Change in expression of TDP43 in various organs during development of ALS model zebrafish.
Amyotrophic lateral sclerosis (ALS) is the devastating motor neuron disease that is characterized by progressive degeneration of both upper and lower motor neuron that control voluntary movement of body. The degeneration of the neurons seen in ALS result in muscle weakness, spasticity and atrophy of both cranial and spinal nerves muscle groups. Since there is often respiratory muscle involvement, aspiration pneumonia is the most common cause of death for the patients with ALS. At present, ALS is invariably fatal disease with no absolute cure and patients usually die within 3-5 years after the clinical onset of symptoms. The mean age of onset of ALS is between 55 and 65 years with slightly more prevalence in male (Male: Female ratio ~ 1.5:1) (1). Even though, incidence rate of ALS are different in different countries of the world, globally average annual incident rate is between 1.5 and 2.5 per 100,000 populations. There has been an increase in death rate of ALS and current international death rates for ALS have be close to 1 per 100,000 population per year(1). Currently, riluzole, an inhibitor of glutamate release, is the only disease modifying treatment available for the disease and can extends life only for couple of months (2,3).
The etiology of ALS is currently unknown. However, approximately 10% of ALS patients have family history for ALS (Familial ALS;FALS) and remaining 90% of case occur sporadically (Sporadic ALS; SALS)(4). Although definitive evidence for environmental factor that cause ALS has remain mostly unknown, the evidence of genetic alternation that cause ALS has been increasing. Till date, only known cause of ALS is mutation in the gene. Mutations in more than 13 different types of genes have already been identified that can cause FALS. FALS is often a Mendelian inheritance with high penetrance, although most cases are autosomal dominant pattern of inheritance, autosomal recessive pedigrees have also been reported (5,6). Even though, FALS are cause due to genetic alternation, FALS are indistinguishable from SALS form histopathological perspective and both the types’ presents with similar sign and symptoms, thus suggesting common intra-cellular processes that lead to the disease symptoms. Among those 13 different types of gene mutation that causes FALS, mutation in Transactive response DNA binding Protein 43kDa (TDP-43) gene is seen in approximately 4% of FALS and 2% of SALS (7).
Transactive response DNA binding protein 43kDa (TDP-43) is a DNA/RNA-binding protein encode by the TARDBP gene on chromosome 1. TDP-43 is an ubiquitously expressed nuclear protein capable of shutting between the nucleus and cytoplasm (8). TDP43 is present in almost all the tissue of a body and have different roles in different tissues (9). Although the precise cellular function of TDP-43 is unknown, TDP-43 has been implicated in regulating of gene transcription (9),alternative exon splicing (10) and mRNA stability (11).
Under normal physiological conditions, TDP-43 resides predominantly in the nucleus where it involved in gene expression. But, in abnormal pathological conditions such as ALS, TDP-43 is mislocalized in the cytoplasm as inclusions body (12,13) . Analysis of TDP-43 in the brain and spinal cord of ALS patients reveled that TDP-43 is pathologically modified and redistribution to the cytoplasm, which is accompanied by loss of normal nuclear function and a toxic gain-of-function in the cytoplasm (14,15). The mislocalization of TDP-43 into cytoplasm is believed to be cause of neuron loss in ALS patients. Moreover, TDP-43 positive inclusions are also found either independent or partially colocalize with the other characteristic inclusion, such as tau, α-synuclei, β-amyloid and polyglutamines, which are found in other neurodegenerative disease such as Alzheimer’s disease, Pick disease and Parkinson’s disease. Interestingly, TDP-43 positive cytoplasmic inclusion are found in almost all ALS patient along with other neurodegenerative disease (16). Although evidence suggest that there is a definitive association between ALS and TDP-43, above observations make it confusing to whether TDP-43 pathology is causative or a secondary response in this disease. Studies done to unravel if TDP-43 is pathology or secondary response to ALS have come with conflicting result. Moreover, the present of TDP-43 in inclusion body of another neurodegenerative has been a mystery. The precise role of TDP-43 in ALS and other neurodegenerative disease is not well known and needs further evaluation.
Study, in the mouse has shown that TDP-43 protein is essential for normal prenatal development. Homozygous loss of TDP-43 in mouse cause early embryo death. But, in heterozygous loss TDP-43 mouse, the TDP-43 protein levels were nearly normal suggesting an auto-regulatory mechanism controlling this protein levels(17,18). Moreover, research on mRNA expression levels of TDP-43 protein in various tissues has shown that TDP-43 plays different roles in different tissue(9). Furthermore, about 40 different mutant in TDP-43 have already been identified so far that is associated with ALS (10). But all this various types of mutations in TDP43 have only affected motor nerve of spinal cord and brain. At the same time, mutation and/or overexpression of TDP-43 has not cause any pathology alternation in other cells and tissue of the body or has been found to be associated with diseases of other organ system. A protein that is so vital for a development of organisms that it’s absent cause death, but when there is mutation in its gene has only abnormalities in nervous system and that abnormalities are evidence after mid-life is yet to be understood.
Moreover, within the nervous system mutation in TDP-43 seems to affect only motor neuron and at the same time spares other neuron such as sensory, autonomic nervous system. And this preference to the motor neuron by mutant TDP-43 is even seen till the late stage of the disease. Physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons is yet to be fully understand. Causative link between TDP-43 positive inclusion and ALS can be well established, if nuclear to cytoplasmic expression of mutant TDP-43 could be study in vivo and in real time. And at the same time, will also be able to understand if TDP-43 pathology is causative or a secondary response to ALS and other neurodegenerative disease.
Transgenic rodent models of ALS have been extremely valuable in providing some insight into biological mechanisms underlying ALS. But, due to difficulty in conducting in vivo real time study with rodent, change in intra cellular expression of TDP-43 has not being well understand. The zebrafish has recently emerged as powerful genetic model system for studying ALS. External development and transparency make it great tool to study the development stages of almost all the organ. External development of its eggs allows easy observation and manipulation of early development process. And, transparency makes is a powerful tool to observe the change at cellular level by using fluorescent reporters. With the help of fluorescent reporter, specific cell type and protein expression within those cells can be easily identify and study in vivo and in real time in zebrafish. In addition, zebrafish is a vertebrate and their nervous system is highly conserved with higher vertebrates including humans and many pertinent feature of the nervous system start to develop within 1 day of development. Moreover, genetic manipulations are comparatively easy in zebrafish. Therefore, zebrafish is a great model system to study the association of TDP-43 and ALS.
In this study, I am trying to understand the change in expression of mutant and overexpressed TDP-43 protein in different tissue of zebrafish. At the same time also will be evaluating the change in expressions of TDP-43 as the zebrafish grow from embryo to adult. I will then compare the change in level of TDP-43 from asymptomatic stage of ALS zebrafish to that of symptomatic stage of ALS zebrafish. In order to conduct this experiment, transgenic zebrafish with human mutant TDP-43 will be created by genetic engineering. Human mutant TDP-43 will be fused with green florescent protein (GFP) before creating transgenic zebrafish. By combining human mutant TDP-43 with GFP will allow easy visualization of TDP-43 protein in zebrafish. Then, image of the fluorescent labeled TDP-43 at different stage of development of zebrafish period will be capture with fluorescent microscope.
1.Logroscino, G., Traynor, B., Hardiman, O., Couratier, P., Mitchell, J., Swingler, R., and Beghi, E. (2008) Descriptive epidemiology of amyotrophic lateral sclerosis: new evidence and unsolved issues. Journal of Neurology, Neurosurgery & Psychiatry 79, 6-11
7.Corrado, L., Ratti, A., Gellera, C., Buratti, E., Castellotti, B., Carlomagno, Y., Ticozzi, N., Mazzini, L., Testa, L., and Taroni, F. (2009) High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis. Human mutation 30, 688-694
8.Winton, M. J., Igaz, L. M., Wong, M. M., Kwong, L. K., Trojanowski, J. Q., and Lee, V. M.-Y. (2008) Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. Journal of Biological Chemistry 283, 13302-13309
9.Ou, S., Wu, F., Harrich, D., García-Martínez, L. F., and Gaynor, R. B. (1995) Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. Journal of virology 69, 3584-3596