MODEL FOR HUMAN NEURODEGENERATIVE DISEASE
The inherent intricacies and neurological complexity of the human brain have hampered faster advances in understanding the molecular mechanisms behind NDs. Much of our current understanding of the molecular basis and the pathophysiology of NDs is offered by the studies in cultured cells and diverse animal models such as yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans), fly (Drosophila melanogaster) and mouse (Mus muluscus) which have been constructed over the years to try to mimic the pathogenesis of NDs. None have been totally successful as the models available so far do not reproduce the full constellation of changes that compose the characteristic neuropathological phenotype seen in human NDs, due to the unique complexities of the human brain. While studies on unicellular models such as yeast or bacteria, as well as immortalised cell lines, primary neuronal cultures have been carried out (Hall and Yao, 2005, Falkenburger and Schulz, 2006, Winderickx et al., 2008), these systems lack intercellular physiological pathways (e.g., neurotransmitter circuitry). In
21 contrast, vertebrate disease models offer in vivo opportunities and extensive similarity to the human brain. However, testing the therapeutic value of small molecules in mice and other mammalian model systems are expensive and require time-consuming experimental designs which can be prohibitive. Recent interests have turned to invertebrate models which bridge the gap between the simplicity of in vitro studies and the complexity of the human brain, and they allow for a more high-throughput and mechanistic approach. C. elegans, in particular, is a powerful model as it lacks many of the complexities of mammalian systems but retains the advantages of an intact organism. It excels in its simplicity, ability to reproduce asexually, fully sequenced genome, short generation time (≈ 3 days) and lifespan (≈ 3 weeks), considerable reproductive capacity (250-300 offspring), economy of testing susceptibility to RNA interference (RNAi), ability to self or cross- fertilise, distinctive behavioural and neuropathological defects, coupled with a surprisingly high degree of biochemical conservation.
Such experimental malleability is particularly advantageous, as C. elegans nervous system maturation and subsequent progressive stages of neuronal degeneration occur over a matter of days and can be assessed in large isogenic populations. The transparency of worms also facilitates the visualisation of any desired neurons and fluorescently-tagged proteins and assessment of the impact of pathogenic proteins within an intact animal in both temporal- and tissue-specific fashions. Recently, optogenetic techniques have been adapted to C. elegans, such that the neuronal activity can be measured by monitoring calcium levels and neurons can be activated by blue light stimulation of channelrhodopsin (Husson et al., 2013). The cell lineage
22 and location of each of the 959 somatic cells which constitute this organism, including the morphology and the complete wiring diagram of the physical and chemical connections between C. elegans’ 302 neurons (White et al., 1976, White et al., 1986, Hall and Russell, 1991), have been fully determined, thereby allowing the dissection of simple and complex behaviours at the level of individual cells and genes (Hobert et al., 2002). Together, the 302 neurons make approximately 5000 chemical synapses, 600 gap junctions, and 2000 neuromuscular junctions (White et al., 1986), thus conferring unparalleled precision and control in the identification and manipulation of neuronal cells (Pouya et al., 2011). Extensive characterisations of whole C. elegans genome have also revealed remarkable homology with the human genome and physiology, with about 70% of human disease-related genes having a clear C. elegans homologue (Kuwabara and O'Neil, 2001), including many that have been implicated in NDs (Table 1.1). Remarkable similarities exist at the molecular and cellular levels between nematodes and vertebrates neurons. For example, ion channels, receptors, classic neurotransmitters [acetylcholine, glutamate, γ- aminobutyric acid (GABA), serotonin, and dopamine (DA)] and vesicular transporters are similar in both structure and function between vertebrate and C. elegans (Hardaway et al., 2012). Physiological processes and signalling pathways such as formation, trafficking, and release of synaptic vesicles, and regulation of nervous system development by common guidance and polarity cues i.e. Netrin, Slt, Wnt, and Par protein complexes (Cáceres et al., 2012) are also highly conserved between the nematode and vertebrates. In addition, C. elegans lacks a vascular system which enables the studying of
23 neuronal damage independently of vascular damage (Duan and Sesti, 2013). C. elegans also mainly encodes a single functional orthologue of the corresponding human gene, thus bypassing the complications of extensive functional redundancy observed among many mammalian proteins to model and shed important light on the specific biological functions of any gene of interest.
Despite its relative neuronal simplicity, C. elegans has different neuronal classes that constitute its rudimentary nervous system, including interneurons, chemosensory, mechanosensory, and thermosensory types; 75 motor neurons innervate the body wall muscles (excluding the head); 56 of these are cholinergic and 19 are GABAergic (Teschendorf and Link, 2009). These neurons enable C. elegans to exhibit a repertoire of simple and well- characterised behaviours which can be experimentally assessed to study specific neuronal dysfunction and degeneration (Figure 1.1), as certain NDs confer differential vulnerability of specific neuronal populations which lead to distinct clinical phenotypes expression; and when combined with biophysical assays can also facilitate the examination of protein solubility in neurons of interest (Teixeira-Castro et al., 2011). Much debate has surrounded the mechanisms underlying age-dependent motor activity decline in C. elegans. Initial detailed examination of representative sensory and motor neurons have previously demonstrated that during neuronal ageing, there is little, if any, neuronal cell death and no signs of any neuronal degeneration despite advanced age and extensive behavioural decline. Instead, C. elegans senescence was attributed to widespread deterioration of surrounding somatic tissues which parallels sarcopenia in vertebrates (Herndon et al.,
24 2002). Recent reports have described progressive morphological changes in C. elegans neurons such as aberrant outgrowths and beading along neuron processes and age associated synaptic deterioration (Tank et al., 2011, Toth et al., 2012). Further functional analysis of the aging nervous system and muscles revealed that motor neurons, not body-wall muscles exhibit progressive functional deterioration, beginning in early life. Lifespan- extending mutations and pharmacological stimulation of synaptic transmission in the aging nervous system was also shown to slow down the rate of functional aging and potentiate locomotion activity in aged worms (Liu et al., 2013). In light of these observations, it is clear that the impact of different challenges i.e. genetic perturbations or exposure to drugs and various external stimuli on the survival and function of defined neuronal populations in C. elegans nervous system can be readily studied. For example, inhibitory GABAergic and excitatory cholinergic motor functions can be assessed by quantifying the motility (amplitude and frequency of body bends on solid surface or in liquid) and foraging behaviour of the worm, respectively. Behaviours such as reduced motility is a typical characteristic of neuromuscular disease and have already been automated to allow for higher throughput analysis (Helmcke et al., 2010). Motor, mechanosensory, chemosensory, DAergic and serotoninergic functions can be evaluated by measuring the pharyngeal pumping rate and behavioural changes in response to mechanical, osmotic, and chemical stimuli. In addition, the ability to generate primary C. elegans neuronal cultures can also expedite the functional analysis of neuron subtypes and allow direct assessment of their
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Figure 1.1: C. elegans nervous system marked by enhanced green fluorescent protein (EGFP) expression (driven by rab-3 promoter) in all worm neurons. Scale bar, 100 μm
26 responses to toxic insults. For all these reasons, C. elegans is a powerful model that can translate findings from basic behavioural and cognitive neuroscience into an improved understanding of NDs in humans, and to assist in expediting translational drug research and clinical testing for the development of new therapeutic interventions against NDs (Leung et al., 2008).