Interneuron dysfunction in amyotrophic lateral sclerosis

Despite more than a century of research, there is still no cure for amyotrophic lateral sclerosis (ALS) and the only available therapeutic extends survival by mere months. The most common motor neuron (MN) disease, ALS is traditionally characterised by selective degeneration of MNs and the systematic destruction of the motor system. However, in the last decade the classification of ALS is evolving from a pure MN disease to be considered instead a multi-system, non-cell autonomous and complex neurodegenerative disease. With new insights into the pathological mechanisms underlying ALS there is increased interest in the regulatory mechanisms that may compromise MN function, in particular those that may contribute to an excitatory and inhibitory imbalance in the disease. Indeed, there is great interest in determining the biological basis for increased cortical hyperexcitability and impaired inhibition identified in the motor cortex of both familial and sporadic ALS patients. It is proposed that altered motor network excitability may be a central pathogenic mechanism in the disease, possibly initiating the final progressive decline of motor neuron function. While intrinsic regulation of the MN is likely implicated in this pathophysiology, loss of inhibitory network function is presumably mediated by intra-cortical inhibitory interneurons; however, the exact cell types responsible are yet to be identified. As such, the intent of this thesis was to examine the role of key inhibitory neuronal populations in the cortex, as they are crucial for normal brain functioning and the balance of excitatory neurotransmission. The current thesis is based upon the hypothesis that the ALS pathogenesis involves cortical interneuron dysfunction‚ÄövÑvp. The current thesis examined the role of cortical interneurons in disease by first establishing a timeline of cortical interneuron involvement in the motor circuitry of the `SOD1^(G93A)` mouse model of ALS. The intent of this initial study was to determine which interneurons are involved in disease, and the time frame of their alteration relative to symptom-onset and motor neuron deficits. Subsequently, the validity of interneuron pathology was established in post-mortem ALS cases. An additional aim of this secondary study was to determine the relationship between interneuron pathology, cortical pathology and clinical characteristics. The final study investigated the potential vulnerability of interneuron populations using an in vitro approach, with electrophysiological techniques designed to explore the innate susceptibility of interneurons in the presence of the `SOD1^(G93A)` mutation. This thesis determined that specific interneuron populations were altered within the motor cortex of the `SOD1^(G93A)` mouse model of ALS. Moreover, a novel timeline of interneuron involvement was identified that included dynamic changes in the density of NPY- and CR-expressing interneuron populations throughout the disease course. Changes originated in the upper cortical layers of the motor cortex from early symptom onset, and progressed to involve the entire motor cortex by endstage. Interneurons were unaltered in the somatosensory cortex, nor other interneuron populations altered in either region, suggesting NPY and CR-interneurons represent a motor-specific inhibitory phenotype early in disease. Interestingly, pathology is found to change throughout disease, suggesting inhibitory involvement may not be a static phenomenon. The validity of interneuron involvement was subsequently investigated using post-mortem ALS cases. Comparing ALS cases and controls revealed changes in CR and NPY interneurons that largely recapitulated the interneuron pathology observed in the `SOD1^(G93A)` mouse model. NPY-pathology was clearly increased in all ALS cases examined, supporting a similar pathogenic process in the motor cortex of ALS patients and the `SOD1^(G93A)` mouse model. However, CR-interneuron pathology was recapitulated in a proportion of ALS cases, suggesting innate differences in the extent of interneuron involvement in individual cases. While no clear link was observed between interneuron pathology and clinical case characteristics, a positive correlation was demonstrated between heterogeneous CRinterneuron pathology, NPY-pathology and pyramidal pathology, which may provide a novel perspective on the neuronal basis of circuit dysfunction in the ALS motor cortex. In support of a role for inhibitory dysfunction in ALS, the examination of cortical interneurons in vitro identified that populations were innately susceptible to the SOD1 mutation, as demonstrated by the alteration of intrinsic electrophysiological properties and morphological development. Collectively, these studies provide strong evidence in support of the hypothesis that the ALS pathogenesis involves cortical interneuron dysfunction.‚ÄövÑvp Herein, evidence is provided for a dynamic and continual influence of interneurons throughout disease, which may include subtype specific dysfunction, initiated during early development, influencing disease-associated circuitry until the final stages of disease. These results highlight the non-cell autonomous nature of ALS, and suggest that further efforts should be made to understand the biological basis of inhibitory deficits in the disease. This will be essential for future efforts aimed at the restoration of normal excitability for the treatment and prevention of ALS, as the efficacy of treatment regimes will likely depend on the extent, type and timing of underlying dysfunction, and therefore pathophysiology, in the disease. Equally, it may also be of great therapeutic benefit to investigate the potential compensatory processes initiated, or contributed to, by these diverse cortical interneuron populations.