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Some of the brain regions Sutcliffe, Thomas, and their colleagues
studied were unaffected and some were very affected—showing
dramatic increases in apoD expression in the disease brains
as compared to the controls.
Significantly, some regions had highly overlapping expression
of apoD in the brains of patients with schizophrenia and bipolar
disorder—the prefrontal cortex, for instance—which
suggests that pathological similarities between the diseases
may account for some of their symptomatic similarities.
But the real strength of the work is that it teased out
some previously unknown differences between the two disorders.
"There are regions that distinguish between the two," says
Thomas.
For example, in the schizophrenic patients, the researchers
found increases of apoD expression in the amygdala, a small
brain region associated with certain types of emotional behavior.
In the bipolar patients, there was no such increase.
However, in the bipolar patients, they found several cortical
regions that were expressing higher levels of apoD than in
the schizophrenic patients, which suggests that the cyclic
nature of bipolar disorder could be due to a cortical imbalance.
These results do not give a complete picture of the mechanisms
of the two diseases, answer all the unanswered questions as
to how they differ, nor identify all the genes involved, but
the data do give solid physiological evidence that the two
diseases differ at the cellular and molecular level.
The study also demonstrates the power of high-throughput
genomic methods to address questions about the fundamental
nature of diseases like bipolar disorder and schizophrenia.
The TOGA® Technology
For these studies, Sutcliffe and Thomas used a PCR-based
method called total gene expression analysis (TOGA®) that
Sutcliffe invented a few years ago. TOGA® is currently
licensed to San Diego biotech company Digital Gene Technologies,
which analyzed the samples in their fully-automated Torrey
Pines facility.
The technology basically divides all the RNA in a tissue
sample into 256 pools, and accounts for all the RNA in each
pool by using polymerase chain reaction (PCR) to amplify them.
First the mRNA in a sample is purified and then an enzyme
is used to create "complimentary" cDNA from the RNA strips,
which is necessary in order to do the PCR.
The cDNAs are then primed—molecules are added that
anneal to the "polyA" repeating track of A nucleotides at
the 5' end (the beginning) of the cDNA. Also at this end is
attached a biotin fragment, which is like a piece of molecular
velcro that allows the cDNA to be fished out later.
The primed cDNAs are then cut with enzymes that recognize
four specific nucleotide bases, and the pieces of cDNA with
the biotin attached are fished out and separated according
to the sequence of four nucleotides adjacent to the cleavage
site. This sounds complicated, but the basic thing to keep
in mind is that this allows the RNA to be divided into 256
pools (4* 4*4* 4 = 256), and identified individually.
Each pool of RNA is then amplified with PCR and the PCR
products are then subjected to capillary electrophoresis,
a technique that essentially separates the pieces based on
their length—the length from the 3' cleavage site to
the poly A tail—and detects them through their fluorescence.
When each lot of cDNA passes by the laser in the capillary
electrophoresis apparatus, a "peak" of fluorescence emission
is detected. The timing of this peak appears on the length
of the original RNA, and it is actually predictable. By counting
the number of bases between the poly A tail and the cleavage
site, and by taking into account the 4 bases adjacent to the
cleavage site, it is possible to know where to expect it.
"For every RNA of known sequence, we know which of the 256
pools will contain that RNA and how long the product will
be," says Sutcliffe.
So when the computers collect an array of bands, these data
can be compared to a list of known sequences of RNA, and candidate
genes can be assigned to them. This is all done automatically.
The technique, then, takes a piece of tissue and returns
a set of data representing which genes are being expressed
in the tissue. Rather than looking for one gene in particular,
the computer provides a range of genes that are active.
The Hypocretins and Narcolepsy
This technology has proven invaluable in other studies that
have originated from Sutcliffe's laboratory. A few years ago,
he and his colleagues found two excitatory neuropeptides expressed
by only about 3,000 neurons in the hypothalamus, the brain
center that governs most aspects of autonomic regulation—such
as aspects of energy metabolism, cardiovascular function,
hormone homeostasis, and sleep-wake behaviors.
These two peptides, now called the hypocretins, are expressed
in neurons with connections to many parts of the brain, from
the cerebral cortex to the base of the spinal cord. Electron
microscopy studies showed these neurons packaging the hypocretins
in vesicles, and the vesicles accumulating at the synapses,
so Sutcliffe and his colleagues arrived at the hypothesis
that the hypocretins are neurotransmitters—neurons fire
action potentials as a result of the release of the peptides,
and these action potentials cause humans and other animals
to wake up.
The name hypocretin is a shortened name for hypothalamus
peptide with a sequence that is related to secretin. It
is a name that is actually attached to two separate, closely-related
peptides that are concentrated in an area of the hypothalamus
that is implicated in arousal, feeding, blood pressure, and
the release of hormones. Not surprisingly, the hypocretins
are important modulators of all of these—especially the
sleep/wake cycle.
In fact, the hypocretins are responsible for narcolepsy.
Narcoleptics suffer hallucinations, loss of muscle control,
and, most notably, frequent sleep "attacks" throughout the
day, even if they are fully rested.
Narcolepsy- is a disease that results from not having hypocretins
or hypocretin receptors. Animals with either hypocretin or
the hypocretin receptor knocked out display signs of narcolepsy,
and humans with narcolepsy have no detectable hypocretins
in their cerebrospinal fluid—there is normally a fixed
amount. The cause of this loss, developed in adolescence,
is not well understood
What is known is that narcolepsy is one of the most common
neurodegenerative disorders—affecting some 250,000 Americans
according to the National Institute of Neurological Disorders
and Stroke. And by continuing to identify, describe, and study
the hypocretins that cause the disease, Sutcliffe hopes to
elucidate the physiological mechanism of narcolepsy and, hopefully,
contribute research that will lead to better treatments.
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