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Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism
SCHRES-07938; No of Pages 5 Schizophrenia Research xxx (2018) xxx–xxx
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Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism Marco Spangaro a,⁎, Marta Bosia a,b, Margherita Bechi a, Mariachiara Buonocore a, Federica Cocchi a, Carmelo Guglielmino a, Laura Bianchi a, Antonella Mastromatteo a, Cristina Lorenzi a, Roberto Cavallaro a,b a b
IRCCS San Raffaele Scientific Institute, Department of Clinical Neurosciences, Milan, Italy Vita-Salute San Raffaele University, Milan, Italy
a r t i c l e
i n f o
Article history: Received 16 October 2017 Received in revised form 27 March 2018 Accepted 24 June 2018 Available online xxxx Keywords: Glutamate uptake EAAT2 Clozapine Cognitive deficit Rehabilitation Psychosis
a b s t r a c t Cognitive deficits represent core features of schizophrenia, affecting quality of life and functioning. The excitatory amino acid transporter 2 (EAAT2) is responsible for the majority of glutamate reuptake and its activity is crucial for glutamatergic neurotransmission, prevention of excitotoxic damage and cerebral metabolism. Different studies reported that EAAT2 rs4354668 (−181 T/G) influences cognitive functions and brain structures in patients with schizophrenia. Specifically, the G allele, linked to lower EAAT2 expression, was associated with impaired prefrontal cognitive performance and reduced grey matter volumes. Cognitive remediation therapy (CRT) is one of the best available tool to treat cognitive deficits in schizophrenia, able to induce a neuroplastic modulation of cognitive functions. The present study aims to investigate the effects of rs4354668 on CRT outcome, also considering possible genotype interaction with antipsychotic (AP) treatment, since EAAT2 expression is negatively influenced by clozapine. We examined rs4354668 in 88 clinically stabilized patients with schizophrenia, treated with CRT and assessed at enrolment, at the end of CRT and after 3 months. We observed greater working memory improvements among patients carrying the T/T genotype, regardless of AP treatment. Moreover, we reported a significant interaction between pharmacological treatment and rs4354668 on executive functions, with greater improvements among T/T patients treated with APs other than clozapine. These observations suggest that impaired EAAT2 expression may attenuate CRT outcome. Moreover, our results indicate the possibility that rs4354668 could also differentially influence the response to CRT depending on the AP treatment. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Abnormalities in cognitive functions are key features of schizophrenia and critical determinants of quality of life and global functioning (Green et al., 2004). To date, cognitive remediation therapy (CRT), is one of the best available and effective tools to treat cognitive deficits and improve general functioning in schizophrenia (Wykes and Spaulding, 2011). However, the reported effects of CRT on cognitive outcome are very heterogeneous and significant predictors are still missing, also due to the lack of knowledge about the underlying neurobiological mechanisms. To date, research has mainly focused on dopaminergic pathway, given its implications in prefrontal cognitive functions and the observation of changes in dopaminergic transmission after CRT ⁎ Corresponding author at: Department of Clinical Neurosciences, IRCCS San Raffaele Scientific Institute, Vita-Salute San Raffale University, Via Stamira d'Ancona 20, 20127 Milano, Italy. E-mail address: [email protected] (M. Spangaro).
(McNab et al., 2009). Different studies reported an influence of a functional polymorphism of the catechol-O-methyltransferase gene (Val158Met) on CRT improvements, also observing an interaction between pharmacological therapy and Catechol-O-methyltransferase (COMT) genotype (Bosia et al., 2007; Bosia et al., 2014; Lindenmayer et al., 2015). Of note, the glutamatergic system is also highly implicated in neuropsychological functioning. Glutamate represents the major excitatory pathway in the central nervous system and it is involved in critical processes such as neural development, synaptic plasticity, learning and memory (Danbolt, 2001; Katagiri et al., 2001). One of the major glutamate modulators is represented by the excitatory amino acid transporter 2 (EAAT2), mainly localized at the plasma membrane of astroglial cells and responsible for N90% of cerebral glutamate uptake (Gegelashvili and Schousboe, 1998; Zhou and Danbolt, 2014). EAAT2 regulates and buffers the amount of synaptic glutamate, limits the spillover and therefore contributes to maintain the signal input specificity and to prevent neuronal damages secondary to glutamate excitotoxicity (Selkirk et al., 2005; Sheldon and Robinson, 2007).
https://doi.org/10.1016/j.schres.2018.06.059 0920-9964/© 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
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Moreover, the transporter also preserves an efficient energy metabolism through a twofold activity: on one side EAAT2 contributes to recycling of neurotransmitter pools by transporting glutamate into astrocytes for its conversion into glutamine (Marcaggi and Attwell, 2004); on the other side it mediates the process of ‘neurovascular coupling’, i.e. the increase in cerebral blood flow following neuronal activation (Robinson and Jackson, 2016). Consistently, it has been demonstrated that EAAT2 also plays a central role in the astrocyte–neuron lactate shuttle (ANLS). In ANLS, glutamate reuptake is coupled to activation of Na+/K + -ATPase that leads to uptake of glucose in astrocytes and production of lactate, which is then shuttled to the neuron. Previous work reported reduced [14C]2deoxyglucose uptake in glutamate transporter 1 (GLT-1, EAAT2 murine homologous) knockout mice. More recently it was found that stimulation of glutamate transporter through ceftriaxone results in widespread increases in glucose metabolism (Stoessl, 2017). Different substances, such as riluzole and ceftriaxone, are known to collaterally raise glutamate reuptake by increasing EAAT2 expression, with neuroprotective effects (Beller et al., 2011; Fumagalli et al., 2008; Kim et al., 2011). Interestingly, Kong and colleagues recently individuated a compound, LDN/ OSU-0212320, that specifically increases the transporter expression. Authors also demonstrated that LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation, hypothesizing possible application of the compound in the treatment of neurodegenerative disorders (Kong et al., 2014). Several studies reported an alteration of EAAT2 in schizophrenia, evidencing deficits of both expression and activity. Indeed, patients with schizophrenia displayed reduced EAAT2 expression in parahippocampal region, hippocampus, superior temporal gyrus and dorsolateral prefrontal cortex (DLPFC) (Ohnuma et al., 1998; Ohnuma et al., 2000). In line with these findings, a post-mortem study revealed an increased mRNA thalamic expression of EAAT2, indicating an abnormality in a glutamatergic corticothalamic circuit (Smith et al., 2001). Interestingly, Shan et al. also reported a lower glycosylation of the transporter, linked to impaired trafficking to plasma membrane and therefore to a lower glutamate reuptake (Shan et al., 2013). Subjects carrying the high-risk glutamate metabotropic receptor 3 haplotype, linked to schizophrenia, showed lower EAAT2 expression in the prefrontal cortex (PFC) and impaired prefrontal cognitive functions. Moreover, through functional magnetic resonance imaging (fMRI) studies, this haplotype has also been associated to dysfunctional activation patterns in the prefrontal cortex and hippocampus during cognitive tasks of verbal list learning and verbal fluency (Egan et al., 2004). The involvement of EAAT2 in the pathogenesis of cognitive deficit in schizophrenia was further supported by Featherstone et al., who showed that subchronic treatment with ketamine in rodents leads to a reduced expression of GLT-1 and to permanent changes in electroencephalography and cognition (Featherstone et al., 2012). EAAT2 transporter expression is modulated by a functional single nucleotide polymorphism (SNP), rs4354668 (−181 T/G), located in the gene promoter region, with the T allele linked to higher EAAT2 expression. Particularly, the G allele determines a consensus binding site for the repressor transcription factor GC-binding factor 2 (GCF2), which leads to increased glutamate concentrations (Mallolas et al., 2006). We previously evaluated possible influences of rs4354668 on prefrontal cognitive performance in a sample of 211 patients with schizophrenia, reporting a disadvantageous effect on executive functions and working memory (WM) among patients carrying the G allele (Spangaro et al., 2012; Spangaro et al., 2014). Interestingly, in a subsequent study we also found that poor WM performance in subjects carrying the EAAT2 G allele was associated with reduced frontal cortical volumes, suggesting the possible presence of an excitotoxic process underlying impaired cognitive performance (Poletti et al., 2014). Similar effects of rs4354668 on prefrontal cognitive performance were recently confirmed in a large-scale study including 3245 patients
and 6154 healthy controls, reporting better executive functions in subjects homozygous for the T allele, compared with the G carriers (Zhang et al., 2015). Given the influences of EAAT2 SNP on basal cognitive functions and brain structure, in this work we retrospectively evaluated possible effects of rs4354668 on CRT response. Considering the previously reported effects of EAAT2 genotype on basal WM and executive functions, we hypothesized that rs4354668 could affect changes of these functions after CRT as well. Moreover, in this study we also investigated possible interactions with pharmacological treatment. In fact, GLT-1 expression and activity is known to be reduced by clozapine treatment, with a dose-dependent reduction of glutamate reuptake (Vallejo-Illarramendi et al., 2005). Therefore we also analyzed possible interactions between EAAT2 genotype and pharmacological treatments on CRT efficacy, hypothesizing a differential outcome depending on antipsychotic treatment (clozapine vs. other antipsychotics). 2. Methods 2.1. Sample In this retrospective study we included 88 clinically stabilized outpatients, recruited at the Psychiatric Rehabilitation Unit of IRCCS San Raffaele Hospital, Milan, Italy. Inclusion criteria were: diagnosis of schizophrenia meeting DSM-IV-TR criteria, age from 18 to 65 years, Intelligence Quotient (I.Q.) N70, treatment with a stable dose of the same antipsychotic in monotherapy since at least 3 months, and good response to treatment, defined as a reduction of 30% or more in Positive And Negative Schizophrenic Symptoms (PANSS) Total Score after 3 months of treatment (Kay et al., 1987). Exclusion criteria were: psychiatric comorbidities, concomitant psychiatric treatments except benzodiazepines, substance abuse, neurological disorders and brain injury. All subjects provided informed consent to a protocol approved by the local Ethical Committee following the principles of the Declaration of Helsinki. 2.2. Genotyping DNA was extracted from whole blood by a manual extraction, using the “Illustra blood genomic Prep Midi Flow kit” (GE Healthcare, Milan, Italy). To identify the EAAT2 polymorphism rs4354668 T/G (DNA forward strand), a standard Polymerase Chain Reaction (PCR) was carried with the following primers: 5′- GCC ACC TGT GCT TTG CTG -3′ and 5′- TGA TGT CAG CTC TCG ACG AA -3′. The PCR was carried out in a 10 μl volume containing 150 ng genomic DNA, 1 μl of 1× Hot Master Taq Buffer with Mg++ (Eppendorf), 0.1 μl of each primer [50 uM], 1 μl of deaza-dNTPs [10 mM], 0,5 μl of Dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, Milan, Italy) and 0.1 μl of Hot Master Taq [5 U/ul] (Eppendorf). After an initial step of 5 min at 94 °C, 35 cycles of amplification (35 s at 94 °C, 35 s at 58 °C, 45 s at 70 °C) and a final extension step of 10 min at 70 °C were performed. An aliquot of PCR product was digested using Msp I (20 U/ul) (New England Biolabs, England, UK) and incubated at 37 °C for 8 h; fragments were separated in agarose gels. Depending on the presence of two or three restriction Msp I sites, either three fragments (allele T) or four fragments (allele G) were produced. 2.3. Assessment Basic clinical and demographic data were collected from clinical reports. Psychopathology was assessed by means of PANSS, administered by trained psychiatrists. The general intellectual level was evaluated with the Wechsler Adult Intelligence Scale – R (WAIS- R), a standardized test designed to measure intelligence in adults.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
M. Spangaro et al. / Schizophrenia Research xxx (2018) xxx–xxx
Neuropsychological measures were evaluated at baseline, at the end of CRT (3 months), and after further three months of follow-up (6 months), in order to analyze both CRT outcome and durability of cognitive improvements. Neurocognitive assessments were performed with the Wisconsin Card Sorting Test (WCST) (Stratta et al., 2004), for evaluation of executive functions (number of perseverative errors), and the Italian Version of the Brief Assessment of Cognition in Schizophrenia (BACS) (Anselmetti et al., 2008; Keefe et al., 2004), a short battery of neuropsychological tests specifically designed in two versions (A and B) to evaluate patients before and after rehabilitation programs, without the results being influenced by recall. It consists of the following tests: verbal memory (words recall); working memory (digit sequencing); token motor task (psychomotor speed and coordination); speed of processing (symbol coding); verbal fluency (semantic and letter production) and planning (Tower of London - ToL). 2.4. Rehabilitative interventions All patients underwent cognitive remediation therapy (CRT), added to standard rehabilitation therapy (SRT). The SRT (1 h, 3 times/week) was focused on the main community goals of social abilities, work, and autonomy, including noncognitive subprograms of IPT (Verbal Communication, Social Skill Training and Problem Solving)(Zimmer et al., 2007), social skills training programs for residential, vocational, and recreational functioning (Roder et al., 2002), and psychoeducation (Bechdolf et al., 2004). The cognitive remediation protocol consisted of three sessions of 1 h each of function-specific computer-aided exercises a week (total 12 weeks, 36 sessions). Computer-assisted neurocognitive exercise was performed employing the Cogpack Software (Marker, 1987– 2007), the program was set for adaptive exercises, based on patients' performances during the course of the session. Sets of exercises were individually created for each patient, starting from baseline performances at neuropsychological assessment. Exercises were administered by trained psychologists whose role was to motivate patients and assist them in completing exercises and trying different strategies, without giving them the solutions to the exercises. 2.5. Data analysis For genetic analysis, as in previous studies (Poletti et al., 2014; Spangaro et al., 2012; Spangaro et al., 2014; Zhang et al., 2015), patients were divided into two groups: the homozygous for the T allele vs G carriers. The rationale is to avoid further levels of factors in the analysis, given the small sample size. To examine the effect of pharmacological treatment, the sample was divided into two groups: subjects treated with clozapine vs subjects treated with other APs, characterized by higher dopamine D2 blocking activity. The rationale of this choice is explained in the introduction and further argued in the discussion. Demographic and clinical characteristics and basal neuropsychological measures were analyzed for group differences by means of analysis of variance (ANOVA) and Chi-Square Test (for dychotomic variables). Repeated Measures Analysis of Covariance (ANCOVA), with genotype (T/T vs G carriers) and treatment (clozapine vs other APs) groups as categorical predictors, age and years of education as covariates, and the WCST and BACS subtests scores as dependent variables was used to evaluate the effects of genotype and treatment on cognitive improvement after CRT and at 3 months follow-up. Post-hoc analyses were performed with Scheffé's method, given the unbalanced group sizes and in order to obtain more conservative results. For the main outcome, the statistical power was calculated with a post-hoc compute achieved power analysis for F-tests. Given the sample size, the number of groups and the number of measurements, we had an excellent power (0.99) to detect a medium effect size (f = 0.25) at a type 1 error level of 0.05, with a critical F of 2.14.
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3. Results The sample was composed of 88 patients, 51 males and 37 females. There were no differences in gender distribution between genotype groups (Chi squared n.s.). DNA analysis showed the following allelic distribution of EAAT2 genotype: 35patients T/T, 37 T/G and 16 G/G, in Hardy–Weinberg equilibrium. Table 1 shows demographic and clinical variables of the sample. No significant differences were found for any of the variables considered between genotype groups. The antipsychotic treatment was distributed as follows: 42 patients were taking clozapine, 46 patients were treated with other antipsychotics (haloperidol, risperidone, olanzapine, paliperidone, aripiprazole). There were no differences in treatment distribution between genotype groups (Chi squared n.s.). Patients treated with clozapine showed lower age (p = .021, F = 5.53) and duration of illness (p = .031, F = 4.82) than subjects treated with other APs. ANOVA revealed no differences at baseline between EAAT2 genotype (T/T vs G carriers) and treatment (clozapine vs other Aps) groups for neuropsychological measures (Table 1). Repeated measures ANCOVA showed significant effects for working memory and for executive functions in the subcomponents of flexibility and planning, while no effect was reported for the other cognitive functions assessed (Table 2). In detail, the analysis revealed a significant main effect (F = 5.27, p = .007) of EAAT2 genotype on “Sequencing” BACS' subtest, a task that evaluates working memory, with no significant effect of covariates and treatment, nor interactions. Post-hoc analysis showed significant differences between baseline evaluation and subsequent assessments only among T/T subjects (3 months: p = .007, 6 months: p = .006), as illustrated in Fig. 1. The whole model explained the 9.1% of WM variability (Adjusted R2 = 0.091). Concerning cognitive flexibility, measured with the WCST perseverative errors score, the ANCOVA revealed a significant treatment*genotype interaction (F = 4.15, p = .018), and a significant effect of age (F = 6.04, p = .017). Sheffé's post-hoc analysis showed significant differences among T/T subjects not treated with clozapine (baseline/6 months: p = .015), as shown in Fig. 2. The whole model explained the 9.8% of executive functions variability (Adjusted R2 = 0.098). Finally, a significant treatment*genotype interaction was found also for measure of planning, assessed with the BACS ToL (F = 3.17, p = .045), with no significant effects of single factors, nor covariates. No significant differences within or between groups were observed with Sheffé's post-hoc analysis.
Table 1 Demographic, clinical and basal cognitive variables of the sample.
Age Years of education Onset Duration of illness PANSS total score WAIS-R Total IQ BACS - verbal memory BACS - working memory BACS – psychomotor coordination BACS - verbal fluency BACS - processing speed BACS – planning WCST - perseverative errors
MEAN S.D.
Therapy (ANOVA: EAAT2 clozapine (ANOVA: TT vs Gcar) vs other APs)
35.01 11.55 23.41 11.34 68.91 85.60 36.35 16.34 67.02 37.55 37.30 12.78 14.10
N.S.⁎ N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
9.61 2.66 6.15 8.27 15.84 10.63 10.52 3.94 14.82 10.97 10.65 4.06 9.94
p = .021, F = 5.53 N.S. N.S. p = .031, F = 4.82 N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
Bold: statistically significant result. ⁎ N.S. = Not Significant.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
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M. Spangaro et al. / Schizophrenia Research xxx (2018) xxx–xxx
Table 2 Results of repeated measures ANCOVA: Effects of EAAT2 rs4354668 (T/T vs G carriers), AP treatment (clozapine vs. other APs) and interaction rs4354668/AP. EAAT2 rs4354668 BACS - verbal memory BACS - working memory BACS – psychomotor coordination BACS - verbal fluency BACS - processing speed BACS - planning WCST - perseverative errors
Antipsychotic (clozapine vs other APs)
Interaction rs4354668/AP
N.S.⁎ N.S. p = .007, F = N.S. 5.27 N.S. N.S.
N.S. N.S.
N.S. N.S. N.S. N.S.
N.S. N.S. p = .045, F = 3.17 p = .018, F = 4.15
N.S. N.S. N.S. N.S.
N.S.
Bold: statistically significant result. ⁎ N.S. = Not Significant.
4. Discussion Analysis of CRT outcome predictors represents one of the main targets in the study and treatment of cognitive deficits in schizophrenia, since it could lead to develop personalized and more effective interventions, as well as to better unravel underlying biological factors (Bosia et al., 2014). To our knowledge, this is the first study to investigate the influence of a genetic polymorphism within the glutamatergic system on CRT outcome. The aim of this research was to evaluate possible effects of EAAT2 rs4354668 on cognitive improvements among subjects affected by schizophrenia, while taking into account the interaction with antipsychotic pharmacological treatment. Our results showed that subjects carrying T/T genotype were associated with better response to CRT intervention on working memory. Specifically, subjects with the T/T genotype showed greater working memory improvement, maintained after 3 months from the end of cognitive rehabilitation. Moreover, we also observed an interaction of EAAT2 genotype with pharmacological treatment on executive functions improvement, with statistically significant changes only among patients homozygotes for the T allele not treated with clozapine. These data suggest that genetic reduction of glutamate transporter expression is associated, in patients with schizophrenia, with a disadvantageous effect on WM improvement. Furthermore, our results indicate the possibility that EAAT2 genotype may also differentially influence the response to CRT depending on the antipsychotic treatment, as supported by the lack of significant differences between genotypes among patients treated with clozapine, a downregolator of EAAT2 expression (Vallejo-Illarramendi et al., 2005). Different studies previously evidenced the influence of rs4354668 on basal prefrontal cognitive function and brain structure, showing
Fig. 2. WCST – number of perseverative errors (executive functions) at baseline, at the end of CRT (3 months) and after 6 months. Patients are divided into four groups: EAAT2 T/T treated with clozapine/other antipsychotics and G carriers treated with clozapine/other antipsychotics.
advantageous effects for patients carrying the T/T genotype, linked to higher transporter expression. It was hypothesized that the presence of the disadvantageous allele could lead to a twofold effect on prefrontal cognitive pathways, acting synergically (Poletti et al., 2014; Spangaro et al., 2012; Spangaro et al., 2014). On one side, reduced transporter expression could determine an inefficient energy metabolism, impairing glutamate recycling and neurovascular coupling (Robinson and Jackson, 2016). On the other, a lower EAAT2 expression could also lead to increased glutamate spillover between synapses, losing signal input specificity, and inducing an excitotoxic neuronal damage (Poletti et al., 2014). Collectively, all these elements could therefore determine disadvantageous effects both on basal cognitive functions and their dynamic modulation, thus decreasing CRT efficacy. Interestingly, executive functions and working memory are the same domains that previously resulted influenced by EAAT2 genotype in the context of basal cognitive studies, suggesting a domain specificity of the transporter (Poletti et al., 2014; Spangaro et al., 2012; Spangaro et al., 2014; Zhang et al., 2015). These hypotheses are speculative and based on preliminary data. However, results of this study further suggest that EAAT2 inefficiency may represent a pharmacological target in the treatment of cognitive deficit of schizophrenia. The results of our study have to be interpreted in the context of some limitations. First, results must be considered as preliminary, because of the relative small sample size. Second, a healthy controls group is lacking and should be included, also given the reported influence of EAAT2 polymorphism on cognitive functioning in the general population. Further studies are needed in order to better understand underlying neurobiological mechanisms and possible interactions with other neurotransmitter systems involved in CRT. Moreover, the inclusion of neuroimaging correlates may be helpful in order to individuate possible neurofunctional and structural targets. Particularly, given the strong association between glutamate transport and glucose metabolism, positron emission tomography and fMRI studies could represent useful tools of investigation (Stoessl, 2017). Finally, our result may be influenced by other unexplored factors, such as environmental elements or other functionally related pathways.
5. Conclusion
Fig. 1. BACS – Sequencing scores (working memory) at baseline, at the end of CRT (3 months) and after 6 months. Patients are divided into two groups: EAAT2 T/T and G carriers.
In conclusion, our findings support the hypothesis that EAAT2 polymorphism, besides basal cognitive function, influences improvement of cognitive functioning after CRT as well. The presence of a high expression T/T genotype was associated with greater improvements in working memory. rs4354668 also resulted to differentially influence CRT improvement in executive functions depending on antipsychotic treatment, probably due to clozapine's downregulation of EAAT2 expression.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
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Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of interest All authors declare that they have no conflict of interest. Contributors Authors Marco Spangaro, Marta Bosia and Roberto Cavallaro elaborated the hypothesis and designed the study. Authors Marco Spangaro and Marta Bosia wrote the manuscript and performed statistical analysis. Authors Mariachiara Buonocore and Margherita Bechi performed the neuropsychological evaluations and intervention. Author Cristina Lorenzi performed DNA analysis. Author Federica Cocchi enrolled the patients and supervised the study design. Authors Carmelo Guglielmino, Antonella Mastromatteo and Laura Bianchi contributed to patient's enrollment. All authors contributed to and have approved the final manuscript. Acknowledgements We would like to thank all the participants who took part in this study and their support persons.
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Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism Marco Spangaro a,⁎, Marta Bosia a,b, Margherita Bechi a, Mariachiara Buonocore a, Federica Cocchi a, Carmelo Guglielmino a, Laura Bianchi a, Antonella Mastromatteo a, Cristina Lorenzi a, Roberto Cavallaro a,b a b
IRCCS San Raffaele Scientific Institute, Department of Clinical Neurosciences, Milan, Italy Vita-Salute San Raffaele University, Milan, Italy
a r t i c l e
i n f o
Article history: Received 16 October 2017 Received in revised form 27 March 2018 Accepted 24 June 2018 Available online xxxx Keywords: Glutamate uptake EAAT2 Clozapine Cognitive deficit Rehabilitation Psychosis
a b s t r a c t Cognitive deficits represent core features of schizophrenia, affecting quality of life and functioning. The excitatory amino acid transporter 2 (EAAT2) is responsible for the majority of glutamate reuptake and its activity is crucial for glutamatergic neurotransmission, prevention of excitotoxic damage and cerebral metabolism. Different studies reported that EAAT2 rs4354668 (−181 T/G) influences cognitive functions and brain structures in patients with schizophrenia. Specifically, the G allele, linked to lower EAAT2 expression, was associated with impaired prefrontal cognitive performance and reduced grey matter volumes. Cognitive remediation therapy (CRT) is one of the best available tool to treat cognitive deficits in schizophrenia, able to induce a neuroplastic modulation of cognitive functions. The present study aims to investigate the effects of rs4354668 on CRT outcome, also considering possible genotype interaction with antipsychotic (AP) treatment, since EAAT2 expression is negatively influenced by clozapine. We examined rs4354668 in 88 clinically stabilized patients with schizophrenia, treated with CRT and assessed at enrolment, at the end of CRT and after 3 months. We observed greater working memory improvements among patients carrying the T/T genotype, regardless of AP treatment. Moreover, we reported a significant interaction between pharmacological treatment and rs4354668 on executive functions, with greater improvements among T/T patients treated with APs other than clozapine. These observations suggest that impaired EAAT2 expression may attenuate CRT outcome. Moreover, our results indicate the possibility that rs4354668 could also differentially influence the response to CRT depending on the AP treatment. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Abnormalities in cognitive functions are key features of schizophrenia and critical determinants of quality of life and global functioning (Green et al., 2004). To date, cognitive remediation therapy (CRT), is one of the best available and effective tools to treat cognitive deficits and improve general functioning in schizophrenia (Wykes and Spaulding, 2011). However, the reported effects of CRT on cognitive outcome are very heterogeneous and significant predictors are still missing, also due to the lack of knowledge about the underlying neurobiological mechanisms. To date, research has mainly focused on dopaminergic pathway, given its implications in prefrontal cognitive functions and the observation of changes in dopaminergic transmission after CRT ⁎ Corresponding author at: Department of Clinical Neurosciences, IRCCS San Raffaele Scientific Institute, Vita-Salute San Raffale University, Via Stamira d'Ancona 20, 20127 Milano, Italy. E-mail address: [email protected] (M. Spangaro).
(McNab et al., 2009). Different studies reported an influence of a functional polymorphism of the catechol-O-methyltransferase gene (Val158Met) on CRT improvements, also observing an interaction between pharmacological therapy and Catechol-O-methyltransferase (COMT) genotype (Bosia et al., 2007; Bosia et al., 2014; Lindenmayer et al., 2015). Of note, the glutamatergic system is also highly implicated in neuropsychological functioning. Glutamate represents the major excitatory pathway in the central nervous system and it is involved in critical processes such as neural development, synaptic plasticity, learning and memory (Danbolt, 2001; Katagiri et al., 2001). One of the major glutamate modulators is represented by the excitatory amino acid transporter 2 (EAAT2), mainly localized at the plasma membrane of astroglial cells and responsible for N90% of cerebral glutamate uptake (Gegelashvili and Schousboe, 1998; Zhou and Danbolt, 2014). EAAT2 regulates and buffers the amount of synaptic glutamate, limits the spillover and therefore contributes to maintain the signal input specificity and to prevent neuronal damages secondary to glutamate excitotoxicity (Selkirk et al., 2005; Sheldon and Robinson, 2007).
https://doi.org/10.1016/j.schres.2018.06.059 0920-9964/© 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
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Moreover, the transporter also preserves an efficient energy metabolism through a twofold activity: on one side EAAT2 contributes to recycling of neurotransmitter pools by transporting glutamate into astrocytes for its conversion into glutamine (Marcaggi and Attwell, 2004); on the other side it mediates the process of ‘neurovascular coupling’, i.e. the increase in cerebral blood flow following neuronal activation (Robinson and Jackson, 2016). Consistently, it has been demonstrated that EAAT2 also plays a central role in the astrocyte–neuron lactate shuttle (ANLS). In ANLS, glutamate reuptake is coupled to activation of Na+/K + -ATPase that leads to uptake of glucose in astrocytes and production of lactate, which is then shuttled to the neuron. Previous work reported reduced [14C]2deoxyglucose uptake in glutamate transporter 1 (GLT-1, EAAT2 murine homologous) knockout mice. More recently it was found that stimulation of glutamate transporter through ceftriaxone results in widespread increases in glucose metabolism (Stoessl, 2017). Different substances, such as riluzole and ceftriaxone, are known to collaterally raise glutamate reuptake by increasing EAAT2 expression, with neuroprotective effects (Beller et al., 2011; Fumagalli et al., 2008; Kim et al., 2011). Interestingly, Kong and colleagues recently individuated a compound, LDN/ OSU-0212320, that specifically increases the transporter expression. Authors also demonstrated that LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation, hypothesizing possible application of the compound in the treatment of neurodegenerative disorders (Kong et al., 2014). Several studies reported an alteration of EAAT2 in schizophrenia, evidencing deficits of both expression and activity. Indeed, patients with schizophrenia displayed reduced EAAT2 expression in parahippocampal region, hippocampus, superior temporal gyrus and dorsolateral prefrontal cortex (DLPFC) (Ohnuma et al., 1998; Ohnuma et al., 2000). In line with these findings, a post-mortem study revealed an increased mRNA thalamic expression of EAAT2, indicating an abnormality in a glutamatergic corticothalamic circuit (Smith et al., 2001). Interestingly, Shan et al. also reported a lower glycosylation of the transporter, linked to impaired trafficking to plasma membrane and therefore to a lower glutamate reuptake (Shan et al., 2013). Subjects carrying the high-risk glutamate metabotropic receptor 3 haplotype, linked to schizophrenia, showed lower EAAT2 expression in the prefrontal cortex (PFC) and impaired prefrontal cognitive functions. Moreover, through functional magnetic resonance imaging (fMRI) studies, this haplotype has also been associated to dysfunctional activation patterns in the prefrontal cortex and hippocampus during cognitive tasks of verbal list learning and verbal fluency (Egan et al., 2004). The involvement of EAAT2 in the pathogenesis of cognitive deficit in schizophrenia was further supported by Featherstone et al., who showed that subchronic treatment with ketamine in rodents leads to a reduced expression of GLT-1 and to permanent changes in electroencephalography and cognition (Featherstone et al., 2012). EAAT2 transporter expression is modulated by a functional single nucleotide polymorphism (SNP), rs4354668 (−181 T/G), located in the gene promoter region, with the T allele linked to higher EAAT2 expression. Particularly, the G allele determines a consensus binding site for the repressor transcription factor GC-binding factor 2 (GCF2), which leads to increased glutamate concentrations (Mallolas et al., 2006). We previously evaluated possible influences of rs4354668 on prefrontal cognitive performance in a sample of 211 patients with schizophrenia, reporting a disadvantageous effect on executive functions and working memory (WM) among patients carrying the G allele (Spangaro et al., 2012; Spangaro et al., 2014). Interestingly, in a subsequent study we also found that poor WM performance in subjects carrying the EAAT2 G allele was associated with reduced frontal cortical volumes, suggesting the possible presence of an excitotoxic process underlying impaired cognitive performance (Poletti et al., 2014). Similar effects of rs4354668 on prefrontal cognitive performance were recently confirmed in a large-scale study including 3245 patients
and 6154 healthy controls, reporting better executive functions in subjects homozygous for the T allele, compared with the G carriers (Zhang et al., 2015). Given the influences of EAAT2 SNP on basal cognitive functions and brain structure, in this work we retrospectively evaluated possible effects of rs4354668 on CRT response. Considering the previously reported effects of EAAT2 genotype on basal WM and executive functions, we hypothesized that rs4354668 could affect changes of these functions after CRT as well. Moreover, in this study we also investigated possible interactions with pharmacological treatment. In fact, GLT-1 expression and activity is known to be reduced by clozapine treatment, with a dose-dependent reduction of glutamate reuptake (Vallejo-Illarramendi et al., 2005). Therefore we also analyzed possible interactions between EAAT2 genotype and pharmacological treatments on CRT efficacy, hypothesizing a differential outcome depending on antipsychotic treatment (clozapine vs. other antipsychotics). 2. Methods 2.1. Sample In this retrospective study we included 88 clinically stabilized outpatients, recruited at the Psychiatric Rehabilitation Unit of IRCCS San Raffaele Hospital, Milan, Italy. Inclusion criteria were: diagnosis of schizophrenia meeting DSM-IV-TR criteria, age from 18 to 65 years, Intelligence Quotient (I.Q.) N70, treatment with a stable dose of the same antipsychotic in monotherapy since at least 3 months, and good response to treatment, defined as a reduction of 30% or more in Positive And Negative Schizophrenic Symptoms (PANSS) Total Score after 3 months of treatment (Kay et al., 1987). Exclusion criteria were: psychiatric comorbidities, concomitant psychiatric treatments except benzodiazepines, substance abuse, neurological disorders and brain injury. All subjects provided informed consent to a protocol approved by the local Ethical Committee following the principles of the Declaration of Helsinki. 2.2. Genotyping DNA was extracted from whole blood by a manual extraction, using the “Illustra blood genomic Prep Midi Flow kit” (GE Healthcare, Milan, Italy). To identify the EAAT2 polymorphism rs4354668 T/G (DNA forward strand), a standard Polymerase Chain Reaction (PCR) was carried with the following primers: 5′- GCC ACC TGT GCT TTG CTG -3′ and 5′- TGA TGT CAG CTC TCG ACG AA -3′. The PCR was carried out in a 10 μl volume containing 150 ng genomic DNA, 1 μl of 1× Hot Master Taq Buffer with Mg++ (Eppendorf), 0.1 μl of each primer [50 uM], 1 μl of deaza-dNTPs [10 mM], 0,5 μl of Dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, Milan, Italy) and 0.1 μl of Hot Master Taq [5 U/ul] (Eppendorf). After an initial step of 5 min at 94 °C, 35 cycles of amplification (35 s at 94 °C, 35 s at 58 °C, 45 s at 70 °C) and a final extension step of 10 min at 70 °C were performed. An aliquot of PCR product was digested using Msp I (20 U/ul) (New England Biolabs, England, UK) and incubated at 37 °C for 8 h; fragments were separated in agarose gels. Depending on the presence of two or three restriction Msp I sites, either three fragments (allele T) or four fragments (allele G) were produced. 2.3. Assessment Basic clinical and demographic data were collected from clinical reports. Psychopathology was assessed by means of PANSS, administered by trained psychiatrists. The general intellectual level was evaluated with the Wechsler Adult Intelligence Scale – R (WAIS- R), a standardized test designed to measure intelligence in adults.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
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Neuropsychological measures were evaluated at baseline, at the end of CRT (3 months), and after further three months of follow-up (6 months), in order to analyze both CRT outcome and durability of cognitive improvements. Neurocognitive assessments were performed with the Wisconsin Card Sorting Test (WCST) (Stratta et al., 2004), for evaluation of executive functions (number of perseverative errors), and the Italian Version of the Brief Assessment of Cognition in Schizophrenia (BACS) (Anselmetti et al., 2008; Keefe et al., 2004), a short battery of neuropsychological tests specifically designed in two versions (A and B) to evaluate patients before and after rehabilitation programs, without the results being influenced by recall. It consists of the following tests: verbal memory (words recall); working memory (digit sequencing); token motor task (psychomotor speed and coordination); speed of processing (symbol coding); verbal fluency (semantic and letter production) and planning (Tower of London - ToL). 2.4. Rehabilitative interventions All patients underwent cognitive remediation therapy (CRT), added to standard rehabilitation therapy (SRT). The SRT (1 h, 3 times/week) was focused on the main community goals of social abilities, work, and autonomy, including noncognitive subprograms of IPT (Verbal Communication, Social Skill Training and Problem Solving)(Zimmer et al., 2007), social skills training programs for residential, vocational, and recreational functioning (Roder et al., 2002), and psychoeducation (Bechdolf et al., 2004). The cognitive remediation protocol consisted of three sessions of 1 h each of function-specific computer-aided exercises a week (total 12 weeks, 36 sessions). Computer-assisted neurocognitive exercise was performed employing the Cogpack Software (Marker, 1987– 2007), the program was set for adaptive exercises, based on patients' performances during the course of the session. Sets of exercises were individually created for each patient, starting from baseline performances at neuropsychological assessment. Exercises were administered by trained psychologists whose role was to motivate patients and assist them in completing exercises and trying different strategies, without giving them the solutions to the exercises. 2.5. Data analysis For genetic analysis, as in previous studies (Poletti et al., 2014; Spangaro et al., 2012; Spangaro et al., 2014; Zhang et al., 2015), patients were divided into two groups: the homozygous for the T allele vs G carriers. The rationale is to avoid further levels of factors in the analysis, given the small sample size. To examine the effect of pharmacological treatment, the sample was divided into two groups: subjects treated with clozapine vs subjects treated with other APs, characterized by higher dopamine D2 blocking activity. The rationale of this choice is explained in the introduction and further argued in the discussion. Demographic and clinical characteristics and basal neuropsychological measures were analyzed for group differences by means of analysis of variance (ANOVA) and Chi-Square Test (for dychotomic variables). Repeated Measures Analysis of Covariance (ANCOVA), with genotype (T/T vs G carriers) and treatment (clozapine vs other APs) groups as categorical predictors, age and years of education as covariates, and the WCST and BACS subtests scores as dependent variables was used to evaluate the effects of genotype and treatment on cognitive improvement after CRT and at 3 months follow-up. Post-hoc analyses were performed with Scheffé's method, given the unbalanced group sizes and in order to obtain more conservative results. For the main outcome, the statistical power was calculated with a post-hoc compute achieved power analysis for F-tests. Given the sample size, the number of groups and the number of measurements, we had an excellent power (0.99) to detect a medium effect size (f = 0.25) at a type 1 error level of 0.05, with a critical F of 2.14.
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3. Results The sample was composed of 88 patients, 51 males and 37 females. There were no differences in gender distribution between genotype groups (Chi squared n.s.). DNA analysis showed the following allelic distribution of EAAT2 genotype: 35patients T/T, 37 T/G and 16 G/G, in Hardy–Weinberg equilibrium. Table 1 shows demographic and clinical variables of the sample. No significant differences were found for any of the variables considered between genotype groups. The antipsychotic treatment was distributed as follows: 42 patients were taking clozapine, 46 patients were treated with other antipsychotics (haloperidol, risperidone, olanzapine, paliperidone, aripiprazole). There were no differences in treatment distribution between genotype groups (Chi squared n.s.). Patients treated with clozapine showed lower age (p = .021, F = 5.53) and duration of illness (p = .031, F = 4.82) than subjects treated with other APs. ANOVA revealed no differences at baseline between EAAT2 genotype (T/T vs G carriers) and treatment (clozapine vs other Aps) groups for neuropsychological measures (Table 1). Repeated measures ANCOVA showed significant effects for working memory and for executive functions in the subcomponents of flexibility and planning, while no effect was reported for the other cognitive functions assessed (Table 2). In detail, the analysis revealed a significant main effect (F = 5.27, p = .007) of EAAT2 genotype on “Sequencing” BACS' subtest, a task that evaluates working memory, with no significant effect of covariates and treatment, nor interactions. Post-hoc analysis showed significant differences between baseline evaluation and subsequent assessments only among T/T subjects (3 months: p = .007, 6 months: p = .006), as illustrated in Fig. 1. The whole model explained the 9.1% of WM variability (Adjusted R2 = 0.091). Concerning cognitive flexibility, measured with the WCST perseverative errors score, the ANCOVA revealed a significant treatment*genotype interaction (F = 4.15, p = .018), and a significant effect of age (F = 6.04, p = .017). Sheffé's post-hoc analysis showed significant differences among T/T subjects not treated with clozapine (baseline/6 months: p = .015), as shown in Fig. 2. The whole model explained the 9.8% of executive functions variability (Adjusted R2 = 0.098). Finally, a significant treatment*genotype interaction was found also for measure of planning, assessed with the BACS ToL (F = 3.17, p = .045), with no significant effects of single factors, nor covariates. No significant differences within or between groups were observed with Sheffé's post-hoc analysis.
Table 1 Demographic, clinical and basal cognitive variables of the sample.
Age Years of education Onset Duration of illness PANSS total score WAIS-R Total IQ BACS - verbal memory BACS - working memory BACS – psychomotor coordination BACS - verbal fluency BACS - processing speed BACS – planning WCST - perseverative errors
MEAN S.D.
Therapy (ANOVA: EAAT2 clozapine (ANOVA: TT vs Gcar) vs other APs)
35.01 11.55 23.41 11.34 68.91 85.60 36.35 16.34 67.02 37.55 37.30 12.78 14.10
N.S.⁎ N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
9.61 2.66 6.15 8.27 15.84 10.63 10.52 3.94 14.82 10.97 10.65 4.06 9.94
p = .021, F = 5.53 N.S. N.S. p = .031, F = 4.82 N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
Bold: statistically significant result. ⁎ N.S. = Not Significant.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
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Table 2 Results of repeated measures ANCOVA: Effects of EAAT2 rs4354668 (T/T vs G carriers), AP treatment (clozapine vs. other APs) and interaction rs4354668/AP. EAAT2 rs4354668 BACS - verbal memory BACS - working memory BACS – psychomotor coordination BACS - verbal fluency BACS - processing speed BACS - planning WCST - perseverative errors
Antipsychotic (clozapine vs other APs)
Interaction rs4354668/AP
N.S.⁎ N.S. p = .007, F = N.S. 5.27 N.S. N.S.
N.S. N.S.
N.S. N.S. N.S. N.S.
N.S. N.S. p = .045, F = 3.17 p = .018, F = 4.15
N.S. N.S. N.S. N.S.
N.S.
Bold: statistically significant result. ⁎ N.S. = Not Significant.
4. Discussion Analysis of CRT outcome predictors represents one of the main targets in the study and treatment of cognitive deficits in schizophrenia, since it could lead to develop personalized and more effective interventions, as well as to better unravel underlying biological factors (Bosia et al., 2014). To our knowledge, this is the first study to investigate the influence of a genetic polymorphism within the glutamatergic system on CRT outcome. The aim of this research was to evaluate possible effects of EAAT2 rs4354668 on cognitive improvements among subjects affected by schizophrenia, while taking into account the interaction with antipsychotic pharmacological treatment. Our results showed that subjects carrying T/T genotype were associated with better response to CRT intervention on working memory. Specifically, subjects with the T/T genotype showed greater working memory improvement, maintained after 3 months from the end of cognitive rehabilitation. Moreover, we also observed an interaction of EAAT2 genotype with pharmacological treatment on executive functions improvement, with statistically significant changes only among patients homozygotes for the T allele not treated with clozapine. These data suggest that genetic reduction of glutamate transporter expression is associated, in patients with schizophrenia, with a disadvantageous effect on WM improvement. Furthermore, our results indicate the possibility that EAAT2 genotype may also differentially influence the response to CRT depending on the antipsychotic treatment, as supported by the lack of significant differences between genotypes among patients treated with clozapine, a downregolator of EAAT2 expression (Vallejo-Illarramendi et al., 2005). Different studies previously evidenced the influence of rs4354668 on basal prefrontal cognitive function and brain structure, showing
Fig. 2. WCST – number of perseverative errors (executive functions) at baseline, at the end of CRT (3 months) and after 6 months. Patients are divided into four groups: EAAT2 T/T treated with clozapine/other antipsychotics and G carriers treated with clozapine/other antipsychotics.
advantageous effects for patients carrying the T/T genotype, linked to higher transporter expression. It was hypothesized that the presence of the disadvantageous allele could lead to a twofold effect on prefrontal cognitive pathways, acting synergically (Poletti et al., 2014; Spangaro et al., 2012; Spangaro et al., 2014). On one side, reduced transporter expression could determine an inefficient energy metabolism, impairing glutamate recycling and neurovascular coupling (Robinson and Jackson, 2016). On the other, a lower EAAT2 expression could also lead to increased glutamate spillover between synapses, losing signal input specificity, and inducing an excitotoxic neuronal damage (Poletti et al., 2014). Collectively, all these elements could therefore determine disadvantageous effects both on basal cognitive functions and their dynamic modulation, thus decreasing CRT efficacy. Interestingly, executive functions and working memory are the same domains that previously resulted influenced by EAAT2 genotype in the context of basal cognitive studies, suggesting a domain specificity of the transporter (Poletti et al., 2014; Spangaro et al., 2012; Spangaro et al., 2014; Zhang et al., 2015). These hypotheses are speculative and based on preliminary data. However, results of this study further suggest that EAAT2 inefficiency may represent a pharmacological target in the treatment of cognitive deficit of schizophrenia. The results of our study have to be interpreted in the context of some limitations. First, results must be considered as preliminary, because of the relative small sample size. Second, a healthy controls group is lacking and should be included, also given the reported influence of EAAT2 polymorphism on cognitive functioning in the general population. Further studies are needed in order to better understand underlying neurobiological mechanisms and possible interactions with other neurotransmitter systems involved in CRT. Moreover, the inclusion of neuroimaging correlates may be helpful in order to individuate possible neurofunctional and structural targets. Particularly, given the strong association between glutamate transport and glucose metabolism, positron emission tomography and fMRI studies could represent useful tools of investigation (Stoessl, 2017). Finally, our result may be influenced by other unexplored factors, such as environmental elements or other functionally related pathways.
5. Conclusion
Fig. 1. BACS – Sequencing scores (working memory) at baseline, at the end of CRT (3 months) and after 6 months. Patients are divided into two groups: EAAT2 T/T and G carriers.
In conclusion, our findings support the hypothesis that EAAT2 polymorphism, besides basal cognitive function, influences improvement of cognitive functioning after CRT as well. The presence of a high expression T/T genotype was associated with greater improvements in working memory. rs4354668 also resulted to differentially influence CRT improvement in executive functions depending on antipsychotic treatment, probably due to clozapine's downregulation of EAAT2 expression.
Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059
M. Spangaro et al. / Schizophrenia Research xxx (2018) xxx–xxx
Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of interest All authors declare that they have no conflict of interest. Contributors Authors Marco Spangaro, Marta Bosia and Roberto Cavallaro elaborated the hypothesis and designed the study. Authors Marco Spangaro and Marta Bosia wrote the manuscript and performed statistical analysis. Authors Mariachiara Buonocore and Margherita Bechi performed the neuropsychological evaluations and intervention. Author Cristina Lorenzi performed DNA analysis. Author Federica Cocchi enrolled the patients and supervised the study design. Authors Carmelo Guglielmino, Antonella Mastromatteo and Laura Bianchi contributed to patient's enrollment. All authors contributed to and have approved the final manuscript. Acknowledgements We would like to thank all the participants who took part in this study and their support persons.
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Please cite this article as: Spangaro, M., et al., Neurobiology of cognitive remediation in schizophrenia: Effects of EAAT2 polymorphism, Schizophr. Res. (2018), https://doi.org/10.1016/j.schres.2018.06.059