Inhibition of Vascular Endothelial Growth Factor Receptor 2 Exacerbates Loss of Lower Motor Neurons and Axons during Experimental Autoimmune Encephalomyelitis

Abstract

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory demyelinating and neurodegenerative diseases in the central nervous system (CNS). It is believed that MS and EAE are initiated by autoreactive T lymphocytes that recognize myelin antigens; however, the mechanisms responsible for neurodegeneration in these diseases remain elusive. Data indicate that vascular endothelial growth factor A (VEGF-A) plays a role in the development of MS and EAE. Interestingly, VEGF-A is regarded as a neurotrophic factor in the CNS that promotes neuron survival and neurogenesis in various neurodegenerative diseases by activating VEGF receptor 2 (VEGFR2). In this study, we sought to explore the role of the VEGF-A/VEGFR2 signaling in neurodegeneration in MS and EAE. We showed that the expression of VEGF-A was decreased in the spinal cord during EAE and that VEGFR2 was activated in lower motor neurons in the spinal cord of EAE mice. Interestingly, we found that treatment with SU5416, a selective VEGFR2 inhibitor, starting after the onset of EAE clinical symptoms exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord of mice undergoing EAE, but did not alter Purkinje neuron loss in the cerebellum or upper motor neuron loss in the cerebral cortex. Moreover, SU5416 treatment had a minimal effect on EAE clinical symptoms as well as inflammation, demyelination, and oligodendrocyte loss in the lumbar spinal cord. These results imply the protective effects of the VEGF-A/VEGFR2 signaling on lower motor neurons and axons in the spinal cord in MS and EAE.

Neuron-specific PERK inactivation exacerbates neurodegeneration during experimental autoimmune encephalomyelitis

“…Frozen sections were cut using a cryostat at a thickness of 10 μm. The other half sagittal brain and the caudal half of the lumbar spinal cord (L3-L5) (39) were post fixed in 4% paraformaldehyde for 72 hours, dehydrated through graded alcohols, and embedded in paraffin wax. Paraffin sections were cut using a microtome at a thickness of 5 μm.…”

“…Paraffin sections were cut using a microtome at a thickness of 5 μm. For quantification of cells and axons in the lumbar spinal cords, we counted immunopositive cells or axons in an area of 0.1 mm 2 within the anterior funiculus medially next to the anterior median fissure in the lumbar spinal cord as described in our previous paper (29,30,38,39). For quantitative MBP IHC analysis, we calculated the percentage of the demyelinated area in the lumbar spinal cord by normalizing the demyelinated area against the total white matter area.…”

“…Next, we determined the effects of PERK inactivation on neuron loss in the CNS gray matter of these EAE mice. One of our previous studies demonstrated significant but moderate neuron loss in the CNS gray matter in the MOG-EAE model, including the lumbar spinal cord, primary motor cortex, and cerebellum (39). On the other hand, it has been demonstrated that the efficiency of Cre-mediated recombination in Thy1/ CreER T2 mice is varied in different regions of the CNS, ranging from 35% to 97% (42).…”

“…When young adult female C57BL/6J mice are immunized with MOG 35-55 peptide, the mice display neurological signs of disease starting around postimmunization day (PID) 12, reach the peak of disease around PID 19-26, and then gradually recover from EAE over time. Our previous studies demonstrated axon degeneration in the lumbar spinal cord and neuron loss in the CNS gray matter in the MOG-EAE model at the peak of disease (29,30,39). Therefore, we sought to determine activation of the PERK-mediated ISR in neurons in the MOG-EAE model.Although we have worked in the ER stress field for many years, we could not find any reliable anti-PERK antibody or anti-phosphorylated PERK antibody for Western blot or IHC.…”

“…Evidence suggests activation of the PERK-mediated ISR in neurons in the CNS of MS patients (26,36). We have used myelin oligodendrocyte glycoprotein (MOG) peptide 35-55-induced EAE in C57BL/6J mice (MOG-EAE model) to study the pathogenesis of MS and EAE for many years (28)(29)(30)(31)(37)(38)(39). When young adult female C57BL/6J mice are immunized with MOG 35-55 peptide, the mice display neurological signs of disease starting around postimmunization day (PID) 12, reach the peak of disease around PID 19-26, and then gradually recover from EAE over time.…”