Gene Expression analysis of Motorneurons in Spinal Muscular Atrophy

Published:

Paper: Neural Stem Cell Transplantation can ameliorate the phenotype of a

Mouse model of Spinal Muscular Atrophy

Abstract:

Spinal muscular atrophy is a type of motor neuron disorder. The motor neuron is responsible to affect the normal activities like walking, crawling, head and neck, control and swallowing. Though SMA affects whole body muscles but proximal muscles are affected most severely. SMA is a second most common genetic disorder which causes death in childhood. The best possible approach towards curing SMA is stem cell transplantation. In this article the author examined the therapeutic effects of spinal cord neural stem cell population and their ability to modify SMA phenotype. Microarray technology was used to assess the global gene expression profile of laser-microdissected motoneurons obtained by transplanted and vehicle treated SMA, and wildtype mice.

Introduction:

Spinal muscular atrophy (SMA) is the genetic disorder which leads to death in childhood and it is an autosomal recessive motoneuron disease (MND). Some of the causative factors are mutations and deletions in the survival motor neuron 1 gene (SMN1) mainly in the telomeric copy, it then leads to the depletion in the protein levels of survival motor neurons (SMN).

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Determination of therapeutic potential of neural stem cell and its effects on the SMA phenotype was performed. Transplanted ALDHhiSSClo cells were placed under the arachnoid membrane of the brain and spinal cord into SMA mice. From the performed experiment it was observed that ALDHhiSSClo NSCs are capable of generating some amount of motoneurons upon migrating into parenchyma. Also transplantation improves the functions of motorneurons and mice survival, endogenous motoneurons expression patterns was found modified in wild-type profile.

Figure 1: The figure shows that in in-vitro conditions ALDHhiSSClo are found differentiating into motor neurons. Figure A and B shows expression of GFP protein (green giving rise to complex morphology neurons) figure C, D, and E shows the expression of demonstrates neuronal marker TuJ1 expression by showing neuronal and motor neuronal phenotype. Figure F and G are the images for ChAT and HB9-GFP and figure H and I shows Islet-1.

Purpose:

The main purpose of this study is to examine the microarray data that was analyzed in the article and review the results, intrathecally transplanted ALDHhi SSClo cells and its therapeutic effect on the SMA phenotype, pattern of endogenous motoneurons modification towards the wildtype profile.

Background:

Within SMA parenchyma wide number of neurons and motoneurons can generate with the engraftment of ALDHhiSSClo NSCs. The transplant with NSC subpopulation provides a beneficial effect on SMA mice which gives an inclination towards the therapeutic role of this class of stem cells for developing MND therapies, through neurogenesis and by inducing neuroprotective mechanism. This whole concept was demonstrated by the authors and we analyzed the data with safety.

Methods:

From the author's illustration in the original article different method are used for getting the result of gene expression of laser- microdissected motorneurons in spinal Muscular Atrophy (SMA). The primary method used is data analysis which was collected from GEO website. The microarray data consists of three different group that is, WT, untreated SMA and treated SMA mice. The population of these groups consists of three RNA profiling samples. According to the author's result the analysis was mainly focused upon 2 outputs of Affymetrix genechip arrays: first being "signal call" and second is the "detection call" were generated for every single gene. Observations were analyzed with the help of R free software which was helpful for the generation of heat map.

Another method used on the basis of statistics was Kaplan-Meier survival analysis which was used for comparison of the survival rate between treated and untreated mice. For the statistical analysis researchers used StatsDirect for windows (version 2.6.4). Since the p-value was found to be less than 0.05 so the null hypothesis was rejected.

Results:

Figure 2: The heat map shows the clustar analysis of differentially expressed genes as upregulated is in red and the downregualed gene is in green colour.

A signifcant improvement in survival of treated SMA mice were seen in comparision to the untreated mice. When the survival was prolonged by 5.12 days, it represented 39.26% gain for the lifetime. According to the researchers the maximun survival found out in treated mice was 21 days and in untreated mice it was observed to be 16 days.

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Figure 3: Here is the Kaplan Meier result of SMA mice treated NSCs (Treated SMA), undifferentiated ALDHhiSSClo cells(SMA NSC), ALDHhi SSClo- derived astrocytes(SMA Astro), and primary Fibroblasts(SMA Fibro). Another graph shows statistical analysis of grip time in treated or untreated SMA mice at 12 to 13 days of age.

Survival curve was evaluated for SMA mice that were transplanted with undifferentiated ALDHhiSSClo cells, ALDHhiSSClo- derived astrocytes and murine primary fibroblasts. Primed ALDHhiSSClo NSCs transplanted SMA mice survived longer in comparison to those mice that were grafted with undifferentiated ALDHhiSSClo cells and ALDHhiSSClo astrocytes or primary fibroblasts. Undifferentiated ALDHhiSSClo NSCs transplanted SMA mice survived longer than the mice that received astrocytes and fibroblast, ALDHhiSSClo NSC- derived astrocytes transplanted mice had a longer survival than the ones which received primary fibroblast.

Discussion:

According to the authors of the paper, it is clearly observed that NSC transplantation may ameliorate MND phenotypes. After performing in vivo transplantation it was found that ALDHhiSSClo cells produces neurons and motonuerons. Improvement in the motoneuron phenotype of SMA transplanted mice was observed by the increase in survival and Neuromuscular Function Test. It is demonstrated that ALDHhiSSClo transplantation causes improvement in the survival rate of SMA mice as compared to graft of different cell types like astrocytes and fibroblasts.

It is also confirmed from the cluster and multidimensional scaling that the role of NSC-treated SMA mouse (SMA Tr), untreated SMA mouse (SMA), ALDHhiSSClo-derived astrocytes (SMA astro), and primary fibroblast (SMA fibro) ahs beneficial effects on SMA mice and it also plays an important role in the MND therapy development. Differentially expressed genes are those that have p-value less than 0.05. There is more than one of most over-expressing genes which are meant to be controlled by ALDHhiSSClo stem cells the importance of this is discussed below:

Top 25 up-regulated genes sorted by decreasing format of Fold Change (FC)

ID

logFC

AveExpr

T

P.Value

adj.P.Val

B

1418493_a_at

1.049362

9.622968

5.000895

0.000524

0.881463

-2.60447

1421933_at

1.034395

6.296394

4.451061

0.00121

0.881463

-2.80247

1424092_at

0.915674

7.609384

3.879901

0.003016

0.881463

-3.04569

1422769_at

0.859466

4.730618

3.392571

0.006784

0.881463

-3.28581

1451975_at

0.63

6.682195

4.693363

0.000833

0.881463

-2.7111

1453111_a_at

0.627072

7.819716

4.466262

0.001182

0.881463

-2.79654

1426856_at

0.597576

5.913235

3.952699

0.002679

0.881463

-3.01243

1449030_at

0.58757

5.130291

3.494187

0.005717

0.881463

-3.23324

1426574_a_at

0.580309

7.43272

3.515433

0.005517

0.881463

-3.22241

1428140_at

0.562806

9.162869

4.658598

0.000878

0.881463

-2.7238

1424888_at

0.556697

7.78336

3.830702

0.00327

0.881463

-3.06855

1423651_at

0.556145

9.554304

3.719745

0.003926

0.881463

-3.12125

1452377_at

0.549686

4.00584

3.570037

0.005035

0.881463

-3.19485

1451803_a_at

0.538752

7.074192

5.153253

0.000419

0.881463

-2.55529

1451158_at

0.5344

5.720237

3.634345

0.004523

0.881463

-3.16288

1429907_at

0.529125

5.849073

3.725435

0.003889

0.881463

-3.11851

1426160_a_at

0.499875

5.383448

3.896021

0.002938

0.881463

-3.03827

1425733_a_at

0.497717

5.368413

4.348657

0.001421

0.881463

-2.84314

1422439_a_at

0.487141

7.665937

3.406577

0.006625

0.881463

-3.27849

1460304_a_at

0.485725

5.663614

4.743416

0.000772

0.881463

-2.69305

1425206_a_at

0.484932

4.410244

3.869576

0.003068

0.881463

-3.05046

1450679_at

0.482207

4.372527

4.857052

0.00065

0.881463

-2.65308

1421064_at

0.464742

7.615341

3.439148

0.006271

0.881463

-3.26155

1456109_a_at

0.460573

6.805319

3.50293

0.005634

0.881463

-3.22878

Acknowledgments:

We are grateful for the work performed by the authors of the original article, for providing with the information needed for carrying out our project. We also thank Professor Guenter Tusch for providing us with an opportunity to learn and guide us through successful completion of our project.