Perturbed iron biology in the prefrontal cortex of people with schizophrenia.

Despite loss of grey matter volume and emergence of distinct cognitive deficits in young adults diagnosed with schizophrenia, current treatments for schizophrenia do not target disruptions in late maturational reshaping of the prefrontal cortex. Iron, the most abundant transition metal in the brain, is essential to brain development and function, but in excess, it can impair major neurotransmission systems and lead to lipid peroxidation, neuroinflammation and accelerated aging. However, analysis of cortical iron biology in schizophrenia has not been reported in modern literature. Using a combination of inductively coupled plasma-mass spectrometry and western blots, we quantified iron and its major-storage protein, ferritin, in post-mortem prefrontal cortex specimens obtained from three independent, well-characterised brain tissue resources. Compared to matched controls (n = 85), among schizophrenia cases (n = 86) we found elevated tissue iron, unlikely to be confounded by demographic and lifestyle variables, by duration, dose and type of antipsychotic medications used or by copper and zinc levels. We further observed a loss of physiologic age-dependent iron accumulation among people with schizophrenia, in that the iron level among cases was already high in young adulthood. Ferritin, which stores iron in a redox-inactive form, was paradoxically decreased in individuals with the disorder. Such iron-ferritin uncoupling could alter free, chemically reactive, tissue iron in key reasoning and planning areas of the young-adult schizophrenia cortex. Using a prediction model based on iron and ferritin, our data provide a pathophysiologic link between perturbed cortical iron biology and schizophrenia and indicate that achievement of optimal cortical iron homeostasis could offer a new therapeutic target.

Increased peripheral inflammation in schizophrenia is associated with worse cognitive performance and related cortical thickness reductions.

While the biological substrates of brain and behavioural changes in persons with schizophrenia remain unclear, increasing evidence implicates that inflammation is involved. In schizophrenia, including first-episode psychosis and anti-psychotic naïve patients, there are numerous reports of increased peripheral inflammation, cognitive deficits and neuropathologies such as cortical thinning. Research defining the relationship between inflammation and schizophrenia symptomatology and neuropathology is needed. Therefore, we analysed the level of C-reactive protein (CRP), a peripheral inflammation marker, and its relationship with cognitive functioning in a cohort of 644 controls and 499 schizophrenia patients. In a subset of individuals who underwent MRI scanning (99 controls and 194 schizophrenia cases), we tested if serum CRP was associated with cortical thickness. CRP was significantly increased in schizophrenia patients compared to controls, co-varying for age, sex, overweight/obesity and diabetes (p < 0.006E-10). In schizophrenia, increased CRP was mildly associated with worse performance in attention, controlling for age, sex and education (R =- 0.15, p = 0.001). Further, increased CRP was associated with reduced cortical thickness in three regions related to attention: the caudal middle frontal, the pars opercularis and the posterior cingulate cortices, which remained significant after controlling for multiple comparisons (all p < 0.05). Together, these findings indicate that increased peripheral inflammation is associated with deficits in cognitive function and brain structure in schizophrenia, especially reduced attention and reduced cortical thickness in associated brain regions. Using CRP as a biomarker of peripheral inflammation in persons with schizophrenia may help to identify vulnerable patients and those that may benefit from adjunctive anti-inflammatory treatments.

Quetiapine and aripiprazole signal differently to ERK, p90RSK and c-Fos in mouse frontal cortex and striatum: role of the EGF receptor.

BACKGROUND: Signaling pathways outside dopamine D2 receptor antagonism may govern the variable clinical profile of antipsychotic drugs (APD) in schizophrenia. One postulated mechanism causal to APD action may regulate synaptic plasticity and neuronal connectivity via the extracellular signal-regulated kinase (ERK) cascade that links G-protein coupled receptors (GPCR) and ErbB growth factor signaling, systems disturbed in schizophrenia. This was based upon our finding that the low D2 receptor affinity APD clozapine induced initial down-regulation and delayed epidermal growth factor receptor (EGFR or ErbB1) mediated activation of the cortical and striatal ERK response in vivo distinct from olanzapine or haloperidol. Here we map whether the second generation atypical APDs aripiprazole and quetiapine affect the EGFR-ERK pathway and its substrates p90RSK and c-Fos in mouse brain, given their divergent agonist and antagonist properties on dopaminergic transmission, respectively. RESULTS: In prefrontal cortex, aripiprazole triggered triphasic ERK phosphorylation that was EGFR-independent but had no significant effect in striatum. Conversely quetiapine did not alter cortical ERK signaling but elevated striatal ERK levels in an EGFR-dependent manner. Induction of ERK by aripiprazole did not affect p90RSK signaling but quetiapine decreased RSK phosphorylation within 1-hour of administration. The transcription factor c-Fos by comparison was a direct target of ERK phosphorylation induced by aripiprazole in cortex and quetiapine in striatum with protein levels in temporal alignment with that of ERK. CONCLUSIONS: These data indicate that aripiprazole and quetiapine signal to specific nuclear targets of ERK, which for quetiapine occurs via an EGFR-linked mechanism, possibly indicating involvement of this system in its action.

Gene expression changes in Brodmann's Area 46 differentiate epidermal growth factor and immune system interactions in schizophrenia and mood disorders.

How early in life stress-immune related environmental factors increase risk predisposition to schizophrenia remains unknown. We examined if pro-inflammatory changes perturb the brain epidermal growth factor (EGF) system, a system critical for neurodevelopment and mature CNS functions including synaptic plasticity. We quantified genes from key EGF and immune system pathways for mRNA levels and eight immune proteins in post-mortem dorsolateral prefrontal (DLPFC; Brodmann's Area (BA) 46) and orbitofrontal (OFC; BA11) cortices from people with schizophrenia, mood disorders and neurotypical controls. In BA46, 64 genes were differentially expressed, predominantly in schizophrenia, where attenuated expression of the MAPK-ERK, NRG1-PI3K-AKT and mTOR cascades indicated reduced EGF system signalling, and similarly diminished immune molecular expression, notably in TLR, TNF and complement pathways, along with low NF-κB1 and elevated IL12RB2 protein levels were noted. There was nominal evidence for altered convergence between ErbB-PI3K-AKT-mTOR and TLR pathways in BA46 in schizophrenia. Comparatively minimal changes were noted in BA11. Overall, distinct pathway gene expression changes may reflect variant pathological processes involving immune and EGF system signalling between schizophrenia and mood disorder, particularly in DLPFC. Further, the abnormal convergence between innate immune signalling and candidate EGF signalling pathways may indicate a pathologically important interaction in the developing brain in response to environmental stressors.

Clozapine induction of ERK1/2 cell signalling via the EGF receptor in mouse prefrontal cortex and striatum is distinct from other antipsychotic drugs.

Treatment resistance remains a major obstacle in schizophrenia, with antipsychotic drugs (APDs) being ineffective in about one third of cases. Poor response to standard therapy leaves the APD clozapine as the only effective treatment for many patients. The reason for the superior efficacy of clozapine is unknown, but as we have proposed previously it may involve modulation of neuroplasticity and connectivity through induction of interconnected mitogenic signalling pathways. These include the mitogen-activated protein kinase-extracellular signal regulated kinase (MAPK-ERK) cascade and epidermal growth factor (EGF)/ErbB systems. Clozapine, distinct from other APDs, induced initial inhibition and subsequent activation of the ERK response in prefrontal cortical (PFC) neurons in vitro and in vivo, an action mediated by the EGF receptor (ErbB1). Here we examine additionally the striatum of C57Bl/6 mice to determine if clozapine, olanzapine, and haloperidol differentially regulate the ERK1/2 pathway in a region or time-specific manner conditional on the EGF receptor. Following acute treatment, only clozapine caused delayed striatal ERK phosphorylation through EGF receptor phosphorylation (tyrosine 1068 site) and MEK that paralleled cortical ERK phosphorylation. Olanzapine induced initial pERK1-specific blockade and an elevation 24-h later in PFC but had no effect in the striatum. By contrast, haloperidol significantly stimulated pERK1 in striatum for up to 8 h, but exerted limited effect in PFC. Clozapine but not olanzapine or haloperidol recruited the EGF receptor to signal to ERK. These in-vivo data reinforce our previous findings that clozapine's action may be uniquely linked to the EGF signalling system, potentially contributing to its distinctive clinical profile.

Brain changes in NF-κB1 and epidermal growth factor system markers at peri-pubescence in the spiny mouse following maternal immune activation.

Environmental risk factors that operate at foetal or neonatal levels increase the vulnerability to schizophrenia, plausibly via stress-immune activation that perturbs the epidermal growth factor (EGF) system, a system critical for neurodevelopment. We investigated potential associations between environmental insults and immune and EGF system changes through a maternal immune activation (MIA) model, using the precocial spiny mice (Acomys cahirinus). After mid-gestation MIA prepubescent offspring showed elevated NF-κB1 protein in nucleus accumbens, decreased EGFR in caudate putamen and a trend for increased PI3K-110δ in ventral hippocampus. Thus, prenatal stress may cause a heightened NF-κB1-mediated immune attenuation of EGF system signalling.

The Impact of Childhood Adversity on Cognitive Development in Schizophrenia.

Childhood adversity, such as physical, sexual, and verbal abuse, as well as neglect and family conflict, is a risk factor for schizophrenia. Such adversity can lead to disruptions of cognitive function during development, undermining intellectual capabilities and academic achievement. Schizophrenia is a neurodevelopmental disorder that is associated with cognitive impairments that may become evident during childhood. The Australian Schizophrenia Research Bank database comprises a large community cohort (N = 1169) in which we previously identified 3 distinct cognitive groups among people with schizophrenia: (1) Compromised, current, and estimated premorbid cognitive impairment; (2) Deteriorated, substantial decline from estimated premorbid function; and (3) Preserved, performing in the normal cognitive range without decline. The compromised group displayed the worst functional and symptom outcomes. Here, we extend our previous work by assessing the relationship among these categories of cognitive abilities and reported childhood adversity in 836 patients and healthy controls. Exploratory factor analysis of the Childhood Adversity Questionnaire revealed 3 factors (lack of parental involvement; overt abuse; family breakdown and hardship). People with schizophrenia reported significantly more childhood adversity than healthy controls on all items and factors. People with schizophrenia in the compromised group reported significantly more lack of parental involvement and family breakdown and hardship and lower socioeconomic status than those in the deteriorated group. The cognitive groups were not related to family history of psychosis. These findings identify specific social and family factors that impact cognition, highlighting the important role of these factors in the development of cognitive and functional abilities in schizophrenia.

Widespread Volumetric Reductions in Schizophrenia and Schizoaffective Patients Displaying Compromised Cognitive Abilities.

Objective: Progress toward understanding brain mechanisms in psychosis is hampered by failures to account for within-group heterogeneity that exists across neuropsychological domains. We recently identified distinct cognitive subgroups that might assist in identifying more biologically meaningful subtypes of psychosis. In the present study, we examined whether underlying structural brain abnormalities differentiate these cognitively derived subgroups. Method: 1.5T T1 weighted structural scans were acquired for 168 healthy controls and 220 patients with schizophrenia/schizoaffective disorder. Based on previous work, 47 patients were categorized as being cognitively compromised (impaired premorbid and current IQ), 100 as cognitively deteriorated (normal premorbid IQ, impaired current IQ), and 73 as putatively cognitively preserved (premorbid and current IQ within 1 SD of controls). Global, subcortical and cortical volume, thickness, and surface area measures were compared among groups. Results: Whole cortex, subcortical, and regional volume and thickness reductions were evident in all subgroups compared to controls, with the largest effect sizes in the compromised group. This subgroup also showed abnormalities in regions not seen in the other patient groups, including smaller left superior and middle frontal areas, left anterior and inferior temporal areas and right lateral medial and inferior frontal, occipital lobe and superior temporal areas. Conclusions: This pattern of more prominent brain structural abnormalities in the group with the most marked cognitive impairments-both currently and putatively prior to illness onset, is consistent with the concept of schizophrenia as a progressive neurodevelopmental disorder. In this group, neurodevelopmental and neurodegenerative factors may be important for cognitive function.

Accelerated Gray and White Matter Deterioration With Age in Schizophrenia.

OBJECTIVE: Although brain changes in schizophrenia have been proposed to mirror those found with advancing age, the trajectory of gray matter and white matter changes during the disease course remains unclear. The authors sought to measure whether these changes in individuals with schizophrenia remain stable, are accelerated, or are diminished with age. METHOD: Gray matter volume and fractional anisotropy were mapped in 326 individuals diagnosed with schizophrenia or schizoaffective disorder and in 197 healthy comparison subjects aged 20-65 years. Polynomial regression was used to model the influence of age on gray matter volume and fractional anisotropy at a whole-brain and voxel level. Between-group differences in gray matter volume and fractional anisotropy were regionally localized across the lifespan using permutation testing and cluster-based inference. RESULTS: Significant loss of gray matter volume was evident in schizophrenia, progressively worsening with age to a maximal loss of 8% in the seventh decade of life. The inferred rate of gray matter volume loss was significantly accelerated in schizophrenia up to middle age and plateaued thereafter. In contrast, significant reductions in fractional anisotropy emerged in schizophrenia only after age 35, and the rate of fractional anisotropy deterioration with age was constant and best modeled with a straight line. The slope of this line was 60% steeper in schizophrenia relative to comparison subjects, indicating a significantly faster rate of white matter deterioration with age. The rates of reduction of gray matter volume and fractional anisotropy were significantly faster in males than in females, but an interaction between sex and diagnosis was not evident. CONCLUSIONS: The findings suggest that schizophrenia is characterized by an initial, rapid rate of gray matter loss that slows in middle life, followed by the emergence of a deficit in white matter that progressively worsens with age at a constant rate.

Clozapine bioactivation induces dose-dependent, drug-specific toxicity of human bone marrow stromal cells: a potential in vitro system for the study of agranulocytosis.

Clozapine, an atypical antipsychotic drug effective in treatment of refractory schizophrenia causes potentially life-threatening agranulocytosis. The drug undergoes bioactivation to a toxic, chemically reactive intermediate with capacity to target stromal cells, central components of the bone marrow microenvironment implicated in neutrophil development. To identify possible mechanisms underpinning disruption of stroma as a site of drug bioactivation, toxicity was induced in vitro. Therefore metabolite generation procedures utilizing HOCl or HRP-H(2)O(2) as primary components involved in clozapine metabolism were adapted for stromal culture and coupled with viability determinations. Drug oxidation by HOCl was less toxic to stromal cells than HRP-H(2)O(2) based methods. More specifically, clozapine bioactivation by HRP-H(2)O(2) caused dose-dependent inhibition of stromal viability at therapeutically relevant concentrations. Differences in susceptibility of HAS303 and LP101 cells to the clozapine nitrenium ion were also evident. Stromal cell death was attributed to clozapine in the presence of a complete metabolising system comprising HRP and H(2)O(2). In the absence of a complete metabolising system clozapine was not cytotoxic. For LP101 cells, drug plus HRP (minus H(2)O(2)) also induced toxicity. Importantly, other antipsychotic drugs including risperidone, olanzapine and haloperidol when bioactivated, were not cytotoxic, indicating system specificity for clozapine. Exogenous GSH, N-acetylcysteine, l-ascorbic acid, catalase, and sodium azide afforded protection to cells whereas S-methylGSH, GSSG, ketoprofen and proadifen did not. Thus functional data derived from the in vitro stromal system defined in these studies may enable further investigation of the mechanisms subserving stromal impairment in clozapine-induced agranulocytosis and direct attention to improved methods for its prevention.

Neuregulin-1 and schizophrenia in the genome-wide association study era.

Clinical and pre-clinical evidence has implicated neuregulin 1 (NRG1) as a critical component in the pathophysiology of schizophrenia. However, the arrival of the genome-wide association study (GWAS) era has yielded results that challenge the relevance of NRG1 in schizophrenia due to the absence of a genome-wide significant NRG1 variant associated with schizophrenia. To assess NRG1's relevance to schizophrenia in the GWAS era, we provide a targeted review of recent preclinical evidence on NRG1's role in regulating several aspects of excitatory/inhibitory neurotransmission and in turn schizophrenia risk. We also present a systematic review of the last decade of clinical research examining NRG1 in the context of schizophrenia. We include concise summaries of genotypic variation, gene-expression, protein expression, structural and functional neuroimaging as well as cognitive studies conducted during this time period. We conclude with recommendations for future clinical and preclinical work that we hope will help prioritize a strategy forward to further advance our understanding of the relationship between NRG1 and schizophrenia.

The impact of premorbid and current intellect in schizophrenia: cognitive, symptom, and functional outcomes.

BACKGROUND: Cognitive heterogeneity among people with schizophrenia has been defined on the basis of premorbid and current intelligence quotient (IQ) estimates. In a relatively large, community cohort, we aimed to independently replicate and extend cognitive subtyping work by determining the extent of symptom severity and functional deficits in each group. METHODS: A total of 635 healthy controls and 534 patients with a diagnosis of schizophrenia or schizoaffective disorder were recruited through the Australian Schizophrenia Research Bank. Patients were classified into cognitive subgroups on the basis of the Wechsler Test of Adult Reading (a premorbid IQ estimate) and current overall cognitive abilities into preserved, deteriorated, and compromised groups using both clinical and empirical (k-means clustering) methods. Additional cognitive, functional, and symptom outcomes were compared among the resulting groups. RESULTS: A total of 157 patients (29%) classified as 'preserved' performed within one s.d. of control means in all cognitive domains. Patients classified as 'deteriorated' (n=239, 44%) performed more than one s.d. below control means in all cognitive domains except estimated premorbid IQ and current visuospatial abilities. A separate 138 patients (26%), classified as 'compromised,' performed more than one s.d. below control means in all cognitive domains and displayed greater impairment than other groups on symptom and functional measures. CONCLUSIONS: In the present study, we independently replicated our previous cognitive classifications of people with schizophrenia. In addition, we extended previous work by demonstrating worse functional outcomes and symptom severity in the compromised group.

Clozapine regulation of p90RSK and c-Fos signaling via the ErbB1-ERK pathway is distinct from olanzapine and haloperidol in mouse cortex and striatum.

Treatment of the positive psychotic symptoms of schizophrenia with standard antipsychotic drugs (APDs) is ineffective in a proportion of cases. For these treatment resistant patients the alternative is the APD clozapine which is superior to other agents but carries serious side effects. Why clozapine is uniquely effective is unknown, but we have previously postulated may involve G-protein coupled receptor (GPCR) and epidermal growth factor (EGF) receptor (ErbB1) transactivation signaling to the mitogen-activated protein kinase-extracellular signal regulated kinase (MAPK-ERK) cascade. This was based upon clozapine induced initial down-regulation and delayed ErbB1 mediated activation of the cortical and striatal ERK response in vivo distinct from other APDs. This study investigated if modulation of the ErbB1-ERK1/2 pathway by clozapine, olanzapine and haloperidol affected expression of the ERK substrates p90RSK and c-Fos, factors that regulate transcription of proteins associated with neuroplasticity and synapse formation in C57Bl/6 mice. In cortex and striatum, acute clozapine treatment induced biphasic p90RSK phosphorylation via MEK that paralleled ERK phosphorylation independent of EGF receptor blockade. By contrast, olanzapine and haloperidol caused p90RSK phosphorylation that was not concomitant with ERK signaling over a 24-hour period. For c-Fos, clozapine elevated expression 24h after administration, a timeframe consistent with ERK activation at 8h. Alternatively, haloperidol stimulation of c-Fos levels limited to the striatum was in accord with direct transcriptional regulation through ERK. The unique spatio-temporal expression of downstream nuclear markers of the ErbB1-ERK pathway invoked by clozapine may contribute to its effectiveness in treatment resistant schizophrenia.

Clozapine-induced ERK1 and ERK2 signaling in prefrontal cortex is mediated by the EGF receptor.

The atypical antipsychotic drug clozapine is effective in treatment-refractory schizophrenia. The intracellular signaling pathways that mediate clozapine action remain unknown. A potential candidate is the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK-ERK) cascade that links G-protein-coupled receptor and ErbB growth factor signaling systems, thereby regulating synaptic plasticity and connectivity, processes impaired in schizophrenia. Here, we examined how clozapine differentially modulated phosphorylation of the MAPK isoforms, ERK1/ERK2 in primary murine prefrontal cortical neurons compared to the typical antipsychotic drug haloperidol. While clozapine and haloperidol acutely decreased cortical pERK1 activation, only clozapine but not haloperidol stimulated pERK1 and pERK2 with continued drug exposure. This delayed ERK increase however, did not occur via the canonical dopamine D(2)-Gi/o-PKA or serotonin 5HT(2A)-Gq-phospholipase-C-linked signaling pathways. Rather, epidermal growth factor (EGF) receptor signaling mediated clozapine-induced ERK activation, given dose-dependent reduction of pERK1 and pERK2 stimulation with the EGF receptor inhibitor, AG1478. Immunocytochemical studies indicated that clozapine treatment increased EGF receptor (Tyr1068) phosphorylation. In vivo mouse treatment studies supported the in vitro findings with initial blockade, subsequent activation, and normalization of the cortical ERK response over 24 h. Furthermore, in vivo clozapine-induced ERK activation was significantly reduced by AG1478. This is the first report that clozapine action on prefrontal cortical neurons involves the EGF signaling system. Since EGF receptor signaling has not been previously linked to antipsychotic drug action, our findings may implicate the EGF system as a molecular substrate in treatment-resistant schizophrenia.