This document describes a study investigating changes in acid sphingomyelinase (ASM) activity levels in Niemann-Pick disease (NPD) and the effects of cyclodextrin (CD) treatment. The study aims to determine if reduced ASM activity in human NPD cells is due to decreased levels of SMPD1 mRNA, which encodes ASM, and if CD treatment alters ASM activity in hamster cells by changing SMPD1 mRNA levels. Primers were designed and tested to quantify SMPD1 mRNA levels in CD-treated hamster cells and human wildtype and NPD cells using quantitative RT-PCR. Initial results showed the primers perform efficiently between 59.9-

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Niemann-Pick is a group of genetic disorders
causing lysosomal disorders due to mutations in
three genes: SMPD1, NPC1 and NPC2. Lack of acid
sphingomyelinase, the SMPD1 gene product, which
causes accumulation of sphingomlyelin in cells.
Sphingomyelin Ceramide + Phosphorylcholine
NPC1 and NPC2 gene products are responsible for
the transport of cholesterol from the lysosome to
the plasma membrane. Mutations in either of
these genes causes accumulation of cholesterol in
lysosome, resulting in an autosomal recessive
disease; Niemann-Pick Type C (NPC). Despite
having a wild type copy of SMPD1, acid
sphingomyelinase (aSMase) activity has been
observed to be reduced in Type C. Reduced
aSMase is also observed when Chinese Hamster
Ovarian (CHO) cells are treated with cyclodextrin
(CD). Currently, CD can be used to treat
individuals with NPC by removing cholesterol from
lysosomes. CDs are cyclic molecules derived from
starch. Beta (β) cyclodextrin treated cells are
used in current study.
Reduced aSMase activity could result via SMPD1
gene regulation. Previous studies showed no
difference in SMPD1 splicing between CD treated
and control CHO cells (unpublished, Chamberlain,
et al.). SMPD1 mRNA is alternatively spliced, but
no condition-specific isoforms were found.
Another level of gene regulation at which this
reduction in activity might be obtained is by
altering the steady state level of SMPD1 mRNA
through transcriptional downregulation or
enhanced mRNA decay.
INTRODUCTION
Niemann-Pick disease is a group of genetic disorders due to
mutations in three genes. Type A and B are caused by mutations
SMPD1, while type C and D are caused by mutations in NPC1 and
NPC2. SMPD1 encodes a protein known as acid sphingomyelinase
(ASM) that catalyzes sphingomyelin into ceramide and also
influences cholesterol levels. Human Niemann-Pick type C cells have
been shown to have a reduced level of ASM activity compared to
wildtype cells. Currently, the drug cyclodextrin (CD) is used to treat
NP disease. The current study has two goals: 1) To determine if
changes in the SMPD1 mRNA steady–state level in human NPC cells
may be responsible for the reduction in ASM activity, and 2) to
determine if cyclodextrin treatment is able to alter the ASM activity
level by altering the steady-state level of SMPD1 mRNA in wildtype
hamster cells. To test both of these hypotheses, SMPD1 mRNA levels
are being quantified by quantitative reverse transcription-
polymerase chain reaction (qRT-PCR) in human wildtype and NP cells
and in hamster cells treated with CD or a control. First, primers for
hamster SMPD1 and two hamster reference genes were designed.
Next, end point gradient RT-PCR was performed on control RNAs
isolated from hamster cells to identify the appropriate annealing
temperature and the presence of a single product. Results show
that the appropriate annealing temperature range for all three
genes is from 59.9-63°C and also confirm the presence of a single
product at these temperatures. The next steps will involve designing
and testing primers for the human cell experiments and performing
real time RT-PCR on RNA from hamster cells treated with and
without CD treatment and from wildtype and NP mutant human
cells.
ABSTRACT
Quality Check of Primers and Determination
of Annealing Temperature
DISCUSSION
MTSU Biology Department, Molecular Biosciences Program, MSPS Program
Snehal Sant, Matthew Fuller, Nicholas Chamberlain, and Rebecca Seipelt-Thiemann
Investigating Acid Sphingomyelin Activity Changes in
Niemann-Pick Disease and Treatment
REFERENCES
Aqul, A., Liu, B., Ramirez, C. M., Pieper, A. A., Estill, S. J., Burns, D.
K., ... & Dietschy, J. M. (2011). Unesterified cholesterol
accumulation in late endosomes/lysosomes causes
neurodegeneration and is prevented by driving cholesterol export
from this compartment. The Journal of Neuroscience, 31(25),
9404-9413.
Chen, F. W., Li, C., & Ioannou, Y. A. (2010). Cyclodextrin induces
calcium-dependent lysosomal exocytosis. PLoS One, 5(11),
e15054.
Nakashima, M., Kudoh, T., Sukegawa, K., Maruyama, K., & Orii, T.
(1986). Metabolism of Sphingomyelin in Cultured Skin Fibroblasts
from Patients with Different Types of Niernann-Pick Disease. The
Tohoku journal of experimental medicine, 148(4), 365-371.
Vanier, M. T. (2010). Niemann-Pick disease type C. Orphanet
journal of rare diseases, 5(1), 1.
MATERIALS AND METHODOLOGY
Next Step: Quantitation of SMPD1 mRNA levels in CHO +/- CD treated and human NP
and wildtype cells
FURTHER WORK
• Category of samples :
- CHO-K1 NPC cells treated with CD : Experimental
- CHO-K1 NPC cells not treated with CD : Control
• Primers :
- Cg_SMPD : Amplify SMPD1 gene
- Cg_Eif3i : Amplify Eif3i gene site (Reference gene)
- Cg_Hirip : Amplify Hirip gene site (Reference gene)
• The following protocol was used to check the end point
annealing temperatures of the primers and presence of a
single product:
1) Reverse Transcription Polymerase Chain Reaction (RT-PCR):
- RT Phusion® (Thermo Scientific)
- cDNA synthesis from mRNA transcripts in control sample
2) Gradient Polymerase Chain Reaction (Gradient PCR):
- Phusion gradient protocol
- To determine annealing temperature for primers
- PCR for a sample carried out at different temperatures
ranging from 41.8°C-63°C
3) Agarose Gel Electrophoresis :
- To run PCR products on gel and visualize PCR results
- 1.5% agarose gel
- Run Eletrophoresis at 100V for 45minutes
- Visualized using UVSMPD1
RESULTS
Figure 1: Gradient Endpoint RT-PCR for SMPD1, Eif3i and Hirip
using primers SMPD1_ASIII, Cg_Eif3i and Cg_Hirip
respectively. Annealing temperatures were (low) 41.8C
44.8C, 49.3C, 55.2C, 59.9C, 63.0 C (high).
SMPD1Eif3i
Hirip
Annealing
Temperature
Gel electrophoresis results for end-point gradient
PCR confirm that primers for all three gene sites
(Cg_SMPD1, Cg_Eif3i and Cg_Hirip) are in working
condition at the given temperatures. Higher
temperatures (59.9-63.0°C) were observed to have
less variance in terms of band patterns compared to
the lower annealing temperatures, which suggests
that the primers perform with the highest efficiency
at these higher temperatures by binding specifically
to the gene site of interest.
Further steps in the project include designing and
testing primers for human NPC cells and comparing
expression of SMPD1 by performing qRT-PCR on CHO
cells treated and non-treated with CD, as well as
wildtype and NP human cells. The work will also
consist of studying expression of SMPD1 expression
in wildtype and mutant NPC human fibroblast cells.
Figure 3.
2-Hydroxypropyl-
β-cyclodextrin
(Structure
courtesy of
abcam®)
Reverse Transcription Polymerase Chain Reaction
(RT-PCR):
- RT/PCR reactions using the above samples will be
performed using BioRad iScript SYBR green system
- First, primer efficiency experiments will be
performed using serial dilutions of template
samples for each primer set
- Next, qRT-PCR reactions will be performed in
triplicate on 3 replicates of each experimental type
to obtain average CT values for each
treatment/cell type.
- Changes in gene expression will be calculated
using both the DD CT and the standard curve
methods.
- Some example data is shown in Figure 3.
Figure 2. Example qRT-PCR graphed results for mutant and normal cells
for SMPD1, LAMP1, LAMP2, and GLB1.
Cell Gene Ct Mean* St dev SEM Change 2|x|= Abundance**
NPC1 GLB1 25.177 0.285 0.117 control
WT GLB1 26.568 0.754 0.308
NPC1 LAMP1 24.015 0.215 0.088 - 0.72 2.14 WT is 2.1x
moreWT LAMP1 24.697 0.129 0.053
NPC1 LAMP2 27.583 0.348 0.142 - 1.11 1.65 WT is 1.7x
moreWT LAMP2 28.253 0.449 0.183
NPC1 SMPD1 26.007 0.197 0.081 - 1.30 2.47 WT is 2.5x
moreWT SMPD1 26.488 0.222 0.091
Example Table. Relative Abundance of Steady
State mRNA Levels
*mean of 6 samples
** X is calculated by subtracting the mean GLB1 value
from either WT or NPC and taking the difference in the
average cycle number of between WT and NPC for each
gene. This value is then used as the exponent to
determine the relative abundance.
Figure 4. Human SMPDI Protein Structure Diagram .
Diagram includes protein domains and modification sites, as
well as predicted regions of disorder and homology to other
species (UniProtKB).
MOBI