HONG KONG – Japanese researchers have determined for the first time the detailed atomic structure of a key molecule involved in intracellular transmission, which should contribute to development of better therapeutic approaches for neurodegenerative diseases such as Alzheimer's.

Determining the molecular structure of the inositol triphosphate receptor (IP3R) has long been considered a major goal in biomedical research, due to its strategic intracellular role in transmitting signals controlling multiple cell functions.

The work was conducted in Wako City, Saitama, at the RIKEN Brain Science Institute under the direction of Katsuhiko Mikoshiba, a professor and senior team leader of the Laboratory for Developmental Neurobiology, where the IP3R gene was first cloned in 1989.

Inositol 1,4,5-trisphosphate (IP3) is an intracellular signaling molecule that binds to the IP3 receptor (IP3R) to release calcium ions (Ca2+) from intracellular stores such as the endoplasmic reticulum (ER). These IP3R-embedded Ca2+ stores play pivotal parts in processes including neural communication, differentiation, plasticity and metabolism.

Of the three genes identified, the brain-dominant type 1 IP3R (IP3R1) was shown to be causative for degenerative diseases such as spinocerebellar ataxia and Gillespie syndrome, and to regulate the cellular processes implicated in the etiology of neurodegenerative disorders, including Alzheimer's disease.

"We identified three IP3R genes by cDNA cloning. Among these three isoforms, genetic analysis of cells from patients showed the IP3R1 gene was causative for spinocerebellar ataxia and Gillespie syndrome. Other evidence regarding the pathophysiology of degenerative diseases was obtained by analysis of cells from patients with Huntington's and Alzheimer's diseases," Mikoshiba told BioWorld Today.

"We found the IP3 receptor was involved directly with the apoptosis and autophagy known to be implicated in neurodegenerative diseases, including Alzheimer's," he said.

"In addition, in cells from patients with Huntington's disease, we showed that environmental stress resulted in neurodegeneration through IP3R and IP3R-associated binding proteins, in some cases associating with mitochondria, which also applies to other neurodegenerative diseases, including Alzheimer's," said study first author Kozo Hamada, a researcher in Mikoshiba's laboratory.

"Other groups have also demonstrated that IP3R directly associates with the presenilin that is known to be causative for familial Alzheimer's disease and to control the degeneration of neurons," Hamada noted.

"Moreover, using IP3R knockout mice mating with the amyotrophic lateral sclerosis [ALS] mouse models, we recently showed that IP3R is directly involved in senescence and ALS. IP3R is therefore highly correlated with neurodegenerative diseases, so better treatments might be possible if we could find a tool to manipulate IP3R function," said Mikoshiba.

Although the important roles of IP3R in normal and disease conditions are relatively well known, understanding how IP3 signals trigger opening of the Ca2+ channel was poor, the researchers reported in the April 18, 2017, early online edition of the Proceedings of the National Academy of Sciences.

The new IP3R1 crystal structure reveals details regarding its function. The IP3R1 molecule is 20 nm in diameter and contains an IP3 binding site and a Ca2+ channel pore. However, how IP3-binding opens the channel has been unknown.

X-ray crystallography of the large cytosolic domain of IP3R1 in the absence and presence of IP3 identified a long-range mechanism involving an IP3-dependent global movement of part of the receptor called the curvature alpha-helical domain, which forms a bridge between the cytosolic and channel domains.

Mutagenesis of that bridge using amino acid substitution revealed the essential role of a molecular leaflet structure that relays IP3 signals to the channel, which may help explain how long-range coupling from IP3 binding to the Ca2+ channel occurs, which will aid future rational design of drugs targeting the receptor, possibly allowing a more diverse range of therapies.

"The importance of this leaflet structure was previously unknown until we demonstrated the function and structure of the IP3R1. We can now search for molecules that associate with the leaflet structure to enhance or suppress IP3R1 activity. We would then be able to design more efficient drugs that could modify the function of IP3R more efficiently," said Mikoshiba.

Those new molecular structural findings may also clarify the roles played by IP3R in cellular senescence and tumor suppression linked to selective vulnerability of cancer cells.

"It is already known that IP3Rs including IP3R1 are involved in senescence. If IP3R and associated molecules are found to be involved in apoptosis, which is closely involved with tumor formation, molecular manipulation of IP3R function might modulate senescence or tumor formation," noted Mikoshiba.

Surprisingly, the study findings also clarify a role for IP3R in the function of pathogenic unicellular organisms like Trypanosoma cruzi, the parasite of Chagas disease, and T. brucei, which causes trypanosomiasis.

"Our initial idea was to investigate the structure and function of IP3R in unicellular animals, since multicellular organisms are complex [and] can sometimes be difficult to understand," Mikoshiba explained.

"We cloned IP3R of T. cruzi and discovered that IP3R integrity was essential for cellular survival. Although the amino acid sequence was not identical to that of mammalian IP3R, the leaflet sequence was nevertheless highly conserved, suggesting structural insights that may assist in drug discovery," he said.

"If future testing showed this sequence was indeed essential for T. cruzi survival, this would mean this leaflet structure was an important basic mechanism of cellular access," he added. "Drugs could then be designed to modulate its function specifically to the parasite, which would be an efficient treatment for trypanosomiasis."

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