Enzyme Indicates Alzheimer

Identified molecule that may diagnose disease years before it manifests itself

MIGUEL BOYAYANPatient with Alzheimer: objective of the study is not treatment, but to prevent the disease from manifesting itselfMIGUEL BOYAYAN

A team from the School of Medicine of the University of São Paulo (FM-USP) has achieved an important victory in the fight against the great challenge of Alzheimer’s disease: understanding the mechanism of the degeneration of the brain that characterizes the disease, in order to establish an earlier and accurate diagnosis. Wagner Farid Gattaz, Cássio Bottino and Orestes Forlenza, from the Institute of Psychiatry, have identified an enzyme – phospholipase A2 – which they hope may be an efficient biological marker for the early detection of the disease, or even the key to its cure. Starting from the measurement of this enzyme in the blood, they intend to work towards the prevention of Alzheimer several years before the disease manifests itself.

At the moment, although it is based on clinical evidence, the diagnosis of Alzheimer is given as “possible” or “probable”. To be really certain, only with an autopsy, when the brain reveals its terrible state of degradation: atrophied cerebral tissue, entangled nervous fibers, neurons invaded by plaques of beta-amyloid protein. The researchers believe that the first symptoms of Alzheimer’s disease, which usually appear after the age of 60, are the culminating point of a silent process that begins 20 to 30 years before – but that can leave traces in the blood right from the start.

Gattaz examined the blood of healthy old people and compared it with samples collected from sufferers of Alzheimer and of slight cognitive impairment [RS5] (SCI) – a disturbance characterized by just a failing memory. The two groups showed a reduction in the levels of phospholipase A2 (or PLA2), an enzyme that works on the metabolism of the phospholipids, components of the cellular membrane. In addition, the more damage to the functions of the brain, the lower the levels of phospholipase.

Schizophrenia and dementia
The first hypotheses about the action of phospholipase are from the 80’s, when Gattaz was at Heidelberg University, Germany, studying the mechanisms of schizophrenia. Phospholipase was little known then. Besides being produced as a digestive enzyme by the pancreas, it was discovered that it is present in all cells, including neurons. “The membrane of a neuron is made up of a double layer of phospholipids. It is in this layer, at the border of the cell with the external surroundings, where the neuroreceptors are – which are responsible for the transmission of information from one neuron to another. Altering the composition of this membrane by the action of enzymes, you alter its architecture, and, consequently, the action of the neuroreceptors”, Gattaz explains.

His hypothesis was originally to conceive schizophrenia as a disease of the pancreas, not of the brain. He supposed that it could have a mechanism similar to other neuropsychiatric conditions, not originating primarily from the brain, but from the peripheral metabolism – like phenylketonuria, a liver disease that stems from the lack of an enzyme and which leads to disturbances in the maturing of the nervous system. In the case of schizophrenia, no lack of phospholipase, but rather an excess of it.

It did not take long for Gattaz to turn his attention to Alzheimer’s disease, for having a feature that is the opposite of schizophrenia: it is very rare for a schizophrenic to develop this kind of dementia (deterioration of the brain). Even when there are serious failings in memory and comprehension, post-mortem examinations of the brain do not show deposits of the beta-amyloid protein – the senile plaques that are typical marks of Alzheimer. If there was an increase in the levels of phospholipase in schizophrenics, it was appropriate to check this in patients with Alzheimer. “We found that phospholipase A2 decreased in the brain, and that the decrease was related to the severity of the lesion of the brain: the lower the level of the enzyme, the greater the occurrence of senile plaques.”

A parallel was found between the decrease of the enzyme in the brain and in the blood: “We evaluated the presence of phospholipase A2 both in brain tissue post-mortem and in the platelets. The presence of phospholipase in the blood makes it possible to extrapolate to the activity of the brain, facilitating the researcher’s access”. A potential biological marker for Alzheimer’s disease had been discovered.

Suspect allele
It is not the only marker being investigated. In a group of patients he following for almost five years, Cássio Bottino tested a potential genetic marker, the e4 allele. Located in chromosome 19, e4 is one of the three forms of the gene that codifies apolipoprotein E, or ApoE (the others are the e2 and e3 alleles). ApoE is a protein of the plasma connected with the transport of cholesterol to the liver, brain and other tissues. According to several research studies, it so happens that the carriers of the e4 allele have more chances of developing Alzheimer’s disease and tend to show the symptoms earlier than the carriers of the e2 and e3 alleles.

In the typing for the e4 allele of 20 control patients (healthy old people), plus 41 sufferers fromAlzheimer’s and 21 with SCI, Bottino confirmed studies that indicate for the elderly who are carriers of the e4 allele a risk 2.4 time greater for developing Alzheimer’s. “In the patients with SCDI the risk was slightly increased. It did not acquire statistical significance, but points to a tendency”, he reveals.

This genetic marker, though, is far from allowing a good diagnosis. “The statistical risk – two and a half times – is very small when compared with markers of other diseases, where you find an increase of 100, 1,000 times”, Gattaz concedes.

Affected neurotransmitter
Phospholipase A2, though, has encouraged researchers with the prospects of being more than a marker, rather a substance involved in the very origins of the disease. “For Alzheimer’s disease”, says Gattaz, “everything indicates that this enzyme has a double meaning: it contributes both towards the formation of senile plaques and to a decrease in the cholinergic activity – the release of the acetylcholine neurotransmitter”.

The release of acetylcholine, a neurotransmitter that is fundamental for the workings of the brain, shrinks in those suffering from Alzheimer. For this reason, the medicines most used act in a way that increases its release in the brain. Among them, there are substances that are precursors of acetylcholine and others that inhibit acetylcholinesterase – an enzyme that degrades this neurotransmitter. They are symptomatic treatments that improve the quality of life of about 70% of the patients.

It so happens that a decrease in the phospholipase A2 enzyme also damages the formation of acetylcholine: “Phospholipase releases choline from the cell membrane to form acetylcholine. A decrease in the activity of this enzyme would therefore result in the worsening of the cholinergic deficit that already exists with Alzheimer’s disease. This deficit is primarily a consequence of the massive death of cholinergic neurons in regions of the brain connected with memory”. Another mechanism that may suffer the influence of this enzyme is the very formation of the senile plaques, according to Gattaz’s hypothesis.

He explains that the beta-amyloid protein, which forms the plaques, is cut out of a larger protein called APP – amyloid precursor protein – which is anchored to the membrane of the nerve cell. APP is metabolized or digested by the alpha-secretase enzyme, which normally breaks it in the middle. However, a decrease in phospholipase results in a reduction of the metabolism of phospholipids. This means that there is an increase in the quantity of phospholipids in the membrane.

The alteration in the architecture of the membrane also change the cleavage (break) points of the APP. Now, the alpha-secretase that used to break it is no longer active. It is then cleaved by a beta-secretase, and afterwards by a gama-secretase, which will release the entire beta-amyloid molecule – free, therefore to form the plaques.

World-wide target
“It would be too optimistic to think that phospholipase is the only cause of Alzheimer”, Gattaz ponders. “However, as it has a direct implication with the production of senile plaques and with cholinergic neurotransmission, we may perhaps come to regard it as a disease in itself – in the same way that glucose, for example, is not just a diagnostic marker for diabetes, but the disease itself. We could then developalternative therapeuticstrategies,to prevent the formation of plaques. This, at least, is what we wish.”

It is a wish shared with groups of researchers from all over the world. By different approaches, the formation of beta-amyloid plaques is one of the favorite targets of work for those seeking to fight Alzheimer at its source. Even a vaccine against this protein has now been developed. In 1999, the American laboratory Elan Pharmaceuticals created a vaccine with small, synthesized doses of beta-amyloid. The preliminary results were heartening: in rats, antibodies against the protein were formed. Shortly afterwards, researchers from the Harvard School of Medicine used the same principle to create an immunizer in the form of a nasal spray, also tested in rats.

Another world-wide line of research into beta-amyloid follows the direction of preventing the aggregation of the protein. A team from the departments of Medical Biochemistry and of Anatomy from the Federal University of Rio de Janeiro, coordinated by biochemist Sérgio Teixeira Ferreira, has developed a medicine based on nitrophenols, to block the aggregation of beta-amyloid in cultures of neuronal cells and in rats. The study was published on March 20th, 2001, in the Faseb Journal, a publication of the Federation of American Societies of Experimental Biology of the United States.

Besides investigating the origin of the disease, world-wide research into Alzheimer is looking for better methods for diagnosis and for new drugs. The Institute of Psychiatry of the FM-USP is also investing in this field. At the Neurosciences Laboratory, under Gattaz’s coordination, Orestes Forlenza’s team is doing neuron cultures and tests on guinea pigs, which receive substances to inhibit or to stimulate phospholipase.

Inaugurated in 1999, the laboratory is one of those that benefited from the funds from FAPESP’s Infrastructure Program: well equipped for analyses in neurochemistry, molecular biology and genetics, it allows the researchers to act on other fronts, such as functional neuro-imaging exams, which can show the regions of the brain where metabolism is low, and magnetic resonance exams, in which any reduction in the structures of the brain can be seen. Bottino noted, for example, that the comparison of the volumetric measurements of the tonsils, hippocampus and para-hippocampal gyrus makes it possible to separate bearers of light to moderate Alzheimer’s disease for healthy persons with an accuracy of 88%.

SCI and Alzheimer
Also promising are the results achieved with sufferers from a slight cognitive disorder, a disturbance that is still little studied. “SCD is usually diagnosed in people over the age of 50 who complain of problems with the memory and show worse results in cognitive tests, when compared with healthy old people, but they still are not affected in their day-to-day activities. Some of them improve as time goes by, but 5% to 10% evolve into Alzheimer”, Bottino reveals.Patients with SCD had lower levels of phospholipase A2 when compared with the control group, but higher levels than the group with Alzheimer. This data made it possible to draw up a graph in which SCD occupies the median range.

The group with SCD is, however, very heterogeneous: some had values very similar to normality, while others bordered the levels of dementia. “That is why we want to do a follow-on study with a larger sample. As 10% of those suffering from SCD evolve into dementia, we need a sample with 100, 150 persons. We will then be bale to investigate whether the patients who are nearer the bottomofthe graph are thosewho will evolve into Alzheimer. It would be more a confirmatory study of the predictive value of the enzyme, and this is what is driving us now”, Gattaz advises. “This is the medicine of the 21st century. Out objective is not to treat anyone, but to prevent them from becoming ill”.

The Projects
1. Metabolism of Phospholipids in Schizophrenia and in Alzheimer’s Disease (nº 97/11083-0); Modality Thematic project; Coordinator
Wagner Farid Gattaz – School of Medicine at USP; Investment R$ 1,590,193.43
2. Dementia of the Alzheimer Type, Slight Cognitive Disorder and Normal Aging: Longitudinal Study of Clinical, Neuropsychological, Genetic, Neurochemical and Neuroimaging Aspects (nº 99/00740-5); Modality Regular line of research benefits; Coordinator Cássio Machado de Campos Bottino – FM/USP; Investment R$ 102,961.39 and US$ 86,119.09