Latent Cytomegalovirus Infection of the Central Nervous System
It has been proposed that some cases of schizophrenia result
from a latent viral infection; one that may have been acquired in vitro. A
primary goal of this laboratory is to study a human neurotropic virus known to
establish latent infections and examine its effects on the cells of the central
nervous system (CNS). Learning more about the pathogenesis of a latent
virus infection in the CNS should lead to a better understanding of how, at a
cellular level, a virus infection could lead to schizophrenia. In the current
study, human cytomegalovirus (HCMV) has been used as a model of a human virus
known to establish latent infections in utero. Congenital (in
utero) HCMV infection has been associated with late manifestations of
disorders of neuronal migration and cortical organization. A latent
infection of human neuronal precursor cells has been established in vitro.
Immunofluorescence assay results indicate that only the major
immediate early region of the viral genome is being transcribed in this latent
state. However, infectious virion production can be induced if latently
infected cells are treated with retinoic acid, which causes the cells to
differentiate. Characterization of the latent infection from both the
virus and the cellular aspect will be discussed. Future plans include
investigation of up or down regulation of some cellular proteins reportedly
important in the pathogenesis of schizophrenia, e.g., neural cell adhesion
molecule.
Determination of Reverse Transcriptase (RT) Activity in
Clinical and Post-Mortem Samples Obtained from Individuals with Schizophrenia
and Bipolar Disorder
Our laboratory is interested in studying the role of
retroviruses in the etiology of neuropsychiatric illnesses such as schizophrenia
and bipolar disorder. Retroviruses may represent an important link between
genetic and environmental factors, as this would account for horizontal and
vertical transmission, and these viruses may be involved with the disease
process in subpopulations affected with these serious mental illnesses. An
important component of infectious retroviruses is that they contain the reverse
transcriptase (RT) enzyme, and consequently they can be detected by assays for
RT activity.
In this study, we set out to examine the levels of RT activity
in various samples, e.g. cerebrospinal fluid (CSF) from first episode patients
with schizophrenia, post-mortem cisternal fluids and various brain regions from
individuals with schizophrenia, bipolar disorder, depression without psychosis,
as well as unaffected individuals. Before proceeding with these
experiments, it is important to determine the baseline RT activity present in
these samples, which may be due to the presence of endogenous
retroviruses. We have modified an ultrasensitive RT assay called
product-enhanced RT (PERT; Pyra et al., PNAS 91:1544-1548, 1994), by adding a
second round of polymerase chain reaction (PCR) amplification. When the
resulting PCR products are visualized with the nucleic acid dye SYBR-green
(Molecular Probes) and analyzed with the Fluorimager SI (Molecular Dynamics) we
are able to detect as little as 10-7 units of Moloney murine leukemia
virus-reverse transcriptase (MMLV-RT) activity in the PERT assay. With
this PERT assay the background RT activity in the cerebellum of unaffected
individuals (n=10) was found to be less than 10-4 units. Data
on the RT activity in the cerebellum from the affected groups, as well as the
CSFs from the first episode patients with schizophrenia, will be presented.
Simultaneous Analysis of Expression Levels of Many Genes in
Post-Mortem Brain Tissue From Individuals with Schizophrenia and Bipolar
Diseases
Kia Faridi
The analysis of expression levels of genes in the post-mortem
brains of individuals with schizophrenia and bipolar disease is a potentially
important method in understanding the molecular basis of these diseases.
The simultaneous analysis of levels of a large number of genes is particularly
valuable, allowing the evaluation of functionally related gene groups.
We used the Clontech Atlas Human I cDNA Expression Array to
simultaneously analyze levels of 588 genes. Total RNA was extracted from
cerebellar brain tissue. This RNA was used to generate 32P-labelled
cDNA probes, which were hybridized to the membrane array. Autoradiograph
images of each array were then analyzed using ImageQuaNT software (Molecular
Dynamics) generating a value for each gene. A total of 22 samples were
analyzed, including four brains from schizophrenics, six from bipolar
individuals, five from clinically depressed individuals, and seven normal
controls. ANOVA analysis was performed to compare the levels of each gene
between the four disease groups. Several genes were found to be
significantly differentially expressed in patients and controls. In addition for
each disease, total RNA from four samples was pooled, and this mixture was
hybridized to the array. The largest differences were seen in levels of glutathione
S-transferase microsomal (raised in schizophrenic and bipolar) and glutathione
S-transferase theta-1 (depressed in schizophrenic).
Protein Alterations in the Postmortem Frontal Lobes of
Individuals with Severe Psychiatric Disorder
Nancy L. Johnston
Two-dimensional gel electrophoresis is an objective means to
compare the levels of individual proteins in different samples. We
analyzed 89 postmortem frontal cortex brain regions from individuals with
schizophrenia, bipolar disorder, major depression and unaffected normal
controls. We used a multivariate analysis to identify changes specific to
psychiatric disease. Eight spots were altered, with 6 decreasing and 2
increasing in level in one or more illnesses. Four of those which were
decreased were variants of glial fibrillary acidic protein (GFAP).
Ubiquinone cytochrome c reductase complex protein 1 was reduced in depression,
and dihydropyrimidinase related protein 2 was reduced in all three
illnesses. Carbonic anhydrase 1 was elevated in depressed individuals and
a second as yet unidentified protein was elevated in all three diseases.
The GFAP protein formed a family of spots that likely include
modified and unmodified forms of this protein. It is noted that two forms
were significantly decreased in all disorders while the other two were only
altered in non-psychotic depression. The pattern of change was distinct
between the three diseases and could not be attributed to any known confounding
variables. Two-dimensional gel electrophoresis offers a sensitive and
viable means to measure changes in levels and modifications that would not be
detected by one-dimensional electrophoresis or analysis of the corresponding
nucleic acids.
Detection of Retroviral RNA in the CSF of Individuals with
Schizophrenia
Håkan Karlsson
We have explored the hypothesis of retroviral involvement in the
etiology of schizophrenia by testing 18 CSF’s obtained from first-episode
individuals with schizophrenia-spectrum disorder and 18 CSF’s from individuals
without psychiatric disease.
RNA’s were isolated from pelleted CSF’s reverse transcribed, and
subjected to PCR using primers directed towards a conserved region of the
retroviral pol-gene (Tuke et al, Acta Neurol Scand 1997;169:16-21).
A PCR-product was detected in seven of the 18 schizophrenia
samples and in three of the 18 control samples. Subsequent cloning and
sequencing of these products showed that at lest six samples from the
individuals with schizophrenia contained a single retroviral species, showing
90-95% homology to the multiple sclerosis associated retrovirus (Perron et al,
PNAS 1997;94:7583-7588). Individual control samples were found to contain
multiple species, homologous to human endogenous retroviral sequences (including
multiple sclerosis associated retrovirus), probably originating from
contaminating human nucleic acids.
These results document the presence of specific retroviral
particles in the CSF of acutely ill individuals with schizophrenia. The
retroviral particles may have arisen from the differential activation of
endogenous sequences or may be the result of exogenous retroviral infection.