Carlo Colantuoni

Gene Expression Profiling in Postmortem Rett Syndrome Brain: Differential Gene Expression and Patient Classification

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.

Expression of GABA Signaling Molecules KCC2, NKCC1, and GAD1 in Cortical Development and Schizophrenia

GABA signaling molecules are critical for both human brain development and the pathophysiology of schizophrenia. We examined the expression of transcripts derived from three genes related to GABA signaling [GAD1 (GAD67 and GAD25), SLC12A2 (NKCC1), and SLC12A5 (KCC2)] in the prefrontal cortex (PFC) and hippocampal formation of a large cohort of nonpsychiatric control human brains (n = 240) across the lifespan (from fetal week 14 to 80 years) and in patients with schizophrenia (n = 30–31), using quantitative RT-PCR. We also examined whether a schizophrenia risk-associated promoter SNP in GAD1 (rs3749034) is related to expression of these transcripts. Our studies revealed that development and maturation of both the PFC and hippocampal formation are characterized by progressive switches in expression from GAD25 to GAD67 and from NKCC1 to KCC2. Previous studies have demonstrated that the former leads to GABA synthesis, and the latter leads to switching from excitatory to inhibitory neurotransmission. In the hippocampal formation, GAD25/GAD67 and NKCC1/KCC2 ratios are increased in patients with schizophrenia, reflecting a potentially immature GABA physiology. Remarkably, GAD25/GAD67 and NKCC1/KCC2 expression ratios are associated with rs3749034 genotype, with risk alleles again predicting a relatively less mature pattern. These findings suggest that abnormalities in GABA signaling critical to brain development contribute to genetic risk for schizophrenia.

SNOMAD (Standardization and NOrmalization of MicroArray Data): web-accessible gene expression data analysis

SNOMAD is a collection of algorithms for the normalization and standardization of gene expression datasets derived from diverse biological and technological sources. In addition to conventional transformations and visualization tools, SNOMAD includes two non-linear transformations which correct for bias and variance which are non-uniformly distributed across the range of microarray element signal intensities: (1). Local mean normalization; and (2). Local variance correction (Z-score generation using a locally calculated standard deviation).

High throughput analysis of gene expression in the human brain

The human brain is thought to have the greatest complexity of gene expression of any region of the body, reflecting the diverse functions of neurons and glia. Studies of gene expression in the human brain may yield fundamental information about the phenotype of brain cells in different stages of development, in different brain regions, and in different physiological and pathological states. As the human genome project nears completion, several technological advances allow the analysis of thousands of expressed genes in a small brain sample. This review describes available sources of human brain material, and several high throughput techniques used to measure the expression of thousands of genes. These techniques include expressed sequence tag (EST) sequencing of cDNA libraries; differential display; subtractive hybridization; serial analysis of gene expression (SAGE); and the emerging technology of high density DNA microarrays. Measurement of gene expression with microarrays and other technologies has potential applications in the study of human brain diseases, including cognitive disorders for which animal models are typically not available. Gene expression measurements may be used to identify genes that are abnormally regulated as a secondary consequence of a disease state, or to identify the response of brain cells to pharmacological treatments. J. Neurosci. Res. 59:1–10, 2000

Cardiac Resynchronization Therapy Corrects Dyssynchrony-induced Regional Gene Expression Changes on a Genomic Level

Background—Cardiac electromechanical dyssynchrony causes regional disparities in workload, oxygen consumption, and myocardial perfusion within the left ventricle. We hypothesized that such dyssynchrony also induces region-specific alterations in the myocardial transcriptome that are corrected by cardiac resynchronization therapy (CRT). Methods and Results—Adult dogs underwent left bundle branch ablation and right atrial pacing at 200 bpm for either 6 weeks (dyssynchronous heart failure, n=12) or 3 weeks, followed by 3 weeks of resynchronization by biventricular pacing at the same pacing rate (CRT, n=10). Control animals without left bundle branch block were not paced (n=13). At 6 weeks, RNA was isolated from the anterior and lateral left ventricular (LV) walls and hybridized onto canine-specific 44K microarrays. Echocardiographically, CRT led to a significant decrease in the dyssynchrony index, while dyssynchronous heart failure and CRT animals had a comparable degree of LV dysfunction. In dyssynchronous heart failure, changes in gene expression were primarily observed in the anterior LV, resulting in increased regional heterogeneity of gene expression within the LV. Dyssynchrony-induced expression changes in 1050 transcripts were reversed by CRT to levels of nonpaced hearts (false discovery rate <5%). CRT remodeled transcripts with metabolic and cell signaling function and greatly reduced regional heterogeneity of gene expression as compared with dyssynchronous heart failure. Conclusions—Our results demonstrate a profound effect of electromechanical dyssynchrony on the regional cardiac transcriptome, causing gene expression changes primarily in the anterior LV wall. CRT corrected the alterations in gene expression in the anterior wall, supporting a global effect of biventricular pacing on the ventricular transcriptome that extends beyond the pacing site in the lateral wall.

Prominent hippocampal CA3 gene expression profile in neurocognitive aging

Research in aging laboratory animals has characterized physiological and cellular alterations in medial temporal lobe structures, particularly the hippocampus, that are central to age-related memory deficits. The current study compares molecular alterations across hippocampal subregions in a rat model that closely mirrors individual differences in neurocognitive features of aging humans, including both impaired memory and preserved function. Using mRNA profiling of the CA1, CA3 and dentate gyrus subregions, we have distinguished between genes and pathways related to chronological age and those associated with impaired or preserved cognitive outcomes in healthy aged Long-Evans rats. The CA3 profile exhibited the most prominent gene expression differences related to cognitive status and of the three subregions, best distinguished preserved from impaired function among the aged animals. Within this profile differential expression of synaptic plasticity and neurodegenerative disease-related genes suggests recruitment of adaptive mechanisms to maintain function and structural integrity in aged unimpaired rats that does not occur in aged impaired animals.

Olfactory cells via nasal biopsy reflect the developing brain in gene expression profiles: Utility and limitation of the surrogate tissues in research for brain disorders

Human olfactory cells obtained by rapid nasal biopsy have been suggested to be a good surrogate system to address brain disease-associated molecular changes. Nonetheless, whether use of this experimental strategy is justified remains unclear. Here we compared expression profiles of olfactory cells systematically with those from the brain tissues and other cells. Principal component analysis indicated that the expression profiles of olfactory cells are very different from those of blood cells, but are closer to those of stem cells, in particular mesenchymal stem cells, that can be differentiated into the cells of the central nervous system.

Negative symptoms of schizophrenia correlate with impairment on the University of Pennsylvania smell identification test.

Deficits in odor identification have been most frequently described in schizophrenia (SZ). A relationship between dysfunction in odor identification and negative symptoms of SZ has also been reported. Furthermore, deficit SZ (a subtype of the illness with primary, enduring negative symptoms) has been found to be associated with a particularly poor performance on odor identification tests indicating that deficits in smell identification could be differentially expressed in some subtypes of SZ. We describe correlations of performance on smell identification with positive and negative symptoms of SZ. Patients with SZ (n=15) and normal controls (n=19) were tested by the University of Pennsylvania Smell Identification Test (UPSIT). Psychopathology was assessed with the Scales for the Assessment of Positive and Negative Symptoms (SAPS and SANS). SZ patients performed more poorly on the UPSIT test than did normal controls. Consistent with previous findings, we observed a correlation of SANS with UPSIT performance. In particular, specific subdomains of SANS, such as blunted affect, apathy and anhedonia, were associated with odor identification deficits. Furthermore, UPSIT score predicts these subdomains of negative symptoms. No correlation was observed between positive symptom and odor identification deficits. Our study further reinforces a relation between olfactory identification deficit and negative symptoms in SZ and suggests that smell identification could be a candidate endophenotype relevant to negative symptoms of SZ.

Individual differences in neurocognitive aging of the medial temporal lobe

A wide spectrum of outcomes in the cognitive effects of aging is routinely observed in studies of the elderly. Individual differences in neurocognitive aging are also a characteristic of other species, such as rodents and non-human primates. In particular, investigations at behavioral, brain systems, cellular and molecular levels of analysis have provided much information on the basis for individual differences in neurocognitive aging among healthy outbred rats. These findings are likely to be relevant to an understanding of the effects of aging on the brain, apart from neurodegenerative conditions, such as Alzheimer’s disease, which do not naturally occur in rodents. Here we review and integrate those findings in a model supporting the concept that certain features of cognitive decline are caused by distributed alterations in the medial temporal lobe, which alter the information processing functions of the hippocampal formation. An additional emerging concept from this research is that preserved abilities at older ages may depend on adaptive changes in the hippocampal system that distinguish successful aging.

Rapid encoding of new information alters the profile of plasticity-related mRNA transcripts in the hippocampal CA3 region.

A theoretical framework for the function of the medial temporal lobe system in memory defines differential contributions of the hippocampal subregions with regard to pattern recognition retrieval processes and encoding of new information. To investigate molecular programs of relevance, we designed a spatial learning protocol to engage a pattern separation function to encode new information. After background training, two groups of animals experienced the same new training in a novel environment; however, only one group was provided spatial information and demonstrated spatial memory in a retention test. Global transcriptional analysis of the microdissected subregions of the hippocampus exposed a CA3 pattern that was sufficient to clearly segregate spatial learning animals from control. Individual gene and functional group analysis anchored these results to previous work in neural plasticity. From a multitude of expression changes, increases in camk2a, rasgrp1, and nlgn1 were confirmed by in situ hybridization. Furthermore, siRNA inhibition of nlgn1 within the CA3 subregion impaired spatial memory performance, pointing to mechanisms of synaptic remodeling as a basis for rapid encoding of new information in long-term memory.