Lack of GDAP1 Induces Neuronal Calcium and Mitochondrial Defects in a Knockout Mouse Model of Charcot-Marie-Tooth Neuropathy

Abstract

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca ²⁺ inflow through store-operated Ca ²⁺ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca ²⁺, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria–endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Author Summary

Charcot-Marie-Tooth (CMT) disease is an inherited motor and sensory peripheral neuropathy. Mutations in the GDAP1 gene cause either an axonapathy or an myelinopathy that can be transmitted recessively or dominantly to offspring. GDAP1 is located in the mitochondrial outer membrane and seems to participate in the mitochondrial network dynamics. To investigate the biological and functional consequences of lack of GDAP1 and to gain insight into the pathophysiology of the GDAP1-related neuropathies we have generated a Gdap1 knockout mouse. Characterization of this model revealed that the absence of GDAP1 induces a peripheral neuropathy with loss of motor neurons and abnormal neuromuscular junctions. We also observed defects in embryonic motor neurons and adult dorsal root ganglia sensory neurons derived from affected animals. Specifically, cultured motor neurons showed large and abnormal mitochondria, dilated perinuclear space and endoplasmic reticulum, changes in acetylation of cytoskeletal α-tubulin and calcium depletion. We propose that pathophysiology of GDAP1-associated recessive CMT neuropathy may be the consequence of abnormal calcium homeostasis and changes in the mitochondrial network biology and mitochondria–endoplasmic reticulum interactions. Our findings may be also relevant to understand the role of GDAP1 in relation to other neuropathy-related mitochondrial proteins such as mitofusin 2.