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The interaction between the nervous system and the voluntary muscles of the body to perform the day to day errands or tasks are co-ordinated by the motor neurons present in the spinal cord and the central nervous system. Upper motor neurons transmit message from the brain to spinal cord which is then transmitted to particular muscles or effector cells by the lower motor neurons. But in case of Amyotrophic Lateral Sclerosis which is a dreadful disease that results in the gradual loss of motor neuron. Within 3-5 years after the onset of this disease exhibit signs of muscle weakness and progressive death of motor neurons. Even now after the astonishing advances in the field of medical sciences, the therapeutic options still remain limited for ALS patients. Predominantly, ALS is classified into two types namely Familial ALS (FALS) and Sporadic ALS (SALS). FALS are inherited in an autosomal dominant fashion whereas SALS is thought to occur due to a combination of genetic, epigenetic and environmental factors which are still unknown. It is evident that mutations in superoxide dismutase (SOD1) gene cause a common familial form of ALS (nearly 20% of FALS cases) (Deng et al., 1993; Valdmanis and Rouleau, 2008). Sod1 gene encodes for the enzyme superoxide dismutase which acts as a powerful antioxidant and protects the cells from damage caused by toxic free radicals (superoxide). Zebrafish is widely used as the animal model to study neurological diseases because it exhibits high regenerative capacity and therefore believed to be able to regenerate motor neurons. Based on this my project involves analysing the degenerative and regenerative modification that occurs in SOD1 zebrafish model of ALS. The future prospects of this project are to identify the regenerative mechanism in zebrafish and extend its application to human models, in turn providing a new therapeutic approach to treat ALS.
KEYWORD: ALS, SOD1 mutation, FALS, SALS, motor neuron degeneration and regeneration, transgenic zebrafish.
Motor neurons play a vital role in communication between the nervous system and the voluntary muscles of the body. Upper motor neurons transmit message from the brain to spinal cord which is then transmitted to particular muscles or effector cells by the lower motor neurons. However, in case of Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease), a gradual degeneration of these neurons is observed. As a result of which the muscles are unable to perform any function. ALS is one of the desolating neurodegenerative diseases first described by Jean-Martin Charcot in 1869 (French neurologist) that is distinguished by the onset of motor neuron loss in the brain and spinal cord, resulting in paralysis and premature death (FitzGerald, 2007 ; Rocha et al., 2005). The statistical analysis of ALS shows that the incidence of this motor neuron disease is uniform across population of Caucasian origin or most people in Western Europe and North America (USA and Canada). The reported incidence of ALS is 1.6 per 100,000 each year (Hirtz et al., 2007). Till date the suggested treatment for ALS approved by FDA is riluzole which extends the life of the patient's by only few months (Bensimon et al., 1994). Clinically ALS is of two types; familial (FALS) which is due to a mutation in a specific gene and forms 10% of the total ALS cases (Gros-Louis et al., 2006) and the remaining 90% of ALS cases are due to Sporadic (SALS) which is thought to occur due to a combination of genetic ,epigenetic and environmental factors . The risk of fatality due to onset of ALS is within 1 - 5 years (Don W. Cleveland and Jeffrey D. Rothstein 2001). Nearly one in every 800 death in a given population is due to ALS. In 90 - 95 % of events, there is no visible genetic linkage but in the remaining 5 - 10 cases the disease of ALS is inherited in a dominant manner. Genetically FALS are transmitted in an autosomal dominant pattern, in which twelve loci and eight genes have been associated as one of the causes (Dion et al., 2009). The symptoms of lower motor neuron damage includes weakness, atrophy and facilitation of skeletal muscle and the symptoms of upper motor neuron damage are spasticity, increased muscle stretch reflexes, combination signs of both these damages are frequently observed in ALS patients. Denervation of the respiratory muscles and diaphragm may lead to lethality consequences. After the advance in the field of genetics and biochemical studies it is recognised that at least 30 % of small inter neurons degeneration present in the motor cortex and spinal cord contributes to onset of ALS (Shook 2009). No single test is available to diagnose ALS in the early stages and so it is advised to undertake a combination of neurological examination and diagnostic studies (Pioro 2009). It has been assessed that to diagnose the onset symptoms of ALS and confirm its presence in a patient takes more than 12 months. This is because ALS is an uncommon disease and its instance in patients exists in a variety of ways. This all reasons can be a cause to delay in referral from initial care to a neurologist (Shook and Pioro 2009). Combination of genetics, pathological and biochemical studies suggest that the disease is due to oxidative damage caused by mutation in sod1 gene (Boillee 2006). But not all SOD1 mutation is a causative agent of this disease and carrier patients of such mutation can endure to be asymptomatic throughout life (Andersen, 2006). The pivotal gene for ALS was 1st discovered in the year 1993. The detection of copper / zinc superoxide dismutase gene with many possible mutations in the axon was considered as the relevant factor to ALS (Rosen, 1993). Action of sod1 gene as follows:
Superoxide dismutase is an enzyme expressed by the sod1 gene which converts the oxygen free radical specifically superoxide (Oâ‚‚Î‡ â») produced during normal cellular metabolism.
This enzyme converts 2 molecules of superoxide and a single molecule of hydrogen into hydrogen peroxide (Hâ‚‚Oâ‚‚) and molecular oxygen (Shaw 1999, Boillee 2006).
In this way this enzyme plays a vital role in the removal of toxic free radicals from the cell and converts it into inert molecules that are non toxic to the molecular environment in the cell.
Other enzymes such as catalase convert hydrogen peroxide into water and thereby detoxify all the free radicals formed during a cellular metabolism (Dion et al., 2009)
The reaction of the above enzyme is as follows:
Oâ‚‚Î‡ â» + Oâ‚‚Î‡ â»+ 2 Hâº_SOD1_→ Hâ‚‚Oâ‚‚ + Oâ‚‚ _Catalase_→ Hâ‚‚O
It is evident that mutations in superoxide dismutase (SOD1) gene cause a common familial form of ALS (nearly 20% of FALS cases) (Deng et al., 1993; Valdmanis and Rouleau, 2008). More than 150 mutations in the SOD1 gene may lead to the occurrence of FALS (Turner and Talbot, 2008). Missense mutation is the most common mutation in SOD1, which results in a change of one nucleotide and thus the codon codes for a different amino acid (example: The G93R point mutation) (Elshafey et al., 1994; Andersen, 2006; Read and Donnai, 2007). Regeneration event is wide spread throughout the animal kingdom but the core principles of biology are fundamental. In reality, regeneration phenomena may be an ancestral trait that has been lost due to varying degrees in different lineages (Sanchez Alvarado 2000). As mammal's exhibit limited regeneration capacity, my aim is to study the degeneration and regeneration in the inter neurons of the spinal cord in GFAP-GFP: SOD1 transgenic embryo as well as adult zebrafish model of ALS .This project involves the use of zebrafish as the model that has the same basic genetic tool kit to achieve regeneration and compare this vertebrate model with the sod1 overexpressing ALS patients. The zebrafish is an extensively used organism for modelling neurological diseases because of the following reasons; firstly they are vertebrates like mice and humans. Secondly, large number of transparent embryos can be obtained. They have a simple genetic makeup helping us to determine the genes involved and compare them to the equivalent genes within the human genome. Furthermore, zebrafish enable us to study transgenic and targeted knockout models of motor neuron diseases (McWhorter et al., 2003; Lemmens et al., 2007; Boon et al., 2009; Kabashi et al., 2010). In ALS it is observed that the spinal cord anatomy of the zebrafish is quite simple, containing the primary motor neurons and the secondary motor neurons in each hemisegment trunk. Zebrafish has the capacity to regain functional recovery within 6 weeks after the spinal damage caused due to injury (Becker et al., 2004). Since, zebrafish has high regenerative capacity; it is believed that it might be able to regenerate motor neurons. As we know, in mammalian ALS the activation of regenerative sign is not very amenable due to their poor regenerative capacity. Based on this project, we believe that production of regenerative mechanism using sod1 zebrafish model will allow replacement of lost neurons and thereby providing a novel therapeutic approach to treat ALS and other neurodegenerative disorder. In addition they are vertebrates and have a simple genetic make up, enabling us to determine the genes involved and compare them to the corresponding genes within the human genome. Based on this my project involves analysing the degenerative and regenerative
BODY OF THE ESSAY
In order to gain an understanding of SOD1 mutation which is the most common factors responsible for onset of ALS, its progression were studied using a mouse model (Turner and Talbot, 2008). In the muscle section of motor axons of mouse expressing the ALS-linked SOD1-G93A mutation determined the interaction between the enzyme and mitochondria of motor fibres occurring in ALS (Sotelo-Silveira, 2009). Mouse model had proved to be beneficial in studying the onset of ALS but there are some drawbacks, such as some highly inbred lines of mice express high levels of mutant protein which may affect the phenotype and disease progression. Another approach to treat ALS is by using stem cells therapy. Stem cells may prove to be effective in providing a treatment to cure ALS. Stem cells can be differentiated to generate large quantities of motor neurons that can be used for studying glia mediated mechanism. Stem cell therapy may provide a mean to replace lost spinal motor neurons and thereby potentially recover paralysis signs in ALS. However, transplanted motor neurons show a high susceptibility to the toxic ALS microenvironment, a condition similar to endogenous motor neurons. Therefore, these are the major shortcomings faced in the development of motor neuron replacement based cell therapies for recovery in ALS. Other experimental models like Drosophila and C.elegans were used to study ALS and had been evident in over expressing human SOD1 mutations but failed to display motor neurons loss, paralyses or premature death which are reflected in human situation (Oeda et al., 2001; Watson et al., 2008). As we know mammalian spinal cord does not regenerate or replace the lost neurons, as a result of injury or disease but some sort of regeneration has been witnessed in vertebrate animals such as salamander, planaria and zebrafish. Through the findings of Ching-Ling Lien et al., 2006, it is evident that adult zebrafish regenerate up to 20% of the heart ventricle when damaged. Transgenic SOD1 zebrafish model of ALS that demonstrate many aspects of human disease were produced to study the cellular stress using a reliable marker HSP70 (Heat shock protein) which can be easily identify by chemical and genetic modifiers of ALS (Tennore Ramesh et al., 2010; Natasha Hannah Redhead 2010). To understand the normal expression levels and patterns of HSP70, that plays an essential part in the cells machinery for protein folding and helps to protect cells from cellular stress in different stages of zebrafish model. The HSP70 promoter is strongly upregulated by heat stress and toxic chemicals. Transgenic zebrafish embryo are fixed at different periods and then immunostained with HSP70 primary antibody and observed for high level of HSP70. To demonstrate the response caused in the stress cell due to degeneration of motor neurons which are characterised by heat shocking the endogenous heat shock protein 70 that acts as a marker. Heat shock response was visible in the embryonic skin, muscle and spinal cord during the initial stages of SOD1 mutation. The heat shock response is visible at early embryonic stages which is up regulated in the inter neuron implying that these damages may initiate disease ALS. DsRed is an efficient marker used for studying heat shock response. The advantage of DsRed is that the result can be obtained in a small time period. Therefore enabling researcher to carry experiment in short space of time and is a reliable for HSP promoter (Natasha Hannah Redhead, 2010). It was clearly demonstrated that motor neurons showed less significant stress level at early stages, there was another cell type located in the dorsal region of spinal cord expressing DsRed. Thus, these other cell types can be referred as inter neurons which are damaged and lost at early stages of ALS and the motor neurons are the one which are affected at latter stages. Findings of Tennore Ramesh, Alison N. Lyon, Ricardo H. Pineda, Chunping Wang, Paul M. L. Janssen, Benjamin D. Canan, Arthur H.M. Burghes and Christine E. beattie reveals phenotypic hallmarks of motor neuron disease in SOD1 mutant zebrafish. In this study they constructed to generate lines of zebrafish SOD1 gene and flanking sequences nearly 16 kb long. They also constructed lines expressing the G93R mutation and compared it with lines expressing wild - type sod1 gene. Transgenic fish expressing mutant SOD1 were constructed using bacterial artificial chromosome (BAC) containing the sod1 gene. The G93R mutants were obtained by point mutation in the sod1 gene, replacing glycine 93 to arginine (Elshafey et al., 1994). To detect the transgene expression heat shock protein 70 promoter and a reliable marker DsRed fluorescent protein was used. Changes in the neuromuscular junction were observed at larval and adult fish stages. The examination of neuromuscular junctions was carried using SV2 as a pre-synaptic marker and α-bungarotoxin (BTX) as a post synaptic marker.
Fig (1). G93R transgenic model exhibit motor neuron loss of the spinal cord at disease extremity.
Anterior spinal cord section of G93R adult fish and nTg control compared using ChAT marker for immunohistochemical staining. Arrows denote decreased number of positively labelled motor neuron loss due to degeneration.
The graph illustrates the number of ChAT positive motor neurons.
The states of the art in the field are conferred below,
To study the degeneration of motor neurons in zebrafish model of ALS:
Generation of a transgenic SOD1 zebrafish model displaying the phenotypic characteristic of ALS.
Motor neuron disease is a chronic disease in which the clinical symptoms are initiated before the onset of the disease. It causes cellular dysfunction which leads to the formation of cellular stress in the body in turn leading to a proteotoxic environment that merge with the pathogenesis conditions of both sporadic and familial forms of ALS. Generation of transgenic zebrafish was achieved using the wild - type SOD1 gene, point mutations G93R and genetically recombineered into a BAC vector containing the zebrafish promoter for heat shock protein. Multiple lines of transgenic fish expressing the G93R mutations and its comparison with the wild - type SOD1 was detected using heat shock promote to induce DsRed expression. These expression levels are comparable to SOD1 mutant levels in FALS patients and similar abnormalities in neuromuscular junction (NMJ) were observed in the larval and adults stages in the mutant. Muscle weakness and atrophy was demonstrated by adult mutant fishes during the early stage in a swim endurance test. The above tests exhibited that a cellular stress is induced in mutant SOD1 by induction of heat shock response.
Fig (2). Endurance test demonstrating muscle neuron weakness and loss in G93R mutant fish. The velocity rate for swimming of the WT, nTg and G93R fish are compared for an established time period. (A) shows that WT and nTg shows similar performance in the swim test. (10 months age fish) (B) G93R and nTg control shows a considerable difference in their performance. (12months) (C) Distressed G93R is unable to swim but the control nTg shows no significant difference. (16 months) (D) WT which is unaffected and compared to nTg controls exhibit similar endurance in swim test. (24 months)
Generation of endogenous hsp induction in DsRed expressing neurons.
The activity of endogenous hsp 70 gene supported the induction of DsRed in the mutants; this demonstrated a high copy number of the hsp 70-DsRed transgene expression signal by a greater number of neurons exhibiting DsRed expression. Thus the colocallization of hsp 70 in cells expressing DsRed helps in screening the genes that regulate heat shock response due to SOD1 mutation (i.e. toxicity of mutant sod1 gene).
Knock down of SOD1 expression in zebrafish results in modification of heat shock response.
Modification of the heat shock response by knock down of sod1 gene can be achieved by two ways such as, by reduction in SOD1 protein expression ultimately results in reduced induction of mutant SOD1 hsp 70 and by reduction in mutant SOD1 hsp 70 induction using an ion channel modulator. The former way confirms that hsp 70 inductions is due to mutant SOD1 expression and reveals that DsRed induction is independent of heat shock in the wild type SOD1 transgenic lines. The later ways demonstrate that neuronal activity is excited due to oxidative stress which is an often observed cause of neurotoxicity in ALS.
To study regenerative capacity of motor neurons in embryo and adult zebrafish:
The mammalian spinal cord is incapable of generating motor neurons that are damaged due to injury or neurodegenerative disease like ALS, muscle dystrophy and many more. It is demonstrated that the adult zebrafish has spinal cord regeneration capabilities (Michell M. Reimer et al., 2008). The finding suggests that a lesion created in the spinal cord of the model zebrafish, result in the development of progenitor cells. A crucial event in neural development is the point at which differentiation of neurons occurs that is neurons become competent to extend long axons. The olig2: egfp transgene present in the transgenic zebrafish model expressed mainly in the unlesioned spinal cord of adult zebrafish. Ependymo radial glial cell lines are found to be expressed in ventrodrsal position present in the central canal that correspondence to expression domain of these genes in the advancing neural tube (Michell M. Reimer and Becker et al., 2008).
Thus the plasticity of the progenitor cells aid in regenerating motor neurons and also exhibiting few markers for terminal differentiation and integration of adult neurogenesis.
Induction of ventricular proliferation due to spinal lesion
The evident of newly generated cells in the spinal cord region was analysed by using immunohistochemical detection at specific interval of injected BrdU. After two weeks the lesioned spinal region exhibited very few numbers of newly generated cells in the spinal tissue. Thus revealing a significant number of cell proliferation in the ventricular zone. The spinal cross section exhibited white matter tracks filled with myelin debris of degenerating fibres, the cytoarchitecture of the lesion spinal cord which is similar in the ALS patients (Becker and Becker, 2001).
Fig (3). Regeneration of motor neurons in the lesioned spinal cord.
Translucent appearance indicates the formation of new neurons in the lesion site.
Dissected horizontal view of lesion spinal cord obtained by electro microscopy, ax → axon present in lesion site SC → remyelinated Schwann cells
GFP labelled cells of transgenic lines exhibited an increase in the number of differentiating motor neurons
The generation of new motor neurons in the core region of the lesion site were examined by expression of GFP labelled motor neurons in the presence of HB9 promoters (Flanagan-Steet et al., 2005). The ventral horn region of the unlesioned HB9: GFP animals showed few large motor neurons and smaller motor neurons (Michell et al., 2008). Immunohistochemical technique showed a significant increase in the number of differentiating motor neurons in the lesioned animals then from those in the unlesioned control using the protein HB9 in this technique. Double labelling of antibody in HB9: GFP transgenic model exhibited heterogeneous motor neurons which were expressed by markers, on the other hand unlesioned spinal cord showed no expression of double labelled motor neurons in model animals (Tsuchida et al., 1994; William et al., 2003). The presence of ependymo-radial cells which were similar in characteristic to stem cells showed slow proliferation (Chapouton et al., 2006)
Newly generated motor neurons demonstrated terminal differentiation.
ChAT marker was used for detecting the small HB9:GFPâº neurons showed significant differentiation of the newly formed motor neurons in the lesioned spinal cord. Triple labelling of BrdU, ChAT and SV2 markers revealed the presence of generated terminal differentiation of new motor neurons. These motor neurons were integrated into the spinal circuitry of the lesioned spinal cord and the motor neurons regenerator cells proliferated around the lesioned area. Thus the spinal lesion activated the formation of motor neurons in the spinal cord of adult zebrafish. It is also demonstrated that newly produced motor neurons do not exhibit normal cytoarchitecture but in fact add these neurons in the pre-existing spinal tissue present near the spinal lesion. The work of (Michell et al., 2008) suggest that (Olig2âºependymo-radial) glial cells have the characteristics of neuronal stem cells which serve as the progenitor cells which induce the growth and lineage tracing of spinal motor neurons. Therefore, Olig2âºependymo-radial glial would be similar to radial glial cell type in many species such as in mammals and adult zebrafish (Pinto and Gotz 2007). However, the study showed considerate increase in number of adult neuronal regeneration that is they have the potential of self-renewal. Detection of the terminal differentiated neuron cells revealed that they were conserved and integrated into spinal network. These observations suggest that motor neurons undergo successive morphological differentiation and gene expression. This indicated that there is significant plasticity of adult spinal progenitor cells in the fully mature spinal cord. The work concluded that regeneration of motor neurons after a lesion triggers the activation of massive stem cell or progenitor cells and thus building network integration of motor neuron in spinal cord of zebrafish.
Fig (4). Cross section of the lesion spinal cord exhibit generation of new motor neurons around the lesioned area. Images are obtained by confocal microscopy.
Lesioned area shows the presence of HB9:GFP_/BrdU.
Lesioned area shows HB9_ but not the unlesioned spinal cord. The ventricular zone shows the presence of olig2: GFP progenitor cells (arrowhead).
The above mentioned discussion is the state of the art in this field. My hypothesis is to investigate and identify the cause of degenerative and regenerative modifications occurring in SOD1 zebrafish model of ALS so that we can elaborate genetics and pharmacological screens to trigger the regenerative capacity.
In this project, we would create transgenic zebrafish harbouring a fragment of GFAP gene driving GFP which would be crossed with SOD1 mutant transgenic fish. These fishes would carry the transgene expression similar to the endogenous gene expression induced in the developing and regenerating motor neurons in the nervous system. In addition to this the tg [GFAP-GFP: SOD1] exhibit degeneration of neurons due to sod1 gene mutation of ALS. Although, GFP expression of the newly generating cells is expressed more during the embryonic period and gradually decline during maturation of the nervous system. Degeneration of motor neurons can be observed in adult transgenic zebrafish by using Green Fluorescence protein expression at multiple stages of development. We are also going to study the degeneration of inter neurons during the post embryonic stages and a comparison of GFP expression would be carried out for transgenic embryos and adult fish lines with the wild - type sod1 gene in the control fish. It is expected that early developmental stage when compared using immunohistochemistry with an antibody against the GFAP regulatory element expression of the GFAP-GFP: SOD1 transgene may overlapped with endogenous GFAP protein distribution. Expression of degenerated motor neurons is found to be lessening during early embryogenesis because at time of embryo development there is more of generation of the newly formed motor neurons to develop the spinal cord. Thus GFP fluorescence facilitates the morphological and functional analysis of motor neuron cells and precursor cells in GFAP-GFP: SOD1 transgenic fish.
Neuronal stress behaviour and death in embryo and adult SOD1 zebrafish model demonstrate the incidence of degeneration of motor neurons:
To measure the total fluorescence of live embryos and also to detect the intensity of induction of the stress response in mutant zebrafish embryo quantitative fluorescence assay was used. The co-ordinating signals from the central pattern generator of zebrafish spinal cord which plays an important role in controlling the swimming behaviour of zebrafish. The expression of the spinal neurons were studied by using specific markers such as Choline acetyl transferase (ChAT), GFP and HB9 to examine the live embryos showing mutant SOD1 stress response by technique such as confocal fluorescent and immunohistochemistry of transgenic embryos and adult.
To study degeneration we will employ TUNEL staining of motor neurons to compare a substantial numbers of new motor neurons that are generated during a spinal injury. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining is used for detection and quantification of apoptic cell at cellular level. This technique involves labelling of both double stranded and single stranded DNA formed during apoptosis. They are identified by labelling free 3'-OH terminal with modified nucleotides in an enzymatic reaction. The assay depends on the presence of nicks in the DNA which can be demonstrated by terminal deoxynucleotidyl transferase, this enzyme catalyses the addition of dUTPs. Then the dUTPs are labelled with a secondary marker which is detected by immunohistochemistry for GFP labelled nuclei. Sections of double labelling were analysed in fluorescence microscopy. Negative control of the TUNEL method can be obtained by exclusion of the terminal nucleotide transferase and detection of highly localised cell death as positive control. My project involves the study of the degenerated neurons by TUNEL staining method. The principle of this technique is described as the degenerated neurons of the zebrafish model of ALS shows the activation of nucleases that eventually degrade the nuclear DNA of this died neurons into fragments which serves as the essential element for this assay and also making it most reliable method for detecting the lost or dead neurons. The degenerated motor neurons exhibit a large number of free 3' hydroxyl end, these hydroxyl groups serve as the initiating point for terminal deoxynucleotidyl transferase (TdT), which adds the deoxyribonucleotide in a template independent manner. Addition of the deoxythymidine analogue 5-bromo-2'-deoxyuridine, 5'-tri'phosphate (BrdUTP) to the terminal deoxynucleotidyl transferase reaction serves to label the disrupted sites. Once BrdU is incorporated into DNA, the presence of BrdU can be detected by anti BrdU antibody using standard immunohistochemical techniques. This method of labelling DNA is to study and quantify the degenerated neurons in the zebrafish model is stated as TUNEL staining (APO - BrdU TUNEL assay kit).
In vivo application of time lapse imaging has helped to advance our understanding of ALS and also to investigate how motor neurons degenerate in disease. This technique also aids in understanding the motor axons attempt to recuperate for the loss of neuron signalling from the brain. The observation of high resolution images may reveal two distinct populations of motor neurons such as one group with axon branches demonstrating their degeneration and one that is simultaneously compensating reinnervation. Thus the time lapse imaging may prove to be useful for understanding the degenerative process that follows spinal cord injury or disease and correspondingly evaluates therapies specified at enhancing regeneration. Time lapse imaging of the spinal cord of the transgenic zebrafish would be carried out for a period of 24 hours. This technique would help us to study the regenerative capacity in the live transgenic fish and comparing it with newly formed cells in the control (wild - type sod1 gene fish). It is expected that many newly generating neurons would be visible within 6 to 24 hours after the onset of ALS sign. Result of these observations would suggest that this type of time lapse imaging can be used for understanding the degenerative process that follow spinal cord injury or loss due to over expression of mutant sod1 gene and also evaluating therapies intended at enhancing degeneration.
We use BrdU (thymidine analogue bromodeoxyuridine) to study the distribution of regenerative neuron cells in the intact SOD1 mutant zebrafish, as well as to demonstrate the migration and differentiation of motor neurons. Bromodeoxyuridine (5-bromo-2-dioxieuridine) is incorporated into the new DNA produced during S phase. Treatment of cells within a short period of time with BrdU enables us to label newly synthesising cells. Then the labelled cells are fixed using a chemical fixative in order to detect presence of BrdU in the cell. The fixative also permits the access of a secondary flurochrome labelled antibody that specifically binds to the BrdU containing cells. The result of this is that all the proliferating cells can be clearly differentiated and quantitated. Single positive BrdU cells are advantages to study mutant SOD1 over expression as this technique exhibit large number of labelled cells found in the regenerating tissue in turn demonstrating that the progeny were actively dividing prior to degeneration formulate a major contribution to the regenerating tissue (Newmark and Sanchez Alvarado, 2000; Eisenhoffer et al., 2008). This labelling technique helps us to label specifically the regenerative neuron cells. Thus it enables us to study the problems of vertebrate regeneration and the control of neuron cell proliferation.
Choline Acetyl Transferase (ChAT) is an enzyme that catalyses the biosynthesis of acetyl choline which is a neurotransmitter that controls the signal transduction at the neuro muscular junction. Acetyl choline is present in the cholinergic neurons which plays an essential role in the fundamental brain processes. Modification in cholinergic neurons may lead to ALS and its loss may cause other neurological disorders such as Alzheimer's diseases, Huntington's diseases and schizophrenia. For detection of cholinergic neurons ChAT is consider to be the most specific marker. Northern blot or in situ hybridisation technique may be employed to identify the ChAT positive neurons present in the spinal cord of the model zebrafish. Currently acetyl cholinesterase histochemistry is extensively used for localizing cholinergic neurons to study the regenerative capacity of zebrafish model. Immunohistochemical localization of ChAT using monoclonal antibodies suggests that, choline acetyl transferase is more specific marker than acetyl cholinesterase for cholinergic neurons and the presence of ChAT positive cells demonstrate the regeneration of cholinergic neurons in transgenic embryo and adult zebrafish model.
Effective GFP identification of progenitor cells is crucial for studying regeneration in transgenic zebrafish model of ALS. And the probable results of this technique are as follows:
Embryonic period: During embryogenesis GFP expressions of regenerated cells are thought to be observed in the ventricular zone. HSP70 promoter which strongly expresses GFP may also be observed in ventricular zone of spinal cord. GFP expression in radial glia would be beneficial for analysing the most initial event of neurogenesis. The BrdU positive cells are assume to be large in number during the developmental stages compared to the adult fish. But there is no significant occurrence of degeneration of motor neurons in this stage; this can also be demonstrated by time lapse imaging of the spinal cord. Therefore, there are no remarkable differences observed in embryos of transgenic line and control.
Adult (and neonate) period: During the adulthood GFP expression is assumed to be observed in the cerebellum but to a lesser extend. On comparing the transgenic GFAP-GFP: SOD1 mutant fish with the wild - type control, will reveal the presence of newly generated motor neurons increasing number in transgenic lines than the control. Thus the results indicate that GFP expression generally occurs where neurogenesis is undergoing. The BrdU positive cells are rarely observed during this stage, but when compared with the control numbers of positive cells are relatively greater than the normal condition. During the adult stage there would be a significant degeneration of motor neurons which could be analysed by presence of ChAT positive cells. Therefore newly generated motor neurons are capable of generating their axons out of the spinal cord in zebrafish model. Muscle endurance analysis of the control and the mutant revealed normal muscle force during the beginning. However, towards the end the mutant exhibited decreased endurance in the swim TUNEL test. This observation suggests that there could be motor neuron loss and muscle atrophy which is similar signs seen in ALS patients.
Neuromuscular junction defects may be identified during larval stages and also in adult fishes. Maintenance of muscle integrity will suggest that there is a neural defect in general which affected the muscle movement. This study provides a novel vertebrate model to study familial ALS that displays the feature of motor neuron disease. Therefore, zebrafish are an exceptional model organism which have conserved vertebrate nervous system employed in studying neurological disease. Zebrafish also illustrates innate nervous system regenerative capacity in addition to their genetic and vertebrate central nervous system architecture. In this project we evaluate various potential to induce neuronal regeneration and examine neuro degeneration. Zebrafish has many essential characters that make it a potent resource for testing hypothesis and determining therapeutic targets in functional central nervous system regeneration. The optical clarity and rapid progress of zebrafish provides the adeptness to follow the experimental events as they occur in life or in developing embryos. These oviparous zebrafish has the advantage to externally lay fertilized eggs which develop as transparent embryo and allow us to observe and compare the stages with the vertebrate development. Zebrafish also carry a fluorescent reporter gene that aids in analysing living tissue and visualizing the changes in gene expression caused due to a mutation or detailed morphogenetic movements. It also has an innate regenerative capacity that is evident by the work done on the regeneration of heart and retina ( ). It is important to know that embryonic, larval and adult regeneration are very different in zebrafish when compared with mammals. Also the ratio of differentiated to undifferentiated cells is different to over all scale. Regeneration of motor neurons depends on the proliferation of stem or progenitor cells. Another approach to treat ALS are the stem cells derived motor neuron which may provide an invaluable tool and develop as a combined therapy for which drugs can be screened to assess their ability to prevent motor neuron death. Optimistically combine therapies designed to protect ALS patients will aid conserve the remaining endogenous motor neurons and enhance the proliferation of newly generated motor neurons. To over come the draw back of stem cell derived therapies, which can be achieved by slowed disease progression in order to decrease the toxicity of the micro environment around the spinal cord and thus helps in replacing the damaged motor neurons using a variety of potential stem cell source and enabling ALS patients to begin their road towards recovery of motor function. Till today great strides have been done in understanding the regenerative capacity of vertebrates and the complex cell interaction that are involved in motor neuron regeneration, very insignificant knowledge about the definitive source of regeneration and regenerative cells fate are known. Unfortunately mammals can regenerate only certain type of organs like liver and kidney to some extend, our regenerative power is comparatively far less notable. Therefore the work done in this project suggests that, if we can identify the factors responsible for the degeneration of motor neurons. Also find the elements that protect certain motor neurons from degenerating or induce their regeneration. The future aspect of regeneration research is enlivening and full of promises; the continuous progress in this field will be achieved when animal model uses the distinct mechanism to reach the underlying principal of gene network common to regeneration capacity. Further more, to promote an innate or introduce the new regenerative capacities in mammalian cell lines. In this project we discuss the rational behind those factors which are the possible reasons for clinical failure and ALS identify the factors that protect certain motor neurons from degeneration and induce their regeneration. Hence, it might be possible to design therapeutic strategies that may be used in the treatment of ALS or motor neurodegenerative disease.