Last week I attended the 2016 Mucolipidosis Type IV (MLIV) conference in Atlanta. It was organized and hosted by the MLIV Foundation. The attendees were about 50/50 MLIV scientists and MLIV families/patients. This is a small and very tight knit community. The MLIV scientists are highly collaborative, and most of the talks included unpublished data. I was struck by their openness to sharing new data, ideas, and reagents. Some of this openness is probably motivated by the wonderful folks at the MLIV Foundation, and their mission to find a therapy. Interactions between scientists and families likely also promotes a focus on therapy research. Meeting patients and their families makes the cause personal — the scientists had a strong desire to carry out research that could have a direct impact. In fact, there were 5 talks presented on exploring therapeutic approaches with small molecules, gene therapy, and bone marrow transplant. According to Rebecca Oberman, the foundation’s executive director, there were no therapeutic approaches presented 2 years ago. Everyone agreed that it was really Rebecca’s hard work that drove this rapid focus and progress on treatments. She’s a wonderful advocate for the MLIV families, rare disease research in general, and works tirelessly for the cause. I also admired her cat wrangling skills at the meeting to get people where they needed to be…it’s not easy to get a group of scientists away from a plate of cookies.
The following is a background on MLIV and a discussion of how Drosophila and C. elegans could be used to discover small molecule therapeutics for the disease.
Mucolipidosis IV (MLIV) is one of the ~50 lysosomal storage diseases. The patients present by age 1 year with psychomotor delay and visual impairment. An MRI can reveal a thin corpus callosum, blood tests will show high levels of circulating gastrin due to achlorydia (absence of hydrochloric acid in the stomach’s gastric secretions), and their cells have lysosome-derived vacuoles with autofluroescent material called lipofuscin. Patients can exhibit spasticity, ataxia, hypotonia, and an inability to walk independently. They often have corneal clouding, retinal degeneration, atrophy of the optic nerve, and can become blind by age 15. Their achlorydia can cause an iron deficiency and anemia because of poor dietary iron absorption. Most patients tend to reach the cognitive, speech, and motor skill levels corresponding to age 15 months, and then the condition stabilizes — or progresses very slowly –throughout the first 3 decades of life. A 5 patient study over 3 years has found a trend toward progressive cerebellar atrophy and gradual reduction in white matter. Patients with the severe form (95%) of MLIV survive to adulthood, though have a shortened lifespan. Treatments are symptomatic, with physical therapy to aid in spasticity and ataxia.
Approximately 100 patients have been diagnosed with MLIV. That’s likely an underestimate because the disease is often mistaken for cerebral palsy. Diagnosis is further complicated in milder variants, which can present with one or more of the aforementioned conditions. MLIV is included with Gaucher, Tay-Sachs, and Niemann-Pick A and B as commonly occurring lysosomal storage diseases in the Ashkenazi Jewish population. About 75% of described patients with MLIV are of this ancestry.
The gene mutated in MLIV is MCOLN1/TRPML1 (hereafter TRPML1). Trpml1 is a six transmembrane domain bearing protein that behaves as a cationic channel in the late endosomes/lysosomes (LE/L) (Figure 1). A pore for cation passage is between the 5th and 6th transmembrane domains. To date, there’s evidence for passage and/or regulation of Ca2+, K+, Na+, H+, Zn2+, Fe2+, and Mn2+ ions in the LE/L.
Figure 1. The Trpml1 cationic channel. The pore for passage of cations is between transmembrane domains 5 and 6. N and C terminal tails reside within the cytoplasm. From Ehud Goldin, Susan Slaugenhaupt, Janine Smith and Raphael Schiffmann’s chapter on MLIV in OMMBID.
Trpml1 functions to ensure the formation of lysosomes from hybrid LE/L organelles (Figure 2). Trpml1 also regulates lysosomal pH, by being a leak channel to H+ ions. In trpml1 mutant cells, lysosome pH is reduced, lysosomal lipases don’t function properly, and lipid hydrolysis is reduced. Trpml1 also regulates autophagy, a lysosome-dependent process that is utilized by cells to breakdown macromolecules and organelles to repurpose their constituents during times of starvation. In trpml1 mutant cells, autophagy substrates accumulate. The absence of these cellular processes in trpml1 mutant cells almost certainly explains the lysosomal storage phenotypes.
Figure 2. Trpml1 promotes formation of lysosomes from LE/L hybrid organelles. Primary lysosomes (LYS) bud from hybrid organelles (HO), which have late endosomal (LE) and lysosomal traits. In trpml1 mutant cells, scission of primary lysosomes from the HO does not occur. This leads to the generation of large vacuoles (LV) in trpml1 mutant cells. From this review on TRP channels where it was adopted from this fine work in C. elegans.
The cup-5 mutant worm phenocopies MLIV pathology
Cup-5 (“coelomocyte uptake defective-5”) C. elegans mutants were discovered in a genetic screen for genes that regulate endocytosis in coelomocytes. Cup-5 is the only worm trpml family member (humans have 3: trpml1, 2, and 3). Like MLIV patient-derived cells, cup-5 mutant coelomocytes have large intracellular vacuoles with LE/L traits, and cup-5 mutant embryos are loaded with acidic organelles (Figure 3). In addition, their coelomocytes accumulate hybrid organelles that share LE and L traits. Johnny Fares and his lab at the University of Arizona showed that Cup-5 promotes formation of lysosomes from these hybrid organelles. In Cup-5’s absence, the hybrid organelles grow in size (Figure 2). They then showed that this model holds true for the function of Trpml1 in human cells. Like patient derived cells, the MLIV worms are defective in autophagy — their accumulation of the autophagosome marker LC3 possibly indicates a block in the formation of autolysosomes.
Figure 3. Cup-5 mutant embryos accumulate acidic organelles. Embryos were stained with lysotracker red, a common stain to visualize LE/L.
The trpml mutant fly phenocopies MLIV pathology
Flies also have only one member of the Trpml family, and the gene was accordingly named trpml. In 2008, mutations were introduced into trpml by Kartik Venkatachalam in Craig Montell’s lab at Johns Hopkins. (Using flies, Craig Montell cloned the first TRP channel in 1989). Trpml mutant flies have pathologies like those in MLIV. The LE/L compartment accumulates lipofuscin, which are aggregates of lipids, proteins, and heavy metals that autofluoresce. Like MLIV patients, Trpml flies exhibit motor defects (Figure 4) and neurodegeneration. Cells of trpml flies are also defective in autophagy, with a failure in amphisome fusion to lysosomes.
Figure 4. Trpml mutant flies are defective in the climbing assay. Wild type flies (left) climb to the top of the tubes, as expected. Trpml flies (right) linger at the bottom, consistent with CNS and motor defects. This is our video, but this was originally demonstrated by Kartik Venkatachalam in 2008.
Suitability of MLIV to high throughput screening in model organisms at Perlstein Lab
At Perlstein Lab, we generate model organisms (yeast, worms, flies, and zebrafish) that carry mutations in genes mutated in human disease. Then, we screen for small molecules that can reverse phenotypes of these model organisms. Small molecules that can reverse the cellular defects of one or more model organism have the potential to be the basis as a disease therapy. From a practical standpoint, we move forward with the phenotypes that are most amenable to high throughput screening. For instance, in our NPC screen, the flies and worms mutated for npc1 were developmentally delayed. We sought small molecules that could restore size and developmental rate to normal. Cup-5 mutant worms have been documented to have embryonic lethality and the embryonic cells are filled with acidic vesicles (Figure 3). Reversal of one of these two phenotypes could potentially serve as the readout of a high throughput genetic screen. Like with any new model system, we’d need to establish new methods and genetic strains, and prove that these phenotypes are robust enough to limit the number of false positives/negatives. A nice feature of the cup-5 mutants is that methylpyruvate, a small molecule that is a TCA substrate, can provide partial rescue of embryonic lethality. Methylpyruvate may therefore serve as a positive control to ensure that assays are performing properly. It also provides peace of mind that other small molecules that can rescue cup-5 mutants can be discovered in a large scale screen.
The Drosophila model of MLIV has nearly 100% lethality at the pupal stage. Pupae are inactive and require autophagy to provide nutrients. The pupal lethality of trpml mutants is likely due to defective autophagy. A high protein diet during the larval stage can partially rescue the pupal lethality. We believe that a high throughput screening assay can be developed to find small molecules that rescue the pupal lethality. Below is a 96 well plate where larvae were cultured to adulthood. Six wells have adults that eclosed from the pupal cases (Figure 5).
Figure 5. Flies cultured to adulthood in a 96 well plate.
The worm and the fly models of MLIV also provide the other aforementioned phenotypes that are reminiscent of those occurring in patients and patient cells. Those phenotypes (defective autophagy, numerous vacuoles that are LE/L in nature, neurodegeneration) can be used in secondary assays to validate our hits. We can also test whether hits will rescue phenotypes found in patient derived cells. Lastly, the MLIV mouse model nicely recapitulates features of MLIV. Trpml1 mutant mouse neurons and other cells have inclusions typical of MLIV. The mice have high levels of circulating gastrin, consistent with achlorydia, and exhibit retinal degeneration. The trpml1 mutant mouse would serve as a pre-clinical validation for any small molecules we discover. Like NPC, the case for using model organisms to discover small molecules with therapeutic potential for MLIV is strong.
These are great reviews on MLIV that were drawn from in the background section: