Inhibitory regulation of motor neurons : identifying new target mechanisms for motor neurons disease

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease and is characterised by the selective degeneration of both upper and lower motor neurons in the motor cortex, brain stem and spinal cord. Traditionally considered a pure motor neuron disease, increasing evidence highlights the overwhelming failure of cortical inhibitory interneurons to appropriately support motor neuron function and health in the ALS brain. The destabilisation of interneuron function and associated hyperexcitability of the motor cortex is one of the earliest pathophysiological events, however, little is known about the vulnerability of interneuron populations. Interneurons are a diverse group of neurons with distinct molecular and morphological features, therefore, this thesis focuses on two specific populations, the parvalbumin and neuropeptide Y (NPY) interneurons. Recent studies highlight the failure of parvalbumin interneurons to regulate inhibition in the ALS brain, while changes to the regulation of NPY interneurons progress with disease. This suggests that these two populations could be important therapeutic targets to improve support for motor neurons in ALS. Therefore, the overall aim of this thesis was to determine the structural and molecular changes to parvalbumin and NPY interneurons that contribute to disease, and to uncover new therapeutic targets for correcting failed inhibition in the ALS brain. The first aim of this thesis was to characterise parvalbumin interneuron pathology in the ALS motor cortex, and determine whether a structure specific to these cells, called the perineuronal net, underlies their vulnerability in this disease. The second aim was to determine if there are changes to NPY interneuron signaling in the motor cortex of an ALS model and in the post-mortem human ALS brain. Subsequently, the third aim focused on investigating the effects of exogenous NPY treatment on the excitability and health of ALS cortical neurons, to determine whether modifying NPY levels could be a viable approach to support cortical motor circuit function in ALS. Collectively, these works substantially expand on the knowledge of interneuron dysfunction in ALS and highlight how the vulnerability of select interneuron populations can significantly contribute to the ALS disease pathogenesis, but also how this may be harnessed to ameliorate disease.
For Aim 1, cortical tissue from male SOD1^G93A mice and C57BL/6 controls was collected at a presymptomatic, symptomatic, and late-symptomatic stage of disease, and stained for parvalbumin interneurons, perineuronal nets, and markers of GABAergic function and synaptic transmission. In Aim 2, post-mortem motor cortex brain tissue of ALS patients and healthy controls, and embryonic cortical neurons from YFPxSOD1 ^G93A mice and wild type YFP littermates were collected. In addition, the brain tissue of YFP-HxSOD1 ^G93A mice and YFP-H littermates were collected at a presymptomatic and late-symptomatic stage of disease.
These tissues were immunolabelled for postsynaptic NPY-Y1 receptor to compare the density of this receptor on upper motor neurons in ALS. To investigate the therapeutic potential of NPY in Aim 3, embryonic cortical neuron cultures from YFPxSOD1 ^G93A, YFPxTDP-43^A315T and YFP wild type littermates were treated with full-length NPY. Multi-electrode arrays were utilised to measure cortical network excitability, and western blotting and immunocytochemistry techniques were used to compare changes in the expression of proteins associated with NPY signaling, the intrinsic apoptotic pathway and changes to cellular morphology via measures of neurite complexity. In addition, patch clamp electrophysiology of male C57BL/6 cortical brain tissue was performed to measure the effect of NPY on the intrinsic excitability of layer 5 inhibitory and excitatory neurons.
Vulnerability of parvalbumin interneurons was evident in presymptomatic SOD1 ^G93A mice but not symptomatic disease stages. Increased inhibitory connections onto parvalbumin interneurons, reduced parvalbumin immunoreactivity, reduced perineuronal nets, reduced parvalbumin interneurons and reduced parvalbumin contacts to upper motor neurons were all observed in the motor cortex of presymptomatic SOD1 ^G93A mice. However, reduced parvalbumin interneuron and perineuronal net density was also observed in the SOD1 ^G93A motor cortex at a late-symptomatic stage of disease. In Aim 2, analysis of NPY-Y1 receptor density revealed increased density of these receptors localised to the soma of upper motor neurons in post-mortem ALS patient motor cortex and SOD1 ^G93A cortical excitatory neurons in vitro. Furthermore, the density of NPY-Y1 receptors was changed on the apical processes of SOD1 ^G93A upper motor neurons in presymptomatic and late-stage symptomatic motor cortex. In Aim 3, treatment with exogenous NPY in vitro reduced cortical network hyperexcitability of SOD1 ^G93A cortical neuron cultures and had minor effects on the neurite complexity of SOD1 ^G93A cortical neurons, but did not affect intrinsic apoptotic pathway activation or downstream NPY signaling pathways. Analysis of TDP-43^A315T cortical excitatory neurons showed no changes to the density of NPY-Y1 receptors compared to controls. Similarly, NPY had no effect on apoptosis or downstream NPY signalling pathways in the TDP-43^A315T model in vitro. In addition, investigations using patch clamp electrophysiology indicated that NPY modifies the intrinsic firing properties of inhibitory interneurons and pyramidal neurons to collectively suppress network excitability in the motor cortex.
The findings from this thesis demonstrate further evidence for interneuron vulnerability as an early event in the ALS disease course. Interestingly, the majority of changes to parvalbumin interneurons appeared to be absent after the onset of motor symptoms. This novel finding suggests that there is functional compensation occurring to substitute early interneuron dysfunction in the ALS brain. This thesis also demonstrates, for the first time, differential expression of a major NPY receptor involved in the control of neuronal excitability on motor neurons in both the ALS patient motor cortex and the SOD1 ^G93A animal model of ALS. Furthermore, investigations using NPY as a treatment demonstrate that broad activation of NPY pathways ameliorates hyperexcitability in the SOD1 ^G93A model of ALS, highlighting the capability to increase inhibitory pathways as a method to prevent pathogenic excitability in this disease. Together, this work provides evidence for several novel opportunities to target inhibition as a method for improving the health of cortical networks and further demonstrates the intricate pathophysiology of interneuron subpopulations in ALS