ACERULOPLASMINEMIA: A NEW POTENTIAL TREATMENT FROM UNISR
A relatively young disease (described for the first time in Japan in 1986) of which in 2015 there were only 71 cases worldwide: aceruloplasminemia is a pathology classified as “extremely rare” which, according to a study conducted on the Japanese population, affects one out of 2 million individuals. A new approach based on the replacement of the damaged or absent enzyme could lead to an improvement of neurological symptoms and a potentially more effective treatment of the pathology. To explain to us the study published in the prestigious journal EMBO Molecular Medicine is Dr. Alan Zanardi, a PhD in Molecular Medicine at the Vita-Salute San Raffaele University and currently a PostDoc Fellow at the Proteome Biochemistry Laboratory directed by Dr. Massimo Alessio, where the research was conducted.
CERULOPLASMIN AND ACERULOPLASMINEMIA
Aceruloplasminemia is a disease caused by the lack of ceruloplasmin, a protein produced by the liver and the central nervous system, which release it respectively into the blood and the cerebrospinal fluid, the liquid that permeates the central nervous system protecting it from any trauma. Normally cells contain iron, which is important for carrying out different intracellular processes and it is exchanged with the external environment to maintain physiological levels. In healthy subjects, when the intracellular iron is in excess in its reduced form (Fe2+), it is released into the extracellular space, where ceruloplasmin converts it to its oxidized form (Fe3+), allowing its recycling. In patients, ceruloplasmin is not produced or it loses its enzymatic activity: Fe2+ is no longer released from the cell and it accumulates inside it, causing toxicity and cell death. In patients suffering from aceruloplasminemia, iron accumulation occurs in central nervous system, liver and pancreas; the pathology begins around 30-40 years of age with diabetes and anemia (the iron “trapped” inside the cells is no longer available for the other cells), which towards the age of 50 are added neurological symptoms that are the consequence of progressive neurodegeneration.
THE INTUITION FOR THIS RESEARCH
Being implicated in neurodegenerative processes, lack of enzymatic activity of ceruloplasmin has been extensively studied in other neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease. Dr. Zanardi says: “The idea of this study arises from a previously published research: when injected into the peritoneum [the membrane that surrounds the abdominal organs and covers the walls of the abdomen, Editor’s Note] of aceruloplasminemic mice, i.e. without ceruloplasmin, this enzyme was able to reach the central nervous system”.
Current therapies are mainly based on iron chelation treatments, which consist of drugs that bind and eliminate excess iron. These are partially effective in controlling iron deposition at the systemic level, but they are ineffective in treating neurological symptoms, since the chelators are unable to enter the brain. “Following this research we were interested in seeing whether, after repeated administration, ceruloplasmin accumulated in the brain, it could maintain a stable level of presence, and especially if it was working”.
OUR RESEARCHERS’ STUDY
“Our study was performed on knock-out mice for ceruloplasmin, i.e. mice in which this protein is absent”, explains Dr. Zanardi. In human pathology, the iron accumulation causes loss of dopaminergic neurons in brain and Purkinje neurons in cerebellum, resulting in motor coordination impairment. “Before starting the treatment, we performed behavioral tests on mice, which reaffirmed – just as for human disease – the presence of motor deficits”. Every 5 days for 2 months the mice were treated with intraperitoneal administration of purified ceruloplasmin. “The very simple idea is to do an enzyme replacement therapy: we supply the missing protein”.
What were the results?
“Behavioral tests performed two months after the administration of ceruloplasmin have shown that the treated mice far improved their performance, recovering – at least partially – their motor function”. Like human disease, in aceruloplasminemic mice there is a loss of dopaminergic neurons in the brainstem and Purkinje neurons in the cerebellum: almost total survival of Purkinje neurons has been promoted following treatment, which may explain the partial motor recovery of treated mice. “Through biochemical analyzes we have also confirmed that this recovery is associated with an increase in the presence of ceruloplasmin and its ferroxidase activity, at the level of the nervous system”.
Despite the great homology, injecting a protein into a foreign organism could generate an immune reaction, however: “By evaluating the production of antibodies against the human protein, we observed that they are not neutralizing, i.e. they recognize the foreign protein, without inactivating it”.
He continues: “Histochemical analyzes on the brains of aceruloplasminemic mice have found a strong accumulation of iron in the area of the choroid plexus, the structure that produces and secretes the cerebrospinal fluid. The accumulation of iron in this area is justified for the choroid plexus cells are the ones that first pick up the iron from the bloodstream, transporting it inside the cerebrospinal fluid. In our work we have shown that the treatment with ceruloplasmin significantly reduces the accumulation of iron in these cells, mobilizing it from the choroid plexus of treated mice and making it available to other cells”.
There remains only some uncertainty about the way in which ceruloplasmin enters the brain. “Since in healthy mice ceruloplasmin does not have access to the brain, our hypothesis is that in sick mice it could enter due to a damage of the blood-liquor barrier. It could be a consequence of the disease: the mouse is without ceruloplasmin, which causes an accumulation of iron in the cells of the choroid plexus, which reflects in damage to the barrier impermeability”.
WHAT FUTURE PERSPECTIVES?
Dr. Zanardi finally reflects: “Given the rarity of the disease, it is very difficult to find pharmaceutical companies willing to invest in the development of an effective treatment. Waiting for a pharmaceutical product, the idea is to subject the patients to a fresh frozen plasma administration [a blood product obtained from the liquid portion of whole blood that contains all the coagulation factors, but without red and white blood cells, Editor’s Note]”. Most of the patients living in Italy are being treated at the San Gerardo Hospital in Monza, under the guidance of Prof. Alberto Piperno, among the authors of the study: “In collaboration with colleagues at the San Gerardo Hospital, we would like to select blood samples more enriched in ceruloplasmin and treat these patients, mimicking a temporary enzyme replacement therapy”.
In future plans there would be to treat patients in advance, to try to anticipate the onset of the neurological component of the disease: “We have already confirmed that the ceruloplasmin injected intraperitoneally reaches the brain: an idea could be to carry out a gene therapy directed to the liver, which produces ceruloplasmin in physiological conditions. The gene therapy would have two main advantages: the protein would be physiologically produced by the patient, who therefore should not inject himself with it, and it would hopefully solve the problem of cost, a non-marginal aspect when dealing with such rare diseases”.