A Neuroligin-1 mutation associated with Alzheimer's disease produces memory and age-dependent impairments in hippocampal plasticity.

Alzheimer's disease (AD) is characterized by memory impairments and age-dependent synapse loss. Experimental and clinical studies have shown decreased expression of the glutamatergic protein Neuroligin-1 (Nlgn1) in AD. However, the consequences of a sustained reduction of Nlgn1 are unknown. Here, we generated a knockin mouse that reproduces the NLGN1 Thr271fs mutation, identified in heterozygosis in a familial case of AD. We found that Nlgn1 Thr271fs mutation abolishes Nlgn1 expression in mouse brain. Importantly, heterozygous Nlgn1 Thr271fs mice showed delay-dependent amnesia for recognition memory. Electrophysiological recordings uncovered age-dependent impairments in basal synaptic transmission and long-term potentiation (LTP) in CA1 hippocampal neurons of heterozygous Nlgn1 Thr271fs mice. In contrast, homozygous Nlgn1 Thr271fs mice showed impaired fear-conditioning memory and normal basal synaptic transmission, suggesting unshared mechanisms for a partial or total loss of Nlgn1. These data suggest that decreased Nlgn1 may contribute to the synaptic and memory deficits in AD.

Selective expression of the neurexin substrate for presenilin in the adult forebrain causes deficits in associative memory and presynaptic plasticity.

Presenilins (PS) form the active subunit of the gamma-secretase complex, which mediates the proteolytic clearance of a broad variety of type-I plasma membrane proteins. Loss-of-function mutations in PSEN1/2 genes are the leading cause of familial Alzheimer's disease (fAD). However, the PS/gamma-secretase substrates relevant for the neuronal deficits associated with a loss of PS function are not completely known. The members of the neurexin (Nrxn) family of presynaptic plasma membrane proteins are candidates to mediate aspects of the synaptic and memory deficits associated with a loss of PS function. Previous work has shown that fAD-linked PS mutants or inactivation of PS by genetic and pharmacological approaches failed to clear Nrxn C-terminal fragments (NrxnCTF), leading to its abnormal accumulation at presynaptic terminals. Here, we generated transgenic mice that selectively recreate the presynaptic accumulation of NrxnCTF in adult forebrain neurons, leaving unaltered the function of PS/gamma-secretase complex towards other substrates. Behavioral characterization identified selective impairments in NrxnCTF mice, including decreased fear-conditioning memory. Electrophysiological recordings in medial prefrontal cortex-basolateral amygdala (mPFC-BLA) of behaving mice showed normal synaptic transmission and uncovered specific defects in synaptic facilitation. These data functionally link the accumulation of NrxnCTF with defects in associative memory and short-term synaptic plasticity, pointing at impaired clearance of NrxnCTF as a new mediator in AD.

Non-productive angiogenesis disassembles Aß plaque-associated blood vessels.

The human Alzheimer's disease (AD) brain accumulates angiogenic markers but paradoxically, the cerebral microvasculature is reduced around Aß plaques. Here we demonstrate that angiogenesis is started near Aß plaques in both AD mouse models and human AD samples. However, endothelial cells express the molecular signature of non-productive angiogenesis (NPA) and accumulate, around Aß plaques, a tip cell marker and IB4 reactive vascular anomalies with reduced NOTCH activity. Notably, NPA induction by endothelial loss of presenilin, whose mutations cause familial AD and which activity has been shown to decrease with age, produced a similar vascular phenotype in the absence of Aß pathology. We also show that Aß plaque-associated NPA locally disassembles blood vessels, leaving behind vascular scars, and that microglial phagocytosis contributes to the local loss of endothelial cells. These results define the role of NPA and microglia in local blood vessel disassembly and highlight the vascular component of presenilin loss of function in AD.

Proteolytic Processing of Neurexins by Presenilins Sustains Synaptic Vesicle Release.

Proteolytic processing of synaptic adhesion components can accommodate the function of synapses to activity-dependent changes. The adhesion system formed by neurexins (Nrxns) and neuroligins (Nlgns) bidirectionally orchestrate the function of presynaptic and postsynaptic terminals. Previous studies have shown that presenilins (PS), components of the gamma-secretase complex frequently mutated in familial Alzheimer's disease, clear from glutamatergic terminals the accumulation of Nrxn C-terminal fragments (Nrxn-CTF) generated by ectodomain shedding. Here, we characterized the synaptic consequences of the proteolytic processing of Nrxns in cultured hippocampal neurons from mice and rats of both sexes. We show that activation of presynaptic Nrxns with postsynaptic Nlgn1 or inhibition of ectodomain shedding in axonal Nrxn1-ß increases presynaptic release at individual terminals, likely reflecting an increase in the number of functional release sites. Importantly, inactivation of PS inhibits presynaptic release downstream of Nrxn activation, leaving synaptic vesicle recruitment unaltered. Glutamate-receptor signaling initiates the activity-dependent generation of Nrxn-CTF, which accumulate at presynaptic terminals lacking PS function. The sole expression of Nrxn-CTF decreases presynaptic release and calcium flux, recapitulating the deficits due to loss of PS function. Our data indicate that inhibition of Nrxn processing by PS is deleterious to glutamatergic function.SIGNIFICANCE STATEMENT To gain insight into the role of presenilins (PS) in excitatory synaptic function, we address the relevance of the proteolytic processing of presynaptic neurexins (Nrxns) in glutamatergic differentiation. Using synaptic fluorescence probes in cultured hippocampal neurons, we report that trans-synaptic activation of Nrxns produces a robust increase in presynaptic calcium levels and neurotransmitter release at individual glutamatergic terminals by a mechanism that depends on normal PS activity. Abnormal accumulation of Nrxn C-terminal fragments resulting from impaired PS activity inhibits presynaptic calcium signal and neurotransmitter release, assigning synaptic defects to Nrxns as a specific PS substrate. These data may provide links into how loss of PS activity inhibits glutamatergic synaptic function in Alzheimer's disease patients.

Lack of replication of previous autism spectrum disorder GWAS hits in European populations.

Common variants contribute significantly to the genetics of autism spectrum disorder (ASD), although the identification of individual risk polymorphisms remains still elusive due to their small effect sizes and limited sample sizes available for association studies. During the last decade several genome-wide association studies (GWAS) have enabled the detection of a few plausible risk variants. The three main studies are family-based and pointed at SEMA5A (rs10513025), MACROD2 (rs4141463) and MSNP1 (rs4307059). In our study we attempted to replicate these GWAS hits using a case-control association study in five European populations of ASD patients and gender-matched controls, all Caucasians. Results showed no association of individual variants with ASD in any of the population groups considered or in the combined European sample. We performed a meta-analysis study across five European populations for rs10513025 (1,904 ASD cases and 2,674 controls), seven European populations for rs4141463 (2,855 ASD cases and 36,177 controls) and five European populations for rs4307059 (2,347 ASD cases and 2,764 controls). The results showed an odds ratio (OR) of 1.05 (95% CI = 0.84-1.32) for rs10513025, 1.0002 (95% CI = 0.93-1.08) for rs4141463 and 1.01 (95% CI = 0.92-1.1) for rs4307059, with no significant P-values (rs10513025, P = 0.73; rs4141463, P = 0.95; rs4307059, P = 0.9). No association was found when we considered either only high functioning autism (HFA), genders separately or only multiplex families. Ongoing GWAS projects with larger ASD cohorts will contribute to clarify the role of common variation in the disorder and will likely identify risk variants of modest effect not detected previously. Autism Res 2017, 10: 202-211. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Dominant-negative mutation p.Arg324Thr in KCNA1 impairs Kv1.1 channel function in episodic ataxia.

BACKGROUND: Episodic ataxia type 1 is a rare autosomal dominant neurological disorder caused by mutations in the KCNA1 gene that encodes the α subunit of voltage-gated potassium channel Kv1.1. The functional consequences of identified mutations on channel function do not fully correlate with the clinical phenotype of patients. METHODS: A clinical and genetic study was performed in a family with 5 patients with episodic ataxia type 1, with concurrent epilepsy in 1 of them. Protein expression, modeling, and electrophysiological analyses were performed to study Kv1.1 function. RESULTS: Whole-genome linkage and candidate gene analyses revealed the novel heterozygous mutation p.Arg324Thr in the KCNA1 gene. The encoded mutant Kv1.1 channel displays reduced currents and altered activation and inactivation. CONCLUSIONS: Taken together, we provide genetic and functional evidence that mutation p.Arg324Thr in the KCNA1 gene is pathogenic and results in episodic ataxia type 1 through a dominant-negative effect. © 2016 International Parkinson and Movement Disorder Society.

A truncating mutation in Alzheimer's disease inactivates neuroligin-1 synaptic function.

Neuroligins (NLs) are cell-adhesion proteins that regulate synapse formation and function. Neuroligin 1 (NL1) promotes the formation of glutamatergic synapses and mediates long-term potentiation in mouse models. Thus, altered NL1 function could mediate the synaptic and memory deficits associated with Alzheimer's disease (AD). Here, we describe a frameshift mutation, c.875_876insTT, in the neuroligin 1 gene (NLGN1) in a patient with AD and familial history of AD. The insertion generates a premature stop codon in the extracellular domain of NL1 (p.Thr271fs). Expression of mutant NL1 shows accumulation of truncated NL1 proteins in the endoplasmic reticulum. In hippocampal neurons, the p.Thr271fs mutation abolishes the ability of NL1 to promote the formation of glutamatergic synapses. Our data support a role for inactivating mutations in NLGN1 in AD. Previous studies have reported rare mutations in X-linked NLGNL3 and NLGNL4 genes in patients with autism, which result in the inactivation of the mutant alleles. Therefore, together with a role in neurodevelopmental disorders, altered NL function could underlie the molecular mechanisms associated with brain diseases in the elderly.

Neurexin dysfunction in adult neurons results in autistic-like behavior in mice.

Autism spectrum disorders (ASDs) comprise a group of clinical phenotypes characterized by repetitive behavior and social and communication deficits. Autism is generally viewed as a neurodevelopmental disorder where insults during embryonic or early postnatal periods result in aberrant wiring and function of neuronal circuits. Neurexins are synaptic proteins associated with autism. Here, we generated transgenic ßNrx1ΔC mice in which neurexin function is selectively impaired during late postnatal stages. Whole-cell recordings in cortical neurons show an impairment of glutamatergic synaptic transmission in the ßNrx1ΔC mice. Importantly, mutant mice exhibit autism-related symptoms, such as increased self-grooming, deficits in social interactions, and altered interaction for nonsocial olfactory cues. The autistic-like phenotype of ßNrx1ΔC mice can be reversed after removing the mutant protein in aged animals. The defects resulting from disruption of neurexin function after the completion of embryonic and early postnatal development suggest that functional impairment of mature circuits can trigger autism-related phenotypes.

Rare variants analysis of neurexin-1ß in autism reveals a novel start codon mutation affecting protein levels at synapses.

Neurexins are synaptic plasma membrane proteins encoded by three genes (NRXN1, -2, -3) with alternative promoters. Mutations in neurexin genes have been identified in different neurodevelopmental disorders, including autism. Recently, two point mutations altering the translation initiation site of NRXN1ß (c.-3G>T and c.3G>T) have been described in patients with autism and mental retardation. In this study, we analyzed the NRXN1ß gene in a sample of 153 patients with autism. We report the identification of a novel mutation, c.3G>A (p.Met1), affecting the translation initiation site. Expression analysis showed that the c.3G>A mutation switches the translation start site of NRXN1ß to an in-frame downstream methionine and decreases synaptic levels of the mutant protein in cultured neurons. These data reinforce a role for synaptic defects of NRXN1ß in neurodevelopmental disorders.

Genetic study of neurexin and neuroligin genes in Alzheimer's disease.

The interaction between neurexins and neuroligins promotes the formation of functional synaptic structures. Recently, it has been reported that neurexins and neuroligins are proteolytically processed by presenilins at synapses. Based on this interaction and the role of presenilins in familial Alzheimer's disease (AD), we hypothesized that dysfunction of the neuroligin-neurexin pathway might be associated with AD. To explore this hypothesis, we carried out a meta-analysis of five genome-wide association studies (GWAS) comprising 1, 256 SNPs in the NRXN1, NRXN2, NRXN3, and NLGN1 genes (3,009 cases and 3,006 control individuals). We identified a marker in the NRXN3 gene (rs17757879) that showed a consistent protective effect in all GWAS, however, the statistical significance obtained did not resist multiple testing corrections (OR = 0.851, p = 0.002). Nonetheless, gender analysis revealed that this effect was restricted to males. A combined meta-analysis of the former five GWAS together with a replication Spanish sample consisting of 1,785 cases and 1,634 controls confirmed this observation (rs17757879, OR = 0.742, 95% CI = 0.632-0.872, p = 0.00028, final meta-analysis). We conclude that NRXN3 might have a role in susceptibility to AD in males.

Mutations affecting synaptic levels of neurexin-1ß in autism and mental retardation.

The identification of mutations in genes encoding proteins of the synaptic neurexin-neuroligin pathway in different neurodevelopmental disorders, including autism and mental retardation, has suggested the presence of a shared underlying mechanism. A few mutations have been described so far and for most of them the biological consequences are unknown. To further explore the role of the NRXN1ß gene in neurodevelopmental disorders, we have sequenced the coding exons of the gene in 86 cases with autism and mental retardation and 200 controls and performed expression analysis of DNA variants identified in patients. We report the identification of four novel independent mutations that affect nearby positions in two regions of the gene/protein: i) sequences important for protein translation initiation, c.-3G>T within the Kozak sequence, and c.3G>T (p.Met1), at the initiation codon; and ii) the juxtamembrane region of the extracellular domain, p.Arg375Gln and p.Gly378Ser. These mutations cosegregate with different psychiatric disorders other than autism and mental retardation, such as psychosis and attention-deficit/hyperactivity disorder. We provide experimental evidence for the use of an alternative translation initiation codon for c.-3G>T and p.Met1 mutations and reduced synaptic levels of neurexin-1ß protein resulting from p.Met1 and p.Arg375Gln. The data reported here support a role for synaptic defects of neurexin-1ß in neurodevelopmental disorders.

Presenilin/γ-secretase regulates neurexin processing at synapses.

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing ß-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect ß-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS-/- cells rescues the processing of ß-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD.

Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons.

Gene transfer methods for efficient co-expression of exogenous proteins in neurons are crucial tools towards the understanding of the molecular basis of the central nervous system. Lentiviruses are retroviral vectors that can transduce a wide variety of cells including differentiated neurons. In this work, we have generated lentiviral vectors containing dual promoters that allow efficient co-expression of exogenous proteins in neurons. We show that insertion of two copies of a human synapsin promoter/WPRE cassette in a single lentiviral vector directs robust co-expression of cDNAs in cultured neurons, while excluding expression in the surrounding glial cells. Furthermore, insertion of the tetracycline-inducible system (Tet-off) controlled by the synapsin promoter results in tightly regulated expression of EGFP when used as a transgene in cultured neurons. Transduction of primary neurons with this inducible system leads to a 100-fold increase in EGFP mRNA levels in the absence of doxycycline. In transduced cultures, EGFP transcription is inhibited within 24h upon addition of doxycycline. The viral systems we developed here provide neuron-specific and regulated expression mediated by single lentiviral vectors and will prove valuable tools for the study of neuronal function.

Silencing of neuroligin function by postsynaptic neurexins.

The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin-neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1beta are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1beta with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.

Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas.

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1alpha-2, podoplanin), a small mucin-type transmembrane glycoprotein originally identified as a cell-surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E-cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell-surface protrusions and reduced Ca(2+)-dependent cell-cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.

Making connections: cholinesterase-domain proteins in the CNS.

Recent studies have highlighted novel functions of a group of cell adhesion molecules during nervous system development. Members of this protein family are characterized by an extracellular domain with sequence homology to cholinesterases and include the neuroligins, synaptic cell adhesion molecules recently implicated in autism, and neurotactin, a cell surface receptor involved in axonal pathfinding. Although these proteins have a structural organization similar to the enzyme acetylcholinesterase, the cholinesterase domain lacks enzymatic activity and functions as a protein-protein interaction motif. This protein family provides a striking example of how the function of a catalytically active domain has evolved to mediate receptor-ligand interactions that regulate morphogenetic processes during development of the nervous system.

Neurexin mediates the assembly of presynaptic terminals.

Neurexins are a large family of proteins that act as neuronal cell-surface receptors. The function and localization of the various neurexins, however, have not yet been clarified. Beta-neurexins are candidate receptors for neuroligin-1, a postsynaptic membrane protein that can trigger synapse formation at axon contacts. Here we report that neurexins are concentrated at synapses and that purified neuroligin is sufficient to cluster neurexin and to induce presynaptic differentiation. Oligomerization of neuroligin is required for its function, and we find that beta-neurexin clustering is sufficient to trigger the recruitment of synaptic vesicles through interactions that require the cytoplasmic domain of neurexin. We propose a two-step model in which postsynaptic neuroligin multimers initially cluster axonal neurexins. In response to this clustering, neurexins nucleate the assembly of a cytoplasmic scaffold to which the exocytotic apparatus is recruited.