Homeoprotein Neuroprotection of Embryonic Neuronal Cells

Abstract

Most homeoprotein transcription factors have a highly conserved internalization domain used in intercellular transfer. Internalization of homeoproteins ENGRAILED1 or ENGRAILED2 promotes the survival of adult dopaminergic cells, whereas that of OTX2 protects adult retinal ganglion cells. Here we characterize the in vitro neuroprotective activity of several homeoproteins in response to H₂O₂ Protection is observed with ENGRAILED1, ENGRAILED2, OTX2, GBX2, and LHX9 on midbrain and striatal embryonic neurons, whereas cell-permeable c-MYC shows no protective effects. Therefore, five homeoproteins belonging to three different classes (ANTENNAPEDIA, PAIRED, and LIM) share the ability to protect embryonic neurons from midbrain and striatum. Because midbrain and striatal neurons do not express the same repertoire of the four proteins, a lack of neuronal specificity together with a general protective activity can be proposed. Interestingly, hEN1 and GBX2 provided protection to primary midbrain astrocytes but not to non-neural cells, including mouse embryo fibroblasts, macrophages or HeLa cells. For the four proteins, protection against cell death correlated with a reduction in the number of H₂O₂-induced DNA break foci in midbrain and striatal neurons. In conclusion, within the limit of the number of cell types and homeoproteins tested, homeoprotein protection against oxidative stress-induced DNA breaks and death is specific to neurons and astrocytes but shows no homeoprotein or neuronal type specificity.

Keywords: DNA damage; homeoprotein; neuroprotection; transcription factor.

Figure 1.

ENGRAILED protection of embryonic midbrain neurons. A, hEN1 dose-dependent survival of embryonic neurons after H₂O₂ oxidative stress. B, Preadsorption of hEN1 with an anti-ENGRAILED antibody abrogates hEN1 neuroprotection. C, hEN1 subjected to repeated freeze–thaw cycles (left) or maintained at 4°C for 6 weeks (right) has significant neuroprotective activity against oxidative stress. D, ENGRAILED internalization and high-affinity DNA binding are necessary for ENGRAILED neuroprotection. LDH assay was used for A–D. ns, nonsignificant, ****p < 0.0001.

Figure 2.

Homeoproteins protect embryonic neurons but not non-neuronal cells in the LDH assay. A, hEN1, mOTX2, GBX2, and hLHX9 protect embryonic ventral midbrain cells against H₂O₂ oxidative stress, while hMYC does not. B, hEN1, mOTX2, GBX2, and hLHX9 protect embryonic striatal neurons against H₂O₂ oxidative stress, while hMYC does not. C, hE1 and hGBX2 protect primary astrocytes against H2O₂ oxidative stress. D, hEN1, hGBX2, and hMYC do not protect fibroblasts against H₂O₂ oxidative stress. E, hEN1, hGBX2, and hMYC do not protect HeLa cells against H₂O₂ oxidative stress. F, hEN1, hGBX2, and hMYC do not protect macrophages against H₂O₂ oxidative stress. G, qRT-PCR reveals the expression of GBX2, LHX9, and OTX2 in embryonic striatum, and LHX9, OTX2, and EN1 in ventral midbrain. ns, nonsignificant, ****p < 0.0001.

Figure 3.

HPs reduce DNA breaks after H₂O₂. A, Cultures of E14.5 ventral midbrain neurons (red) untreated (control) show few bright γH2AX foci (green), while those treated with 100 μm H₂O₂ have numerous foci and those pretreated with mEN1 have only a few. B, Quantification of γH2AX foci. H₂O₂ increases the number of foci from ∼1-2 per neuron to ∼8. mEN1, mOTX2, hGBX2, and hLHX9 reduce the number of foci in a dose-dependent manner. C, D, Inhibition of reverse transcriptase activity protects against H₂O₂ oxidative stress in midbrain (C) and striatal neurons (D). In the control condition, few γH2AX foci are observed in embryonic midbrain neurons, while those challenged with 100 μm H₂O₂ show multiple DNA damaged foci. Pretreatment with 10 μm stavudine or 2.5 nm hEn1 completely blocks the formation of DNA damage foci. ****p < 0.0001.

Figure 4.

hEN1 protects immortalized human dopaminergic neuronal precursors, LUHMES cells, against H₂O₂ oxidative stress by LDH assay. A, Three hundred and 200 μm 6-OHDA reduce the number of LUHMES cells surviving, while 50 nm hEN1 completely protects against this oxidative stress. B, Quantification of γH2AX foci. 6-OHDA increases the number of foci by twofold to fourfold. Preincubation with hEN1 reduces the number of foci to the control level. ns, nonsignificant, ****p < 0.0001.