Molecular Identity of Synapsin in the Locust Brain - Essay Example

Molecular Identity of Synapsin in the Locust Brain Introduction: The Synapsins: The synapsins are a part of a select group of neuron-specific phosphoproteins that modulate neurotransmission across chemical synapses. They are vesicle-associated and are believed to be wide-spread in the central and peripheral nervous system (Keilland et al, 2006; Gitler et al, 2004). Nevertheless, there is recent evidence that the three types – I, II and III – are differentially distributed across the nervous system not only region-wise but also by age. It is believed that synapsin III regulates the growth rate of axons and the size of the growth cones of developing neurons (Feng et al, 2002). There is also evidence that synapsin III is less evident in adults than synapins I and II. Synapsin III presence starts to decline compared to synapsins I and II after the first week postnatal (Feng et al, 2002). Also, while synapsins I and II are predominantly available at presynaptic sites, synapsin III is present at subcellular locations. It is present in cell bodies and growth cones as well as at presynaptic sites (Feng et al, 2002). Also, inhibits the binding of ATP to synapsin III whereas it actually facilitates ATP binding to synapsin I. It does not seriously affect ATP binding to synapsin II (Feng et al, 2002). In true sense, synapsins I and II, with isoforms, have C domains that have high affinity ATP-binding molecules that nevertheless bind differentially to ATP in regard of . Synapsin I has a conserved glutamate residue () that induces it to bind to ATP in the presence of (Hosaka and Sudhof, 1998). In synapsin II, the glutamate residue is substituted by a lysine residue that induces it to bind to ATP without presence. Substitution of by lysine in the same position in synapsin I takes away its dependence and makes it -independent like synapsin II (Hosaka and Sudhof, 1998). It is believed that the synapsin affinity for ATP is highly instrumental in their attachment to presynaptic vesicle exostructure and a large part of their functional capabilities Hosaka and Sudhof, 1998). Synapsins are considered essential for intense vesicle recruitment during sustained transmission across relevant synapses (Kielland et al, 2006). They are believed to have multiple functions within presynaptic terminals. Besides recruiting synaptic vesicles (SVs) they also regulate their fusion (Gitler et al, 2004). It is believed that the synapsins are implicated in synaptic plasticity. Synaptic vesicles from the reserve pool go through a process of docking and priming before exocytosis. It is believed that synapsins, after recruiting the SVs to the reserve pool, also function in the docking process. Inactivation of the synapsin genes demonstrate that transfer of the SVs from the reserve to the docking pool is inhibited and, in consequence, SVs from the docked pool are released more preferentially and facilely (Kielland et al, 2006). This last may demonstrate that synapsins are not necessary for fast transmission and their inactivation may actually assist in such fast neurotransmission by making the SVs already incumbent in the docked pool preferentially available for speedy release (Kielland et al, 2006). It is notable in this context that the entire functional gamut of these phosphoproteins still remains unexplored thoroughly. Synapsins bind to phospholipids and protein constituents of the SVs, as well as to components of the cytoskeleton, such as actin, tubulin and spectrin (Gitler et al, 2004). To date they have been implicated in neuronal development, synaptogenesis and maintenance of mature synapses (Gitler et al, 2004). Presently, three basic types of synapsins I, II and III have become evident. Synapsin I and II are expressed homologously by their isoforms synapsins Ia and Ib and IIa and IIb through alternate splicing from two similarly named genes while synapsin III, a relatively new find, is also expressed by a similarly named gene (Hosaka and Sudhof, 1998). There is evidence that the multiple isoforms bind differentially with some isoforms like 1a performing only when there is adequate coexpression of the other isoforms (Gitler et al, 2004). Also, the binding domains, most commonly domains B, C and E, have variable tasks. For example, though domain A is not necessary for binding, its presence enhances targeting. Domain D inhibits targeting but this inhibition is overcome by the implication of domain E. In domain location, the vertebrate synapsins I and II, with isoforms, have amino-terminal domains, the short A and B domains and the highly conserved long C domain, that are common to all these four isoforms. There are also the divergent carboxyl-terminal domains (domains D to I-) that are differentially combined in the four isoforms (Hosaka and Sudhof, 1998). The long amino-terminal C domain has been conserved through evolutionary processes in both vertebrate and invertebrate synapsins and is thus construed to be the central functional domain. The C domain constitutes more than half of the synapsin sequences (Hosaka and Sudhof, 1998). The invertebrate synapsin gene, found in Drosophila first, expresses two protein products that differ at the carboxyl terminus (Hosaka and Sudhof, 1998). Locusta migratoria: Locusta migratoria is a native of sub-Saharan Africa. It is a poikilotherm and cannot adjust to large temperature variations in its environment. It is chosen as the animal of study here because it has a relatively simple nervous system that allows able examination at the cellular levels in live animals and semi-intact preparations (Rodgers et al, 2006). Also, locusts and other such large orthoptherans have a unique nervous system, in addition to having a simple one, in which the brain and the ventral cord are functionally comprised of individual neurons in complex circuitry. These neurons are easily identifiable by their morphology and physiological characteristics (Watson and Schurmann, 2002). This is of especial use to researchers because complex circuitry that subserve known behaviour and other patterns can be studied in context of individual neurons that serve that particular circuitry. Often, in large animals, such study is only possible on sets of neurons comprised in complex circuitry. This essentially does not allow structural disclosure of individual neurons in the circuit. Also, the synaptic distribution of the neurons can easily be revealed in the orthopteran system as it is large enough and close enough to the body surface compared to other insect classes with nervous systems conducive to study of individual neurons (Watson and Schurmann, 2002). This is of particular use again because the orthopteran system is closely related to that of diptera and, though the dipteran system cannot be so easily studied, it is more susceptible to genetic manipulation which often unveils characteristics that are common not only to both insect systems but also to those of other animals (Watson and Schurmann, 2002). In effect, study of the orthopteran system produces morphological implications on individual neuron basis while that of the dipteran system produces genetic implications. The close association between the two systems thus produces, in conjunct study, a combined library of phenotypic and genotypic materials on nervous systems that prove invaluable (Watson and Schurmann, 2002). In the orthopteran system the synapses, which are of two types – the more prolific chemical and the less evident electrotonic or gap, are served by pre- and post synaptic regions that are asymmetrically electron-dense (Watson and Schurmann, 2002). Also, in the central nervous system of insects, the neurons are monopolar and have one primary neurite that emerges from the cell-body region (the cortex) into the soma-free synaptic area (the neuropile) (Lohr et al, 2002). In the neuropile, the neurite branches out and forms axons and other branched structures that associate with presynaptic and postsynaptic sites in yet unknown manner (Lohr et al, 2002). In the larger insects like the orthopteran the neurites are specialized with either presynaptic or postsynaptic associations or they may also be associated with both (Lohr et al, 2002). In smaller insects like the drosophila (diptera) such association is not easily manifested and it is unclear how the organization lies (Watson and Schurmann, 2002). It is notable that, during the development of the nervous system in any animal, the synapses have to differentiate and position themselves atypically and accurately to be functionally viable (Lohr et al, 2002). Lohr et al, 2002, seems to have derived morphological facts that are unique to synaptic placements at specific locations along neurites. It seems that these synapse development processes are evidently intrinsic and intracellular-driven and do not depend in large part upon any extracellular cues (Lohr et al, 2002). For example, mechanisms that drive synapse placement along nuerites in the CNS are evidently different from those that drive such placement in neuromuscular junctions (Lohr et al, 2002). Also, there is evidence that some of these intracellular mechanisms do derive cues in part from extracellular structures or mechanisms (Lohr et al, 2002). This is of immense interest to neuroscience as mechanisms for synapse placement at particular regions of the CNS can explain numerous essential features inherent to the system. Yet, there is still a lot of study to be done in this area and it still remains unclear how mechanisms and components, both intracellular and extracellular, that participate in neuronal differentiation and development organise synapse placement along neurites. This is in more so context of specific neurites and specific synapse types (Lohr et al, 2002). It is also notable that studies like this in which the molecular identities of the synapsins are being implicated can assist immensely in identifying and understanding such organisational components and processes. This is since the synpasins are unique neuronal proteins that are extensively available throughout the CNS, with a few exceptional regions, and are easily identifiable because of their unique response mechanisms. Immunohistochemical Analysis The research project will undertake an immunohistochemistry analysis wherewith the expressed synapsin phosphoproteins in locust (Locusta migratoria) neuronal brain cells, fully differentiated so that synapses are definitely in place, will be localised using the principle that antibodies bind specifically to antigens in biological tissues (IHC World, 2007). There are two manners in which this antibody-antigen reaction can be visualised. In the first method, the antibody is conjugated to an enzyme, such as a peroxidase, that catalyses a colour-producing reaction (also called immunoblot staining (IHC World, 2007). In the second method the antibody is tagged to a fluorospore, such as FITC and rhodamine, producing a reaction that induces fluorescence. This is also called immunofluorescence analysis (IHC World, 2007). Immunofluorescence is a much more sensitive process with possibilities of multiple protein interactions. Specific methods such as Western Blot analysis use electrophoresis to separate the test proteins, the synapsin brain phosphoproteins in this study, on polyacrylamide gels and electroblot these proteins on a protein binding membrane. The membrane captures the proteins and immobilises them. Specific anti-synaptin antibodies, tagged with fluorescent spores like fluorescein isothiocynate (FITC)-dextran, induce fluorescence by reaction with the expected protein product (synapsins in this study) and are focused on by special high-calibre microscopes and quantified by special software like TINA (Rivera, 2004). The proteins may be expressed first with suitable mutated primer sections using methods of identification and purification such as reverse transcriptase polymer chain reaction (RT-PCR) analysis (Rivera, 2004). More details of the process is provided in the research report which follows. Gel Electrophoresis Technique: 1.5 g agarose in 100µl of buffer was taken. Of this 0.75 g of agarose and 50 ml of 1xTBE was taken in a conical flask and the contents were boiled in microwave till the mixture was fully dissolved. The dissolved mixture was then taken off the microwave and cooled to in a water bath to approximately 50oC. The partly cooled mixture was then poured on both sides of a taped gel tray, which had combs to allow well formation. After some time, when the gel had set, the tapes and combs were taken off and the gel tray was placed in the electrophoresis machine. TBE buffer was placed on the gel till it wholly covered the gel. Marker X was used as molecular weight marker. 1µg of the marker consisting of 2µl of loading buffer, 7µl of water and 3µl of Marker X was loaded onto the gel in the machine. The Locusta migratoria brain neuronal cultures, fully differentiated, were also loaded onto the gel. For each machine run, 10µl of brain cell culture, together with 5µl water and 5µl loading buffer was used. Electric current at 100V was applied. The run was for one and half hours and it was made sure that the products did not run off the gel during this time. Staining was done with ethidium bromide applied for 30 minutes. After staining, the stained gels were observed under UV light to assess degree of protein expression. Photographs were taken of the expression patterns on the stained gels. Results: The synapsins in the locust brain cells comigrated with the anti-synapsins through the gel for a distance of ~45, ~60 and ~77mm. It is construed that the three synapsins – I, II and III in that particular order - migrated through the gel for the corresponding distance of ~45mm, ~60mm and ~77mm. It is construed that synapsin I (~70kDa), synapsin II (~75kDa) and synapsin III (~80kDa) (Baumann et al, 2004) are evident in the gel plate but there is evidence of a fourth synapsin in arthropods like Drosophila that is much heavier at ~140kDa (Baumann et al, 2004). This fourth member is not evident in this gel study and it is also not clear from any published literature if this member is at all present in larger insects like the orthopterans and, more specifically in Locusta migratoria. The derived molecular weights do signify from present literature that these molecular units correspond to the three synapsins and there is also some indistinct evidence from the 1st and 2nd products, the synapsins I and II, that there are two bands instead of one. This may be evidence of the two isoforms each of these synapsins – Ia, Ib, IIa and IIb. Nevertheless, this cannot be stated clearly as such distinction requires more elaborate analysis that is not done in this study. Discussion: It is evident from the study that all three synapsins have been found in Locusta migratoria brain cell and it is also evident that the two isoforms each of the first two synapsins – Ia, Ib, IIa and IIb – are presently, though the last indistinctly so. It is notable that synapsin III, which is not manifested in large quantities in differentiated cells as it is necessary more for cell growth and differentiation than neurotransmission (Feng et al, 2002), is also evident in the L. migratoria brain cells, which are fully differentiated, and it is recommended that further more elaborate study be made to quantify this find and determine whether, really, synapsin III is present in the L. migratoria differentiated brain cells in similar quantities as the other two synapsins with their isoforms. In this context it is notable that not only synapsin III is required for cell differentiation and growth but the other synapsins, specifically synapsin IIa, are also similarly implicated (Kao et al, 2002). There is definite evidence that synapsin IIa has a phosphorylation site on one of its short amino-terminal domain, signified Domain A, that, when blocked by antibodies, inhibits neurite outgrowth in X. laevis embryonic neurons (Kao et al, 2002). This actually indicates that this specific phosphorylation site on the A domain is instrumental in neuronal development. It is also evidient that this phosphorylation process, Kao et al, 2002, believe and have proved, actually mediate the cAMP (cyclic amino butyric acid) pathway that actuates neurite outgrowth. The short A domain is a highly conserved one of 29-30 residues length and is evident in a wide range of animal synapsins, including the vertebrates. It seems that this domain is similar to the longer C domain (also amino-terminal one) that is also highly conserved across a wide range of species (Hosaka and Sudhof, 1998) and is the prime functional domain of tis group of neural phosphoproteins. Thus, it now does not seem strange that all three forms of synapsins was found in the differentiated L.migratoria brain cell. Conclusion: This study is considered important because the synapsins are highly implicated in neurotransmission, synaptogenesis and maintenance of mature synapses. There is also possibility that synapins like the IIa isoform, through initiation of selective phosphorylation, can assist in neuronal repair when necessary and such a therapeutic mean will become available to a wide range of species as the involved synapsin domain, the short amino-terminal A, is conserved in most such species. Yet it is admitted in this study that there is still a lot to know of the synapsins and their mechanisms of action, whether in neurotransmission, synaptogenesis or maintenance of mature synapses. It is also probable that synapsins are involved in some manner in the placement of neuron specific synapses, a process that is still unclear to this day. References: Baumann, Otto, et al, 2004, Dopaminergic and serotonergic innervation of cockroach salivary glands: distribution and morphology of synapses and release sites, Journal of Experimental Biology, 207, 2565-2575; 2004. Gitler, Daniel, et al, Molecular Determinants of Synapsin Targeting to Presynaptic Terminals, The Journal of Neuroscience, April 7, 2004, 24(14); 3711-3720. Hosaka, Masahiro, and Sudhof, Thomas C., Synapsins I and II are ATP-binding Proteins with Differential Regulation, Kao, Hung-The, et al, A protein kinase A-dependent molecular switch in synpasins regulates neurite outgrowth, Nature Neuroscience, Vol. 5; No. 5; May 2002. Keilland, Anders, et al, Synapsin Utilization Differs among Functional Classes of Synapses on Thalamocortical Cells, The Journal of Neuroscience, May 24, 2006, 26(21); 5786-5793. Lohr, Robert, et al, Compartmentalization of Central Neurons in Drosophila: A New Strategy of Mosaic Analysis Reveals Localization of Presynaptic Sites to Specific Segments of Neurites, The Journal of Neuroscience, December 1, 2002; 22(23); 10357-10367. Rodgers, Corinne I., et al, Photoperiod induced plasticity of thermosensitivity and acquired thermotolerance in Locusta migratoria, The Journal of Experimental Biology, 2006, 209; 4690-4700. Watson, Alan Hugh David, and Schurmann, Friedrich-Wilhelm, Synaptic Structure, Distribution and Circuitry in the Central Nervous System of the Locust and Related Insects, Microscopy Research and Technique, 56: 210-226; 2002. Read More