Niemann-Pick C disease is an inherited autosomal recessive disorder typified by a cholesterol accumulation defect. This manifests as a progressive and eventually fatal neurodegenerative disease. The disease arises due to mutations in the Niemann-Pick C (NPC) gene, which normally encodes a ~1300 amino acid protein consisting of specific functional domains. This gene has been found to be evolutionarily conserved in most organisms.
This conservation of the gene, combined with the severe deficits associated with its malfunction led researchers to hypothesize that its loss in other organisms too would result in abnormal development.
In the year 2000, Mary Sym and colleagues at Exelixis studied a nematode model, called Caenorhabditis elegans, lacking the orthologous NPC gene. Before I move ahead on this topic, let me introduce to you the humble soil-dwelling nematode, Caenorhabditis elegans or C. elegans:
Although its name is quite a mouthful, this transparent nematode (or worm) is only 1mm long and thrives on bacteria. Don’t let its diminutive size fool you. C. elegans has been responsible for some seminal scientific discoveries: programmed cell death and RNA interference were first demonstrated in this model organism. Both these findings led to Nobel Prizes for their discoverers. C. elegans also made the news when nematode specimens were transported into space and were later found surprisingly to have survived the Columbia disaster in early 2003.
The transparent 1mm body of the C. elegans is particularly suited to the visualization and study of cells, neuronal circuits and organelles with the use of fluorescent reporters (e.g. green fluorescent protein, mcherry or red fluorescent protein, etc). Further, the life cycle, number of cells and developmental fate of every cell in the worm has been deciphered, giving a whole new meaning to the phrase, ‘I know you inside out:’
A lot of what we know about basic biological processes in larger animals, from mice to humans, has been demonstrated in C. elegans as well. This is because there are numerous similarities in the genomes of C. elegans and larger animals. Nearly 35% of C. elegans genes have human homologs. Surprisingly, human genes have been shown to substitute for C. elegans genes when introduced into the C. elegans genome. The converse has also been demonstrated, i.e, C. elegans genes have similar functionality to their homologous human genes.
This evolutionary conservation thus enables the study of critical human genes in a simple model like C. elegans, which brings us back to NPC. So, in the year 2000, Mary Sym et al, discovered two genes in the C. elegans genome with remarkable similarity to human NPC gene. They created nematode models bearing deletions in the NPC-related gene 1, or ncr-1 and NPC-related gene 2, or ncr-2. In humans, the lack of NPC results in a cholesterol trafficking deficit with widespread accumulation or trapping of cholesterol in lysosomes. In C. elegans bearing mutations in ncr-1 and ncr-2, similar cholesterol abnormalities were observed. When grown in an environment deprived of cholesterol, worms were unable to survive long and succumbed to death in premature larval stages. Furthermore, the ability of the surviving worms to reproduce was affected, reflected as reduced numbers of progeny. These defects were pronounced in worms that bore deletions in ncr-1. Deletions in the ncr-2 gene did not produce as dramatic an effect.
However, when both ncr-1 and ncr-2 deletions were present in the same animal, the double mutant displayed a striking developmental defect — inappropriate dauer formation. The figure below displays the dauer stage of worms:
The dauer stage is a growth-arrested minimal life stage that worms enter when conditions for growth are compromised. Normally, wildtype worms enter the dauer stage when presented with environmental hardships such as starvation, infection, and over-crowding. Dauers exit from this dormant stage when conditions become favorable again, that is when food is presented again or infection cleared. The double mutants, however, showed greater proclivity to enter the dauer stage compared to normal worms.
A follow-up study in 2004 by Jim Thomas’ group showed that ncr-1 and ncr-2 functioned in a hormonal branch of dauer development upstream of the critical genes daf-9 and daf-12. While these genes have known human orthologs, it is unclear what the equivalent of this hormonal pathway in humans is. By fusing the ncr-1 promoter region to fluorescent reporter genes, the Thomas group showed that ncr-1 gene was expressed in numerous cell types, all rich in cholesterol, illustrative of a role in cholesterol trafficking. Importantly, the paper showed the presence of the ncr-1 genes in a defined population of cells called the XXX cells. The XXX cells in C. elegans function as the gateway for entry/exit to dauer. Most genes critical for this process are also encoded in these cells. A similar finding has been reported in a Drosophila model of NPC as well. You can read about the NPC fly model here.
The fact that cholesterol accumulates in lysosomes of NPC models combined with findings that cholesterol deficiency also exacerbates disease pathology does present a conundrum of sorts. However, both findings can be likened to two sides of a coin — perhaps it is a cholesterol trafficking defect that leads to its accumulation and trapping in the lysosomes?
Consistent with previously published reports, in our hands too, both nematode and Drosophila NPC models show rescue when supplemented with cholesterol and 7-dehydrocholesterol respectively. Could the answer to this devastating disease then lie in a simple supplementation of good ol’ fat? Sounds too good to be true, doesn’t it? Cholesterol does play an important role in numerous metabolic pathways, so perhaps the answer might lie in a molecule with a similar structure?
At Perlstein Lab, we’re utilizing a parallel model organism drug screening platform to find answers to these questions. Working with Knudra Transgenics, we’ve generated additional mutant nematode models to replicate the spectrum of mutations associated with NPC in humans. We have validated that our NPC nematode models also display developmental delays when grown in an environment deprived of cholesterol, as shown here:
Given the evolutionarily conserved nature of this defect, we are now embarking on chemical library screens to determine if we can find a molecule that will rescue the NPC phenotype across our model organism platform. Along the drug screening route, we hope that our parallel model organism approach will enable us to discover and investigate the pathway or pathways that are critical to this disease. Stay tuned for further updates!
1) Sym M., Basson M., and Johnson C., (2000). A model for Niemann-Pick type-C disease in the nematode Caenorhabditis elegans. Curr Biol. (10). 527-530
2) Li J., Brown G., Aillon M., Lee S., and Thomas J., (2004). NCR-1 and NCR-2, the C.elegans homologs of the human Neimann Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathway. Development. 131(22):5741-5752
3) Hu P.J., Dauer (August 8, 2007), Wormbook, ed. The C.elegans Research Community, Wormbook, doi/10.1895/wormbook.1.144.1, http://wormbook.org