Does cortical pathology spread through the corticomotor system in ALS, and can we stop this spread?
Creators: name: Reale, Laura
Publisher: name: University of Tasmania
Year: 2024

description: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that involves the selective degeneration of both the upper and lower motor neurons of the corticomotor system. There are currently no effective therapeutic interventions available for ALS, and approximately 90-95% of ALS cases are seemingly sporadic with no known cause. The other 5-10% are attributed to heritable familial mutations, such as point mutations in the gene that encodes the RNA/DNA binding protein TDP-43. Although, it is unknown where within the corticomotor system pathology originates, or how it spreads through the system, the vast majority of sporadic and familial ALS cases are characterised by the presence of pathological TDP-43. This pathological TDP-43 is mislocalised out of the nucleus into the cytoplasm of upper and lower motor neurons, making this protein a major pathological hallmark of disease. Understanding how this protein causes network-wide dysfunction of the corticomotor system will be critical in devising effective therapeutic interventions to prevent ALS.<br>The research presented in this thesis, aimed to determine whether TDP-43-mediated pathology in the motor cortex was sufficient to cause corticomotor system neurodegeneration. Cortical hyperexcitability is consistently observed in people with ALS and appears to be an early event in disease, yet it is unknown how early changes in cortical excitability and pathological TDP-43 cause corticomotor system demise. Understanding whether cortical TDP-43 pathology can cause a spread of pathogenic changes throughout the corticomotor system, that is associated with early changes in cortical excitability, will fill a significant gap in the literature, and furthermore will be a vital link in designing therapeutic treatments aimed at stopping this spread.<br>Determining whether driving TDP-43 pathology in the motor cortex caused functional and excitatory changes in the cortex and widespread corticomotor system dysfunction, was the focus of the experiments described in chapter 3. We used a transgenic mouse line that expressed an inducible form of TDP-43 that was targeted to the cytoplasm by the conditional deletion of the nuclear localisation sequence from the gene (TDP-43ΔNLS). The resulting protein replicates the pathological cytoplasmic build-up of TDP-43 observed in ALS. TDP-43ΔNLS was expressed in excitatory forebrain neurons, under the CaMKIIα gene promoter. Twenty days of mislocalised TDP-43 expression was sufficient to cause layer V excitatory neurons in the motor cortex to become hyperexcitable. By 30 days, lower motor neuron number was significantly decreased in the lumbar spinal cord ventral horn, however, cell loss was selective, with a significant reduction in lumbar regions 1-3, but a sparing of lower motor neurons in lumbar regions 4-6. This reduction was associated with alterations in the expression of lumbar pre-synaptic excitatory and inhibitory proteins, where the number of excitatory synaptic puncta was increased in all lumbar regions, while the number of inhibitory synaptic puncta was increased in lumbar regions 4-6 only. These data reveal a differential regional vulnerability of lower motor neurons to cortical pathology. This vulnerability was likely due to an increase in the number of excitatory synapses to the spinal cord, driving excitotoxicity in lumbar regions 1-3 that was successfully offset by an increase in the number of inhibitory synapses in lumbar regions 4-6. This study provides evidence that TDP-43 mediated pathology, initiated in the cortex, spreads through corticofugal tracts and identifies a potential pathway for therapeutic intervention in ALS, involving the selective manipulation of cortical and spinal cord excitability.<br>Selectively targeting and modulating cortical excitability may be a beneficial therapeutic intervention in disease, therefore, chapter 4 aimed to do this by employing repetitive transcranial magnetic stimulation (rTMS). rTMS is a safe and non-invasive technique that uses electromagnetic induction to modulate neuronal excitability. rTMS was administered to the motor cortex or spinal cord of TDP-43ΔNLS mice as an intermittent theta burst stimulation (iTBS) to induce neuronal excitation, or a continuous theta burst stimulation (cTBS) to enhance neuronal inhibition, or no stimulation (sham). When compared with sham stimulation, 3.5 weeks of iTBS or cTBS delivery to the motor cortex did not alter the intrinsic electrophysiological properties of excitatory layer V pyramidal neurons. However, cTBS partially restored the expression of post-synaptic receptor subunits in the cortex. Neither iTBS or cTBS impacted rotarod performance, but cTBS improved hindlimb paralysis. When the treatment period was extended to 4.5 weeks, the maximum firing frequency of layer V pyramidal neurons was reduced following cTBS, which partially rescued cortical hyperexcitability in this model. When rTMS was instead applied to lumbar regions 1-3, 2 weeks of cTBS partially protected the number of lower motor neurons compared to sham or iTBS mice; but was insufficient to rescue motor function. These data suggest that if refined, rTMS may be a beneficial therapeutic intervention to protect against the dysfunction caused by mislocalised TDP-43, when applied judiciously at the correct time point and to the correct region.<br>To determine whether rTMS may also be effective in familial presentations of ALS, chapter 5 of this thesis delivered rTMS to mice carrying a familial TDP-43A315T variant. This protein is expressed prenatally; therefore, TDP-43 pathology is widespread through the corticomotor system prior to rTMS intervention. Delivering cortical iTBS for 5.5 weeks appeared to slow the rate of motor function decline compared to cTBS and sham mice, however, the effect of iTBS appeared to be transient. By end-stage disease, cTBS mice had more severe hindlimb paralysis relative to sham or iTBS mice, and a decreased survival rate relative to sham treated mice. rTMS also altered descending corticofugal inputs to lower motor neurons, as both iTBS and cTBS mice had fewer lower motor neurons compared to sham mice. This study suggests that rTMS may not be beneficial at later disease stages, and that an earlier intervention is more effective.<br>Excitability alterations in ALS are evolving, with periods of regional hyper- and hypo- excitability observed at different disease stages. Data in chapter 3 shed insight into early pathological changes that occur due to cortical mislocalised TDP-43 and reveal how disease may spread through the corticomotor system. This timeline of changes identified regional differences in vulnerability and highlights the need for tailored interventions to different regions of the corticomotor system. Data presented across chapter 4 and 5 suggest that rTMS can be used to modulate excitability, however, the timing, region and type of stimulation, need to be carefully considered for future clinical trials.