What is contained in cell biology

Small cause - tragic effect

"Lorenzo's Oil" is the name of a film that tells the true story of a child with a mysterious illness. His ailments begin with mild imbalance and end with severe brain damage. The medical name of Lorenzo's disease is "adrenoleukodystrophy," a rare hereditary disorder that researchers now know to be due to the malfunctioning of tiny bodies called peroxisomes inside the cell. Dariush Fahimi and Eveline Baumgart from the Institute of Anatomy and Cell Biology explain what peroxisomes are, what significance they have for human health and disease, and what methods scientists are using today to better understand peroxisomal diseases and develop new treatment concepts.

"Microbodies", tiny bodies, they called their discoverers in the mid-1950s. With the help of the electron microscope, the scientists had tracked down the inconspicuous phenomena inside the cell. For several decades the small organs of the cell, they are called cell organelles, led a "Snow White existence" - until their vital importance was recognized. Today the tiny bodies are called "peroxisomes". The cell organelles received this name from the Belgian biochemist Christian de Duve: He proved that the peroxisomes contain certain enzymes, so-called oxidases, which produce hydrogen peroxide. They also contain the enzyme catalase, which breaks down the peroxides. In 1974 de Duve received the Nobel Prize for the discovery of peroxisomes and the first description of other tiny cell particles, the lysosomes. In the 1960s, Dariush Fahimi, one of the authors of this article, developed a method at Harvard Medical School in Boston to detect catalase in peroxisomes using light and electron microscopy. The systematic application of this method showed that the peroxisomes are indispensable components of every cell.

It is now known that peroxisomes are a functionally and morphologically heterogeneous group of related cell organelles that occur in all nucleated cells (eukaryotes) and, in addition to the enzymes mentioned for hydrogen peroxide metabolism, have other enzymes for the breakdown of various fats (lipids). In addition, the peroxisomes are involved in the biosynthesis of complex fats (so-called plasmalogens), cholesterol and bile acids. Under the light microscope, the peroxisomes are found particularly numerous in the liver and kidneys; but they also occur with different frequencies in other organs. If you look at the peroxisomes with an electron microscope, you can see that a finely granulated inner matrix is ​​enclosed by a simple shell, a membrane. The matrix can contain different crystalline inclusions in different species. Some cells harbor very long, tubular peroxisomes that can reach a length of several microns. In most cells, however, the peroxisomes are spherical, around 0.2-0.5 microns in diameter.

It is noticeable that the liver peroxisomes of some vertebrates, especially rodents, can be greatly increased by treatment with xenobiotics (foreign substances). Such foreign substances are referred to as "peroxisome proliferators". They include plasticizers from the plastics industry (phthalates), lubricating oils and other petroleum products as well as various lipid-lowering pharmaceuticals (fibrates), aspirin and the body's own inflammatory messenger substances such as prostaglandins and leukotrienes. These observations suggest that peroxisomes play an important role in the breakdown of certain environmental toxins. Indeed, our research shows that peroxisomes in the digestive gland of sea clams increase significantly when exposed to environmental toxins. We are currently conducting a study together with scientists from the University of Bilbao, Spain, to determine what significance peroxisome proliferation could have as a bio-indicator for marine pollution.

In medicine, the peroxisomes became interesting in 1973. At that time it was found that patients with a severe genetic disease associated with malformations of the nervous system, liver and kidney (cerebro-hepato-renal syndrome, CHRS) do not have peroxisomes in their cells. Although the peroxisome-typical enzyme catalase was biochemically detectable in the liver of these patients, no peroxisomes could be detected cytochemically. Because of this discrepancy, the first case reports were treated with skepticism. In the mid-1980s, new biochemical methods confirmed the role of peroxisomes in the development of this disease. The discovery of other diseases led to the term "peroxisomal diseases". Today fifteen very different diseases are grouped under this heading.

The absence of peroxisomes in patients with such diseases aroused the interest of cell biologists. They wanted to research where the peroxisomes actually come from and how they are formed. Right at the beginning of the cell biological investigations it was found that - although peroxisomal diseases and especially the CHR syndrome are fatal for humans - the individual cells of these patients, such as connective tissue cells, can continue to live in cell culture despite defective peroxisomal functions. This made it possible to use cell culture models to study the biogenesis of peroxisomes and to explain how peroxisomal diseases arise. Cell cultures of yeast cells, for example the brewer's yeast Saccharomyces cerevisiae, have proven to be very successful cell systems. Interestingly, the results of research with yeast cells regarding the biogenesis of peroxisomes can largely be transferred to humans: the corresponding genes were largely preserved during the evolution of the organisms.

Where do the peroxisomes come from?

In the 1970s, scientists believed that peroxisomes were formed when small vesicles bud from the endoplasmic reticulum (ER), a branched, tubular transport system within the cell. It turned out, however, that peroxisomal proteins are synthesized on free ribosomes (the "workbenches" of protein synthesis in the cell). Our electron microscopic investigations proved that the membrane structures, which were previously thought to be connections to the endoplasmic reticulum, contain peroxisomal proteins and are parts of a new peroxisome. In some fast-growing cells, peroxisomes can take on very long tubular forms that are joined together, for what has been suggested the term "peroxisomal reticulum". Today it is clear that all peroxisomal matrix proteins arise on free ribosomes that are not bound to the endoplasmic reticulum and are only later transported into the peroxisomes. For import into the matrix of the peroxisomes, these proteins receive a signal sequence that works like a zip code: By binding to specific receptors ("anchor points") in the cytoplasm and in the peroxisome membrane, they bring these proteins into the matrix of the peroxisomes.

There are two such peroxisomal "targeting" signals (PTS): a very simple one (PTS 1), which consists only of the three amino acids serine, lysine and leucine (abbreviated as SKL), and a second, more complex PTS 2. In contrast to Proteins of the mitochondria (the "powerhouses" of the cell), which are imported in a similar way, the peroxisomes can even import folded and oligomerized proteins. Even gold particles with a diameter of nine nanometers coated with SKL-containing peptides are still smuggled in. The exact mechanism for the import of such large particles through the peroxisome membrane is unclear. It is assumed, however, that the peroxisomes take up the proteins by invagination of their membrane (endocytosis) or with the help of adjustable pores in their membrane.

So far, around 20 different proteins have been identified that are involved in the biogenesis of peroxisomes and are known as "peroxins" (Pex1p to Pex20p). The corresponding genes are called "PEX genes". The protein Pex5p, for example, is responsible for importing the SKL-containing matrix proteins (PTS1 receptor), while the protein Pex7p is the corresponding receptor for the PTS2-containing proteins. In the event of changes or mutations in the genes for these receptor molecules, the matrix proteins cannot be imported into the peroxisomes; despite their normal structure, they are broken down in the cytoplasm. In the affected cell, all peroxisomal functions collapse. Indeed, in CHR syndrome, the most severe peroxisomal disease in humans, the Pex gene is mutated; however, other Pex5 genes can also be defective.

It is still unknown where the membrane of the peroxisomes comes from. While the membrane lipids are probably produced at the endoplasmic reticulum, the exact mechanism by which the membrane proteins are incorporated is still unclear. Recently, peroxins have been identified that appear to be localized in smaller vesicles (bubbles) that may originate from the endoplasmic reticulum. That is why there has been intense discussion recently as to whether the endoplasmic reticulum is perhaps necessary for the formation of the peroxisome membrane.

The current idea of ​​the biogenesis of peroxisomes looks like this: The role of the endoplasmic reticulum has not yet been clearly clarified; however, the concept of the formation of new peroxisomes from existing particles is generally accepted. The peroxisomal proteins are synthesized in the cytoplasm on free ribosomes and provided with appropriate target signals ("zip codes") in order to get into the peroxisome. While the peroxisomes in most mature cells have a spherical shape under normal conditions and multiply by division (constitutive path), after strong stimulation (for example by growth factors or xenobiotics) they can take on long tubular shapes, which then break up again into small spheres ( induced path). The failure of certain peroxins leads to disturbances in the formation and import of matrix proteins. The ability to divide and reproduce (proliferation) are also impaired.

When the peroxisomes are absent

The diseases that are attributed to functional disorders of the peroxisomes today are divided into two groups:
• peroxisomal biogenesis disorders in which several functions of the peroxisomes are disturbed and
• Peroxisomal single enzyme defects in which only a single peroxisomal protein appears to be changed.
While morphologically recognizable peroxisomes are absent in the first category, they are microscopically detectable in the second group.

An example of a disease in which an important function of the peroxisomes is disturbed is adrenoleukodystrophy. This inherited disease (the altered gene is on the X chromosome) is the most common and best characterized peroxisomal disease with an incidence of 1 : 20,000 to 1: 100,000 births. It is characterized by severe damage to the "protective sheaths" (myelin sheaths) of the nerves. The damage occurs primarily in the white matter of the brain, hence the name leukodystrophy. In addition, the function of the adrenal cortex (Adreno -...) is severely impaired in the patient. The first cases of X-linked adrenoleukodystrophy (X-ALD) were described as "bronze disease and sclerosing encephalomyelitis" as early as 1923. However, the relationship to the disturbed lipid metabolism only became clear fifty years later, when crystalline inclusions were found in the adrenal cortex of patients, which had formed due to the deposition of large amounts of long-chain fatty acids. Since similar inclusions also occur in another form of peroxisomal disease, a defect in the peroxisomes in ALD was suspected, but initially ruled out because these patients had morphologically identifiable peroxisomes in the liver. It was only when the exclusive oxidation of long-chain fatty acids in peroxisomes was demonstrated in the mid-1980s that it was discovered that X-ALD could be a single enzyme defect in the peroxisomes.

The gene for X-ALD was identified on chromosome Xq28 in 1993. The gene product (ALD protein = ALDP) is a transporter protein in the peroxisome membrane. It transports the long-chain fatty acids into the matrix so that they can be broken down there. Mutations in the ALDP lead to different manifestations of the disease. Their first symptoms can appear in infancy, adolescence or adulthood. In general, the earlier symptoms of ALD appear, the more severe the course of the disease. In extreme forms, the disease can lead to death in childhood. In this clinical picture, the long-chain fatty acids, which cannot be broken down in peroxisomes, are deposited on the myelin sheaths of the nerves and cause inflammation there. This destroys the white matter of the brain, causing the various symptoms of the disease.

The film "Lorenzo's Oil", which is based on a true case, shows the development of neurological symptoms such as balance disorders, numbness and cramps in a very intelligent school child named Lorenzo. In the film, Lorenzo's parents try to find out the cause of the disease and possible treatment methods through self-study and committed initiative. Although the parents manage to normalize the blood level of the "toxic" lipids with a special diet of unsaturated fatty acids (glycerol trioleate and glycerol trierucate = Lorenzo's oil), the severe brain damage that occurred in the acute phase of the disease remains unchanged. The only reliable form of therapy for the severe forms of X-ALD appears in the future to be gene therapy - i.e. the repair or replacement of the defective gene. In laboratory tests, a French working group succeeded in correcting the abnormal breakdown of fatty acids in connective tissue cells in patients with X-ALD by transferring a healthy gene over several months. It remains to be seen whether this will also work for patients.

In the last two years, three groups worldwide (one group works at the Center for Molecular Biology in Heidelberg, ZMBH) have succeeded in switching off the X-ALD gene and thus developing a "knockout" mouse model for X-ALD. The animals store the abnormal fatty acids in various organs - such as the brain and adrenal glands - but they do not develop any inflammatory reactions in the nervous system. Accordingly, in contrast to X-ALD patients, they also show no neurological deficits.

The second example of a peroxisome disease is Cerebro-Hepato-Renal Syndrome (CHRS). It is also known as Zellweger syndrome after its first description, Hans Zellweger (an American of Swiss descent). In this rare disease, the biogenesis of the peroxisomes is disturbed. Because all peroxisomal metabolic pathways fail completely, the affected children usually die during the first year of life. The infants often have a typical tower skull with wide open gaps in the bones (fontanelles) and generally show reduced muscle tension (generalized hypotension). CHRS is a multi-organ disease complex characterized by developmental disorders in the brain associated with seizures, blindness and deafness and severe generalized hypotension, as well as chronic jaundice with liver fibrosis / cirrhosis and kidney cysts. In addition, developmental disorders of the skeleton, the genitals and a failure of the adrenal functions can occur. It is still unclear which of these symptoms are primarily caused by poor peroxisome functions and which are caused by toxic metabolic products. In any case, this deadly disease shows the vital importance of peroxisomes for humans.

Unfortunately, the therapy options for patients with Zellweger Syndrome are only very limited, as pronounced developmental disorders and deformities are already present at the time of birth. In the future, however, gene therapy could possibly be considered, since peroxisomal biogenesis diseases are based on individual defective genes, the failure of which could be corrected by transferring healthy genes.

In the last decade, the technical requirements for breeding "knock out" mice as animal models for human diseases caused by defects in individual genes have been developed or significantly improved. Since the elucidation of peroxisomal biogenesis and the genes involved has only progressed in the last few years, hardly any such mouse models for peroxisomal diseases have so far been established. The "knock out" mouse model for Zellweger syndrome described below is the first animal model for a peroxisomal biogenesis disorder.

To breed this "Zellweger mouse", we switched off the gene for the most important import receptor for peroxisomal matrix proteins (PTS1 receptor = Pex5p). This gene is also known to be defective in a subgroup of Zellweger patients. The characterization of 196 mouse fetuses confirmed a clear autosomal recessive inheritance of the PEX5 defect.In the animals lacking both genes (homozygous = homozygous negative animals, PEX5 - / -), neither messenger ribonucleic acid (mRNA) nor the protein for Pex5p could be detected in the liver. This proves the successful "knock out" of the PEX5 gene. All newborn Zellweger mice could be differentiated from the heterozygous (heterozygous: they have a healthy and a diseased gene, the healthy gene balancing out the diseased) "healthy" animals due to their growth disturbances already observed in the mother's uterus. In addition, the Zellweger mice, which lack both genes, show general muscle weakness and lack of muscle tension (hypotension), which are comparable to the symptoms of patients with Zellweger syndrome. A clear sign of weakened muscles is the inability to suckle, which leads to the death of these mice within 72 hours of birth.

Although the results of "knock out" mice are not generally comparable with the corresponding human clinical picture, the clinical symptoms of the PEX5 - / - mice almost completely agree with the symptoms of patients with Zellweger syndrome, as are the biochemical-serological symptoms Parameters and the pathological changes. The mice are therefore an ideal animal model to better understand the development of peroxisomal biogenesis diseases and to develop new therapy concepts.

Prof. Dr. H. Dariush Fahimi and Priv.-Doz. Dr. Eveline Baumgart
Institute for Anatomy and Cell Biology, Im Neuenheimer Feld 307, 69120 Heidelberg,
Department of Medical Cell Biology,
Telephone (0 62 21) 54 86 56