Despite heterogeneity in the potential mechanisms of ALS (Bruijn et al 2004, Goodall & Morrison 2006, Rothstein 2009), current studies lend support to differences in excitatory and inhibitory receptor profiles as key determinants of MN vulnerability and potential for survival.
In ALS, not all MNs degenerate, rather there is spectrum of vulnerability in MN subtypes. Specific MNs are spared, such as the oculomotor and abducens populations responsible for preservation of eye movements in patients (Mannen et al 1977). Recent gene expression studies have explored the normal gene profiles of the subpopulations of MNs known to be either vulnerable or resilient to ALS (Brockington et al 2013). These studies have identified a number of factors that may contribute to differential vulnerability, including differences in genes implicated in ubiquitin-dependent proteolysis, mitochondrial function, immune system function and the extracellular matrix. However, the most striking differences were present in genes associated with functions in synaptic transmission, in particular genes responsible for the GABA and glutamate receptor subunits. In disease-resistant oculomotor neurons an upregulation of six different GABAA receptor units was identified compared to vulnerable spinal MNs (Brockington et al 2013). AMPA receptor subunits were also differentially altered with upregulation of GluR1 and GluR2 subunits. Indeed, the expression of GABAA receptor density ratios and synaptic and extrasynaptic GABAA receptors were altered in a separate gene study assessing differential features of ALS resistant
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MNs (oculomotor, trochlear and abducens) compared with vulnerable MNs (facial, hypoglossal and trigeminal nerve) (Lorenzo et al 2006, Wijesekera & Leigh 2009). There are a number of reasons why these differing receptor profiles may produce differential vulnerability in the ALS disease setting, as discussed below.
A baseline increase in GluR2 subunits in resilient MNs may maintain vital AMPA receptor calcium impermeability. The loss of the GluR2 subunit (Burnashev et al 1992, Lomeli et al 1994), causes AMPA receptors to become permeable to calcium (Van Damme et al 2002, Isaac et al 2007), resulting in increased burden on downstream cell signaling cascades (Rothstein 2009). In ALS, defects in the RNA-editing of the GluR2 subunit are identified in sporadic patients (Kawahara et al 2004) and genetically engineered mice lacking ADAR, the enzyme responsible for normal GluR2 editing, develop symptoms suggestive of MN degeneration (Hideyama et al 2010). Interestingly, ADAR2 has been identified as a site of TDP-43 misprocessing in sporadic ALS patient MNs (Yamashita & Kwak 2014), which may suggest a pathological convergence of the proposed RNA-editing dysfunction in ALS on this GluR2 subunit.
GABAA receptors subunit composition determines the localisation of the receptor as well as its distinct pharmacological and electrophysiological properties thus differential GABAA subunit expression will alter GABAergic receptor function (Sieghart & Sperk 2002, Sigel & Steinmann 2012, Labrakakis et al 2014). Furthermore an increase in GABAA receptors may allow for a greater extent of GABAergic influence and protection from altered neuronal activity such as excessive glutamate. This is supported by observations of reduced levels of the neurotransmitter GABA in ALS patients (Foerster et al 2012) and of the GABAA receptors alpha1-subunit in the motor cortex of patients (Petri et al 2003). Indeed, the hyperexcitability that develops in ALS (Eisen et al 1993) can be reversed by Diazepam, a prominent GABAA receptor agonist (Caramia et al 2000), indicating that loss of inhibitory input may be driving the hyperexcitability in this disease.
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THESIS HYPOTHESIS AND AIMS
Hyperexcitability and excitotoxicity are increasingly implicated in the pathogenesis of ALS, yet studies to investigate the pathological alterations to interneurons, a key regulator of excitability, are fragmentary. The great diversity of interneurons in the CNS has likely contributed to the failure to fully explore the role of this cell type in disease processes, with most studies focusing on pyramidal and motor neuron populations. There is accumulating evidence for a role of interneurons in ALS, however, this evidence is limited and a systematic investigation is required. This thesis aims to establish the role of cortical interneurons in ALS by firstly establishing a time course of interneuron pathology in the motor neuron circuitry of a well-established ALS mouse model. The validity of interneuron pathology is subsequently determined in human post-mortem ALS cases, examining neuronal pathology in key disease-associated cortical regions. Finally, the vulnerability of interneuronal populations is assessed in vitro in order to develop insight into the pathogenicity of interneurons in disease.
Aim 1: Determine cortical interneuron involvement in the SOD1G93A mouse model of ALS. Hypothesis 1: The inhibitory motor neuronal circuit is central to disease in ALS, evidenced by specific interneuronal pathology in the motor cortex.
Investigations focusing on interneuronal populations in the cortex are few and incomplete, despite increasing evidence of aberrant excitation in ALS. The first aim of this thesis will be to investigate if specific interneuron populations are altered in the cortex of the SOD1G93A mouse model of ALS. In addition, interneuron populations will be assessed at defined stages relative to disease progression, as less is known about the potential timing of inhibitory involvement in the disease. This will be completed by systematic assessment of specific interneuronal markers in cortical lamina utilising immunohistochemical and confocal microscopy techniques.
Aim 2: Establish if specific interneuron pathology is recapitulated in human post-mortem ALS cortical tissue. Hypothesis 2: Specific interneuron populations affected in the SOD1G93A
mouse are affected in ALS patients and are associated with the extent of cortical pathology.
Inhibitory circuit dysfunction during early hyperexcitability is an increasingly recognised event in the ALS patient cortex. Further, GABAergic dysfunction and glutamatergic overload occur concurrently in the ALS motor cortex. Hence the inhibitory circuit is likely implicated in hyperexcitability and excitotoxicity in ALS. However, a cellular basis for inhibitory alterations in the ALS cortex remains to be determined. Therefore, Aim 2 will determine if interneuron
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pathology identified in Aim 1 is present in post-mortem ALS cortical tissue. The extent of interneuron pathology will be established in individual cases and compared to cortical pathology after optimisation of relevant immunohistochemical markers.
Aim 3: Identify the effect of the SOD1G93A mutation on interneuron development in vitro. Hypothesis 3: The SOD1 mutation will affect the intrinsic function of interneurons and their normal development in vitro.
In ALS there is potential for early interneuron dysfunction to initiate, or respond to, changes in excitability. However, as interneurons are primarily considered an adaptive cell type, little is known about their innate vulnerability in disease. Despite studies that demonstrate motor neurons on an ALS genetic background have altered excitability and neurite development in vitro, it is unknown if interneurons are affected similarly. Therefore, using an in vitro primary culture model, Aim 3 will investigate the affect of the SOD1 mutation on normal interneuron function and neurite development, by examining intrinsic electrophysiological properties and neurite arborisation patterns.
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