II

fraction from cerebrospinal fluids (CSFs) from cases of first-break psychoses

and controls.  Pellets from low speed centrifugation of CSFs were used as

the inoculum on two different New World monkey cell lines.  Cells were

serially subcultured for up to twelve passages.  Samples for analysis by

polymerase chain reaction (PCR), reverse transcriptase assay, differential

display, and electron microscopy were taken at regular intervals.  Results

from analyses of cells initially inoculated with CSFs from psychosis patients

(case) were compared to those of cell initially inoculated with CSFs from

patients suffering from pseudotumor cerebri (control) or to results from

mock-inoculated cells (mock).  Electron microscopy of the fourth passage

cells inoculated with one case CSF reveals extracellular as well as

intracellular viral particles and a budding virion that resemble a C-type

retrovirus.  No particles (intra- or extracellular) were seen in controls

or mock at equivalent passages. Electron micrographs of negatively stained

purified virus show virions with morphology consistent with C-type retroviruses.

PCR analysis of particles from cases yielded sequences that suggest the presence

of a member of the Multiple Sclerosis associated retrovirus (MSRV)/ERV-9 group

of endogenous human retroviruses.  Association of the particles with

disease state of the patients has not been conclusively established but will be

in the pathogenesis of schizophrenia.  We investigated the transcription of

RNA in the new world monkey NZP-60 cell line (ATCC: CRL-1924) which, after being

cocultured with cerebrospinal fluid obtained from an individual with recent

onset schizophrenia (S-CSF), contained retroviral particles as visualized by

electron microscopy.  This cell line was selected since new world monkeys

are not endogenously infected with the human endogenous retroviruses (HERV)-W,

polymerase chain reaction (PCR) in order to characterize the RNA transcription

of cells inoculated with S-CSF.  We serially passaged S-CSF inoculated

NZP-60 cells 9 times in parallel with mock infected cells.  RNA was

extracted, converted to cDNA, and amplified using a series of differential

display primers (Delta Differential Display kit by Clontech).  Initial

analysis has identified a number of amplified products that are differentially

expressed in S-CSF and mock inoculated cells.  The nucleotide and predicted

amino acid sequence and analysis of these differentially expressed products are

ongoing.  These studies may identify viral and cellular RNA transcripts

retroviruses in the etiology of schizophrenia and bipolar disorder. Retroviruses

may represent an important link between genetic and environmental factors, as

this would account for both horizontal and vertical transmission, and these

viruses may be involved with the disease process in subpopulations affected with

these serious mental illnesses.  The reverse transcriptase (RT) enzyme is

an important component of infectious retroviruses, and as a result they can be

samples, e.g. cerebrospinal fluid (CSF) from patients with recent onset

schizophrenia, and post-mortem cerebellum obtained from individuals with

schizophrenia, bipolar disorder, depression without psychosis, as well as

unaffected individuals. We have modified an RT assay called product-enhanced RT

(PERT; Pyra el at, PNAS 91:1544-1548, 1994) to measure RT activity in our

samples.  Using this PERT assay, a significant increase in CSF RT activity

(p=0.0002) was found in patients with recent onset schizophrenia (n=18) when

compared to unaffected individuals (n=18).  RT activity was not detected in

CSF from a neurological control group with Alzheimer’s Disease (n=12). 

Post-mortem cerebellar RT activity was also significantly increased in the

schizophrenic (p=12; n=12) and the non-psychotic depression (p=0.021; n=11)

groups in comparison to the unaffected group (n=12).  CSF from selected

patients with recent onset schizophrenia were used to inoculate a New World

monkey cell line (OMK), and the culture media were assayed by the PERT method.

Our preliminary in vitro data indicate that there is an increase in RT activity

(approx. 10-fold over background), which suggests the presence of a replicating

several known diseases and may be of interest in schizophrenia research,

particularly in view of the neurodevelopmental models of the disease. 

Patterns of genomic instability are frequently associated with repetitive

elements.  Line-1 elements are repetitive elements of the retrotransposon

class found in mammalian DNA at copies of approximately 600,000 per haploid

genome (Smit, 1996), most of which are inactive as retrotranspons, but still may

facilitate recombination and genomic arrangements in a sequence-specific manner. 

Although it is possible that the large numbers of Line-1 elements have little

significance to genomic function, and represent “relic” material, it

is nevertheless tempting to speculate that such a large component of the

mammalian genome serve some function, and perhaps even an important function, in

the normal brain.  Thus, prior to comparing the status of Line-1 elements

between genomic samples from normals and patients with schizophrenia, a study

was undertaken to assess the properties of Line-1 elements in different regions

of the same human brain.  Inverse PCR was selected as the initial method to

screen two brain regions, the pons and the hippocampus, as well as DNA from an

indicate differential sensitivity of Line-1.2 to the restriction enzyme Msp1

between DNA from the pons, the hippocampus, and the HFF cells.  The

difference may derive from variation in patterns of methylation, as Msp1 is

inhibited by methylation of the outer C 1 the CCGG recognition site. 

Alternatively, the difference may result from structural variations in the DNA,

for example, a single stranded region in the Line-1.3 element that would prevent

Msp1 from cutting.  In addition to following up on this finding, future

studies will look at other regions of the Line-1.3 sequence, specifically

focusing on integration sites that are unique to one brain region and not the

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