Huntington’s disease is a rare, debilitating, and insidious neurological disease. Patients usually begin exhibiting symptoms in their 30s or 40s and die before they reach 60. Caused by a faulty Htt gene, which encodes the huntingtin protein, it affects 5–10 in every 100,000 people in Europe, where it is most common, and leads to the destruction of neurons that produce gamma-aminobutyric acid (GABA). Neural imaging of Huntington’s patients indicates that the death of these cells—the GABAergic neurons—intensifies in early adulthood and progresses for almost a decade until the first symptoms appear: loss of motor control, cognitive difficulties, and behavioral changes, such as depression. The disease was described in detail in 1872 by American physician George Huntington (1850–1916). Now, a century and a half later, researchers from the Institute of Chemistry (IQ) at the University of São Paulo (USP) are helping us understand why it is so devastating. From the very beginning of their lives, people with Huntington’s may have fewer GABAergic neurons, which are then destroyed at an accelerated pace.
The suspicion that there may be less of these neurons in the brains of people with Huntington’s is the result of experiments on rodent and human cells, carried out by a team led by IQ-USP’s Alexander Henning Ulrich, a German biochemist who lives in Brazil. For almost 15 years, Ulrich and his colleagues have investigated the chemical and biological phenomena that transform the immature and versatile cells of an embryo into the specialized cells of an adult. The tests are conducted on rodent embryonic cells, which behave similarly to human cells, or on adult human cells chemically treated to function like embryos. The researchers grow them in small vials containing nutrients to keep them alive, and then add or remove compounds that decide the cell’s outcome in an attempt to discover when and how the compounds act in a healthily developing organism.
Using this technique, Ulrich and biologist Talita Glaser found evidence that embryonic cells with genetic alterations typical of Huntington’s do not always go through the maturation stages that generate GABAergic neurons. The results, presented in the journal Molecular Psychiatry on April 29, reinforce a recent idea in neurology: the protein made by altered versions of the huntingtin gene impair brain development from birth, not just in adulthood as was previously thought. “These findings have significant consequences,” says Glaser, who is currently doing a postdoctoral fellowship at Ulrich’s laboratory. “If a treatment is found to slow the progression of the disease, it will probably only produce the desired effect if it is administered very early in life.” Current therapies are only capable of relieving the symptoms.
“This study is important because it suggests that the formation of these neurons is already altered in the embryo, and proposes the mechanism by which this occurs,” says neurologist Raphael Machado de Castilhos, from the Hospital de Clínicas in Porto Alegre (HCPA), linked to the Federal University of Rio Grande do Sul (UFRGS). Castilhos and his colleagues treat people with Huntington’s, and estimate that the disease may be less frequent in Rio Grande do Sul than in Europe—there is no data on the rest of Brazil. “I am not surprised that the production of these neurons may be different in individuals with the disease,” he says.
Neurons begin to form in humans in around the fourth week of gestation, when the embryo is just a few millimeters long. To simulate the phenomena that occur in this phase, Glaser isolated rodent embryo stem cells capable of originating different tissues and stimulated them to multiply. As they proliferate in the lab, they form a kind of miniature embryo and stop at the neural precursor stage, when they still maintain the ability to divide, but can only generate neurons and other central nervous system cells (astrocytes, oligodendrocytes, and microglia). The scientist then induced the neural precursors to transform into neurons and recorded the results.
The neural precursors only became neurons when calcium ion concentrations within them increased in specific episodes. Oscillations of a very particular profile were needed to transform the neural precursors into GABAergic neurons: sequences of four to five peaks that were repeated about 30 times in an hour. At each peak, the ion level was tripled for 15 seconds. This oscillation pattern activated the ASCL1 gene for a certain period of time, which in turn triggered other genes that determine the cell’s fate. “The pattern directed stem cells to become GABAergic neurons,” says Ulrich.
How these calcium oscillations are controlled remains to be discovered. The ion exists in concentrations thousands of times higher outside the cell than inside, and it can only increase in the intracellular medium thanks to proteins that open the cell’s internal reservoirs or create channels in the membrane. Among the hundreds of molecules that can perform these functions, the USP team found that the actions of two—VGCC-L from the first group and P2Y2 from the second—were particularly important to the formation of GABAergic neurons.
Glaser and Ulrich then decided to investigate how these neurons are produced during embryonic development. Using patient cells treated to behave like embryos, the biologists stimulated them to transform into neurons. It did not work. There were no calcium spikes and no GABAergic neurons were created. “The altered form of huntingtin somehow disrupts the functioning of the P2Y2 receptor,” says the researcher.
“Knowledge of this mechanism can be used to select drugs with the potential to slow down the evolution of Huntington’s disease,” says Portuguese neurologist Rodrigo Cunha of the University of Coimbra, a specialist in neurodegenerative diseases. “It is not a cure or a total understanding of the disease, but it is a step toward it.”
Purine and kinin receptors as targets for study and therapeutic intervention in neurological diseases (no. 18/07366-4); Grant Mechanism Thematic Project; Principal Investigator Alexander Henning Ulrich (USP); Investment R$5,250,547.27.
GLASER, T. et al. ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington’s disease: A novel approach for cell clock research. Molecular Psychiatry. Apr. 29, 2020.