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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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Schematic depiction of brain regions exhibiting elevated apoD expression in schizophrenia and bipolar disorder. Click to enlarge.