CASPASE-3-MEDIATED PROTEOLYSIS OF AMYLOID PRECURSOR PROTEIN AND THE PRODUCTION OF AMYLOID-BETA IN HUMAN ACUTE AND CHRONIC COMPRESSIVE MYELOPATHY

Introduction: This study aimed to analyse immunohis-tochemically the proteolysis of Amyloid Precursor Protein (APP) using Caspase-3-mediated APP proteolytic peptide (CMAP), beta-Amyloid (Aβ) and Active Caspase-3 in post-mortem human specimens in acute and chronic compressive myelopathy. Compressive myelopathy, occurring through traumatic fracture/dislocation of vertebrae, iatrogenic injury, cervical spondylotic myelopathy (CSM), or metastatic tumour, causes much socio-economic and emotional disability for patients as well as physical consequences. In such conditions, APP is recognised as an early and specifi c marker of axonal injury. The proteolysis of APP in both acute and chronic compressive myelopathy has not yet been described. Studies analysing axonal injury after brain trauma suggest a role for Caspase-3 in the cleavage of APP 1 . In addition, Caspase-3-mediated cleavage of APP has been found to be associated with the formation of Aβ, a neurotoxic protein thought to contribute to cell death in Alzheimer’s disease 2 . Furthermore, A? may subsequently encourage activation of Caspases −2, −3, and −6, the major effector molecules in apoptosis 2 . The current study addressed two hypotheses; that APP provides a substrate for the Caspase-3 enzyme, and, that this event is associated with Aβ production in the compressed spinal cord. Methods: Spinal cord material from 17 patients with documented SCI was analysed. The spatial distribution of cellular immunoreactivity was qualitatively assessed in injury due to trauma (n=5), iatrogenic event (n=1), CSM (n=6) and metastatic tumour (n=5). Morphological, immunohistochemical and immunofl uorescent techniques were used to investigate APP proteolysis. Results: Caspase-3, APP, CMAP and Aβ were present in anterior horn cells of the grey matter and axons of the white matter. An association was found between neuronal immunoreactivity and that of axons in motor tracts. Dual-immunolabelling revealed axonal co-localisation of CMAP with Aβ and Caspase-3 with Aβ. Although CMAP was present in axons which were immunoposi-tive for APP, an inverse relationship was found as each marker was limited to its own, distinct region, consistent with the theory that CMAP actively cleaves APP. In neurons, co-localisation occurred between Caspase-3 and Aβ, and CMAP with Aβ. No neuronal co-localisation was shown between CMAP and APP in the acute and chronic state. Discussion: Caspase-3 appears likely to contribute to the proteolytic cleavage of APP in compressive myelop-athy. CMAP was associated with the production of Aβ as demonstrated using single and dual immunolabelling. Furthermore, evidence is given for the association of Caspase-3 itself with the neurotoxic peptide, Aβ. It is possible that activation of Caspase-3 via these secondary mechanisms may trigger the advancement of the apoptotic cascade with the subsequent demise of the cell.