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

Accepted Manuscript A Truncating Mutation in Alzheimer’s Disease Inactivates Neuroligin-1 Synaptic Function Enriqueta Tristán-Clavijo, Rafael J. Camacho-Garcia, Estefanía Robles-Lanuza, Agustín Ruiz, Julie van der Zee, Christine Van Broeckhoven, Isabel Hernandez, Amalia Martinez-Mir, Francisco G. Scholl PII: S0197-4580(15)00450-9 DOI: 10.1016/j.neurobiolaging.2015.09.004 Reference: NBA 9381 To appear in: Neurobiology of Aging Received Date: 26 June 2015 Revised Date: 2 September 2015 Accepted Date: 3 September 2015 Please cite this article as: Tristán-Clavijo, E., Camacho-Garcia, R.J., Robles-Lanuza, E., Ruiz, A., van der Zee, J., Van Broeckhoven, C., Hernandez, I., Martinez-Mir, A., Scholl, F.G., A Truncating Mutation in Alzheimer’s Disease Inactivates Neuroligin-1 Synaptic Function, Neurobiology of Aging (2015), doi: 10.1016/j.neurobiolaging.2015.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 A Truncating Mutation in Alzheimer’s Disease Inactivates Neuroligin-1 Synaptic Function Enriqueta Tristán-Clavijoa,b, Rafael J. Camacho-Garciaa, Estefanía RoblesLanuzaa,b, Agustín Ruizc, Julie van der Zeed,e, Christine Van Broeckhovend,e, Isabel Hernandezc, Amalia Martinez-Mira,*, Francisco G. Scholla,b,* aInstituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain bDepartamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain cResearch Center and Memory Clinic. Fundació ACE. Institut Català de Neurociències Aplicades, Barcelona, Spain dNeurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Universiteitsplein 1, Antwerp, Belgium eLaboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium *Corresponding authors at: Instituto de Biomedicina de Sevilla (IBiS), Avda Manuel Siurot s/n, 41013 Sevilla, Spain, Tel.: +34 955923042; fax: +34 955923101. E-mail address: [email protected] (F.G. Scholl), [email protected] (A. Martinez-Mir). MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2 ABSTRACT Neuroligins 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 showed accumulation of truncated NL1 proteins in the ER. 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 neuroligin function could underlie the molecular mechanisms associated with brain diseases in the elderly. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 1. Introduction Alzheimer’s disease (AD) is a neurodegenerative illness characterized by progressive memory loss in the patients. Increasing evidence suggests that synapses are a main pathological target in AD (Selkoe, 2002; Spires-Jones and Hyman, 2014). Indeed, synapse loss is the best pathological correlate of cognitive dysfunction in AD patients (DeKosky and Scheff, 1990; DeKosky, et al., 1996; Masliah, et al., 2001; Terry, et al., 1991). Therefore, perturbation of synaptic function is a key event for memory decline in AD. Neuroligins (NLs) are postsynaptic proteins that regulate excitatory and inhibitory synapse formation and function by trans-synaptic interaction with their presynaptic receptors, such as neurexins (Dean, et al., 2003; Scheiffele, et al., 2000; Sudhof, 2008). In humans, NLs are coded by five genes located in autosomic (NLGN1 and NLGN 2) and sexual chromosomes (NLGN3, NLGN4 and NLGN4Y). NLs have received especial attention since the identification of mutations in patients with autism spectrum disorders (ASD). Bourgeron and colleagues identified one missense and one frameshift mutation in X-linked NLGN3 and NLGN4 in two pairs of brothers with ASD, respectively (Jamain, et al., 2003). Following this initial finding, rare mutations in NRXN1, NLGN3 and NLGN4 have been described in patients with neurodevelopmental diseases, including autism and intellectual disability (Camacho-Garcia, et al., 2012; Laumonnier, et al., 2004; Lawson-Yuen, et al., 2008; Rabaneda, et al., 2014; Yan, et al., 2005; Zhang, et al., 2009). Although the number of identified mutations in NLGN genes is low, functional characterization of the mutant NL3 and NL4 proteins have helped to identify a convergent disease mechanism. Autism-associated mutations in NLGN3 and NLGN4 often induce the MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 4 accumulation of the mutant proteins in the ER and impair their synaptogenic activity (Chih, et al., 2004; Chubykin, et al., 2005; Zhang, et al., 2009). Therefore, mutations in NGLN genes are so far restricted to neurodevelopmental disorders. NL1 localizes at glutamatergic postsynaptic terminals and expression of NL1 in neurons promotes the formation of glutamatergic synapses (Chih, et al., 2005; Scheiffele, et al., 2000; Song, et al., 1999). Moreover, NL1 and neurexins are substrates for presenilins (PS), a γ-secretase component frequently mutated in familial AD (Peixoto, et al., 2012; Saura, et al., 2011; Suzuki, et al., 2012). Based on these data, defects in NL1 function might underlie synaptic and memory deficits associated with AD (Bie, et al., 2014; Martinez-Mir, et al., 2013; Sindi, et al., 2014). However, the role of NLGN genes in AD is unclear since no mutations have been described in patients. Here we report a frameshift mutation in NLGN1 gene in a familial case of AD. The frameshift mutation truncates the protein at the extracellular domain, induces ER accumulation, and inhibits the induction of glutamatergic synapses. These data extends the role for mutations in NLGN genes to AD. 2. Subjects and Methods The coding region of the NLGN1 gene was sequenced in a group of 192 patients with AD using Haloplex target enrichment (Agilent Technologies, USA) and a MiSeq sequencing platform (Illumina, USA). The c.875_876insTT NLGN1 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 mutation was confirmed by Sanger sequencing in the index case (for a full clinical description of the patient, refer to Supplementary Material). For transfection in N2A and COS cells, cultures were transfected with Lipofectamine 2000 (Invitrogen, USA) and analyzed 24-48 hours later. Hippocampal cultures were obtained from E18-E19 rat brains and maintained in in neurobasal medium (Invitrogen, USA) containing B27 (LifeTechnologies, UK), Glutamax and penicillin/streptomycin (Invitrogen, USA). Neurons were transfected at 9-11 days in vitro and analyzed 48 hours later by immunofluorescence. The following primary antibodies were used for immunodetection: mouse anti-calnexin (Thermo Scientifc, USA), rat anti-HA (Roche, Switzerland), rabbit anti-vGlut1 (Synaptic Systems, Germany). 3. Results We identified a two base-pair insertion (c.875_876insTT; p.Thr271fs) in NLGN1 in a patient with AD (Fig. 1A). The index case experienced the first events of memory loss at 65, when she was still occupationally active, but no objective changes were observed in a neurological and a neuropsychological exploration. Her medical history was otherwise unremarkable. After retirement, she suffered from depression and progressive memory loss. The patient received a diagnosis of AD at the age of 71 (Supplementary Material). MRI showed cerebral atrophy with biparietal predominance (Supplementary Fig.1). Genetic screening of AD-associated genes, including PS1, PS2 and APP, and NLGN2, NLGN3 and NLGN4 genes revealed no further mutations in the patient. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 6 Her mother and three of her brothers had AD (Fig. 1B). She married with a first cousin and had five children, three of whom were diagnosed with schizophrenia, ADHD and bipolar disorder (Fig. 1B). Based on the role of mutations in NLGN3 and NLGN4 in neurodevelopmental disorders, we analyzed the presence of the NLGN1 p.Thr271fs mutation in the progeny. The mutation was absent in the father and in the two participating daughters, one with ADHD and another one with no mental disease (Fig. 1B). The rest of the offspring declined to participate, which precluded the analysis of the mutation in these family members. Although a role of the mutation cannot be excluded in the non-participating offspring, the absence of p.Thr271fs mutation in the daughter with ADHD might suggest an effect of consanguinity on the progeny with mental disorders. The two-base pair insertion c.876_877insTT affects residue Thr271 of NL1, which is located upstream of the alternatively spliced site B (Fig. 1C). The insertion predicts a premature STOP codon at position 284 for NL1 isoforms lacking the B site (NL1 284X) or at position 293 for NL1 isoforms containing the B site (NL1 293X) (Fig. 1D). The predicted truncated proteins lack the dimerization domains of NLs (Dean, et al., 2003). To analyze the effect of the mutation on NL1 function, we generated expression vectors for NL1 284X and NL1 293X proteins tagged at the N-terminus with a HA-epitope. As controls, we used expression constructs for HA-tagged NL1 (+B) and NL1 (-B), containing or lacking the B site respectively. In Western blot experiments of transfected N2A cells, the HA antibody detected bands of ∼130 kDa for the wild type NL1 proteins, corresponding to mature proteins (Fig. 1E). In contrast, the p.Thr271fs mutation resulted in truncated proteins of ∼ 30 kDa for NL1 284X and NL1 293X MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 7 (Fig. 1E). Since NL1 284X and NL1 293X contain the signal sequence but lack the transmembrane domain, we analyzed the presence of the mutant proteins in the cell media. We found that NL1 284X and NL1 293X could be detected in the media of transfected cells, suggesting that mutant NL1 proteins can be incorporated into the secretory pathway (Fig. 1E). The fact that NL1 proteins were detected as faster migrating bands in the media than in the cell lysates likely reflects the shedding of the ectodomain of the mature NL1 (Fig. 1E) (Peixoto, et al., 2012; Suzuki, et al., 2012). We further studied the subcellular localization of wild type and mutant NL1 proteins by immunofluorescence. Wild type NL1 (-B) and NL1 (+B) were localized at the cell surface of transfected COS cells, as indicated by the HAstaining at the cell periphery (Fig. 1F). In contrast, mutant NL1 284X and NL1 293X did not show localization at the plasma membrane, but were concentrated in an intracellular compartment (Fig. 1F). Co-staining experiments with the ER marker calnexin showed a substantial colocalization with NL1 284X and NL1 293X proteins (Fig. 1F). These data indicate that the AD-associated mutation p.Thr271fs results in mutant NL1 proteins that accumulate in the ER and do not reach the cell surface. NL1 promotes the formation of glutamatergic synapses when transfected into neurons (Chih, et al., 2006; Song, et al., 1999). We therefore analyzed whether NL1 2846X and NL1 293X can induce synapse formation in cultured neurons. With this aim, we co-transfected hippocampal neurons with GFP alone or with wild type or mutant NL1-expressing vectors. The density of glutamatergic synapses on the transfected neurons was analyzed with the glutamatergic marker vGlut1. As previously reported, NL1 (-B) and NL1 (+B) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 8 showed a dendritic localization and increased the density of vGlut1-positive synapses on the transfected dendrites (Fig. 2). Notably, the synaptogenic activity of NL1 was abolished by the p.Thr271fs mutation. NL1 284X and NL1 293X accumulated mostly in the soma of transfected neurons, consistent with the ER accumulation detected in COS cells (Fig. 2). In dendrites, NL1 284X and NL1 293X were expressed at low levels in dendritic segments proximal to the soma. Importantly, NL1 284X and NL1 293X proteins failed to induce synaptic differentiation, as the mutant NL1 proteins did not increase the number of vGlut1-positive terminals formed on the surface of the transfected dendrites. These data indicate that the p.Thr271fs mutation in NLGN1 results in the production of truncated NL1 proteins that accumulate in the ER and fail to promote the formation of glutamatergic synapses. 4. Discussion Our study supports a role for NL1 in the etiology of AD. We identified a frameshift mutation in NLGN1 in a familial case of AD. The p.Thr271fs mutation results in the generation of NL1 proteins truncated at the extracellular domain, which accumulate in intracellular compartments of neurons. Mutant NL1 proteins fail to reach the plasma membrane and to induce the formation of glutamatergic terminals in hippocampal neurons. Thus, the frameshift mutation identified here in AD results in the inactivation of the mutant NLGN1 allele. It is puzzling to note that the effect of the mutation in NLGN1 in AD parallels that of mutations in X-linked NLGN genes identified in ASD, as rare MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 15 fear memory in the amygdala of adult animals. Proc. Natl. Acad. Sci. USA 105, 9087-92. 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MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT • We have identified a frameshift mutation in NLGN1 in Alzheimer’s disease. • The mutation results in the accumulation of truncated neuroligin-1 in the ER. • The AD-associated mutation abolishes the synaptogenic activity of neuroligin-1. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Supplementary Material Case report Woman, 71 years old at first visit. She had a degree in nursing (midwife) and was married with a first cousin. Her mother and three siblings had dementia. A son has schizophrenia and two daughters have bipolar disorder and ADHD. Six years before diagnosis, when still occupationally active, she complained of memory loss. However, no objective changes were observed in a first neurological and neuropsychological exploration. She suffered form depression at 65 years old. She had had a very active life and her family thought that the problem was a consequence of retirement. During the visit of diagnosis, her family referred that she had lost their executive ability, her memory had deteriorated and she had great difficulty with linguistic expression. She showed disorientation, even at home. The family also referred acalculia, apraxic and agnosic symptoms. She was aware of her illness and had significant distress about the possibility of her offspring suffering the disease. Neurological exploration: No focal signs. Normal extrapyramidal features and walking. MMSE: 20. Blessed: 5/0/1. NPI-Q Severity 3, NPI-Q stress 5. Neuropsychological exploration: The exploration was performed following the neuropsychological battery NBACE (Alegret, et al., 2012). The patient was right-handed and had mild disorientation in all three areas (time, place and person). The patient showed marked impairment in attention and learning abilities. Verbal retention was impaired, as well as the imitation motor praxis, visuospatial and visual complex gnosis. The inhibition of automatic responses and abstract reasoning was also impaired. Anomic spontaneous language, phonetic and semantic verbal MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT fluencies, as well as ideomotor and constructive praxis, were impaired. Repetition, comprehension and writing were relatively preserved. Time orientation: 4/5, place orientation: 4/5, personal orientation: 3/5, learning ability: WMS-III: 1,4,3,3 . Retention WMS-III: 0/12. Recognition: 17/24. Boston denomination: 7/15. Poppel gnosis: 10/10. Praxis: 8/12. Phonetic verbal fluency: 7/10. Phonetic semantic fluency : 6/12. Clock test: 3/7. 15-Object test: 8/15. The patient received a diagnosis of Alzheimer’s Disease according to the new NIA-AA criteria (McKhann, et al., 2011). Clinical evolution was defined by rapidly impairment in praxis and gnosis. Supplementary Fig. 1. 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