPs in the KOs. Conversely, tetrodotoxin treatment to block voltage-gated Na+ channels/spiking neurons did not differentially affect OPs in WT and KO mice. Conclusions: The decreased scotopic b-wave and decreased responses to increased stimulation frequencies are consistent with signaling malfunctions at photoreceptor and inner retinal ribbon synapses. Because phototransduction in the photoreceptor outer segments is unaffected in the KOs, their supernormal OPs presumably result from a dysfunction in retinal synapses. The relatively mild ERG phenotype in KO mice, particularly in the photopic range, is probably caused by compensatory mechanisms in retinal signaling pathways.

Polarized targeting of neurexins to synapses is regulated by their C-terminal sequences.

Two families of cell-adhesion molecules, predominantly presynaptic neurexins and postsynaptic neuroligins, are important for the formation and functioning of synapses in the brain, and mutations in several genes encoding these transmembrane proteins have been found in autism patients. However, very little is known about how neurexins are targeted to synapses and which mechanisms regulate this process. Using various epitope-tagged neurexins in primary hippocampal neurons of wild-type and knock-out mice in vitro and in transgenic animals in vivo, we show that neurexins are trafficked throughout neurons via transport vesicles and the plasma membrane insertion of neurexins occurs preferentially in the axonal/synaptic compartment. We also observed that exit of neurexins from the ER/Golgi and correct targeting require their PDZ-binding motif at the C terminus, whereas two presumptive ER retention signals are inactive. The ubiquitous presence of neurexin-positive transport vesicles and absence of bassoon colabeling demonstrate that these carriers are not active zone precursor vesicles, but colocalization with CASK, RIM1alpha, and calcium channels suggests that they may carry additional components of the exocytotic machinery. Our data indicate that neurexins are delivered to synapses by a polarized and regulated targeting process that involves PDZ-domain mediated interactions, suggesting a novel pathway for the distribution of neurexins and other synaptic proteins.