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A jigsaw puzzle in construction

Proteins lead to the belief that schizophrenia is a biological disorder

reproductionWho am I? (detail) – Duane Michals, 1955reproduction

Schizophrenia is nothing new to the viewers who watched the soap opera “Caminho das Índias,” aired by TV Globo from January to September 2009. The character Tarso, played by actor Bruno Gagliasso, hears voices, believes that someone implanted a chip under his skin to steal his thoughts, imagines that he is going to dissolve and loses control of himself during violent crises. These symptoms are typical of this mental disorder that afflicts one out of every 100 people – it is estimated that there are approximately 1.8 million schizophrenics in Brazil. Biologist Daniel Martins-de-Souza, currently a post-doctorate researcher at Germany’s Max Planck Institute, identified a series of proteins involved in the biochemical mechanisms of schizophrenia that have helped understand the details of how this disorder causes all the symptoms referred to above.

During his doctorate studies at the Biochemistry Department of the State University of Campinas/ Unicamp, Souza examined the proteins produced in the brains of seven healthy people and nine people with schizophrenia. “Each region of the brain expresses thousands of different proteins,” he says. “We were able to reduce this number to the couple of dozen proteins related to the disorder.” These proteins appear in altered quantities in brains of schizophrenic patients and may be able to provide valuable clues on how schizophrenia appears and manifests itself. The study was coordinated by biologist Emmanuel Dias Neto from the Neurosciences Lab of the Institute of Psychiatry/Ipq. The Institute is a department of the University of São Paulo/USP’s Hospital das Clínicas teaching hospital of the Medical School/FMUSP. The project was funded by FAPESP and by the Associação Beneficente Alzira Denise Hertzog da Silva/Abadhs association.

Focusing on the damage that schizophrenia causes to the brain, Souza selected the regions known to be related to the disorder: the pre-frontal cortex responsible for certain kinds of memory, differentiations in contradictory thoughts, establishment of the concepts of right and wrong, social behavior and expression of personality; the Wernicke region, a part of the cortex linked to speech, language and communication; the front lobe, related to cognitive and affective processes. This distribution of the affected regions attests to the complexity of schizophrenia, a word that means “splitting of the brain.”

Several research teams from around the world have concentrated on analyzing the genetic alterations associated with the disorder, but Souza is in favor of focusing on proteins, the product of these altered genes. “The proteins are the actual players that act on the organism,” he says, as a more active gene does not necessarily translate into a higher concentration of the protein whose production this gene commands. “We have confirmed previous findings and added proteins that had not been taken into consideration.” This year, the findings have already resulted in four scientific articles. He is now concentrating on some of these altered molecules to see how they affect the development of the disorder.

A comparison of the regions of the brain altered by this mental disorder is a complex task. “Most of the proteins appear in distinct quantities in the different parts of the brain,” says Souza. His intention is not to characterize how each part of the brain functions, but rather see what the parts have in common and what can act as the marker of this disorder, which differentiates patients form healthy people. “This can help us understand schizophrenia,” he believes. He started looking for the proteins – something akin to searching for specific stars in a starry sky – in the proteomic laboratory at Unicamp, which is headed by José Camillo Novello and Sérgio Marangoni.

With promising results in his hands, the researcher went to the Max Planck Psychiatry Institute in Germany in search of a more sensitive method that would allow the detection of even tiny concentrations of proteins: the shotgun proteomic analysis, a method which is not used in Brazil yet. This more refined method uses a small quantity of proteins to distinguish samples of healthy brains from brains afflicted by schizophrenia.

Detailed exam
One half of the alterations detected in the IPq group is related to energy production in cells. One series of proteins involved in the degradation of glucose and in the production of adenosine triphosphate /ATP, the molecule that provides energy to cells, appears in lower quantities in the brains of schizophrenics. There are signs that the metabolism of glucose is damaged in schizophrenia; however, researchers do not know whether this is the cause of the disorder or a consequence of the treatment. In Souza’s opinion, the findings tend towards the first option. “The proteins that we identified show that the degradation of glucose is altered due to the action of specific enzymes.” But the issue is far from being clarified. Wagner Gattaz, director of the IPq and clinical advisor of the study, explains that all patients were on medication that affected brain activity. “The possibility that this medication influenced part of our results cannot be discarded,” he states.

reproductionWho am I? (detail) – Duane Michals, 1955reproduction

Souza detected high contents of proteins that fight oxidative stress. This indicates that, in addition to reducing the use of glucose, metabolism alterations generate more free radicals, causing damage to the brain cells. He explains that the energy generating process itself, inside the cells” mitochondria, produces oxidative molecules. When the concentration of these molecules – the free radicals – reaches a specific level, the stress is so great that the mitochondria break and free radicals spread throughout the cell.

The detailed method also allowed the researcher to observe a decrease in the quantity of proteins produced in the oligodendrocytes. These are important cells because they produce myelin, a substance that coats the neuron projections – the nerve cells responsible for transmitting information. Without the myelin, the nerves are similar to live wires that let electric power leak along the way. “Our findings suggest that alterations occur in the two markers linked to the oligodendrocytes,” Souza says. Three of these proteins – the basic myelin protein, transferine, and myelin glycoprotein – had already been associated with another mental disorder, namely multiple sclerosis. This finding suggests that, like multiple sclerosis, some symptoms of schizophrenia can be caused by the degeneration of the nervous system.

The ability of the nerves to transmit information is also affected by calcium. By detecting the alterations in the production of several proteins, Souza observed that the brain cells of schizophrenics absorb more calcium. This important signalizer of several cell functions also regulates the action of enzymes that degrade the myelin; this is why a concentration imbalance can lead to significant losses of the nerve functions. Calcium also controls the functioning of dopamine receptors, a neurotransmitter whose production is excessive in schizophrenia. Souza’s findings have helped describe a chain that leads to the excessive activity of dopamine. Psychiatrists treat this excess activity of dopamine with medication that blocks the dopamine-activated receptors.

Proteins are also related to other aspects of schizophrenia that deserve a more detailed investigation, such as the relationship of the disorder with the immune system (epidemiological studies have shown that people whose mothers had had the flu during pregnancy have a higher risk of developing schizophrenia) and with the structure of cells. One-fourth of the proteins produced in bigger or smaller quantities participate in the formation of the cytoskeleton, the modification of which affects the shape of cells and the neurons’ ability to transmit information. The alterations are found in specific molecules that are exclusive to astrocytes, one of the nerve cell types that form the structure of the brain and maintain the structure where the neurons fit in. Although the effect on the structure of some cells is flagrant in schizophrenia and helps clarify the related biology, Souza is not going to investigate this as a marker for diagnosis. “Any kind of disease causes alterations in the cytoskeleton,” he says.

Back from Germany after concluding his post-doctorate studies, Souza is now looking for altered quantities of these same cells in the blood and in cerebrospinal fluid, the fluid that surrounds the brain and the spinal cord. This is the only way – in view of the fact that taking brain samples of a live person is far from being a trivial task – in which it will be possible to develop a diagnostic test that could complement the clinical exam in cases where this disorder has not completely manifested itself.

In spite of these promising results, protein analysis has to be viewed cautiously. “No biochemical exam by itself can detect schizophrenia,” emphasizes psychiatrist Helio Elkis, from USP’s Department of Psychiatry and coordinator of the IPq’s Programa de Esquizofrenia/Projesq, Schizophrenia Project. In his opinion, the only safe diagnosis is a clinical evaluation that adheres strictly to well-defined international criteria, which include psychotic symptoms, such as deliriums and hallucinations; denials, which involve the loss of feeling, difficulty in making decisions, and lack of interest; disorganized thoughts that make it difficult to understand what the patient is trying to say; anxiety and depression; and cognitive disturbances.

Head in slices: Imaging reveals an accumulation of calcium in the typical brain of Fahr´s disease, the symptoms of which can be confused with those of schizophrenia

matheus oliveira/ufpe Head in slices: Imaging reveals an accumulation of calcium in the typical brain of Fahr´s disease, the symptoms of which can be confused with those of schizophreniamatheus oliveira/ufpe

In his opinion, the credibility of Souza’s work is reinforced by the diagnosis of patients whose brains were examined , which followed international criteria and includes an extended clinical follow-up. But he emphasizes that many things have to happen before the measuring of these proteins will help in diagnosing a psychiatric disorder. “Once these markers are identified, we will need to conduct tests on a large group to compare the molecular results with the clinical ones.”

In view of such a multi-dimensional disorder, the more tools become available the easier it will be to elucidate its biological functioning and, who knows, deal with it. These tools can come from unexpected sources, such as another disorder that provokes similar effects to those of schizophrenia. “The study of other disorders with psychotic symptoms can help understand schizophrenia,” argues neuropsychiatrist João Ricardo Oliveira, from the Federal University of Pernambuco/UFPE. He studied genes linked to schizophrenia approximately 10 years ago, when he was still an undergraduate medical student. Now he is a specialist in Fahr’s disease, in which the accumulation of calcium in various parts of the brain causes a variable combination of symptoms such as Parkinson’s, tremors, cognitive difficulties, psychosis and mood swings. “When the first symptom is psychosis, Fahr’s disease is often treated as being schizophrenia,” he says.” In these cases, medication has no effect and the mistaken diagnosis is only discovered when the calcification of the brain is seen in CAT scans.

His group is now studying the genetics and calcification patterns of Fahr’s disease. Recently, the group reported the genetic influence in the disease, based on a study of identical twins: the accumulation of calcium appeared at the same time and evolved in a similar way, affecting the same regions of the twins’ brains, according to an article published this year in “Parkinsonism and Related Disorders.” In Oliveira’s opinion the analysis of how genetic composition and calcium deposition patterns cause different symptoms can help understand schizophrenia and various other mental disorders. Oliveira has samples from approximately 15 families.

This task demands multiple approaches. While they commemorate tangible results, researchers still see a long path that needs to be followed. To confirm the meaning of the alterations observed by the group from IPq, it will be necessary to show that these alterations are specific to schizophrenia and detect whether any of them result from the treatment and not from the disorder. “The specificity of the findings can only be clarified if, in a future study, we compare the brains of schizophrenics and those of the healthy control group with the brains of a third group, which comprises individuals under psychiatric control (for examples, patients with bipolar disorder),” Gattaz explains. Emmanuel Dias Neto adds that “For years we have tried to over-simplify things. The time has come to look at these issues in terms of their true complexity, by examining the metabolic pathways rather than isolated markers – if these markers actually existed, they would already have been identified.”

The project
Metabolism of phospholipids in neuropsychiatric diseases (nº 02/13633-7); Modality Theme Project; Coordinator Wagner Farid Gattaz – USP; Investment R$ 1.803.528,52

Artigos científicos
MARTINS-DE-SOUZA, D. et al. Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia. Journal of Psychiatric Research. v. 43, n. 11, p. 978-986. jul. 2009.
MARTINS-DE-SOUZA, D. et al. Proteome analysis of schizophrenia patients Wernicke’s area reveals na energy metabolism dysregulationBMC Psychiatry. v. 9, n. 17. abr. 2009.
MARTINS-DE-SOUZA, D. et al. Prefrontal cortex shotgun proteome analysis reveals altered calcium homeostasis and immune system imbalance in schizofrenia. European Archives of Psychiatry and Clinical Neuroscience. v. 259, n. 3, p. 151-163. abr. 2009.
MARTINS-DE-SOUZA, D. et al. Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis. Journal of Neural Transmission. v. 116, n. 3, p. 275-289. mar. 2009.