Cytoplasmic TDP-43 accumulation drives changes in C-bouton number and size in a mouse model of sporadic Amyotrophic Lateral Sclerosis

Abstract

An altered neuronal excitability of spinal motoneurones has consistently been implicated in Amyotrophic Lateral Sclerosis (ALS) leading to several investigations of synaptic input to these motoneurones. One such input that has repeatedly been shown to be affected is a population of large cholinergic synapses terminating mainly on the soma of the motoneurones referred to as C-boutons. Most research on these synapses during disease progression has used transgenic Superoxide Dismutase 1 (SOD1) mouse models of the disease which have not only produced conflicting findings, but also fail to recapitulate the key pathological feature seen in ALS; cytoplasmic accumulations of TAR DNA-binding protein 43 (TDP-43). Additionally, they fail to distinguish between slow and fast motoneurones, the latter of which have more C-boutons but are lost earlier in the disease.To circumvent these issues, we quantified the frequency and volume of C-boutons on traced soleus and gastrocnemius motoneurones, representing predominantly slow and fast motor pools respectively. Experiments were performed using the TDP-43ΔNLS mouse model that carries a transgenic construct of TDP-43 devoid of its nuclear localization signal, preventing its nuclear import. This results in the emergence of pathological TDP-43 inclusions in the cytoplasm, modelling the main pathology seen in this disorder, accompanied by a severe and lethal ALS phenotype.Our results confirmed changes in both the number and volume of C-boutons with a decrease in number on the more vulnerable, predominantly fast gastrocnemius motoneurones and an increase in number on the less vulnerable, predominantly slow soleus motoneurones. Importantly, these changes were only found in male mice. However, both sexes and motor pools showed a decrease in C-bouton volume. Our experiments confirm that cytoplasmic TDP-43 accumulation is sufficient to drive C-bouton changes.

“Peptidergic modulation of motor neuron output via CART signaling at C bouton synapses”

“…However, differences in ion channels that control the recruitment and firing rates of fast and slow motor neurons are beginning to be described and could contribute to their differential modulation 25,28 . Further, recent evidence points to greater C bouton innervation of motor neurons that innervate fast compared to slow twitch muscles; a difference that is most pronounced in male mice 63 , and is in line with behavioral deficits that we observe in CART knockout animals. Regarding CART receptors, recent studies have pinpointed the G-Protein-Coupled-Receptor (GPCR) Gpr160 as a putative receptor for the CART peptide, proposing that CARTp/Gpr160 signaling in the dorsal horn of the spinal cord mediates neuropathic pain through a c-fos and ERK/CREB pathway 64 .…”

“…This finding is perhaps not entirely unexpected, given that sex differences regarding CART expression during a stressful motor demanding task have been previously observed 61 . Also, male specific changes of the C bouton synapse have been observed in disease 62,63 . Although there are currently limited tools (conditional knock out mice, agonists, antagonists) available to study CART’s specific role in motor control, it is possible that this behavioral deficit might be due to the loss of CART in other areas of the CNS.…”

Synaptic Expression of TAR-DNA-Binding Protein 43 in the Mouse Spinal Cord Determined Using Super-Resolution Microscopy

“…This was apparent using both an anti-pTDP-43 antibody and an anti-TDP-43 aptamer, visualised with a range of high-resolution and super-resolution microscopy techniques. While there is considerable literature on the role of TDP-43 in synaptic function in health and disease (Bak et al, 2022;Dyer et al, 2021;Fogarty et al, 2016;Godena et al, 2011;Jiang et al, 2019), our findings offer substantial subsynaptic characterisation of TDP-43 that support the conclusion that TDP-43 may have direct implications on synaptic function. The clusters of TDP-43 at synapses appear to be diffraction limited in size -measuring 60nm in diameter using AD-PAINT, or just over 100 nm in diameter using g-STED microscopy.…”

“…It could be that TDP-43 pathology and synaptopathy in SOD1 mice occur through independent mechanisms, despite the fact that TDP-43 is present at synapses and that other findings from human post-mortem studies suggest a correlation between TDP-43 pathology and synaptic loss (Henstridge et al, 2018). Similarly, other synapse subtypes received by motor neurons are also subject to changes, including inhibitory glycinergic synapses (Allodi et al, 2021) and modulatory cholinergic synapses (Bak et al, 2022; Herron & Miles, 2012; Witts et al, 2014). The approaches used in this study would be highly suitable for identifying nanoscopic TDP-43-associated synaptopathy in other such synapse subtypes in ALS.…”

“…There is extensive literature on synaptic dysfunction associated with mutations in the SOD1 gene (Allodi et al, 2021;Avossa et al, 2006;Bączyk et al, 2020;Broadhead et al, 2022;Fogarty, 2019;Fogarty et al, 2015;Herron & Miles, 2012;Naumenko et al, 2011;Rei et al, 2020). Finally , structural and functional synaptic changes have been demonstrated in animal models with TDP-43 mutations (Bak et al, 2022;Dyer et al, 2021;Heyburn & Moussa, 2016;Jiang et al, 2019) and human iPSC neurons from patients with TDP-43 mutations (Devlin et al, 2015), while our findings, along with other published results, support synaptic roles for TDP-43 based on its presynaptic and postsynaptic expression (Johnson et al, 2009;Kao et al, 2015;Wong et al, 2022;Zacco et al, 2022). There is also evidence that misfolding of TDP-43 and FUS proteins may induce pathological misfolding of SOD1 through prion-like mechanisms (Pokrishevsky et al, 2016).…”