I hold a PhD in the field of neuroscience from a UK University. I have a Master’s degree in Molecular Medicine from the same university and hold a B.Tech degree in Biotechnology. Throughout my academic career, I have been exposed to various fields related to the biological sciences including; immunology, animal biotechnology, neuroscience, molecular and cellular biology and have maintained my position in the top five percent of the class at each stage of my career.
The molecular basis of neurodegenerative disorders: proposing suitable methods for testing a given hypothesis
Hereditary Spastic Paraplegia (HSP) is a neurodegenerative disorder of the upper motor neurons where the degeneration of longer corticospinal motor neurons is observed towards the distal end. This observed pattern has been the subject of research in areas like axonal transport and protein trafficking mechanisms taking place in the neurons; disturbances of these functions may be the cause of the disorder (Corby and Proukakis, 2002). The peculiar degeneration of neurons with longer axons leads to the development of symptoms such as muscle spasticity and hyper reflexes in the lower limbs of the body (Fink, 2003). Numerous genes responsible for HSP have been mapped and the disorder is considered to be genetic, having all forms of inheritability patterns. One of the recently discovered causative defective genes is the atlastin on Chr 14 (Chr 14q11–q21) (Zhao et al., 2001).
Genetically defined HSPs are referred to by the term ‘Spastic Gait’ and the gene loci responsible are designated ‘SPG’ followed by a number. The SPG3A HSP refers to the autosomal dominant form of HSP that is a result of mutations in the atlastin gene. The disease, in this case, has an early onset (typically in children under ten years) and accounts for up to ten percent of the HSP cases (Fink, 2003).
The SPG3A gene, whose coding sequence is distributed along 14 axons, was mapped by Zhao et al. in 2001. The gene encodes a cytosolic protein, atlastin, of 558 amino acid residues and 63.5 KDa; it has three prominent conserved motifs – P loop, RD loop and the DxxG domain. This arrangement of the peptides confers the protein functioning and activities that are similar to the guanylate binding protein (GBP) thereby making it vulnerable for GTPase activity (Zhao et al., 2001). Though there is considerable homology between GBP 1 and atlastin, the differences in the active site (the presence of an RD loop in case of atlastin) has altered the functioning of the protein when compared to that of its homolog – GBP 1. The expression of atlastin is said to be predominant in the cortical area of the brain and the hippocampus. It is also expressed in other tissues of the body such as the adrenals, kidneys, testes, lungs and liver etc. (Zhu et al., 2003).
The molecular functioning and properties of atlastin were elucidated by Zhu and his group (Zhu et al., 2003). Various protein interaction studies and localisation of the patterns of atlastin indicate its relationship with the Golgi apparatus. The presence of both N terminal and C terminal (unlike the GTPase family of proteins) has lead researchers to hypothesise that atlastin may be an integral transmembrane protein (Zhu et al., 2003). Studies of the localisation of atlastin have pointed out its association with the Golgi, ER, neurites, Dynamin etc. indicating that there is strong evidence that the protein might be involved in the trafficking and processing of the proteins that are mediated between the Endoplasmic Reticulum (ER) and the Golgi for transportation, i.e. transport across short distances in the cell body (Zhu et al., 2003; Namekawa et al., 2007).
Endoplasmic Reticulum (ER) and the Golgi Apparatus
The ER – Golgi uses a sophisticated system for its trafficking process. Numerous components like actin, microtubule (Apodaca, 2001), dynactin, dynein (LaMonte et al., 2002), etc. tend to play a role in the process. The Golgi apparatus is the integral packaging unit of a cell that targets macromolecules (especially proteins synthesised by the Rough Endoplasmic Reticulum (RER)) for modification, intracellular and intercellular transport. The Golgi performs this function by compartmentalising the proteins in vesicles and transporting them to target destinations. The enzymes in the Golgi ensure that this trafficking is smooth and continuous. Vesicle coat proteins (COPI, COPII and clathrin) coat the vesicles containing proteins and regulate them through a protein sorting mechanism. An active component of the vesicle trafficking system is the GTPases which are known for the budding/fusion of vesicles from the Golgi ribbons. The Golgi responds to ER proteins by transporting them in both anterograde and retrograde directions (Pelham, 2001).
Golgi, ER and Atlastin
In neurons, this ER – Golgi trafficking is important in the transport of neurotrophic factors and the transmission of impulses along the axons: hence a disturbance in the trafficking cascade may lead to the impairment of other related cellular processes including axon growth, maintenance and development. It has a relationship with another protein that is involved in axonal transport and maintenance: spastin. Researchers have speculated about its role and the molecular mechanisms involved in the peculiar degeneration of longer axons in HSP (Sanderson et al., 2005; Evans et al., 2006; Zhu et al., 2006). Spastin (SPG4 – autosomal dominant HSP) is another protein that plays an important role in regulating the microtubules which are the pathways for transportation of cellular components between the cell body and the axon (Svenson et al., 2001).
Namekawa and co-workers confirmed the relationship of atlastin with the Golgi by performing immunoprecipitation reactions with vesicle family proteins. Mutant atlastin was found to be the cause of improper Golgi morphology and impaired budding of vesicles, leading to a disorganised trafficking process (Namekawa et al., 2007). This evidence supports the notion that atlastin, with its expression and localisation patterns, is indeed a protein that interacts/interferes with the neurons of the ER – Golgi by regulating intracellular trafficking. Mutations in the protein may result in defects in the Golgi apparatus and its function, culminating in the loss of maintenance of axonal growth and development (Duncan and Goldstein, 2006; Evans et al., 2006; Zhu et al., 2006). Currently, nine mutations have been reported to be responsible for this autosomal dominant form of HSP (Zhao et al., 2001; Rainier et al., 2006) resulting from this gene. The given hypothesis studies and correlates this finding with the aim of elucidating the possible molecular basis of the disease with respect to atlastin.
Verification of the given hypothesis can initially be done using an in vitro model to identify the characteristic features which result from the different mutations. This can be done as follows:
Mutations in the case of the atlastin gene could be of two types with respect to the disease phenotypes that have been suggested in the hypothesis:
- Loss of function mutation or ‘amorphic mutation’ may be the result of haploinsufficiency of the gene where the amount of normal gene product is incapable of coping with the loss of the product resulting from the mutant allele. This loss of function can be assayed by tracking the trafficking capabilities of the Golgi and ER in the cytoplasm, using suitable markers.
- Dominant negative mutation or ‘anti-morphic mutations’ are mutations where the loss of function of the gene is due the disruptions caused in the gene product, thereby impairing its further activities in the cell which ultimately leads to the malfunctioning of a cascade process in certain cases.
In vitro analysis by the use of RNAi
In order to examine the possible effects of the mutation, an RNAi approach in neuronal cell lines would prove to be an initial step. Short interfering RNAs (siRNA) can be introduced, using suitable plasmid, into neuronal cell lines such as the NSC-34 to study the gene silencing effects of atlastin in the cells (Zhu et al., 2006). Utilising suitable fluorescent tagged markers for the Golgi apparatus, the effect of the vesicle transport system can be deduced with this model. Also, staining procedures like the Golgi staining technique can also be employed for analysis of the silenced gene’s function (Penny and McCabe, 2005). In addition to this, dominant negative vector constructs comprising all three of the mutant forms of atlastin can be made to express and study their cellular functioning in a similar way (Evans et al., 2006). These data may provide evidence for the dominant negative effect of the gene on the cellular functioning (Gilchrist et al., 1999). Likewise, the cell lines can be used for rescue experiments by using a chemically inducible promoter expressing atlastin in the form of a bicistronic plasmid along with the above mentioned siRNA approach. Alternatively, the use of lentiviral vectors/adeno-associated viral vectors for examining the effect of rescue experiments would be feasible (Pirozzi et al., 2006).
In vivo models and suitable experimental approaches
Animal models for the HSP disease are just beginning to appear. To assess the detrimental effects of atlastin in an in vivo model, it is a primary requisite that a model mimics the human disease as closely as possible. Orso et al, generated a drosophila model for studying HSP that occurs due to mutations in the spastin gene. The developed model is a knockout for spastin that uses the UAS spastin system to silence the expression of the gene in the nerves of the drosophila (Orso et al., 2005).
Drosophila knockout models, as in the above cases, are generally created using an Upstream Activating Sequence – a foreign gene system that allows the gene to be expressed in specific tissues thereby allowing the studies of the disease to be similar to what they would be in humans. The designed model is assessed for the HSP-like symptoms, i.e. retrograde degeneration of long motor neurons, by evaluating the locomotion abilities of the fly. Orso and his co-workers devised a way to do this by examining the upward locomotor capability of the flies, the process of which relates directly to the use of the motor neurons (Penny and McCabe, 2005; Orso et al., 2005).
Thus this model would provide insight into the effect of silencing the gene in an in vivo state. Since atlastin also has a fly homolog in its genome, the same concept can be used for the generation of an SPG3A mutant drosophila model. The dominant negative effect of the atlastin knockout can be confirmed by the analysis of neurons. This can be done by staining for the Golgi using Golgi staining and/or silver nitrate staining. Fluorescent markers specific to components of ER – Golgi trafficking pathways, vis-á-vis ER, ribosomes (Rolls et al., 2002), dynamins, GBP-1, ESCRT I / II (Gill et al., 2007; Filimonenko et al., 2007), etc. can also be used to analyse any internal disruptions that may have arisen due to the knockout, resulting in the disease-like symptoms if the model works, in any way, as hypothesised.
In order to check for the haploinsufficiency phenomenon that may also be the underlying cause of HSP due to SPG3A, transgenic drosophila, which has a heterozygous allele for atlastin, has to be created. This haploinsufficiency model would enable us to study and verify the effects of the mutant gene.
Therefore, the experimental approach for assessing this hypothesis must be categorised in the following three broad categories:
- Wild type (+/+): This will be the homozygous phenotype which has the normal atlastin.
- Haploinsufficient (+/-): The phenotypes generated must be heterozygous for all the three mutations that have been found in atlastin.
- Mutant (-/-): The mutant created should be homozygous for the three mutant forms of atlastin.
By analysing the models using markers for specific proteins such as SNARE, GM130 (Golgi Matrix protein) (Marra et al., 2006), Multivesicular Body (MVBs) proteins, which are routinely processed and transported to local sites in the neurons, provide an indirect measure of the healthy and stable ER-Golgi trafficking process in the cell. Then again, by performing immunoprecipitation reactions with the vesicle family proteins p24/emp/gp25L, which are suitable markers for vesicle formation and budding, we should also be able to evaluate the mechanism which possibly causes the onset of the disease in the model (Namekawa et al., 2007).
Alternative animal models
Though the drosophila model proposed above might prove to be a suitable model for examining the hypothesis, alternative animal models, such as the transgenic mice and C. elegans would also be suitable for the study.
Paraplegin knockout mice have been produced to model the HSP resulting from the SPG7 gene mutations (Pirozzi et al., 2006). SPG3A mutants and haploinsufficient strains could be generated in the same way for modelling the loss of function of atlastin. The assessment of the disease model would be easier than that of drosophila since the motor functions of the animal can be studied using various standard techniques such as:
- Rotarod Tests
- Grip Strength Analysis
- Right Reflex Assessment, etc (adapted from Devon et al., 2006).
An analysis of haploinsufficiency has been possible by using mouse models. A recent research for determining Glut-1 haploinsufficiency using mice could be used to confirm that the same approach is possible to unravel the given hypothesis (Wang et al., 2006). The dominant negative theory could be verified in this in vivo study using a specific RNAi strategy as done by Maeda (Maeda et al., 2005).
In addition to the proposed drosophila and mice models, C. elegans could be also be used initially for the purpose of this research. The C. elegans model provides a live imaging platform for the neurodevelopmental studies (Rolls et al., 2002) and any developmental defects with respect to the mutations in the atlastin can be assayed via this model. The use of GFP tagged neuronal proteins for the studies on developments of the nervous system in the worm has become a widely used protocol.
A possible approach for performing this study using C. elegans, would be to use different fluorescent dyes and/or proteins in the following context –
- A GFP tagged atlastin in its three forms – wild type, haploinsufficient and mutant(s) – could be used to study the expression and localisation pattern in the worm which will provide evidence of the phenotypes being successfully generated.
- Another fluorescent protein tag, such as RFP, can be used to tag the Golgi complex and a co-localisation analysis with the GFP – atlastin will give out the in vivo and biological aspect of the mutation (adapted from Rolls et al., 2002).
- Does it lead to a neurodevelopmental disorder?
- Does it result in a haploinsufficient phenotype where there is a reduced expression of the normal gene affecting the overall cellular mechanisms?
- Has there been impairment in the cellular functions of atlastin, etc.?
The dominant negative phenotype can be confirmed using the above options in a similar manner to that used by Corsi (Corsi et al., 2002) to clarify the dominant negative effects of Twist protein. Zebrafish would also provide a similar platform to monitor the developmental changes, but C. elegans would be a simpler option as only drosophila and transgenic mice would be preferred for studying HSP.
The given hypothesis seems to be appropriate with reference to the research being carried out in the context of atlastin. Until now elucidating the molecular mechanisms, protein interactions and cellular functions of atlastin has been the main focus of the research.
Furthermore animal models for HSP have been difficult to generate and if they are generated, studying a complex human disorder such as HSP has always been a major task. The models and methodologies proposed above to develop the given hypothesis are the reflections of potential experimentation procedures that have been used under similar circumstances and thereby can be used with the given context.
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