Transcriptome studies in the context of disturbed connectivity in schizophrenia

Schmitt, Andrea; Reich-Erkelenz, Daniela; Gebicke-Härter, Peter; Falkai, Peter

doi:10.1590/S0101-60832012005000001

Abstracts

Schizophrenia is a severe neurobiological disease with genetic and environmental factors playing a role in the pathophysiology. Several brain regions have been implicated in the disease process and are connected in complex neuronal circuits. On the cellular and molecular level, affected connectivity between these regions, involving dysfunctional myelination of neuronal axons, as well as alterations on the synaptic level and energy metabolism of neurons leading to disturbances in synaptic plasticity are major findings in post-mortem studies. Microarray studies investigating genome-wide gene expression have contributed to the findings of alterations in complex pathways in relevant brain regions in schizophrenia. Moreover, first Laser-capture Microdissection studies allowed the investigation of gene expression in specific groups of neurons. However, it must be kept in mind that in post-mortem studies confounding effects of medication, mRNA quality as well as the capability of the brain for neuroplastic regenerative mechanisms in individuals with a lifetime history of schizophrenia may influence the complex pattern of alterations on the molecular level. Despite these limitations, hypothesis-free transcriptome studies in brain tissue from schizophrenia patients offer a unique possibility to learn more about underlying mechanisms, leading to new insights in the pathophysiology of the disease.

Schizophrenia; gene expression; microarray; RT-PCR; in situ hybridization; brain regions

Esquizofrenia é uma severa doença neurobiológica com fatores genéticos e ambientais desempenhando um papel na fisiopatologia. Diversas regiões cerebrais têm sido implicadas no processo da doença e estão conectadas em complexos circuitos neuronais. Nos níveis molecular e celular, a conectividade afetada entre essas regiões, envolvendo mielinização disfuncional dos axônios neuronais, bem como as alterações no nível sináptico e metabolismo energético levando a distúrbios na plasticidade sináptica, são os maiores achados em estudos post-mortem. Estudos de microarranjos investigando a expressão gênica contribuíram para os achados de alterações em vias complexas em regiões cerebrais relevantes na esquizofrenia. Além disso, estudos utilizando microdissecção e captura a laser permitiram a investigação da expressão gênica em grupos específicos de neurônios. Entretanto, deve ser mantido em mente que em estudos post-mortem, confusos efeitos de medicação, qualidade de RNAm, bem como capacidade de mecanismos regenerativos neuroplásticos do cérebro em indivíduos com história de vida de esquizofrenia, podem influenciar o complexo padrão de alterações no nível molecular. Apesar dessas limitações, estudos transcriptômicos livres de hipóteses em tecido cerebral de pacientes esquizofrênicos oferecem uma possibilidade única para aprender mais sobre os mecanismos subjacentes, levando a novas ópticas da fisiopatologia da doença.

Esquizofrenia; expressão gênica; microarranjo; RT-PCR; hibridização in situ; regiões cerebrais

^IDepartment of Psychiatry and Psychotherapy, University of Göttingen, Germany

^IIDepartment of Psychiatry, LMU München, Germany

^IIILaboratory of Neuroscience (LIM27), Institute of Psychiatry, University of Sao Paulo, SP, Brazil

^IVDepartment of Psychopharmacology, Central Institute of Mental Health, Mannheim, Germany

Correspondence

ABSTRACT

Schizophrenia is a severe neurobiological disease with genetic and environmental factors playing a role in the pathophysiology. Several brain regions have been implicated in the disease process and are connected in complex neuronal circuits. On the cellular and molecular level, affected connectivity between these regions, involving dysfunctional myelination of neuronal axons, as well as alterations on the synaptic level and energy metabolism of neurons leading to disturbances in synaptic plasticity are major findings in post-mortem studies. Microarray studies investigating genome-wide gene expression have contributed to the findings of alterations in complex pathways in relevant brain regions in schizophrenia. Moreover, first laser-capture microdissection studies allowed the investigation of gene expression in specific groups of neurons. However, it must be kept in mind that in post-mortem studies confounding effects of medication, mRNA quality as well as the capability of the brain for neuroplastic regenerative mechanisms in individuals with a lifetime history of schizophrenia may influence the complex pattern of alterations on the molecular level. Despite these limitations, hypothesis-free transcriptome studies in brain tissue from schizophrenia patients offer a unique possibility to learn more about underlying mechanisms, leading to new insights in the pathophysiology of the disease.

Keywords: Schizophrenia, gene expression, microarray, RT-PCR, in situ hybridization, brain regions.

Introduction

Schizophrenia is a debilitating, chronic neurocognitive disorder affecting mostly young adults. Its prevalence is 1% world-wide independently of cultural identity or ethnicity. The disorder is characterized by various abnormal cognitive, affective and motor behavioral features. Most of the affected subjects (> 80%) are unable to maintain a self-sustaining professional life¹ and their life expectancy is shortened at an average of 10 years². Along with frequent hospitalizations and the progressive neurocognitive impairment it deeply impacts the lives of those afflicted and their families. The cumulated financial burden of European social and health systems is high (€ 35 229 000 000 annually)³.

Despite tremendous advances in science and medicine, the disorder remains mysterious. In the absence of consistent neurobiological markers for schizophrenia, diagnosis still relies on subjective assessment of a cluster of signs and symptoms, based on ICD-10 or DSM-IV-R criteria, and the current pharmacological treatments in most cases are empirically based. The identification of the pathophysiology associated with biological parameters deployable to unequivocally diagnose or evaluate the course and treatment remains challenging.

To date, the hypothesis of schizophrenia basing on multifactorial interactions which impact both early and late brain developmental changes prevails. Several lines of evidence support this neurodevelopmental hypothesis; among them are the association between obstetric complications such as the common factor hypoxia and schizophrenia^4,5. There is strong evidence for a genetic basis for schizophrenia with functional polymorphisms in the promoter region of a gene, resulting in quantitative changes of the amount of gene expression or polymorphisms in the encoding region resulting in qualitative alterations of the gene product. However, abnormal gene-environmental interaction during development might also be due to epigenetic mechanisms on chromatin remodeling, which regulates gene transcription in complex neuronal pathways, and such mechanisms have been demonstrated in schizophrenia^6,7.

Neuronal circuits involved in schizophrenia

Recent investigations suggest that not only a deficit in one circumscribed region is underlying cognitive or positive/negative symptoms in schizophrenia, but alterations in neuronal networks control cognition and higher sensory processing.

Meta-analyses of structural magnetic resonance imaging (MRI) studies reveal gray matter volume deficits in a number of different brain regions in schizophrenic patients. Affected regions are the medial temporal lobe including the hippocampus and entorhinal cortex, the heteromodal association cortex including the prefrontal, parietal, and superior temporal cortex. The degree of grey matter reduction is in the range of 5%-10% in the frontotemporolimbic network^8,9.

Structural MRI and post-mortem studies have shown volume loss in the medial temporal lobe, especially in the hippocampus, as one of the most consistent structural abnormalities, along with deficits in spatial and verbal memory¹⁰. Additionally, post-mortem studies showed volume loss in hippocampal subregions which have been related to positive symptoms^11,12. Newer developments in neuroimaging techniques like diffusion tensor imaging (DTI) afforded the investigation of disturbances in connectivity and neuronal networks. DTI studies of white matter tracts of the fornix body and hippocampus in schizophrenia show decreased fractional anisotropy and support the hypothesis of structural and functional dysconnectivity^13-16 which is correlated with deficits in cognitive function, e.g. verbal declarative memory¹⁷.

The prefrontal cortex has been regarded as a key region for the pathophysiology of the disease due to its involvement in executive function and working memory, which are affected in schizophrenia¹⁸. MRI investigations uncovered reductions mainly in the dorsolateral prefrontal cortex in 70% of the studies repor-ting decreased volumes^19,20. A further sign of early developmental deficit consists in the disturbed gyrification index as a measure of disturbed cortical folding^21-25. A decreased prefrontal glucose uptake and hypofrontality has been related to negative symptoms and cognitive dysfunction^26,27. The anterior cingulate cortex is part of to the medial prefrontal cortex and receives sensory information from insular, temporal and parietal association cortices plus emotional information from the amygdala and orbitofrontal cortex. This brain region is modulator of the dorsolateral prefrontal cortex^28,29 and has been shown to mediate urban upbringing as an environmental risk factor in schizophrenia³⁰. The prefrontal cortex, as much as the superior temporal gyrus, anterior cingulate and inferior parietal cortex, belongs to the heteromodal association cortex, which is responsible for cognitive processing and influences complex beha-vior³¹. A dysfunction in these cortices has been described in chronic schizophrenia patients living in home residencies, a population which serves as basis for most of the post-mortem investigations³². A meta-analysis of fMRI studies of executive function in schizophrenia revealed reduced activation of the prefrontal cortex, anterior cingulate cortex, and thalamus³³.

A reduction in thalamic volumes in schizophrenia has been shown in structural MRI studies^34,35. The thalamus acts as a central relay station, transferring peripheral sensory inputs to the cortex. It plays a critical role in filtering sensory information, in regulating cognitive input to the cortex, and mediating cortico-cortical connections between areas particularly implicated in schizophrenia, such as frontal and temporal regions. In schizophrenia patients, positron emission tomography (PET) studies show a dysfunction of the cortico-cerebellar-thalamic-cortical neuronal circuit contributing to "cognitive dysmetria", i.e. impaired cognition and other symptoms of the disease^36,37. In addition, a disturbed prefronto-parietal-thalamic network has been shown to be involved in disease-related working memory deficits in schizophrenia³⁸. In summary, alterations of connectivity are present in complex neuronal circuits, causing not only a dysfunction in specific brain regions but also basing on deficits in complex pathways on the molecular level.

Principles of gene expression methods in post-mortem tissue

To unravel the neurobiological disturbances underlying altered activation of volume loss of brain regions, during the last years, transcriptome studies using a hypothesis-free approach with microarray investigations of thousands of genes, and hypothesis-driven studies using quantitative real-time polymerase chain reaction (qRT-PCR) or in situ hybridization have been conducted. Each of these methods provides advantages in molecular biology: the high quantity of genes detectable with microarray studies and higher sensitivity in the detection of a limited number of genes in qRT-PCR studies, which makes this method suitable for verification of altered gene expression in microarray experiments. The spatial resolution of subregions, cortical layers or cell populations is the advantage of in situ hybridization investigations. The development of high-throughput technologies as genome-wide microarray, proteomics or recently next-generation sequencing has improved the understanding of the involvement of complex molecular pathways in the pathophysiology of the disease, thus enhancing knowledge about the dysfunction of single molecules³⁹. New microscopic techniques like laser-capture microdissection, used for collection of neuronal cell populations or subregions with preserved mRNA integrity in histological stained sections, provide new insights in the understanding of interactions between cellular and molecular networks^40,41.

However, many samples stemming from human autopsies are in sub-optimal condition for gene expression studies and multiple factors influence the quality of extracted mRNA from brain tissue. They mainly include the agonal state with hypoxia, cause of death and the post-mortem delay. The tissues' pH-level is influenced by these factors and may serve as a marker of subsequent RNA quality. Additionally, individual mRNA transcripts may exhibit different decay rates and half-lifes. Thus, post-mortem delay and pH should be documented and mRNA quality of the tissue should always be determined for example using the Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA)^42,43. Additionally, most of the chronic schizophrenia patients had been treated with antipsychotics for decades, so effects of antipsychotic doses calculated in chlorpromazine equivalents should be correlated statistically to individual mRNA levels⁴⁴.

Disturbed macroconnectivity: evidence from cellular and molecular studies

Disturbed connectivity in schizophrenia is associated with deficits in myelination of neuronal axons. Myelination plays an important role in nerve cell propagation and also integrates the brain's structural synchrony, resulting in functional and structural connectivity. Oligodendrocytes are the myelin-building glia cells in the brain. Other oligodendrocyte functions are trophic signaling to adjacent neurons, synthesis of growth factors, neuronal survival and development, neurotransmission, and synaptic function⁴⁵. Reduced number, dysfunction or death of oligodendrocytes may influence neuronal integrity. In several stereological studies, numbers of oligodendrocytes have been reported to be decreased in the prefrontal cortex and CA4 subregion of the hippocampus^46-48; density maps of oligodendrocytes show lesser clustering in the prefrontal cortex⁴⁷. Structural abnormalities of myelin sheath and regressive changes in oligodendrocytes in the prefrontal cortex and hippocampus may be based on neurodevelopmental disturbances or apoptotic cell death^49-51. Interestingly, the heteromodal association cortex has its main myelination period in young adulthood with first symptoms of schizophrenia appearing⁵². On the molecular level, evidence for disordered myelination has been provided by several microarray studies in brain regions of schizophrenia patients^53-57. Quantitative RT-PCR studies^57,58 confirm the implication of oligodendrocyte and myelin in the pathogenesis by revealing down-regulation of multiple oligodendrocyte and/or myelin-related genes in different brain areas⁵⁹. Additionally, proteomic studies reported oligodendrocyte dysfunction and related disturbances of myelination. Myelin basic protein (MBP) is the major constituent of the myelin sheath produced by oligodendrocytes and the myelin oligodendrocyte glycoprotein (MOG) was found to be downregulated in the prefrontal and temporal cortex, hippocampus and entorhinal cortex, where it was associated with migrational abnormalities of pre-alpha cells^60-63. Additionally the oligodendrocyte marker 2',3' cyclic nucleotide,3'-phosphodiesterase (CNP) was downregulated in these regions and reduced cell number of CNPase-stained neurons has been reported in the prefrontal cortex⁴⁷. Other commonly altered myelin-related genes are peripheral myelin protein 22 (PMP22), myelin-associated Gylcoprotein (MAG), Myelin and leucozytes protein (MAL), transferrin (TF), claudin 1, claudin 5, Proteolipid Protein (PLP), Gelsolin (GSN), Plasmolipin (PLLP, TM4SF11), and quaking (QKI)^56,57,59, which play an important role in neurodevelopment, growth, and maintenance of myelin und axons⁶⁴. These proteins might also be potential biomarkers for schizophrenia⁶⁵. The oligodendrocyte protein ermin was found to be upregulated in the prefrontal cortex and downregulated in the temporal cortex in two used shotgun proteomic studies^66,67. Brockschnieder et al.⁶⁸ concluded that ermin probably contributes to the maintenance and stabilization of the myelin sheath in the adult brain.

Schizophrenia as a disorder of disturbed synaptic plasticity

'Synapses are located on dendritic spines which have been reported to be reduced in the prefrontal cortex of schizophrenic patients⁶⁹. Possibly a hypofunction of the glutamatergic NMDA receptor acting on long-term potentiation disturbs synaptic plasticity⁷⁰. On the molecular level there is strong support for decreased synaptic plasticity, mainly affecting the expression of presynaptic vesicle proteins including the synaptosome-associated protein 25 (SNAP-25) and syntaxin forming the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex^71-79. The trimeric SNARE complex is involved in membrane fusion and exocytosis of neurotransmitters. Levels of SNAP-25 mRNA have been reported to be increased in the superior temporal cortex of the younger group of schizophrenic patients⁷⁹. A microarray study in the prefrontal cortex showed altered expression of genes associated with synaptic vesicle recycling in schizophrenia⁸⁰. Accordingly, on the protein level reduced synaptophysin and SNAP-25 immunoreactivity have been detected in the prefrontal cortex^60,69,81-83. However, results are not consistent and vary between different brain regions^83,84. In the terminal fields of entorhinal cortex projections, SNAP-25 expression was decreased⁸⁵. In the hippocampus it was shown that neuron populations had fewer dendritic spines and reduced dendritic arborizations⁸⁶. This observation was supported by studies detecting molecular markers like microtubule-associated protein 2 (MAP2) and spinophilin^87,88. Evidence for reduced presynaptic markers was also reported. A more consistent finding within the hippocampus is abnormal synaptic connectivity, as shown by decreased expression of the presynaptic proteins synapsin, synaptophysin and SNAP-25⁷³. In addition, disturbances of complexins in the hippocampus were associated with the severity of ante-mortem cognitive impairment⁷⁸. Further insights in disturbed synaptic plasticity provided a microarray study of the superior temporal cortex, showing that decreased expression of immune-related genes may affect synaptic strength and transmission⁴⁴. Altogether, when combining findings on the mRNA and protein level, this strongly supports the notion of regionally distinct expression of synaptic genes.

Altered expression of cytoskeletal proteins in schizophrenia-related brain regions may influence synaptic plasticity during neurodevelopment and in adulthood, with consequences on neurotransmission. In nerve terminals, crystalline mu modulates cytoskeletal proteins⁸⁹. In a proteomic study, this cytoskeletal protein has been reported to be upregulated in the prefrontal cortex in schizophrenia⁶⁶. Increased expression of crystalline mu in schizophrenia has also been reported in transcriptome studies^53,90. However, a more detailed investigation of cytoskeletal genes in post-mortem brain regions is needed. Interestingly, a laser capture microdissection (LCM) study in isolated dentate granular neurons reported decreased expression of genes related to cytoskeletal proteins, synaptic plasticity and energy metabolism⁹¹, thus supporting the hypothesis of disturbed microconnectivity.

Disturbed energy metabolism: evidence from transcriptome studies

In schizophrenia, several altered genes are involved in energy metabolism-related processes of glucose metabolism. For example, hexokinase was found to be upregulated in prefrontal and superior temporal cortex. It participates in different cellular processes, be it glycolysis or fructose, galactose, sucrose, and mannose metabolism⁶⁵. Some other proteins reported to be altered on the protein level are also described as differentially expressed in transcriptome studies. They are involved in energy metabolism such as the mitochondrial precursor of ATP synthase alpha chain, transferrin, and aldolase C^53,92. Neuronal mitochondria are known to produce most of the cell energy. Therefore, alterations in mitochondrial proteins, caused by endogenous or exogenous factors, can result in dysregulation of energy production. Pathways involved in energy metabolism entail internal processes as glycolysis, the Krebs cycle, and oxidative phosphorylation. Changes in mitochondrial oxidative phosphorylation in the brain⁹³ and in platelets⁹⁴ of schizophrenic patients have already been reported. Genes associated with mitochondrial functions essential for the normal development of the brain and synaptic function have also been shown to be abnormal^95,96. Ben-Shachar et al.⁹⁵ found a pathophysiological link between mitochondrial function and schizophrenia. This was demonstrated at the level of mitochondrial function, mitochondrial respiration and complex I activity and at the level of gene and protein expression. The disease also exhibits an abnormal interaction between dopamine and the mitochondria^95,97. Alterations in prohibitin were hypothesized to be involved in disturbed synaptic plasticity⁹⁸. It is known to control histone deacetylation⁹⁹ and can be localized in the inner membrane of mitochondria.

Conclusions

Transcriptome studies state strong evidence for a synaptopathy, deficits in oligodendrocyte function and energy metabolism underlying cognitive deficits and symptom domains in schizophrenia. These may be fundamental for volume deficits in distinct neuronal circuits as well as altered brain activation during cognitive tasks. However, despite these pathophysiological insights, in post-mortem studies confounding effects of medication, mRNA quality, cause of death as well as the capability of the brain for neuroplastic regenerative mechanisms in individuals with a lifetime history of interacting mechanisms by the disease process itself, but also by physical and cognitive activation, may lead to a complex pattern of brain alterations. Despite these limitations, transcriptome and proteome studies of post-mortem tissue offer the unique opportunity of identifying disturbances in complex pathways to unravel the complex picture of the schizophrenia pathophysiology and to develop new neuroprotective treatment strategies.

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