DARPP‐32 and Cdk5 Expression in the Ventral Cervical Spinal Cord of Rats

Abstract

Dopamine‐ and cAMP‐regulated phosphoprotein, 32 kDa (DARPP‐32) is a phosphatase that integrates complex neuronal information, including dopamine responsive neurons in the striatum. Differential regulation of DARPP‐32 phosphorylation is an important CNS mechanism integrating neuronal responses to concurrent activation by diverse neurochemicals, including neurotransmitters, neuromodulators and hormones. When Gs protein‐coupled metabotropic receptors activate adenylyl cyclase and increase cAMP levels, PKA activation phosphorylates DARPP‐32 at Threonine 34; in this state, DARPP‐32 inhibits protein phosphatase‐1 (PP‐1). Conversely, when DARPP‐32 is phosphorylated at Threonine 75 by Cyclin‐dependent kinase 5 (Cdk5), DARPP‐32 becomes a PKA inhibitor. Thus, DARPP‐32 is a dual‐function enzyme that inhibits PP‐1 or PKA, depending on prevailing conditions. Since PKA (Hoffman & Mitchell, Neuroscience, 2013) and PP‐1 (Huxtable et. al., J Appl. Physiol., 2018) are both key regulators of plasticity in the phrenic motor system, we performed preliminary studies to determine DARPP‐32 and Cdk5 expression in phrenic motor neurons or nearby cells. Since cervical spinal injury and/or intermittent hypoxia preconditioning trigger plasticity in the phrenic motor system, we explored if they change expression patterns of DARPP‐32 (n = 3) and/or Cdk5 (n = 15). We used immunofluorescence to detect DARPP‐32 and Cdk5 expression in the cervical spinal cords of rats that received intrapleural cholera toxin B subunit (CtB) injections to identify phrenic motor neurons. We studied intact and chronic (~8 week) C2 spinal hemisection (C2Hx) rats exposed to 4 weeks of normoxia or repetitive acute intermittent hypoxia (10, 5 min episodes of 10.5% O2; 5 min intervals; 4 days/week, 4 weeks; rAIH). Spinal cords were harvested, sectioned, and processed with antibodies for CtB and either DARPP‐32 or Cdk5 (C3‐C5; 40 μm). In all rats, DARPP‐32 was not expressed in motor nuclei, including phrenic motor neurons, or in the substantia gelatinosa of the dorsal spinal cord. Dense labeling was observed in distinct cells distributed throughout the medial grey matter; the size, shape, and distribution of these cells is consistent with tanycytes. Cdk5 expression was observed in many cell types throughout the grey matter, including phrenic motor neurons. Using a custom MATLAB algorithm, phrenic motor neuron somatic and total ventral horn expression was quantified; there were no significant effects of C2Hx or rAIH on staining intensity or distribution. In separate rats (n = 12), daily AIH for 28 days had no detectable effect on Cdk5 mRNA levels in ventral spinal cord homogenates, as assessed by qPCR (p=0.116). Thus, DARPP‐32 is not expressed in motor nuclei of the mid‐cervical spinal cord, making a role in phrenic motor plasticity unlikely; the role of DARPP‐32 in putative spinal tanycytes is unknown. Conversely, Cdk5 expression in phrenic motor neurons suggests potential for roles in plasticity or other motor neuron functions.Support or Funding InformationSupported by: University of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (LLA), NIH OT2OD023854 (SPARC; GSM), NIH R01 69064 (GSM), and the McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.