Frank Middleton - US grants

The basal ganglia and cerebellum are known to receive inputs from widespread regions of the cerebral cortex, such as the prefrontal, posterior parietal, and temporal lobes. According to one viewpoint, these inputs are integrated in the basal ganglia and cerebellum, and directed to the primary motor cortex, or other cortical motor areas, for use in the control of movement. However, Alexander, Delong, and Strick (1986) and Leiner, Leiner, and Dow (1991, 1993), proposed that significant portions of the output from the basal ganglia and cerebellum target the same regions of the frontal lobe which they receive input from. Thus, according to this scheme, it is possible that several non-motor "loops" exist between the prefrontal cortex and the basal ganglia and cerebellum. Despite the absence of direct anatomical evidence for the existence of such loops, there have been many suggestions about their role in a number of neurological and psychiatric disorders. The proposed experiments are designed to examine the possible existence of the prefrontal loops and the potential role of the basal ganglia and cerebellum in cognitive function. First, it will be determined if the basal ganglia and cerebellum have anatomical connections with regions of the prefrontal cortex that are known to be involved in cognitive function. Second, the degree of separation of these pathways from each other and from previously-described pathways that are involved in motor functions will be determined. To address these issues, retrograde transneuronal transport with the McIntyre-B strain of herpes simplex virus type 1 will be used in cebus monkeys. This technique will enable the direct observation of any pathways in the basal ganglia and cerebellum which are directed to the different prefrontal regions.

DESCRIPTION (provided by applicant): The effects of chronic ethanol abuse on the brain and body can have a devastating impact on health and well-being, and predispose individuals toward risky behaviors that might even result in premature death. In order to reduce the prevalence of alcohol abuse, we need to improve our ability to detect the earliest signs of damage to the central or internal organs, which might enable appropriate interventional or therapeutic strategies to be initiated. The goal of this project is to develop screening tools that could have true translational potential for diagnosing when the earliest signs, of ethanol-induced damage to the brain has occurred. This information will be acquired through a careful series of experiments involving laboratory rats that are engaged in drinking behaviors for variable periods of time, beginning either as adults or adolescents, and are determined to have signs of central or internal organ damage. The peripheral blood of these animals will be monitored for signs of liver or heart damage in order to guard against the possibility that the effects we observe in the blood are not specific for the brain. Total RNA will be extracted from the circulating leukocytes to determine those genes with expression patterns that are significantly correlated (both positively and negatively) with RNA levels in three different brain regions (cerebellum, hippocampus, and somatosensory cortex) of the same animal. In the same animals, we will also assess potential liver or cardiac damage by monitoring serum ALT and AST levels. We will use the expression information as background data for a second phase of investigation, involving the collection of peripheral blood and screening of RNA from human subjects who are currently undergoing treatment for alcohol abuse who are positive or negative for signs of CNS, liver, or heart damage. In the end, we hope to identify a small subset of genes with diagnostic and prognostic utility that could be used to identify at risk individuals in order to commence treatment and tailor this treatment toward their specific pattern of alterations.

The CELL/MOLECULAR BIOLOGY CORE provides several of the MAIN and PILOT PROJECTS with state-of-the-art support for their proposals primarily in assessments of fresh (unfixed) specimens. The specific services provided by the CELL/MOLECULAR BIOLOGY CORE include (a) extraction of DMA,RNA, and protein from tissue samples, (b) genotyping, real-time quantitative RT-PCR, (c) Western immunoblotting, (d) cloning and synthesis of cRNA riboprobes, (e) performing radioactive and non-radioactive in situ hybridization, (f) phosphorimaging analysis, (g) HPLC-based neurochemical assays, and (h) laser microdissection. In addition to the methodoligcal expertise, the CORE provides advice and collaboration for the design, implementation, and analyses of cell and molecular biology studies. The CORE is directed by the capable oversight of Drs. Middleton and Vallano. Through careful consultation and experimental design, DEARC Pis will benefit from uniform application of the highest level of quality control. Moreover, the raw data generated by the CELL/MOLECULAR BIOLOGY CORE will be routinely added into the central DEARC database, so that any investigator can analyze them for specific effects and possible correlations with data generated from their studies. The CELL/MOLECULAR BIOLOGY CORE personnel will assist each of the Pi's in this regard, and will also work closely with the ANIMAL and NEUROANATOMY CORES to ensure that the correct tissue samples are being utilized in each assay according to the experimental designs.

The CELL/MOLECULAR BIOLOGY CORE provides several of the MAIN and PILOT PROJECTS with state-of-the-art support for their proposals primarily in assessments of fresh (unfixed) specimens. The specific services provided by the CELL/MOLECULAR BIOLOGY CORE include (a) extraction of DMA, RNA, and protein from tissue samples, (b) genotyping, real-time quantitative RT-PCR, (c) Western immunoblotting, (d) cloning and synthesis of cRNA riboprobes, (e) performing radioactive and non-radioactive in situ hybridization, (f) phosphorimaging analysis, (g) HPLC-based neurochemical assays, and (h) laser microdissection. In addition to the methodoligcal expertise, the CORE provides advice and collaboration for the design, implementation, and analyses of cell and molecular biology studies. The CORE is directed by the capable oversight of Drs. Middleton and Vallano. Through careful consultation and experimental design, DEARC Pis will benefit from uniform application of the highest level of quality control. Moreover, the raw data generated by the CELL/MOLECULAR BIOLOGY CORE will be routinely added into the central DEARC database, so that any investigator can analyze them for specific effects and possible correlations with data generated from their studies. The CELL/MOLECULAR BIOLOGY CORE personnel will assist each of the Pi's in this regard, and will also work closely with the ANIMAL and NEUROANATOMY CORES to ensure that the correct tissue samples are being utilized in each assay according to the experimental designs.

DESCRIPTION (provided by applicant): Understanding the effects of ethanol on neural stem cells is critical to unraveling the etiological knots of deficits associated with fetal alcohol spectrum disorder (FASD). FASD is a compelling problem because it is a major cause of developmental mental dysfunction affecting ~2% of all live births (indeed, it is the chief cause of mental retardation in the USA), and it provides insights into the etiology of other (often co-morbid) mental health disorders (e.g., attention deficit hyperactivity disorder and autism). The composition and size of the brain are established by the proliferation of neural stem cells and by the numbers and lineage of the derivatives. Improper numbers or balance of neuronal subpopulations can underlie mental dysfunction. Thus, we will test the hypothesis that ethanol affects the cycling behavior and fates of cells generated in the developing central nervous system. Cerebral cortex is composed of two types of neurons: excitatory projection neurons (PNs) and inhibitory local circuit neurons (LCNs). These derive prenatally from two distinct proliferative regions. PNs come from the dorsal telencephalon which comprises two zones: the ventricular zone (VZ) and its derivative, the subventricular zone (SZ). Most cortical LCNs are generated in the ventral telencephalon, in the medial ganglionic eminence (MGE). The fates of VZ/SZ and MGE cells are defined in a two-step process. (1) It is decided whether the cells remain in the cycling population and (2) the phenotype (e.g., the type of neuron) is defined. During the previous period of support, we showed that transforming growth factor (TGF) 21 is a key regulator of neural stem cell proliferation. The present project will explore the effects of ethanol on dynamics of cell proliferation and on the definition of cell fate. Specific Aims 1 and 2 will use an in vivo model to determine the effects of ethanol on the cycling activity (cell cycle kinetics and exit) and on the expression/activation of TGF2 receptors by neural stem cells in the cortical proliferative zones. During their development, neural stem cells express a homeobox gene product(s), Pax6 and/or Tbr2. These proteins define the transition from (Pax6+) stem cells in the VZ to an (Tbr2+) intermediate progenitor cell stage. This transition is critical for the development of superficial cortex (e.g., the origin of callosal projections) which is a target of prenatal exposure to ethanol. Specific Aim 3 will use two types of cultures (organotypic slices that retain in vivo-like organization and lines of neural stem cells harvested from the VZ/SZ or MGE) to explore the effects of ethanol on TGF21-regulated cell proliferation and fate decisions. In addition, we will identify genes that are up- and down-regulated and silenced (methylated) by ethanol and/or TGF21. In Specific Aim 4, neural stem cells will be transplanted (homotopically or heterotopically) to determine the effects of ethanol and/or TGF21 on genetic and environmental contributions to determining cycling behavior and to defining cell fate. In concert, the novel Aims will use three complementary models to gain critical insight into mechanisms defining cell proliferation and fate and the effects of ethanol on these critical factors. PUBLIC HEALTH RELEVANCE: Fetal alcohol spectrum disorder affects an estimated 2% of all live births in the United States. One common target of alcohol toxicity is proliferating cells, particularly neural stem cells that give rise to the brain. The present study will explore a mechanism by which alcohol-induced defects result - from changes (a) in the fates of proliferating neural stem cells and (b) in their response to a key regulator of that proliferation, transforming growth factor.

DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorder is common (affects ~2% of all live births) and is a major cause of mental dysfunction. Of the many negative effects that ethanol has on the developing nervous system, the most severe is neuronal death. It is severe because this loss is permanent; with the exception of a couple of sites, post-mitotic neurons in the CNS are not replaced by newly generated ones. Indeed, after the generation of neurons is complete, the nervous system has to decide if and whether damaged neurons can be repaired. If not, they must be eliminated. A key molecule in this decision process is the oncoprotein p53. p53 is critical for maintaining neuronal integrity and resiliency. We will test the hypothesis that developmental exposure to ethanol alters neuronal survival and DNA repair through p53-dependent activities. In the developing nervous system, neuronal death is a natural and critical process. This death appears to be apoptotic and to involve p53. Our preliminary data and the work of others on developing cerebral cortex show that exposure to ethanol during the period of naturally occurring neuronal death causes a dramatic and transient increase in both active caspase 3 expression and terminal uridylated nick-end labeling (TUNEL). On the other hand, our novel data also show that this pattern does not coincide with the ultimate loss of cortical neurons either in time or space. The implication is that ethanol causes DNA fragmentation, but this degradation may not be obligatory for apoptosis. Instead, it may reveal DNA repair mechanisms. p53 is a key player in DNA repair. Three complementary aims will be addressed using p53 deficient mice and cells. (1) Vulnerability of cortical neurons to ethanol will be addressed in long- and short-term in vivo studies. Long-term studies will determine the ethanol-induced loss of neurons in cortical layers occurring in the deficient mice. Complementary studies will examine short-term changes in the expression of presumed death markers (active caspase3 immunoexpression and TUNEL) in cortical layers and the timing of the neuronal loss. (2) The acute genomic responses of p53 deficient mice and cultured neural stem cells to ethanol will be determined. We will identify changes in the expression of transcripts involved in apoptosis and DNA repair. In addition, we will determine the epigenetic effects of ethanol on the silencing of genes through hyper-methylation. (3) Securin is a protein that is regulated by p53 and is pivotal for DNA repair. Preliminary microarray and immunocytochemical data show that it is profoundly affected by ethanol. Thus, we will examine the effects of ethanol on cells deficient of securin in vitro and in vivo after transplantation into layers that are apparently susceptible and refractory to ethanol. These studies will explore two dovetailed responses that developing neurons have to ethanol: DNA repair, and failing that, apoptotic death. We will address critical questions. For example, what defines the susceptibility of a young neuron to ethanol? Can neurons be manipulated to reduce their ethanol vulnerability? Thus, the studies explore two new targets of ethanol, p53 and securin, that are critical for the neuronal survival and integrity. PUBLIC HEALTH RELEVANCE: Fetal alcohol spectrum disorder affects an estimated 2% of all live births in the United States. One common target of alcohol toxicity is differentiating cells, particularly those that are newly integrating into the complex environment of the developing brain. The present study will explore two complementary mechanisms by which alcohol-induced defects result (a) from ethanol-induced neuronal death and (b) from ethanol-altered DNA repair.

The CELL/MOLECULAR BIOLOGY CORE provides several of the MAIN and PILOT PROJECTS with state-of-the-art support for their proposals primarily in assessments of fresh (unfixed) specimens. The specific services provided by the CELL/MOLECULAR BIOLOGY CORE include (a) extraction of DMA, RNA, and protein from tissue samples, (b) genotyping, real-time quantitative RT-PCR, (c) Western immunoblotting, (d) cloning and synthesis of cRNA riboprobes, (e) performing radioactive and non-radioactive in situ hybridization, (f) phosphorimaging analysis, (g) HPLC-based neurochemical assays, and (h) laser microdissection. In addition to the methodoligcal expertise, the CORE provides advice and collaboration for the design, implementation, and analyses of cell and molecular biology studies. The CORE is directed by the capable oversight of Drs. Middleton and Vallano. Through careful consultation and experimental design, DEARC Pis will benefit from uniform application of the highest level of quality control. Moreover, the raw data generated by the CELL/MOLECULAR BIOLOGY CORE will be routinely added into the central DEARC database, so that any investigator can analyze them for specific effects and possible correlations with data generated from their studies. The CELL/MOLECULAR BIOLOGY CORE personnel will assist each of the Pi's in this regard, and will also work closely with the ANIMAL and NEUROANATOMY CORES to ensure that the correct tissue samples are being utilized in each assay according to the experimental designs.

DESCRIPTION (provided by applicant): Our ongoing 20-year family-genetic study of schizophrenia and other psychotic disorders (SCZ) in the isolated population of Palau provides a valuable resource for examining the genetic and epigenetic mechanisms that govern familial transmission of SCZ. We have identified a highly promising copy number variant (CNV) that points to the possible discovery of another Disrupted-In-Schizophrenia locus. This structural variant clearly co-segregates with SCZ in a 5- generation, high-density Palauan family. Genetic, epigenetic, and functional genomic lines of evidence support its relevance for SCZ. The deletion occurs at a 9p24 site that controls histone methylation, an important epigenetic event in glutamatergic gene expression. This epigenetic locus is adjacent to the EAAC1 (excitatory-amino-acid-carrier-1) glutamate transporter gene, which plays an essential role in regulating glutamatergic neurotransmission, a well- recognized component of the pathophysiology of SCZ. The Palauan family with the 9p24 deletion is as large and as densely affected as the Scottish family that led to the discovery of the original DISC1 gene. Our preliminary studies have validated the deletion status in all affected and unaffected family members, and indicated that EAAC1 gene expression is reduced in SCZ family members with the deletion. The goals of the present application are to expand our phenotypic and genotypic assessments to include all members of the extended pedigree (N = ~75), test for co-segregation of the deletion with affection status, and conduct preliminary studies to examine the possible functional significance of the deletion in terms of EAAC1 gene expression and histone methylation levels at the 9p24 locus. Once completed, the study will provide proof of concept for a full-scale R01 study of the functional consequences of this disruption and its potential as a diagnostic biomarker for SCZ and possible target for drug development. Ultimately, this line of research may lead to improved risk prediction and treatment decisions for young high-risk individuals in the prodromal stage of SCZ when preventive intervention can be most effective. PUBLIC HEALTH RELEVANCE: Our proposed study directly addresses the need for research aimed at the prevention and treatment of the most debilitating neuropsychiatric disorders: schizophrenia, depression and bipolar disorder. Using an extraordinary 6-generation SCZ family, we aim to identify risk predictors for psychotic disorders in young people who carry a strong genetic susceptibility for disease development as they enter their adult years. The proposed developmental study represents a step toward the next generation of diagnostics and treatments that aim to identify emerging psychosis in its early prodromal stage when preventive intervention can be most effective. Because post-onset treatment has limited impact on course and outcome, the prodromal phase of psychotic disorders represent an important window of opportunity for early intervention. Preventive intervention programs for young people with prodromal symptoms have been shown to delay or even prevent illness onset and reduce the risk of the crippling functional disabilities associated with psychotic disorders.

The NeuroCore provides DEARC investigators with access to state-of-the-art resources and equipment for conducting molecular, cellular, or neuroanatomical related research. These studies involve quantification, visualization, or manipulation of RNA, DNA, or protein. At every stage of investigation, the NeuroCore personnel will ensure the highest quality of data is established and maintained, that unifonn techniques are applied across projects to maximize data harmonization, and that tissue resources used by investigators are preserved as much as possible for future studies. NeuroCore personnel will also regularly meet with individual DEARC investigators to help plan specific details of their experiments and assist in data analysis and training of Project or Pilot personnel. To accomplish these goals, the NeuroCore is designed to consist of three complementary components, each one directed by a separate investigator. First, a Molecular Component provides laser microdissection, nucleic acid and protein purification and several quantitative measures of gene or protein expression. Second, a Neuroanatomy Component provides immunoflourescent and immunohistochemical techniques to map regional alterations in neuronal and glial phenotypes and changes or adaptations in cells or circuitry due to alcohol exposure. Third, a Cellular Component provides the resoures and expertise for conducting in vitro experiments in primary neuronal, glial, and cell line specific cultures, helps investigators construct and validate appropriate vectors for gene delivery or knockdown experiments, and enables future studies that might involve genetically modified rodent models.

Ant1 is the muscle/heart/central nervous system (CNS) isoform of adenine nucleotide translocase that is primarily involved in ATP/ADP exchange across the inner mitochondrial membrane (IMM). An increasing number of missense mutations in Ant1 are found to cause dominant diseases that affect skeletal muscle and the central nervous system. These diseases are commonly manifested by fractional mtDNA deletions and mild bioenergetic defects. The mechanism of neuromuscular damage in the diseases is poorly understood. Interestingly, our recent studies in yeast and cultured human cells suggested that the mutant Ant1 is misfolded. This leads to cell death by a novel mechanism that we named mitochondrial Precursor Overaccumulation Stress (mPOS). mPOS is characterized by the toxic accumulation and aggregation of un-imported mitochondrial preproteins in the cytosol. These findings led to the central hypothesis that the mutant Ant1 primarily affects mitochondrial protein import. This results in mPOS in the cytosol, which plays an important role in inducing neural and muscular degeneration. Fractional mtDNA deletions occur independent of nucleotide transport activity, likely as a secondary damage collateral to reduced mitochondrial protein import. In this application, we propose to directly test this hypothesis in mouse models. We successfully generated knock-in (KI) mouse lines expressing misfolded variants of Ant1. Preliminary studies indicated that these mice develop phenotypes consistent with neural and muscular degeneration. In Specific Aim 1, we will use these unique experimental models to test the hypothesis that misfolded Ant1 induces neural and muscular degeneration and mtDNA instability independent of nucleotide transport. In Specific Aim 2, we will use various experimental tools that we developed in yeast, cultured human cells and the Ant1-KI mice to test the hypothesis that the misfolded Ant1 (or Aac2 in yeast) causes structural and functional damage to the mitochondrial protein import machinery and induces mPOS in the cytosol. In Specific Aim 3, we will determine the mechanisms that protect cells against Ant1-induced protein import stress and mPOS. Success of the project will establish a mouse model of protein import stress associated with mPOS. Particularly, validation of the mPOS model would help reconciling the mitochondrial and proteostatic pathways in many neural and muscular degenerative diseases. Finally, the results could have important implications for the understanding and therapy of Ant1-induced diseases, as well as many other clinical conditions that directly or indirectly affect mitochondrial protein import.