Determining the Molecular Consequences of Cocaine Abuse
Michael J. Bannon, Ph.D. and Donald Kuhn, Ph.D.
Our studies aim to develop a detailed profile of changes in gene expression occurring within the brains of drug abusers. By comparing cocaine overdose subjects with drug-free matched controls, as well as with cocaine-positive subjects dying of other causes, we may be able to develop forensic assays that assist in determining the cause of death. We obtain, under contract with a medical examiner's office, brain tissue sections retained as part of the normal autopsy process. Samples are checked for pH, photographed, flash-frozen, and entered into a computerized tracking system. Upon receipt of toxicology data and a determination of the cause of death, samples are matched for demographic and other variables prior to analysis. Our initial studies have focused on several candidate genes of interest. For example, expression of both the transcription factor NURR1 and the NURR1-responsive dopamine transporter gene are markedly decreased within the dopamine neurons of cocaine abusers. Our more recent studies utilize microarrays to profile gene expression in defined brain regions and in identified cell types isolated by laser capture microdissection. Postmortem assessment of gene expression in drug abusers' brains may extend our knowledge of the molecular basis of addiction and ultimately suggest novel therapeutic strategies.
Challenges and Research Outcomes in Brain Banking at the Drug Abuse/HIV Interface
Jeanne Bell, M.D., Ray Brettle, Vince Egan, Anthony Busuttil and Peter Simmonds
In 1990 a tissue bank was established in Edinburgh, Scotland, for postmortem samples derived from HIV/AIDS subjects. The aim of this project was to discover whether the effects of HIV in the nervous system were significantly different in drug users (the majority of Edinburgh HIV-positive subjects) compared with non-drug users. From the outset, the practice has been to retain non-CNS as well as CNS postmortem tissue samples in both fresh-frozen and fixed form to investigate tissue distribution and viral load. Appropriate control cases, including HIV-negative drug users and patients dying in the earlier stages of the infection, have been ascertained by collaboration with forensic pathologists.
The past decade has seen not only considerable changes in postmortem practice in the United Kingdom, particularly in public attitudes toward this activity, but also in conceptual and methodological advances that have revolutionized pathology-based research. These have certainly impinged on the work undertaken by the Edinburgh HIV tissue bank. In addition much has been learned about the undertaking of detailed high-risk postmortem examinations, the ethics of research in postmortem tissues, and the establishment of national and international brain bank and research networks. The operation of this bank has survived the introduction of effective combination therapy and the changed perceptions of the AIDS epidemic in United Kingdom.
The formula of combining an active multidisciplinary local research program with supply to other research groups worldwide has continued from the outset. The Edinburgh cohort of drug users showed a peculiarly high prevalence of HIV encephalitis, and this has provided a unique opportunity to study the effects of infection with neurovirulent HIV strains combined with opiate and amphetamine misuse. The change in disease pattern following the introduction of effective therapy has generated new questions that in part may be answerable only by a comparison between current and archival cases. There was a window of opportunity between 1988 and 1996 to lay down a resource of tissue samples from untreated patients, study the pathology and neuropathology of a new disease (HIV), and add to what is known of an older problem (drug misuse). The challenge is to circumvent the limitations of working with human post mortem tissue and to support continued progress by providing a high-quality research resource.
Neuroimaging Studies in HIV and Drug Abuse
Linda Chang, M.D.
Advances: Various combinations of stimulant drug abuse, including methamphetamine, cocaine, and MDMA (or ecstasy) are commonly used among those infected with HIV. The mechanisms of interactions between HIV and psychostimulants on brain injury are not well understood. Decreased N-acetyl compounds, a neuronal marker, and increased myoinositol, a glial marker, on proton MR spectroscopy, have been reported in psychostimulant users as well as those additionally infected with HIV. Likewise, decreases in dopamine transporters and receptors have been reported in methamphetamine users. However, the combined effects of HIV and psychostimulant abuse on dopamine function have not been well studied in humans. Challenges: 1) Many of the drug users are poly-drug users; therefore, in order to better define the interaction or combined effects of certain class of drugs with HIV, extensive screening procedures are needed. 2) Another challenge is to assess and ensure an accurate drug use history in relation to when the imaging studies occurred. These problems are common in drug abuse research involving human subjects that often lead to results that are difficult to interpret. Future Directions: 1) In vivo neuroimaging may guide the neuropathological evaluations, with regards to brain regions, in tissues from the brain banks. 2) Ex vivo measurements may allow detailed neurochemical measurements of the combined effects of chronic psychostimulant abuse and HIV. 3) Classical neurochemicals (such as dopamine, serotonin, etc.) as well as structural neurochemicals (including N-acetylaspartate, choline compounds, myoinositol, glutamate/glutamine, etc.) that are visible on in vivo neuroimaging studies should be evaluated in the brain bank specimens in order to facilitate future in vivo monitoring of treatment effects.
(Studies were supported by NIDA: K-20-00280; R01-DA12734; R01-NS38834; and the GCRC MO1 000425).
Brain Collection in the Republic of Macedonia
Aleksej Duma, M.D., Ph.D.
The Republic of Macedonia has a population of 2.2 million people in an area the size of Vermont (approximately 25,000 square miles). There is a single Institute of Forensic Medicine (Medical Examiners Office); three hospitals for patients with mental diseases; and one Institute for Psychiatry, which is located in Skopje, the capital. One psychiatric hospital is located in Bardovci, a village adjacent to Skopje, and the other two are in the southern portion of the country, approximately 3 hours from Skopje by car. There is universal, centralized health care. Most patients with mental diseases are hospitalized when necessary, and a majority of them spend the last years of their lives in one of the psychiatric hospitals, which together can accommodate approximately 2,000 patients. Autopsies are required by law for all hospital deaths and for all deaths from unnatural causes. Although exceptions are made to this requirement, these conditions allow for a population-based collection of brains from individuals with severe psychiatric illnesses. A strong educational system provides a pool of skilled doctors who have a knowledge of English and an interest in research, and who are willing and able to collect the specimens and clinical information.
Neuronal Loss in Intravenous Drug Users
Ian Paul Everall, Ph.D.
The availability of tissue collections from brain banks has been crucial in furthering our understanding of the differences among brain pathology in different risk groups infected with HIV. First, I will present work that we have undertaken assessing neuronal loss in intravenous drug users as well as data demonstrating the correlation of pathological changes in the HIV-infected brain with cognitive impairment, including its relationship with rising brain viral burden. Second, I will use the examples to highlight the importance of standard protocols for tissue handling as well as the importance of prospectively acquired antemortem clinical data. Third, I will propose that future collections facilitate in the provision of viable human brain tissue to promote important studies into pathological mechanisms as well as allow the development of neuroprotective strategies. I will demonstrate this with our work on fibroblast growth factor and currently available antiretroviral agents.
Diagnosis and Clinical Assessment Protocols for Brain Banks
Therese Garrick, R.N.
Clinical Information and Diagnosis: The clinical diagnosis of cases collected for inclusion in the TRC is given the highest priority. Clearly, a poor diagnostic workup of cases will confound any research data derived from TRC tissues. The older neuropathology literature for schizophrenia research is filled with conflicting studies, in part due to errors in the clinical diagnosis of cases (Powers 1999).
To ensure a comprehensive profile of the cases, a standardized protocol has been developed. Relatives of the deceased complete questionnaires, and hospital medical records are reviewed by a trained clinician with psychiatric experience. The treating medical staff are interviewed where possible. Information relating to the primary psychiatric disorder, developmental history, family history, and the medication and treatment history is collated into a structured and formatted treatment summary. The treatment summary is organized in such a way that it is possible to audit the file for interrater reliability purposes. Pathology, radiology reports, and neuropsychological data are collected when available.
The Diagnostic Instrument for Brain Studies (DIBS) is then applied to this information. The DIBS is a multidiagnostic instrument designed for use with medical records and has proven reliable. To include a case in the TRC, it has to meet both Diagnostic and Statistical Manual for Mental Disorders, 4th edition (DSM-IV) and ICD-10 criteria (non-substance use cases). For the substance use disorder (alcohol) cases, they must meet DSM-1V criteria. Random cases are selected for interrater reliability by an independent clinician. Cases are excluded from the TRC if there is inadequate clinical information to reach criteria using the DIBS.
Tissue Donor Programs: Experience in Australia and internationally indicates that people with brain diseases and their families are interested in organ donation for research. Recently, two brain donor programs have been launched in New South Wales. The "Gift of Hope" TDP, which has a strong focus on schizophrenia and allied disorders (1999), and the "Using our Brains" TDP, which targets a broader group of the community, with a strong emphasis on controls (2002). The outstanding advantage of the TDPs is the ability to compare premortem clinical, neuroimaging information with postmortem findings. The clinical protocol for the TDPs involves initial and annual assessments of the donors providing longitudinal data.
A general medical and treatment profile, family history, the Annetts handedness scale, the Diagnostic Instrument for Psychosis, the Repeatable Battery for the Assessment of Neuropsychological Status, and the National Adult Reading Test are carried out on all donors. Donors may also be invited to participate in an MRI scan.
The Department of Pathology at the University of Sydney will ensure that when a donor dies, rapid notification procedures are in place for the retrieval of the donor's tissue.
NCI Tissue Resources and New Challenges
Thea Kalebic, M.D., Ph.D.
Evolving research challenges and broader use of novel high-throughput technologies in basic and clinical studies impose new challenges for developing tissue repositories. Research studies aimed at validating or discovering clinically useful biomarkers require a large number of specimens and collections of specially processed and preserved tissues. Limitations of tissue availability represent a considerable impediment to progress in cancer research.
The National Cancer Institute (NCI) is using various approaches to increase tissue availability and to anticipate future research needs. It supports the NCI Cooperative Human Tissues Network, Cooperative Breast Cancer Tissue Resource, Clinical Trials Cooperative Groups, AIDS and Cancer Specimen Bank, The Cancer Family Registries, and NCI Specimen Resource Locator. Throughout the United States, more than 300 million specimens are stored in various tissue banks.
Nevertheless, the research community encounters many difficulties in obtaining appropriately collected and stored specimens for basic and translational studies. The most common tissue preservation modality-formalin fixation and paraffin embedding-causes the formation of covalent bonds. As a result, the tissue architecture remains well preserved, which is crucially important for histopathological diagnosis. However, the extraction from fixed tissue of macromolecules, such as RNA and proteins, is rather inefficient. To preserve the structural integrity of the tissue while minimizing RNA, DNA, and protein damage that may occur in the process of preservation is a new challenge for developers of tissue repositories.
NCI, under the leadership of the Resource Development Branch, has been assessing novel technologies for tissue preservation as well as modalities to "unfix" fixed tissue. These and other efforts to improve the availability and access to research-driven tissue collections will be discussed. Also, legal and ethical questions related to the use of human specimens will be addressed.
Cellular and Molecular Studies in Schizophrenia
Joel E. Kleinman, M.D., Ph.D. NIMH, NIH.
Studies in the Section on Neuropathology have sought to address two questions. First, is there evidence for a cellular and molecular neuropathology in schizophrenia? Second, how do recently discovered genetic susceptibility factors effect neuropathology? The focus at the cellular level has been on glutamate pyramidal neurons in the mesial temporal lobe and the dorsolateral prefrontal cortex (DLPFC). At the molecular level the focus has been on synapse formation, plasticity and neurodevelopment. Lastly, we have genotyped postmortem human brains to test whether functional polymorphisms for COMT have predicted effects on mRNA expression in midbrain dopamine neurons.
Data will be presented looking at mRNA expression for neuronal and glial glutamate transporter in mesial temporal lobe and DLPFC of brains of schizophrenics and controls. Decreases in the neuronal glutamate transporter in the mesial temporal lobe in schizophrenics appear to be related to decreases in the glial glutamate transporter in the DLPFC. Similar changes in synaptophysin mRNA are also seen in these regions especially in hippocampal subfields, CA3 and 4.
In an attempt to connect these changes to a developmental hypothesis of schizophrenia, we have studied molecules that effect cortical glutamate neuron development and appear to change expression levels in the DLPFC during adolescence and young adulthood, periods linked to the onset schizophrenia. Three candidate genes, GAP-43, BDNF and dopamine type1 receptors change mRNA expression in the DLPFC of normals at these critical times and are further implicated in the neuropathology of schizophrenia.
Lastly, a functional polymorphism in a gene recently shown to be associated with schizophrenia and normal DLPFC cognitive function, COMT, has been shown to effect tyrosine hydroxylase mRNA expression in midbrain dopamine neurons of normals.
NeuroAIDS and Its Progression In India: A Model for an International CNS Tissue Consortium?
Mahendra Kumar, Ph.D.
It was in 1987 that the first case of HIV-infection was reported from India. Over the years, HIV-infection has spread aggressively and according to recent estimates, India has about 4 million HIV-infected individuals, with their numbers nearly doubling almost every 18 months. At this rate of transmission, it is estimated that in 5-10 years the number of HIV infected individuals in India may exceed the present prevalence of the entire world. The primary risk factor for contracting HIV throughout the country appears to be unprotected heterosexual sex, although in Northeast India, injecting drug use is the major risk factor. The vast majority of seropositive men and women belong to the lower SES group but all strata of the society are infected to some extent. Although all strains of the HIV-1 virus are present In India, clad C seems to be the most dominant strain. HIV-2 infection has also been reported from India and it is estimated that up to about 25% of infected individuals may have co-infections of both HIV-1 and HIV-2. The impact of co-infection of HIV-1/2 on the Central Nervous System is scientifically an area of great interest since it has been reported that HIV-2 infection protects against HIV-1 infection.
It is well established that immediately after infection, HIV crosses the blood-brain-barrier and is localized in very high concentrations in the hippocampus and to variable degrees in other areas of the brain. The hippocampus plays an important role in regulating the various hypothalamic-pituitary-endocrine axes and in cognitive functioning. Our earlier investigations support these concepts. The prevalence of HIV infection is very high in Southern India, particularly in the states of Maharashtra, Tamil Nadu, and Karnataka. Recently, in association with the National Institute of Mental Health and Neurosciences (NIMHANS) located in Bangalore, in the state of Karnataka, we have started investigations on the neurological progression of HIV-1+, HIV-1 /2+, and HIV-2+ in men and women. This is a longitudinal study and each participant is proposed to be followed for three years. Every participant will be evaluated every six months for cognitive functioning, mental health outcomes, as well as immune and neuroendocrine status. We have been able to locate a large cohort of seropositive participants (approximately 5,000) for these studies. At present, there is a scarcity of data on neuropathological findings among seropositive individuals in India. NIMHANS is one of the major centers in India known for carrying out autopsies in HIV seropositive individuals but to the best of our knowledge, there is no data available on cognitive functioning during life among the patients coming for post-mortem. Our present funded study can potentially fill this gap in defining the neurological functioning during life of participants in whom autopsy could be performed. This presentation will attempt to provide a model which could be suitable for such studies in developing countries particularly in India, and will take into consideration the cultural, social, religious and belief systems of that country. (Supported by NIH grants # NS 41205, DA 12792 and DA 13550)
Banking on Brains for Cocaine Abusers
Deborah C. Mash, Ph.D.
The transit of cocaine from source nations in the Caribbean and Central and
South America through the Bahamian corridor to the United States frequently occurs via the Florida coast. Metropolitan Miami-Dade County, Florida, continues to have one of the highest rates of cocaine-related emergency room episodes and cocaine-related deaths in the United States. Based on a retrospective case control analysis of the toxicology reports, scene descriptions, supplemental background information, and autopsy findings, we have developed a cohort of postmortem neuropathological specimens from chronic cocaine abusers.
Postmortem brain specimens from cocaine-related deaths and cocaine intoxication cases are banked with precision and uniformity. Cases are subdivided into three groups of fatal cocaine intoxication: accidental cocaine overdose, acute high-dose toxicity with seizures, and cocaine intoxication presenting with preterminal excited delirium. Retrospective chart reviews are used to exclude cases from neurochemical study based on evidence of significant underlying cardiovascular pathology, cerebrovascular disorders, and polydrug abuse. Structured interviews with informants are done to assess the patterns of cocaine use in the 30 days prior to death and for lifetime exposure. Cocaine and metabolite levels are measured in blood and brain tissue specimens at the time of death. An overview of the approach used to develop a collection of brain tissues from chronic cocaine abusers and age-matched drug-free control subjects will be presented.
Uncovering Transcriptome Differences With DNA Microarrays in Postmortem Tissue:Technical Considerations
Károly Mirnics, M.D.
Postmortem brain tissue contains intact mRNA that can be successfully extracted, reverse transcribed, and analyzed using DNA microarrays. It appears that transcript integrity depends more on the agonal state and circumstances of death than on the postmortem interval (PMI): we routinely isolate minimally degraded RNA from brains with >24 hours of PMI.
The appropriate experimental design is essential to perform meaningful comparisons of gene expression patterns. These paradigms consider the individuality for transcriptome of each investigated subject. Furthermore, in brain areas with high phenotypic complexity, DNA microarrays will not be able to assess the expression of the sparsest transcripts, which become "diluted" beyond detection by more abundant RNA species.
Microarray experiments can be compromised at different stages of this complex procedure. Establishing conservative starting material and data quality checkpoints will ensure that the obtained findings represent true biological differences rather than methodological artifacts.
Combined Effects of Drug Abuse and HIV Infection on the Brain: Correlations Between Experimental, Clinical, and Pathological Studies
Avindra Nath, M.D.
Both HIV infection and drugs of abuse have clearly shown independently to have adverse effects on the brain. Furthermore, experimental studies in vitro and using animal models also clearly demonstrate that the combined effects of these drugs of abuse and HIV infection of HIV proteins causes synergistic neurotoxicity, increased viral replication, and immune activation. However, previous clinical and pathological studies have shown conflicting results. The recent advances in technology and the identification of sensitive objective markers of neurological function should allow us to revisit this issue and resolve these questions. Critical to these issues is the proper banking of nervous system tissues from well-characterized cohorts as well as the development of surrogate markers of drug induced central nervous system injury.
The Role of Butyrylcholinesterase in the Pathogenesis of HIV-1 Dementia
Walter Royal III, M.D.
Cognitive impairment in individuals with HIV-1 (HIV) infection appears to be multifactorial, involving the direct effects of viral proteins and toxicity associated with secreted products of immune cells. In patients with Alzheimer's disease, it has been observed that the enzyme butyrylcholinesterase (BChE) is localized to plaques on immunocytochemical staining of tissue. The association of this finding with clinical dementia in these patients has formed part of the rationale for treatment of Alzheimer's disease patients with the drug rivastigmine, a BChE inhibitor. In addition, BChE has been demonstrated to be involved with the metabolism of cocaine and, therefore, may be important in preventing neurotoxicity from this drug.
To investigate the possible role of BChE in the pathogenesis of HIV dementia, we examined sections of fixed brain from patients with HIV-related cognitive impairment and from control subjects for expression of this enzyme. In this presentation we will discuss data from these studies as well as methods that may be useful in examining the possible role of BChE in the development of cognitive impairment in individuals with HIV infection.
A New Technology for Indepth Gene Expression Profiling
Richard Woychik, Ph.D.
Massively parallel signature sequencing (MPSS) is one of the newest tools available to conduct indepth expression profiling. MPSS is an open-ended platform that analyzes the level of expression of virtually all genes in a sample by counting the number of individual mRNA molecules produced from each gene. There is no requirement that genes be identified and characterized prior to conducting an experiment. MPSS has a routine sensitivity at the level of a few molecules of mRNA per cell, and the data sets are in a digital format that simplifies the management and analysis of the data. Therefore, of the various microarray and nonmicroarray technologies currently available, MPSS provides many advantages for generating the type of complete data sets that will help facilitate experiments in systems biology directed at studying the function of specific regions of the brain.