Homeoprotein Neuroprotection of Embryonic Neuronal Cells

Stephanie E. Vargas Abonce; Mélanie Leboeuf; Alain Prochiantz; Kenneth L. Moya

doi:10.1523/ENEURO.0061-19.2019

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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.
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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.
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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.
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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.

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Table 1.

Statistical power analysis

Data structure		Type of test	Power
Trypan blue	Control vs H₂O₂	Post hoc t test, two-tailed	1.00
Trypan blue	H₂O₂ vs 12.5 nm	Post hoc t test, two-tailed	0.92
Figure 1A	H₂O₂ vs 12.5 nm	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs 2.5 nm		1.000
	H₂O₂ vs 25 pm		1.000
	H₂O₂ vs 12.5 pm		1.00
	H₂O₂ vs 2.5 pm		1.000
	H₂O₂ vs 1.25 nm		1.000
Figure 1B	H₂O₂ vs 12.5 nm	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs 2.5 nm	Dunnett’s multiple comparisons	1.00
Figure 1C	H₂O₂ vs 12.5 nm 1× frozen	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs 2.5 nm 1× frozen		1.00
	H₂O₂ vs 12.5 nm 5× frozen		1.00
	H₂O₂ vs 2.5 nm 5× frozen		1.000
	H₂O₂ vs 12.5 nm 4°C		1.000
	H₂O₂ vs 2.5 nm 4°C		1.000
Figure 1D	H₂O₂ vs 12.5 nm chEn2	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs 2.5 nm chEn2		1.000
	H₂O₂ vs 12.5 nm hEn1Q50A		1.000
	H₂O₂ vs 2.5 nm hEn1Q50A		1.000
	H₂O₂ vs 12.5 nm hEn2		1.000
	H₂O₂ vs 2.5 nm hEn2		1.000
Figure 2A	H₂O₂ vs 12.5 nm hEn1	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs 2.5 nm hEn1		1.000
	H₂O₂ vs 12.5 nm mOtx2		1.000
	H₂O₂ vs 2.5 nm mOtx2		1.000
	H₂O₂ vs 12.5 nm hGbx2		1.000
	H₂O₂ vs 2.5 nm hGbx2		1.000
	H₂O₂ vs 12.5 nm hLhx9		1.000
Figure 2B	H₂O₂ vs 12.5 nm hEn1	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs 2.5 nm hEn1		1.000
	H₂O₂ vs 12.5 nm mOtx2		1.000
	H₂O₂ vs 2.5 nm mOtx2		1.000
	H₂O₂ vs 12.5 nm hGbx2		1.000
	H₂O₂ vs 2.5 nm hGbx2		1.000
	H₂O₂ vs 12.5 nm hLhx9		1.000
	H₂O₂ vs 2.5 nm hLhx9		1.000
Figure 2C	H₂O₂ vs 12.5 nm hEn1	Dunnett’s multiple	1.000
	H₂O₂ vs 2.5 nm hEn1	comparisons	1.000
	H₂O₂ vs 12.5 nm hGbx2		1.000
	H₂O₂ vs 12.5 nm hLhx9		1.00
Figure 3B	H₂O₂ vs 2.5 nm mEn1	Dunnett’s multiple comparisons	1.00
	H₂O₂ vs 1.25 nm mEn1		1.000
	H₂O₂ vs 0.62 nm mEn1		1.00
	H₂O₂ vs 0.31 nm mEn1		1.00
	H₂O₂ vs 3.3 nm mOtx2		1.000
	H₂O₂ vs 1.65 nm mOtx2		1.00
	H₂O₂ vs 0.82 nm mOtx2		1.000
	H₂O₂ vs 0.41 nm mOtx2		1.00
	H₂O₂ vs 2.68 nm hGbx2		1.000
	H₂O₂ vs 1.34 nm hGbx2		1.000
	H₂O₂ vs 0.67 nm hGbx2		1.000
	H₂O₂ vs 0.33 nm hGbx2		1.000
	H₂O₂ vs 2.27 nm hLhx9		1.000
	H₂O₂ vs 1.14 nm hLhx9		1.000
	H₂O₂ vs 0.56 nm hLhx9		1.000
	H₂O₂ vs 0.28 nm hLhx9		1.000
Figure 3C	H₂O₂ vs stavudine	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs hEn1	Dunnett’s multiple comparisons	1.000
Figure 3D	H₂O₂ vs stavudine	Dunnett’s multiple comparisons	1.000
	H₂O₂ vs hEn1	Dunnett’s multiple comparisons	1.000
Figure 4A	−hEN1 vs +hEN1 at 300 μm 6-OHDA	Post hoc t test, two-tailed	1.000
	−hEN1 vs +hEN1 at 200 μm 6-OHDA	Post hoc t test, two-tailed	1.000
Figure 4B	−hEN1 vs +hEN1 at 10 μm 6-OHDA	Post hoc t test, two-tailed	1.000
	−hEN1 vs +hEN1 at 50 μm 6-OHDA	Post hoc t test, two-tailed	1.000