Nora I. Perrone-Bizzozero

Combining fMRI and SNP data to investigate connections between brain function and genetics using parallel ICA

There is current interest in understanding genetic influences on both healthy and disordered brain function. We assessed brain function with functional magnetic resonance imaging (fMRI) data collected during an auditory oddball task—detecting an infrequent sound within a series of frequent sounds. Then, task‐related imaging findings were utilized as potential intermediate phenotypes (endophenotypes) to investigate genomic factors derived from a single nucleotide polymorphism (SNP) array. Our target is the linkage of these genomic factors to normal/abnormal brain functionality. We explored parallel independent component analysis (paraICA) as a new method for analyzing multimodal data. The method was aimed to identify simultaneously independent components of each modality and the relationships between them. When 43 healthy controls and 20 schizophrenia patients, all Caucasian, were studied, we found a correlation of 0.38 between one fMRI component and one SNP component. This fMRI component consisted mainly of parietal lobe activations. The relevant SNP component was contributed to significantly by 10 SNPs located in genes, including those coding for the nicotinic α‐7cholinergic receptor, aromatic amino acid decarboxylase, disrupted in schizophrenia 1, among others. Both fMRI and SNP components showed significant differences in loading parameters between the schizophrenia and control groups (P = 0.0006 for the fMRI component; P = 0.001 for the SNP component). In summary, we constructed a framework to identify interactions between brain functional and genetic information; our findings provide a proof‐of‐concept that genomic SNP factors can be investigated by using endophenotypic imaging findings in a multivariate format. Hum Brain Mapp, 2009.

The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs

The protein that has been designated as GAP-43, B-50, F1 or pp46 is associated with the growth and modulation of neuronal connections. cDNA clones for the rat and human genes were isolated and used to demonstrate that the messenger RNA for the protein is expressed only in neurons, that its overall level is highest in the developing brain, and that in the adult human brain levels of the mRNA are highest in the associative neocortex.

Role of HuD and other RNA-binding proteins in neural development and plasticity.

Transcription factors have traditionally been viewed as the main determinants of gene expression. Yet, in recent years it has become apparent that RNA‐binding proteins also play a critical role in determining the levels of expression of a large number of genes. Once mRNAs are transcribed, RNA‐binding proteins can control all subsequent steps in their function, from alternative splicing and translation to mRNA transport and stability. In the nervous system, a large number of genes are regulated post‐transcriptionally via the interaction of their mRNAs with specific RNA‐binding proteins. This type of regulation is particularly important in the control of the temporal and spatial pattern of gene expression during neural development. This review will discuss the function of the embryonic lethal abnormal vision (ELAV)/Hu family of nervous system‐specific RNA‐binding proteins, with a special emphasis on HuD, a member of this family that controls GAP‐43 mRNA stability and expression. In addition, we will present recent findings on other neural RNA‐binding proteins: the ribonucleoprotein K homology (KH)‐domain proteins, Fragile X mental retardation protein (FMRP), quakinguiable protein (QKI), and Nova‐1. Together with the ELAV/Hu family, these proteins are essential for proper neural development and in some cases for plasticity in the mature brain. The biological significance of these proteins is evident not only by their evolutionary conservation but also by the magnitude of problems arising from autoimmune reactions against them or from mutations affecting their expression or function.

The Elav-like Proteins Bind to a Conserved Regulatory Element in the 3′-Untranslated Region of GAP-43 mRNA

Previous studies have identified three brain proteins (40, 65 and 95 kDa, respectively) that specifically bind to the 3′-untranslated region of GAP-43 mRNA. In this study, using a specific monoclonal antibody, we now show that the 40-kDa proteins are members of the Elav-like protein family. This family of specific RNA-binding proteins comprise three neural specific members called HuD, HuC, and Hel-N1. We have shown that purified recombinant HuD can bind with high affinity to GAP-43 mRNA. In addition, we have mapped the binding site to a highly conserved 26-nucleotide sequence within the regulatory element. The binding of HuD to this site is readily displaced by RNA oligonucleotides encoding other HuD binding sites. We also show that only the first and second RNA binding domains of HuD are required for selective binding to GAP-43 mRNA.

RNA-protein interactions and control of mRNA stability in neurons.

In addition to transcription, posttranscriptional mechanisms play a vital role in the control of gene expression. There are multiple levels of posttranscriptional regulation, including mRNA processing, splicing, editing, transport, stability, and translation. Among these, mRNA stability is estimated to control about 5–10% of all human genes. The rate of mRNA decay is regulated by the interaction of cis‐acting elements in the transcripts and sequence‐specific RNA‐binding proteins. One of the most studied cis‐acting elements is the AU‐rich element (ARE) present in the 3′ untranslated region (3′UTR) of several unstable mRNAs. These sequences are targets of many ARE‐binding proteins; some of which induce degradation whereas others promote stabilization of the mRNA. Recently, these mechanisms were uncovered in neurons, where they have been associated with different physiological phenomena, from early development and nerve regeneration to learning and memory processes. In this Mini‐Review, we briefly discuss the general mechanisms of control of mRNA turnover and present evidence supporting the importance of these mechanisms in the expression of an increasing number of neuronal genes.

Altered levels of the synaptosomal associated protein SNAP-25 in schizophrenia

BACKGROUND Identifying brain changes in schizophrenia has been a major research focus for many years. Although impressive gains have been made in neuroimaging and brain electrophysiology, molecular and cellular markers of schizophrenia have lagged. There are no consistent biochemical markers for schizophrenia pathophysiology and none that reflect treatment course. METHODS Samples were obtained from 25 postmortem schizophrenic brains and 31 nonschizophrenic controls. These samples were processed, and the synaptosomal fraction was isolated. Ten micrograms of protein from each of these samples was solubilized in a sodium dodecylsulfate sample buffer and separated on 10% (wt/vol) polyacrylamide gels. Monoclonal antibody (SMI-81) was incubated with the blots and, using quantitative Western blotting, we measured the relative amounts of SNAP-25 in these samples. RESULTS We report altered levels of SNAP-25 in both the inferior temporal cortex (Brodmann area 20) and prefrontal association cortex (Brodmann areas 9 and 10) in postmortem brains of patients with schizophrenia relative to nonschizophrenic controls. Normal levels of SNAP-25 are noted in schizophrenics in area 17, decreased levels in areas 10 and 20, and an elevated level in area 9. CONCLUSIONS These data support cytoarchitectural observations that the cerebral cortex of schizophrenic patients has extensive pathology. The data presented here, along with data on other brain-specific proteins, indicate a complicated molecular adaptation to the causative factors of schizophrenia.

Chapter 6: The expression of GAP-43 in relation to neuronal growth and plasticity: when, where, how, and why?

Publisher Summary Studies on where and when GAP-43 gets expressed afford a unique opportunity to visualize neuronal differentiation and synaptic organization as they occur in vivo and in culture. Moreover, it is likely that by investigating how the expression of GAP-43 is regulated and how the protein functions physiologically in the cell, a deeper understanding of the molecular mechanisms may gain that underlie growth and plasticity in the nervous system. Prior to their final cell division and process outgrowth, neurons express very little GAP-43 mRNA and protein. This can be seen in embryonic cortical neurons that are grown in culture in the absence of serum, in which case the cells remain viable but fail to extend neurites. During nerve development and regeneration, levels of GAP-43 mRNA increase just prior to the changes in protein levels. A close parallel between levels of the protein and the mRNA is also observed regionally in the human cerebral cortex. Thus, levels of GAP-43 synthesis appear to be determined primarily by the steady-state level of the mRNA.

Genomic Convergence Analysis of Schizophrenia: mRNA Sequencing Reveals Altered Synaptic Vesicular Transport in Post-Mortem Cerebellum

Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.

Altered myelination of the hippocampal formation in subjects with schizophrenia and bipolar disorder

Myelination of the frontal and temporal lobes occurs at a similar time period as symptom onset in schizophrenia. To assess this potential relationship, we compared myelination and oligodendrocyte numbers in the hippocampal formation of controls and matched subjects with schizophrenia and bipolar disorder. The levels and distribution of the myelin marker myelin basic protein (MBP) and the oligodendrocyte marker adenomatous polyposis coli (APC) were measured using immunocytochemistry. MBP immunoreactivity (IR) was increased in several hippocampal subregions of control females versus control males. Female subjects with schizophrenia and bipolar disorder exhibited decreased myelination in the hippocampal formation while male subjects with bipolar disorder showed increased MBP levels in the superior medullary lamina. In contrast, the number of APC immunoreactive cells did not differ in any disorder or region. Our results demonstrate an interaction between gender, mental illness, and myelination, and may be related to cognitive deficits seen in schizophrenia and bipolar disorder.

Novel recognition motifs and biological functions of the RNA-binding protein HuD revealed by genome-wide identification of its targets

HuD is a neuronal ELAV-like RNA-binding protein (RBP) involved in nervous system development, regeneration, and learning and memory. This protein stabilizes mRNAs by binding to AU-rich instability elements (AREs) in their 3′ unstranslated regions (3′ UTR). To isolate its in vivo targets, messenger ribonucleoprotein (mRNP) complexes containing HuD were first immunoprecipitated from brain extracts and directly bound mRNAs identified by subsequent GST-HuD pull downs and microarray assays. Using the 3′ UTR sequences of the most enriched targets and the known sequence restrictions of the HuD ARE-binding site, we discovered three novel recognition motifs. Motifs 2 and 3 are U-rich whereas motif 1 is C-rich. In vitro binding assays indicated that HuD binds motif 3 with the highest affinity, followed by motifs 2 and 1, with less affinity. These motifs were found to be over-represented in brain mRNAs that are upregulated in HuD overexpressor mice, supporting the biological function of these sequences. Gene ontology analyses revealed that HuD targets are enriched in signaling pathways involved in neuronal differentiation and that many of these mRNAs encode other RBPs, translation factors and actin-binding proteins. These findings provide further insights into the post-transcriptional mechanisms by which HuD promotes neural development and synaptic plasticity.