Determining the effects of potential Prader-Willi Syndrome drug treatments

on PC1 gene expression in neuronal-2a cells

Gabriela Hubner
December 8th, 2016
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Abstract
Prader-Willi syndrome (PWS) is a genetic disorder caused by a loss of paternally
expressed genes in an imprinted region of chromosome 15q. Among the PWS phenotypes are
hyperphagic obesity, central hypogonadism and low growth hormone levels. The minimum
critical deletion region of PWS is comprised of the genes SNORD109A, SNORD116, and IPW.
Studies found that mice with only a Snord116 deletion display neuroendocrine features similar to
those of PWS, including defects in prohormone processing of proinsulin, proGHRH, and
proghrelin. The impairments in prohormone processing are likely due to a PC1 deficiency,
caused by low levels of Pcsk1 gene expression. This study used neuro-2a cells to test two
compounds, referred to here as compound X and Y, that could increase PC1 gene expression and
potentially treat PWS phenotypes. Neuro-2a cells were differentiated into neuron-like cells and
treated with the two different compounds. RNA was extracted from the cells and gene expression
was measured using a quantitative Polymerase Chain Reaction (qPCR). Preliminary qPCR
results were statistically insignificant but suggest that compound X did not have an effect on PC1
gene expression, whereas compound Y may have an effect.

Introduction
Prader-Willi syndrome (PWS) is the most common known cause of life-threatening
obesity, affecting approximately 1 in 25,000 live births (Smith, 2003). PWS results from a loss
of paternally-expressed genes at the chromosomal region 15q11.2q13 (Cassidy, Miller &
Driscoll, 2012). The major symptoms of PWS include hyperphagic obesity, hypogonadism, low
growth hormone, hyperghrelinemia, and hypoinsulinemia (Butler, Lee & Whitman, 2006). Many
studies identified PWS microdeletions and Burnett et. al determined the minimum critical
deletion region in PWS, meaning the gene region that, when deleted, is sufficient to cause all of
the major symptoms of PWS. This region contains three non-coding RNA genes, including
SNORD109A, SNORD116, and IPW (Bieth, 2014; Duker, 2010). None of the PWS mouse
models developed obesity, but mice in which the paternal copy of Snord116 was deleted
(Snord116p-/m+) displayed many of the neuroendocrine phenotypes of PWS, including
hyperphagia, low growth hormone levels, decreased body length, hypoinsulinemia, and
hyperghrelinemia (Bervini & Herzog, 2013; Ding, 2008).
Very little is known about the SNORD116 gene or mechanisms by which it influences
biological processes. Although the endocrine features of PWS have been well described, a
molecular mechanism linking these features to the genes deleted in the PWS minimum critical
deletion region has only recently been identified. Burnett, L.C., et al found that the major PWS
neuroendocrine phenotypes can be accounted for by reduced expression of the prohormone
processing enzyme, PC1 (PCSK1) (Burnett, 2016).
Proprotein convertase 1 (PC1) is an enzyme that in humans is encoded by the PCSK1
gene, which plays a role in processing prohormones into mature hormones (Genetics Home
Reference). PC1 is implicated in the conversion of POMC into -MSH, Proghrelin into mature
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ghrelin, proGHRH into growth hormone, ProGnRH into gonadotripin and Proinsulin into insulin
(Burnett, 2016). All of these prohormones and hormones are linked to the endocrine profile of
PWS, and therefore PC1 deficiency could account for the PWS endocrine phenotype. If the PC1
levels increased, the neuroendocrine profile of PWS could potentially be normalized. This
experiment tested two different compounds and measured Pcsk1 gene expression in comparison
to control group.

Methodology
Cell culture & differentiation
Mouse brain neuroblastoma cells (Neuro-2a, CCL-131) were obtained from the
American Type Culture Collection (ATCC, Manassas, VA) and frozen in liquid nitrogen. Cells
were thawed from stock and seeded at a density of 10/mm2 in cell media overnight. Media was
composed of Dulbeccos Modified Eagles Medium (DMEM), 10% Fetal Bovine Serum (FBS),
and 1% Pen Strep. Cells were then incubated at 37oC and fed daily with 10mL of new media
until they appeared confluent. In order to decrease cell density, cells were detached from plate
using trypsin, centrifuged at 0.8 relative centrifugal force (RCF) for 4 minutes and resuspended
in cell media. The cells and media were then transferred to the new plates, media was added, and
the cells were incubated again at 37oC.
To prepare for cell differentiation into neurons, new 6-well plates were coated
with a coating solution composed of sterile water and poly-d-lysine. Plates were incubated for 2
hours, rinsed with Phosphate-buffered saline (PBS) and dried. To begin differentiation, cells
were fed a differentiation media composed of Neurobasal medium, brain derived neurotrophic
factor (BDNF), vitamin C, and DAPT every 24 hours for 12 days. Once differentiated, cells were
imaged under a microscope to check for signs of mature neuronal growth.

Drug administration
After the 12 days of differentiation, cells were starved for 6 hours in order to
minimize exogenous insulin or growth factor signaling. Old media was removed from the plates
and new 1% fatty acid free BSA media was added. The next day cells were washed with PBS
and incubated with the drug at 37 C. Compound X was incubated for 6 hours, whereas
Compound Y for 24 hours. Cells were washed with PBS and removed from plates with lysing
media (QIAzol) and placed into 2mL tubes labeled 1-12. The tubes were then placed on ice to
inhibit RNAse.

qPCR
RNA was extracted from the N2a cells, and RNA concentrations were measured
using a Nanodrop spectrophotometer instrument. The extracted RNA was stored at -80 C
overnight. The RNA (1 g) was then converted into cDNA via reverse transcriptase (Roche Cat.
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No. 05081955001) and diluted in 150L of water. 2 L of the diluted cDNA were added to a
384 well plate along with 8 L of a mastermix solution, which is composed of water, forward
primer, reverse primer and SYBR green (Roche Cat. No. 04707516001). Primers target either the
-Actin gene (control) or Pcsk1 gene messenger RNA. The 384 well plate was placed in a
quantitative Polymerase Chain Reaction (qPCR) instrument (LightCycler 480) which serves to
quantify gene expression. Results were analyzed with delta delta Ct method.

Results
Data was obtained from N2a cells at day 12 of differentiation. The Pcsk1 gene expression
was measured against the housekeeping gene, -actin. The data points show Pcsk1 gene
expression of the drug-administered cells relative to the control (DMSO-only) cells. The results
of the Compound X test showed gene expression was still significantly low at baseline and had a
statistically-insignificant P value of 0.21 (Figure 1A). The results of the Compound Y test
showed gene expression levels significantly higher than that of -actin but had a statistical
significance of P=0.06 (Figure 1B). The qPCR data was inconclusive because it was found to be
insignificant.

Figure 1: Relative Gene expression levels of Pcsk1 gene expression for day 12 differentiated N2a
cells treated with Compound X (A) and Compound Y (B) when compared to the DMSO-only
group. Data points were calculated through qPCR and gene expression is measured against
-actin, a common gene normalizer. Standard error of the mean bars are represented.
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Discussion
The insignificance of the data prevents the drawing of any conclusions about the
importance of the results. qPCR results for both compounds were inconclusive, with P values
above the significant level of 0.05. Despite their insignificance, the results suggest that
Compound X can be ruled out as a drug treatment for PWS. Pcsk1 gene expression baseline
values were significantly low and very similar to the DMSO-only group, suggesting the drug had
no effect on gene expression. This could be due to the short incubation time of only 6 hours. If
this experiment were to be repeated, drug incubation would start during differentiation so as to
increase incubation time and potentially drug effect.
The qPCR results for Compound Y were also statistically insignificant, and certain
numbers did not fall in the expected range for this type of experiment. Due to its low statistical
significance, the experiment would have to be repeated in order for any conclusions to be drawn.
However, the results look more promising, with a clear trend and a larger margin compared to
the DMSO-only group.
It was hypothesized that the compounds would significantly increase Pcsk1 gene
expression. If this had been the case, the data points for both compounds would be closer
together and significantly higher than that of the DMSO-only group.
Experiments with Compound Y will be repeated with a longer drug treatment time. If
compound X or Y is seen to increase gene expression, this finding could lead to further testing in
other cell models and in animal models, which pending results of further in vitro and in vivo
testing may provide scientific justification for a clinical trial and could eventually become a
pharmaceutical treatment for patients with PWS.

Acknowledgements
This research was made possible by the oversight and instruction of Dr. Lisa Cole
Burnett and Dr. Rudolph Leibel, the help of the Columbia University Research Center staff and
the use of their facilities, and the direction and support of Ms. Erin Schmitz at the Packer
Collegiate Institute.

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