Abstract
ABSTRACTAltered cortical excitability and synapse dysfunction are early pathogenic events in amyotrophic lateral sclerosis (ALS) patients and animal models. Recent studies propose an important role for TAR DNA-binding protein 43 (TDP-43), the mislocalization and aggregation of which are key pathological features of ALS. However, the relationship between ALS-linked TDP-43 mutations, excitability and synaptic function is not fully understood. Here, we investigate the role of ALS-linked mutant TDP-43 in synapse formation by examining the morphological, immunocytochemical and excitability profile of transgenic mouse primary cortical pyramidal neurons that over-express human TDP-43A315T. In TDP-43A315T cortical neurons, dendritic spine density was significantly reduced compared to wild-type controls. TDP-43A315T over-expression increased the total levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropinionic acid (AMPA) glutamate receptor subunit GluR1, yet the localization of GluR1 to the dendritic spine was reduced. These postsynaptic changes were coupled with a decrease in the amount of the presynaptic marker synaptophysin that colocalized with dendritic spines. Interestingly, action potential generation was reduced in TDP-43A315T pyramidal neurons. This work reveals a crucial effect of the over-expression mutation TDP-43A315T on the formation of synaptic structures and the recruitment of GluR1 to the synaptic membrane. This pathogenic effect may be mediated by cytoplasmic mislocalization of TDP-43A315T. Loss of synaptic GluR1, and reduced excitability within pyramidal neurons, implicates hypoexcitability and attenuated synaptic function in the pathogenic decline of neuronal function in TDP-43-associated ALS. Further studies into the mechanisms underlying AMPA receptor-mediated excitability changes within the ALS cortical circuitry may yield novel therapeutic targets for treatment of this devastating disease.
Highlights
Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease
TDP-43A315T expression does not significantly affect cortical neuron dendrite or axon outgrowth, but reduces dendritic arbor complexity Synaptic loss is a major feature of neurodegenerative disorders, and to begin examining the effect of mutant TAR DNA-binding protein 43 (TDP-43) on excitability we first examined the effect of TDP-43A315T expression on the outgrowth of axons and dendrites, on which the pre- and postsynaptic compartments are located, respectively
Despite its lower overall presence, accumulations of synaptophysin were frequently observed in YFP:TDP-43A315T neurons (YFP):TDP-43A315T dendrites, and quantification of the size of synaptophysin-immunoreactive particles confirmed a significant increase in the area of synaptophysin puncta in the YFP:TDP-43A315T neuronal network in comparison to YFP:WT controls [F(1,74)=16.30, P=0.001, two-way ANOVA] with no change in NT-GluR1 particle area (Fig. 5F). This data indicates that despite increased overall levels of GluR1, TDP-43A315T causes a decrease in the localization of GluR1 to the synapse and accumulation of synaptophysin at the presynaptic site, potentially driving a reduction in excitability in TDP-43A315T cortical neurons
Summary
Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease. Similarities in pathological hallmarks and the clinical progression of both sporadic and familial forms of ALS have led to the suggestion of a commonality in the final neurodegenerative pathway In recent years, this theory has extended to observations of altered excitability. Clinical electrophysiological studies have identified the phenomenon of cortical hyperexcitability in sporadic and familial forms of ALS, preceding both the onset of clinical symptoms and measurable lower motor neuron dysfunction in patients (reviewed by Geevasinga et al, 2016). This suggests that imbalances in motor cortex excitation are one of the earliest pathological events in the disease (Fogarty, 2018). Animal model and human induced pluripotent stem cell (iPSC) studies indicate that excitability alterations in ALS are a complex and evolving sequence of events, potentially involving both hyperexcitability and hypoexcitability of various neuron and interneuron populations that make up the cortical circuitry and varying at different disease stages (Clark et al, 2015; Geevasinga et al, 2016; Leroy and Zytnicki, 2015; White et al, 2018)
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