Investigating the roles of MARCKS and MARCKS-like 1 proteins in Xenopus laevis spinal cord development and regeneration
El Amri, Mohamed
El Amri, Mohamed
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Publication Date
2024-02-12
Type
Thesis
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Abstract
The identification and characterisation of key molecules that can promote or suppress regeneration has been an elusive, long-sought objective for researchers in spinal cord injury. The myristoylated alanine-rich C-kinase substrate (MARCKS) and MARCKS-like 1 (MARCKSL1) are two homologous proteins that are highly expressed in early developmental nervous tissue, with implications in gastrulation, embryogenesis, brain development, and myogenesis. Sharing conserved domains, both proteins act as primary substrates for protein kinase C (PKC), which enables them to dynamically regulate the actin cytoskeleton, membrane phosphoinositides, and other sub-cellular components. Previously, MARCKSL1 has been shown to promote appendage regeneration in other vertebrate models. Proteomic data from a former study in our lab also indicates that MARCKS and MARCKSL1 are differentially expressed after spinal cord injury (SCI) in regenerative and non-regenerative systems. Yet, there have been no functional studies exploring the roles of MARCKS and MARCKSL1 in spinal cord development and regeneration. This study reveals that marcks and marcksl1 are expressed in various tissues of Xenopus laevis throughout embryonic development, including the spinal cord. Genetic disruption of MARCKS and MARCKSL1 using both CRISPR/Cas9 and morpholino approaches results in a significant reduction in neurite outgrowth and mitotic and neural stem cell activity during spinal cord development, indicating that these proteins have essential and redundant functions during normal spinal cord development. Alternatively, mRNA overexpression of MARCKS and MARCKSL1 further enhances neurite outgrowth and cell proliferation. Pharmacological activation and inhibition of PKC, PIP2 and other signaling pathways in MARCKS and MARCKSL1 CRISPR mutants suggests that the proteins may modulate neurite outgrowth and cell proliferation by two different mechanisms. First, MARCKS and MARCKSL1 can promote cell proliferation and neurite outgrowth through a PIP2-dependent mechanism that is inhibited by PKC and, thus, probably involves unphosphorylated forms of MARCKS and MARCKSL1. Second, targets of PKC phosphorylation, which may include phosphorylated MARCKS and MARCKSL1 can promote cell proliferation and neurite outgrowth through additional mechanisms. This study also indicates that MARCKS and MARCKSL1 are upregulated at 5 days post spinal cord transection (DPT) in Xenopus laevis tadpoles. Here, higher levels of MARCKS and MARCKSL1 can be observed in the ependymal layer, white matter area, and meninges. These findings also demonstrate a general increase in cell proliferation at 2 DPT, followed by a gradual increase of Sox2+ neural progenitor cells along the ependyma, gradually filling the injury gap and posterior injury stump. Following CRISPR mediated knockdown of MARCKS and MARCKSL1, tadpoles show significant delays and deficiencies in behavioural recovery, injury gap closure, proliferative response, and stem cell activation, indicating that MARCKS and MARCKSL1 are required for these processes during spinal cord regeneration. A pharmacological study after spinal cord injury in MARCKS/MARCKSL1-mutant tadpoles indicates that Phospholipase D (PLD) activation significantly rescues regenerative outcomes in the absence of MARCKS and MARCKSL1, suggesting that these proteins may promote spinal cord regeneration via a PLD-dependent mechanism. Taken together, this study provides evidence for novel roles of MARCKS and MARCKSL1 for axon outgrowth and proliferation of neural progenitor cells during spinal cord development and suggests that these proteins are redeployed after spinal cord injury to recapitulate similar functions during spinal cord regeneration.
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NUI Galway