Thesis on the fish known as Paulistinha came first in the X FCM Research Week
Inborn errors of metabolism (IEM) form a group of genetic diseases related to changes in the body's biochemical processes. Most IEMs manifest in the pediatric age group, but they can occur at any age. To date, more than 550 metabolic diseases are known. If considered in isolation, the incidence of each of them is rare.
Biologist Marcella Bergamini de Baptista proposed in her doctoral thesis the use of Dario rerio ou zebrafish, better known as the Paulistinha fish, as an animal model for the study of GM1 gangliosidosis, a neurodegenerative disease caused by a decrease in the enzyme beta-galactosidase, which leads to the accumulation of gangliosides within liposomes. The thesis, supervised by professors Carlos Eduardo Steiner and Cláudia Vianna Maurer Morelli, came in first place during FCM's X Research Week, which brought together more than 140 works developed by research groups from the Faculty of Medical Sciences and Unicamp.
GM1 ganglioside is present in the cell membrane of all vertebrate tissues. The growth and maturation of the central nervous system are influenced by gangliosides. The brain contains the largest amount of GM1, with one to two grams of its composition. “The main characteristic of gangliosidosis is the progressive accumulation of macromolecules. Almost two-thirds of patients present neurodegeneration in a specific region of the brain, before affecting the entire central nervous system”, explains Marcella.
GM1 gangliosidosis is classified into three clinical forms according to age, enzymatic activity and severity. Type I (or infantile form) begins in the sixth month of life and progresses quickly. Patients may experience lack of appetite and difficulty sucking, dysarthria, dysphagia, hand tremors, stiffness in the arms and legs, and difficulty walking.
The main characteristics of type II (or juvenile) are delayed development of the motor and cognitive system, neuronal lipidosis and skeletal dysmorphisms. Type III (or adult) has dystonia – involuntary movement that can occur in any region of the body – as the main neurological manifestation, in addition to skeletal deformities and difficulties in speaking and walking.
“There are currently no effective treatments for GM gangliosidosis, only supportive therapies. Animal models are studied to understand the molecular and cellular bases of the disease, in addition to testing new treatments and medications before using them in humans,” says the biologist.
Similar to humans
Rats and mice have traditionally been used in research on GM1 gangliosidosis. However, these animal models do not allow observation of all clinical aspects of the disease. In this sense, the zebrafish has advantages in genetic manipulation for the study of liposomal storage diseases (LDD).
One of the advantages of zebrafish is to have a large part of organs and systems similar to those of humans – 70% of the genes are similar to the human genome – and its complete genome is available in databases. A couple of fish can have up to 200 embryos per week, which allows a statistically relevant sample to be generated at a low cost.
“The fish are transparent during the initial stages. When there is genetic manipulation in the embryo, the phenotypes tend to manifest themselves during the larval stage, which makes it possible to evaluate in vivo non-invasive. To date, fewer than six DDL have been reproduced in zebrafish. The objective of the research was to characterize the fish for functional studies of the gene glb1”, says the researcher.
Embryos were obtained through natural mating. The biologist used the morpholino (MO) technique, which consists of microinjecting antisense oligonucleotides into fish still in the embryo stage to temporarily silence the expression of the beta-galactosity gene enzyme glb1 in larvae.
The animals were observed daily, from fertilization until the fifth day, when the MO begins to lose its inhibition function. After 96 hours of the fertilization period, cartilaginous skeletal changes became evident. After 120 hours, progression of the malformations was observed in five animals.
“Two days post-fertilization, the face change in embryos zebrafish it was subtle, but suggestively corresponded to the facial thickening observed in patients with GM1 gangliosidosis. The animals also showed altered neurological functions”, informs Marcella Baptista.
According to the Unicamp researcher, for this disease, which can be manipulated in the laboratory, no other animal model showed changes in bone development when having the gene glb1 silenced like zebrafish. So far, the zebrafish has been presented as a future valid animal model for the study of beta-galactosidase.
“A single animal cannot contemplate all the signs and symptoms of the same disease. Therefore, the results of zebrafish complement the research on GM1 gangliosidosis, along with the other animal models studied to date. Despite the good results, further research is needed to gain more complete knowledge regarding the disease in this animal”, says the thesis author.