Calcium-binding proteins as markers and functional determinants of neurons in pain networks

The thesis focuses on the anatomical and cellular distribution of three EF-hand calcium binding proteins, secretagogin, neuronal calcium-binding protein 1 (NECAB1) and NECAB2 in dorsal root ganglia (DRGs) and spinal cord of three species, mouse, rat and human, and their possible roles in pathophysiological pain.

In Paper I and Paper IV, we report that the expression of secretagogin is limited to a small subpopulation of peptidergic neurons in mouse and human DRGs expressing calcitonin generelated peptide (CGRP). Secretagogin is present both in the cell bodies in the DRGs and in the central branches in lamina I of the dorsal horn and in peripheral branches together with CGRP; it thus centrifugally transported. The loss of secretagogin (a knockout mouse) does not affect the development of pain hypersensitivity after nerve injury or experimentally induced inflammation.

In Paper II, we demonstrate a wide expression of NECAB1/2 in many cell bodies in mouse DRGs and in cell bodies/nerve terminals with a wide distribution in different laminae in the spinal cord. NECAB2 is expressed in excitatory neurons in the spinal cord, showing a punctate staining and often co-localization with vesicular glutamate transporter 2 (VGLUT2) and synaptophysin. NECAB2 in DRGs is distinctly down regulated, at both mRNA and protein levels, by peripheral nerve injury.

In Paper III, we show a conserved excitatory property and laminar distribution of NECAB2 in mouse, rat and human spinal cord, while NECAB1 exhibits species diversity with regards to neurochemical properties in mouse and rat spinal cord. NECAB1 is present in oligodendrocytes surrounding axons in the white matter of the human spinal cord. We also reveal a differential expression of NECAB2, calbindin-D28k and calretinin in ependymal cells surrounding/within (human) the spinal central canal when comparing rodents and human.

In Paper IV, we characterize a NECAB2 population in mouse DRGs using a new NECAB2 antibody validated with help of a Necab2 knockout mouse. These NECAB2 neurons cover previously defined the C-low threshold mechanoreceptors (LTMRs) and Aδ D-hair LTMRs. Genetically induced loss of NECAB2 attenuates inflammatory but not neuropathic pain. This may, tentatively, be mediated by modulation of brain-derived neurotrophic factor (BDNF) expressed in DRGs, and through the interaction with its receptor tyrosine receptor kinase B (TrkB) in the spinal cord to modulate spinal glutamatergic neurotransmission.

List of scientific papers

I. Shi TJ, Xiang Q, Zhang MD, Tortoriello G, Hammarberg H, Mulder J, Fried K, Wagner L, Josephson A, Uhlén M, Harkany T, Hökfelt T. Secretagogin is expressed in sensory CGRP neurons and in spinal cord of mouse and complements other calcium-binding proteins, with a note on rat and human. Mol Pain. 2012 Oct 29;8:80.
https://doi.org/10.1186/1744-8069-8-80

II. Zhang MD, Tortoriello G, Hsueh B, Tomer R, Ye L, Mitsios N, Borgius L, Grant G, Kiehn O, Watanabe M, Uhlén M, Mulder J, Deisseroth K, Harkany T, Hökfelt T. Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury. Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):E1149-58.
https://doi.org/10.1073/pnas.1402318111

III. Zhang MD, Barde S, Szodorai E, Josephson A, Mitsios N, Watanabe M, Attems J, Lubec G, Kovács GG, Uhlén M, Mulder J, Harkany T, Hökfelt T. Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord. Brain Struct Funct. 2016 Sep;221(7):3803-23.
https://doi.org/10.1007/s00429-016-1191-3

IV. Zhang MD, Su J, Adori C, Cinquina C, Malenczyk K, Girach F, Peng CG, Ernfors P, Löw P, Borgius L, Kiehn O, Uhlén M, Mulder J, Harkany T, Hökfelt T. A role for the calcum binding protein NECAB2 in inflammatory pain in mouse. [Manuscript]