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Trancriptional control of dendritic arbors morphology in pathogeny and therapy of neuropsychiatric diseases

Final Report Summary - PSYCHIAPROTEGENOMIC (Transcriptional control of dendritic arbour morphology in pathogeny and therapy of neuropsychiatric diseases)

This project was designed to identify the possible alteration of SP4 transcription factor in psychiatric disorders and identify new altered molecular players that contribute to normal dendritic development. Also, this project has also addressed to test the capacity of lithium treatment to modulate dendritic morphology by SP4-dependent mechanism and some SP4-target genes altered in schizophrenia.

Work carried out and main results

(1) Analysis of SP1 and SP4 in post mortem brain tissue of subjects with bipolar disorder (BD) and in response to neuronal activity and lithium (data published in: The transcription factor SP4 is reduced in post mortem cerebellum BD subjects: Control by depolarisation and lithium. Pinacho R., Villalmanzo N., Lalonded J., Haro J.M. Meana J., Gill G. and Ramos B. Bipolar Disorders, (2011),13:474-48)
We hypothesised that SP4 levels may be altered in the brain of BD subjects and regulated by neuronal activity and drug treatment. We analysed messenger ribonucleic acid (mRNA) and protein levels of SP4 and SP1 in post mortem prefrontal cortex and cerebellum of BD subjects and controls. We also examined regulation of SP4 mRNA and protein levels by neuronal activity and lithium in rat cerebellar granule neurons. We report a reduction of SP4 and SP1 proteins, but not mRNAs, in cerebellum of BD subjects. SP4 protein and mRNA were also reduced in prefrontal cortex. Moreover, we found in rat cerebellar granule neurons that under non-depolarising conditions SP4, but not SP1, was polyubiquitinated and degraded by the proteasome while lithium stabilised SP4 protein.

(2) Analysis of SP factors in post mortem brain tissue of subjects with schizophrenia (data in a manuscript submitted and under final revision: Opposite regulation of brain SP1 and SP4 proteins associated to different symptomatology in schizophrenia. Pinacho R., Villalmanzo N., Roca M., Iniesta R., Monje A, Fusté M, Haro J.M. Meana J., Ferrer I. and Ramos B)
In this study, we explore the possibility that SP proteins and some possible target genes NR1, NR2A and DRD2 in cerebellum and prefrontal cortex of subjects with schizophrenia could be associated to positive or negative symptoms. We used immunoblot and quantitative polymerase chain reaction in extracts from post mortem brain of subjects with schizophrenia compared to control individuals. Subjects were evaluated pre mortem by the positive and negative syndrome scale and the clinical global impression schizophrenia scale. We report that cerebellar SP1 and SP4 levels as well as NR2A and DRD2 expression inversely correlated to negative symptoms in schizophrenia subjects. However, SP1 and SP4 protein levels directly associated to positive symptoms in prefrontal cortex, but not NR1, NR2A or DRD2. SP3 gene expression also associated to negative symptoms in cerebellum. None of cerebellar SP proteins were altered in schizophrenia subjects and only SP1 was reduced in prefrontal cortex.

(3) To identify proteomic changes in brain tissue of subjects with schizophrenia compared to healthy individuals which could control dendritic development (data in preparation to submit for publication: Large scale quantitative proteomic analysis in post mortem brain in schizophrenia. Pinacho R., Villalmanzo N., J. J. Meana, J. M. Haro, J. Villén, B. Ramos)

We performed a quantitative proteomic analysis post mortem cerebellum and prefrontal cortex of subjects with schizophrenia in collaboration with Dr Judt Villén (University of Washington, Seattle, USA). This method allows to compare proteomic profiles from samples with the same biological substrate in post mortem tissue without the need of an intermediate standard in vivo peptide labelled. Control lysate contained light endogenous peptides and schizophrenia sample was subject to a chemical reaction in vitro to obtain heavier peptides. We fractionated the peptide samples by HPLC and subsequently analysed by mass spectrometry. This analysis detected parallel peptides corresponding to light (L-control) and heavy (H-schizophrenia) peptides. The ratio H / L provides a quantitative determination of each identified peptide. Distribution of both proteins and peptides versus the ratio of Schizophrenia / control shows similar profiles in prefrontal cortex and cerebellum suggesting that similar proportion of the proteome is altered in both areas. By using this approach we have quantified more than 30 000 total peptides and 2 000 proteins in each brain area. Although proteome from cerebellum and prefrontal cortex share around 60 % of similarity, the percentage of common regulated proteins in both areas was only of 10 % approximately. Gene ontology analysis revealed that gene expression regulation functions are enriched in prefrontal cortex and cerebellum. We have validated 6 candidates in prefrontal cortex in the original samples and confirmed the results in a larger and independent cohort. We are now initiating the testing of an altered candidate in controlling dendritic function.

(4) To investigate whether lithium treatment change dendritic morphology by Sp4-dependent regulation of transcription (unpublished data). We monitored dendrites during the final phases of dendritic maturation in granule neurons treated with lithium under depolarising and non-depolarising conditions. We had not observed any effect of lithium in primary and secondary dendrites of granule neurons during dendritic development neither in response to depolarisation. As I have described in the first section, we have found that lithium partially restored Sp4 protein levels, essential in dendritic maturation, but only in mature neurons. However, at later stages, we still did not observe a reduction of dendritic number after lithium treatment in non-depolarising conditions.

Conclusions

(1) Our studies in BD provides the first evidence of altered SP4 protein in cerebellum and prefrontal cortex in this disorder supporting a possible role of transcription factor SP4 in the pathogenesis of the disease. In addition, our findings that SP4 stability is regulated by depolarisation and lithium in rat cerebellar granule neurons provides a pathway through which neuronal activity and lithium could control gene expression and suggests that normalisation of SP4 levels could contribute to treatment of affective disorders.

(2): Our results in schizophrenia show an opposite brain-regional regulation of SP proteins linked to a different symptomatology and suggest a molecular stratification in brain by symptoms. Cerebellar SP1 and SP4 may cooperate to prevent negative symptoms with a weak contribution of SP3 and through the upregulation of NR2A and DRD2 expression, while these factors may work independently of SP3 in prefrontal cortex to control positive symptoms.

(3) Conclusion 3: Our quantitative proteomic analysis suggests that the panel of protein players altered in cerebellum or in prefrontal cortex of subjects with schizophrenia is different showing the local brain diversity of molecular alterations in this disease. In addition, the fact that gene expression function were enriched in both areas supports the idea that SP4 transcription factor is not the only transcriptional regulator altered in this disorder and the hypothesis that a cellular reprogramming is involved in the etiopathogenia of SZ.

Potential socio-economic impact and use of the project

Psychiatric diseases constitute a relevant health problem in the world. During the last decades, multiple research efforts have been addressed to try to achieve an effective and selective treatment for people who suffer from severe mental disorders as schizophrenia or BD. Although a number of drugs are available for the treatment of these disorders, there are still significant limitations as the treatment of resilient symptoms and a limited efficacy. Our studies have contributed to advance the scientific knowledge in the brain in psychiatric disorders and show an example of how the brain molecular heterogeneity is linked to different clinical presentations in schizophrenia. Thus, our work opens the possibility to develop new and more selective and effective therapeutic strategies where SP proteins could be targeted to compensate a predominant clinical manifestation and / or resilient symptoms. These novel strategies could result in an improvement of the quality of life of patients with schizophrenia or BD and will reduce the massive medical and socioeconomic impact of these diseases.