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Researchers Outline Mechanism of Three Rare Lysosomal Diseases

ARTICLE IN BRIEF

Researchers have uncovered the molecular underpinnings of three neurodegenerative diseases, all caused by loss of function of the same enzyme, FIG4.

WASHINGTON—In a new study one expert called “elegant” and “beautifully done,” researchers have worked out in detail the molecular underpinnings of three neurodegenerative diseases, including one form of Charcot-Marie-Tooth disease (CMT), all caused by loss of function of the same enzyme, FIG4.

The study, presented in a platform talk at the AAN Annual Meeting here in April and published in the April 29 issue of the Journal of Neuroscience, shows that mutations in the FIG4 gene set in motion a series of events that ultimately prevent cleavage of budding lysosomes, and that supplying a synthetic replacement for the normal product of the FIG4 enzyme may be therapeutic.

FIG4 is a phosphatase enzyme whose target is the phospholipid phosphatidylinositol-3,5-bisphosphate (PI3,5P2). PI3,5P2 is found on the membranes of lysosomes, cellular organelles that promote recycling of damaged organelles and other cellular debris.

Figure

DR. MICHAEL E. SHY said regulation of PI3,5P2 has been suspected to be the cause of the three FIG4 diseases, “but Dr. Li has taken this further,” carefully working out the multiple steps in the mechanism. “This is very elegant, beautifully done work,” he said.

Autosomal recessive mutations in the FIG4 gene cause CMT type 4J, Yunis-Varon syndrome with mental retardation and cleidocranial dysplasia, and seizures with cerebral polymicrogyria. All three disorders are characterized by abnormally large lysosomes, but unlike in lysosomal storage disorders such as Tay-Sachs disease, there is no defect in the enzymatic degradation of the lysosome's contents.

“FIG4 deficiency affects a different aspect of lysosomal function, namely membrane trafficking,” said senior study author Jun Li, MD, PhD, an associate professor of neurology at Vanderbilt University School of Medicine in Nashville, TN. Dr. Li's experiments showed that the loss of FIG4 explained the overly large lysosomes through a complex mechanism that ultimately prevents fission of the growing lysosome.

“The lysosome is a dynamic organelle,” fusing with other organelles called endosomes and dividing to counteract its natural growth from engulfing cellular contents, Dr. Li explained. Lysosome fusion and fission are highly regulated to maintain the proper distribution of lysosome sizes. While fusion is a well-recognized and largely understood process, fission is much less so.

“We hypothesized that in the absence of FIG4, and with a deficiency of its target PI3,5P2, we would have normal fusion but abnormal fission,” leading to lysosomal growth, Dr. Li said.

STUDY METHODOLOGY

To test this hypothesis, Dr. Li first developed an automated microscopy system that enabled him to quantify the size of thousands of lysosomes per minute. With first author Jianlong Zou, PhD, now at Sun-Yat Sen University in Guangzhou, China, Dr. Li first examined the rate of lysosomal fusion and fission in fibroblasts. By artificially fragmenting lysosomes and then tracking the growth in size over time, he showed that fusion was normal in homozygous FIG4 mutant cells. Next, he artificially swelled lysosomes and tracked their fission over time. Over the next 15 hours, normal lysosomal size distribution was restored by 83 percent in wild-type fibroblasts but by only 37 percent in mutant cells. “Therefore, lysosome fission is defective, but fusion is OK,” Dr. Li said.

Figure

DR. JUN LI said his experiments showed that mutations in the FIG4 gene set in motion a series of events that ultimately prevent cleavage of budding lysosomes, and that supplying a synthetic replacement for the normal product of the FIG4 enzyme may be therapeutic.

The next piece of the puzzle fell into place when Dr. Li considered that PI3,5P2 is a ligand for a lysosomal calcium channel called TRPML1. This suggested that the FIG4-induced reduction in PI3,5P2 might inhibit the channel's ability to move calcium out of the lysosome. Indeed, he found that lysosomes in FIG4-deficient cultured Schwann cells had very high levels of calcium, and that this could be normalized by replacing the absent PI3,5P2 with a synthetic ligand of the channel, called ML-SA1. This indicated that the sequestration of calcium in the lysosome could be a critical step in disease pathogenesis.

Dr. Li didn't yet know what the effect of calcium sequestration might be, but he did know that a calcium-sensitive protein called dynamin plays an important role in endoplasmic reticulum fission, acting as molecular scissors to snip off budding vesicles. If dynamin also promoted lysosome fission, and if trapping calcium in the lysosome prevented dynamin from doing its job, that would lead to the abnormally large lysosomes seen in FIG4 mutants.

To test whether dynamin dysregulation was involved in FIG4 mutants, he measured the amount of dynamin in FIG4-deficient cells. Not only was there less than the normal level of dynamin present, but the level did not rise in response to artificial induction of lysosome swelling, when the demand for lysosomal fission is high. While the precise mechanism is still under investigation, Dr. Li said, the low level of dynamin in FIG4 mutants suggests that calcium induces transcription of the dynamin gene, rather than just activating pre-existing protein.

In sum, Dr. Li explained, the loss of the FIG4 enzyme means that there is too little PI3,5P2 to open the TRPML1 calcium channels at the lysosome. With the channels closed, calcium can't escape in response to lysosomal swelling, and thus can't trigger the production of dynamin. Without dynamin, lysosome buds can't be snipped off, and the outsized lysosome continues to grow within the cell. How lysosomal overgrowth causes neuronal death or Schwann cell dysfunction is still unknown, and will be the focus of future research, he said.

“Based on this mechanism, if you can reactivate the TRPML1 channels, the lysosome should become smaller,” Dr. Li said, and that is what he showed with ML-SA1, which takes the place of PI3,5P2. In fibroblasts, treatment with ML-SA1 increased dynamin levels and reduced the number of large lysosomes in a dose-dependent manner. In mouse spinal cord preparations, treatment also reduced the percentage of neurons with overly large lysosomes from 45 percent to 10 percent.

Work in living mice is hampered by the rapid mortality of the mutation, Dr. Li noted, but he is now exploring targeting the mutation to Schwann cells or neurons. Combined with the present results, these studies may set the stage for developing a therapy for these rare diseases based on restoring calcium efflux from the lysosome.

It is not clear how a single mutation can cause three distinct neurologic diseases, but Dr. Li hopes to find answers now that the mechanism can be studied in more detail. And while the three syndromes caused by FIG4 mutation are all relatively rare, more cases are likely to emerge, he said. “Because people did not realize there is a such a wide spectrum of the phenotype, I would not be surprised in the next couple of years to find the mutation in subsets of other diseases,” including perhaps dementia.

EXPERT COMMENTARY

“A common theme in many of the genetic peripheral neuropathies is a defect in endosomal trafficking. This is a very nice example of that,” said Michael E. Shy, MD, a professor of neurology and pediatrics at the University of Iowa Carver School of Medicine and director of the university's Charcot-Marie-Tooth Clinic.

Regulation of PI3,5P2 has been suspected to be the cause of the three FIG4 diseases, Dr. Shy said, “but Dr. Li has taken this further,” carefully working out the multiple steps in the mechanism. “This is very elegant, beautifully done work.”

As for therapy, he said, “It makes sense to look at the role of calcium efflux as a treatment strategy in this rare type of CMT. Whether or not it would be effective in other types of CMT or other types of lysosomal storage diseases, it is definitely intriguing to go forward for these three diseases.”

LINK UP TO MORE INFORMATION:

•. Zou J, Hu B, Arpag S, Yan Q, Hamilton A, Zeng YS, Vanoye CG, Li J. Reactivation of lysosomal Ca2+ efflux rescues abnormal lysosomal storage in FIG4-deficient cells http://www.jneurosci.org/content/35/17/6801.short. J Neurosci 2015;35(17):6801–6812