CCP1, a Tubulin Deglutamylase, Increases Survival of Rodent Spinal Cord Neurons following Glutamate-Induced Excitotoxicity

Abstract

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Post-translational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates post-translational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knockdown of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in C. elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knockdown of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries. Significance Statement Combining an in vitro model of the secondary phase of spinal cord injury with shRNA knockdown, we demonstrate that the deglutamylase CCP1 protects neurons from excitotoxic 4 death. Excitotoxicity plays a role in the secondary phase of neuronal injuries, contributing to neurodegeneration. CCP1 function was previously known to be associated with cilia. We provide the first demonstration (to our knowledge) that spinal cord interneurons are ciliated. However, our data suggest that neuroprotection by CCP1 may be independent of cilia in spinal neurons. Our work supports the idea that targeting enzymes that modify tubulins, such as glutamylases and deglutamylases, might be an avenue of treatment for nervous system injuries.

Glutamylation is a negative regulator of microtubule growth

“…, 2012 ). CCP1 also protects spinal cord neurons from excitotoxic injury in rats ( Ramadan et al. , 2021 ).…”

Tubulin Polyglutamylation by TTLL1 and TTLL7 Regulate Glutamate Concentration in the Mice Brain

“…Recently, the "glutamate-BCAA cycle" has been proposed; this mechanism not only supports the synthesis of glutamate, it also provides a complementary mechanism that supplements the action of the glutamate-glutamine cycle in the instantaneous "disposing" of synaptic glutamate [48]. Increased concentrations of glutamate can cause excitotoxicity in neurons, while the cytosolic carboxypeptidase 1 (CCP1) can catalyze deglutamylation to promote neuron survival [49]. Therefore, when Ttll knockout increased the glutamate, the increase of valine and leucine/isoleucine could support the idea of "disposing" of over-released glutamate in Ttll knockout mice brains.…”

Cypin Inhibition as a Therapeutic Approach to Treat Spinal Cord Injury–Induced Mechanical Pain

“…Spinal cord cultures were grown as we previously described (Du et al, 2007;Ramadan et al, 2021). Cells were plated on poly-D-lysine-(0.1mg/ml; Sigma-Aldrich Corp) coated glass coverslips in 24 well plates at a density of 100,000 cells/well.…”