Oligodendrocyte-specific ATF4 inactivation does not influence the development of EAE

PLP/Fv2E-PERK mice [22, 23], ATF4^loxP mice [28, 29], and CNP/Cre mice [30, 31] were on the C57BL/6J background. PLP/Fv2E-PERK mice were maintained by mating with C57BL/6J mice. ATF4^loxP mice were crossed with CNP/Cre mice, and the resulting offspring were further crossed with ATF4^loxP mice to obtain ATF4^loxP/loxP; CNP/Cre mice and ATF4^loxP/loxP mice. Genotypes were determined by PCR from DNA extracted from tail tips as described previously [22, 29, 30]. To determine the deletion of exons 2 and 3 of the Atf4 gene through Cre-Lox recombination in ATF4^loxP/loxP; CNP/Cre mice, genomic DNA was isolated from the indicated tissues and PCR was performed as described in previous papers [28, 29]. To activate Fv2E-PERK in the oligodendrocytes of PLP/Fv2E-PERK mice, the mice were given daily intraperitoneal (i.p.) injections of AP20187 (Ariad Pharmaceuticals, Cambridge, MA) as described in our previous paper [22]; controls were injected with vehicle (4% ethanol, 10% PEG-400, and 2.0% Tween-20 in water) only.

To induce EAE, adult female mice were injected subcutaneously in the flank and at the tail base with 200 μg of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide emulsified in complete Freund’s adjuvant (BD Biosciences, San Jose, CA, USA) supplemented with 600 μg of Mycobacterium tuberculosis (strain H37Ra; BD Biosciences). Two i.p. injections of 400 ng pertussis toxin (List Biological Laboratories, Denver, CO, USA) were given 0 and 48 h later. Clinical scores (0 = healthy, 1 = flaccid tail, 2 = ataxia and/or paresis of hindlimbs, 3 = paralysis of hindlimbs and/or paresis of forelimbs, 4 = tetraparalysis, 5 = moribund or death) were recorded daily as described in our previous papers [22, 31, 32].

All animal procedures were conducted in complete compliance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Minnesota.

Brains harvested from mice were rinsed in ice-cold PBS and were homogenized using a motorized homogenizer as previously described [31‐33]. After incubating on ice for 15 min, the extracts were cleared by centrifugation at 14,000 rpm for 30 min twice. The protein content of each extract was determined by DC Protein Assay (Bio-Rad Laboratories). The extracts (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose. The blots were incubated with a primary antibody against ATF4 (1:4000, Abcam, Cambridge, MA, RRID:AB_940373), CHOP (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, RRID:AB_783507), or β-actin (1:1000, Sigma-Aldrich, St. Louis, MO, RRID:AB_476694), followed by an HRP-conjugated secondary antibody, and, following incubation with the ECL Detection Reagents (GE Healthcare Biosciences, Pittsburgh, PA), the chemiluminescent signal was detected. The intensity of the recorded chemiluminescence signal was quantified using the ImageQuantTL software from GE Healthcare Life Sciences.

Anesthetized mice were perfused through the left cardiac ventricle with 4% paraformaldehyde in PBS. Brains were bisected in the sagittal plane. Both the upper (lumbar 1—lumbar 3) and the lower (lumbar 3–lumbar 5) regions of the lumbar spinal cord were carefully dissected from the vertebra as described in our previous paper [34]. One-half of brains and the spinal cord segments from the lumbar 3 to lumbar 5 were postfixed for at least 48 h in 4% paraformaldehyde in PBS, dehydrated through graded alcohols, and embedded in paraffin. Serial sections of 5 μm thickness were cut. The other half of brains and the spinal cord segments from the lumbar 3 to lumbar 1 were postfixed for 1 h in 4% paraformaldehyde in PBS, cryopreserved in 30% sucrose for 48 h, embedded in OCT compound, and frozen on dry ice. Frozen sections were cut in a cryostat at 10 μm thickness. Immunohistochemistry (IHC) for CC1 (APC7, 1:50; EMD Biosciences, Gibbstown, NJ, RRID:AB_2057371), myelin basic protein (MBP, 1:1000; Sternberger Monoclonals, Berkeley, CA, RRID:AB_10120129), CD3 (1:50; Santa Cruz Biotechnology, RRID:AB_627010), CD11b (1:50; Millipore, Temecula, CA, RRID:AB_92930), NeuN (1:500, Millipore, RRID:AB_2298772), phosphorylated neuro-filament-H (SMI31, 1:1000, Sternberger Monoclonals, RRID:AB_509995), non-phosphorylated neuro-filament-H (SMI32, 1:1000, Covance, San Diego, CA, USA, RRID:AB_509997), CHOP (1:100, Santa Cruz Biotechnology, RRID: AB_783507), calbindin 2 (1:400, Sigma-Aldrich, RRID:AB_476894), and aspartoacylase (ASPA, 1:1000, kindly provided by Dr. M.A. Aryan Namboodiri at Uniformed Services University of the Health Sciences, Bethesda, MD) were performed as described in our previous papers [22, 31, 32, 34]. Signals were detected using Fluorescein (Vector Laboratories, anti-rabbit, RRID:AB_2336197), Cy3 (Millipore, anti-mouse, RRID:AB_11213281, anti-rat, RRID:AB_90854), or enzyme-labeled secondary antibodies (Vector Laboratories, anti-rat, RRID:AB_2336202; anti-mouse, RRID:AB_2313581;, anti-rabbit, RRID:AB_92489). Fluorescent-stained sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and visualized with a Zeiss Axioskop 2 fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY).

To quantify the cells and axons in the white matter of the lumbar spinal cord, we counted immunopositive cells or axons within the anterior funiculus directly medial to the anterior median fissure in the lumbar spinal cord and confined to an area of 0.1 mm², as described in our previous articles [20, 22, 32, 34]. For quantitative MBP IHC analysis in EAE mice, we calculated the percentage of the demyelinated area in the lumbar spinal cord by normalizing the demyelinated area against the total white matter area. The total white matter area and the demyelinated area in the lumbar spinal cord were measured by NIH ImageJ software (http://​rsb.​info.​nih.​gov/​ij/​; RRID:SCR_003070) as described in our previous paper [20, 22, 32].

To quantify the lower motor neurons in the spinal cord segment from the lumbar 3 to lumbar 5, serial sections of 5 μm thickness were cut and every tenth section was immunostained with the NeuN antibody. The anterior horn of the spinal cord was selected for motor neuron counts. Only cells that had a visible nucleolus, the characteristic morphological features of an α-motor neuron, and a minimum diameter of 13.0 μm were counted using the NIH ImageJ software as described in our previous paper [34]. To quantify the Purkinje neurons in the cerebellum, 5 μm thick sagittal brain sections were cut and every tenth section in the series spanning from Bregma lateral 0.12 mm to 0.36 mm were immunostained with the calbindin 2 antibody. Calbindin 2 positive cells were counted in the lobules I/II, III, and IV of the anterior cerebellum as described in our previous paper [34]. To quantify neurons in the primary motor cortex, 5-μm-thick sagittal brain sections were cut and every tenth section in the series spanning from Bregma lateral 1.08 mm to 1.32 mm were immunostained with the NeuN antibody. NeuN-positive cells were counted in the layer V of the primary motor cortex as described in our previous paper [34].

Single-spleen cell suspensions were generated from EAE mice at post-immunization day (PID) 10. The cells were plated in 96-well microtiter plates, treated with MOG35–55 peptide (0, 1, 10, and 100 μg/ml), and incubated at 37 °C and 5% CO₂ as described in our previous papers [22, 31, 32]. After 48 h, 20 μl of BrdU labeling solution (Millipore) was added to the culture media for 24 h. Cell proliferation was determined using the Colorimetric BrdU Cell Proliferation kit (Millipore) according to the manufacturer’s instructions. We quantified the cytokines in the culture supernatants using the ELISA kits (Thermo Scientific) according to the manufacturer’s instructions. For the T cell viability assay, the spleen cells treated with MOG35–55 peptide were incubated at 37 °C and 5% CO₂ for 72 h. Cell viability was determined by the MTT assay kit (Progema) according to the manufacturer’s instructions.

The sample size for each individual experiment is listed in the corresponding figure legend. For experiments that determine the effects of ATF4 inactivation on oligodendrocytes under normal conditions, both male and female mice were used. For EAE experiments, only female mice were used due to the well-known sex differences in MS. EAE clinical score data are presented as mean ± SEM and are compared using a two-way ANOVA with a Sidak’s multiple-comparisons test using GraphPad Prism 6 Software (RRID:SCR_002798). All other data are presented as mean ± SD. For quantitative analyses, multiple comparisons were statistically evaluated by the one-way ANOVA with Tukey’s posttest using GraphPad Prism 6 Software. Comparison of two groups was statistically evaluated by t test using GraphPad Prism 6. P < 0.05 was considered significant.

Seven-week-old female PLP/Fv2E-PERK mice were immunized with MOG35-55 peptide to induce EAE, and then were given i.p. injections of 0.5 mg/kg AP20187 or vehicle daily starting on PID10. Our previous study has demonstrated that AP20187 treatment enhances PERK activation specifically in oligodendrocytes and reduces EAE disease severity and EAE-induced oligodendrocyte apoptosis, demyelination, and axon degeneration, without affecting the inflammatory response [22]. On the other hand, our recent study has demonstrated that the MOG-EAE model displays significant neuron loss in the CNS gray matter, including a significant reduction of lower motor neuron numbers in the lumbar spinal cord, a significant reduction of neuron numbers in the layer V of the primary motor cortex, and a significant reduction of Purkinje neuron numbers in the cerebellum [34]. Herein, we determined the effects of PERK activation in oligodendrocytes on neuron loss in the CNS gray matter during EAE. Quantitative NeuN IHC showed that AP20187 treatment significantly increased lower motor neuron numbers in the lumbar spinal cord (Fig. 1a–c) and neuron numbers in the layer V of the primary motor cortex (Fig. 1d–f) in PLP/Fv2E-PERK mice at the peak of disease, PID 21. Moreover, quantitative calbindin 2 IHC showed that AP20187 treatment significantly increased Purkinje neuron numbers in the cerebellum of PLP/Fv2E-PERK mice at PID 21 (Fig. 1g–i). Collectively, these data suggest that protection of oligodendrocytes resulting from PERK activation led to attenuation of neuron loss in the CNS gray matter of EAE mice.

ATF4 is the master transcription factor of the PERK-eIF2α pathway, which preserves cell viability and function during ER stress by stimulating the expression of numerous cytoprotective genes [12‐14]. It has been demonstrated that neither PERK activation in oligodendrocytes nor PERK inactivation in oligodendrocytes affects the viability or function of oligodendrocytes under normal conditions [21‐23]. Nevertheless, global ATF4-deficient mice have a complex phenotype that includes severe anemia, infertility, microphthalmia, growth retardation, and premature death [35, 36]. Therefore, we generated a mouse model that allowed for inactivation of ATF4 specifically in oligodendrocytes and determined the effects of ATF4 inactivation on oligodendrocytes under normal conditions.

ATF4^loxP mice were crossed with CNP/Cre mice, and the resulting progeny were further crossed with ATF4^loxP mice to obtain ATF4^loxP/loxP; CNP/Cre mice (ATF4 cKO mice) and ATF4^loxP/loxP mice (control mice). ATF4 cKO mice did not display any neurological phenotypes and were indistinguishable from littermate control mice. PCR analysis confirmed the deletion of exons 2 and 3 of the Atf4 gene selectively in the CNS and PNS of ATF4 cKO mice, but not in other organs of ATF4 cKO mice or any organs of control mice (Fig. 2a). Western blot analysis revealed the decreased level of ATF4 in the CNS of ATF4 cKO mice compared to control mice (Fig. 2b, c). IHC for CC1, a marker for oligodendrocytes, showed a comparable number of oligodendrocytes in the CNS of 10-week-old ATF4 cKO mice and control mice (Fig. 2d–f). MBP IHC showed that the degree of myelination in the CNS of 10-week-old ATF4 cKO mice was comparable to control mice (Fig. 2g–j). SMI-31 IHC revealed a comparable number of axons in the lumbar spinal cord of 10-week-old ATF4 cKO and control mice (Fig. 3a–c). NeuN IHC showed that ATF4 inactivation in oligodendrocytes did not significantly affect lower motor neuron numbers in the lumbar spinal cord (Fig. 3d–f) or neuron numbers in the layer V of the primary motor cortex (Fig. 3g–i) in 10-week-old mice. Moreover, calbindin 2 IHC showed a comparable number of Purkinje neurons in the cerebellum of 10-week-old ATF4 cKO mice and control mice (Fig. 3j–l). Therefore, these results suggest that ATF4 inactivation specifically in oligodendrocytes has a minimal effect on the CNS under normal conditions.

Recent studies have demonstrated the cytoprotective effects of the PERK-eIF2α pathway on oligodendrocytes during EAE [15, 18, 19]. Our previous study demonstrated that PERK activation specifically in oligodendrocytes attenuates axon degeneration during EAE [22]. Herein, we also showed that PERK activation specifically in oligodendrocytes reduced neuron loss during EAE. It is generally believed that the protective effects of the PERK-eIF2α pathway on cells are mediated by its master transcription factor ATF4 [12‐14]. Thus, we determined the involvement of ATF4 in the protective effects of PERK activation in oligodendrocytes in EAE using ATF4 cKO mice. Ten-week-old female ATF4 cKO mice and control mice were immunized with MOG35-55 peptide to induce EAE. As expected, control mice exhibited a typical EAE disease course (Fig. 4a). Surprisingly, ATF4 cKO mice displayed a comparable disease course as compared to control mice (Fig. 4a). Western blot analysis showed that the levels of ATF4 and CHOP were significantly increased in the CNS of control mice with EAE at PID21 compared to naïve control mice (Fig. 4b, c). Importantly, the levels of ATF4 and CHOP were significantly decreased in ATF4 cKO mice with EAE at PID21 compared to control mice with EAE (Fig. 4b, c). In accordance with previous studies [20‐23], double immunostaining for ASPA, a marker for oligodendrocytes [22, 37], and CHOP showed that the majority of ASPA-positive oligodendrocytes were positive for CHOP in the lumbar spinal cord of control mice with EAE at P21 (Fig. 4d). Nevertheless, none of the ASPA-positive oligodendrocytes were positive for CHOP in the lumbar spinal cord of ATF4 cKO with EAE at P21 (Fig. 4d). These data demonstrate that ATF4 is activated in oligodendrocytes during EAE and that ATF4 activity is abrogated in oligodendrocytes in ATF4 cKO mice with EAE. Collectively, these results suggest that ATF4 activation in oligodendrocytes does not influence EAE disease severity.

CNS tissues were prepared from these EAE mice at PID21. ASPA immunostaining revealed that there are a few remaining oligodendrocytes in the demyelinated lesions in the lumbar spinal cord of control mice. Unexpectedly, we found that ATF4 inactivation specifically in oligodendrocytes did not significantly change the number of the remaining oligodendrocyte in the demyelinated lesions of EAE mice (Fig. 5a–c). Quantitative analysis of MBP IHC showed that the percentage of area in the white matter of the lumbar spinal cord that was demyelinated in ATF4 cKO mice was comparable with control mice (Fig. 5d–f). Quantitative analysis of SMI-31 immunostaining showed that there was a comparable number of total axons in the demyelinated lesions of ATF4 cKO mice and control mice (Fig. 5g–i). Similarly, quantitative analysis of SMI-32 immunostaining showed that ATF4 inactivation specifically in oligodendrocytes did not significantly alter the number of degenerating axons in the demyelinated lesions of EAE mice (Fig. 5j–l). Moreover, CD3 immunostaining revealed a comparable number of CD3⁺ T cells in the demyelinated lesions in the lumbar spinal cord of ATF4 cKO mice and control mice (Fig. 6a–c). CD11b immunostaining also revealed a comparable number of CD11b⁺ microglia/macrophages in the demyelinated lesions in the lumbar spinal cord of ATF4 cKO mice and control mice (Fig. 6d–f). Taken together, these data suggest that ATF4 activation in oligodendrocytes has no effect on oligodendrocyte loss, demyelination, axon degeneration, and inflammation in the CNS white matter of EAE mice.

We further determined the potential effects of ATF4 inactivation in oligodendrocytes on neuron loss in CNS gray matter. Quantitative NeuN IHC showed that ATF4 inactivation specifically in oligodendrocytes did not significantly alter the number of lower motor neurons in the lumbar spinal cord (Fig. 7a–c) or the number of neurons in the layer V of the primary motor cortex (Fig. 7d–f) in EAE mice at PID 21. Quantitative calbindin 2 IHC also showed that there was a comparable number of Purkinje neurons in the cerebellum of ATF4 cKO mice and control mice (Fig. 7g–i). Moreover, CD3 immunostaining and CD11b immunostaining revealed comparable T cell infiltration and microglia/macrophage activation in the anterior horn of the spinal cord (Fig. 8a, b, g, h, i, n), the layer V of the primary motor cortex (Fig. 8c, d, g, j, k, n), and the molecular layer and internal granular layer of the cerebellum (Fig. 8e–g, l–n) in ATF4 cKO mice and control mice at PID21. Thus, these data suggest that ATF4 activation in oligodendrocytes has no effect on neuron loss and inflammation in the CNS gray matter of EAE mice.

As described above, PCR analysis showed that Atf4 knockout allele was undetectable in the spleen of ATF4 cKO mice. Next, we performed in vitro T cell recall assays to rule out the possibility that T cell priming was altered in ATF4 cKO mice during EAE. Spleen leukocytes generated from ATF4 cKO mice and control mice at PID10 were re-stimulated with MOG 35-55 peptides. As expected, MTT cell viability assay and BrdU cell proliferation assay showed that the abilities of T cells generated from ATF4 cKO mice to survive and proliferate were not significantly changed compared to those from control mice (Fig. 9a, b). Similarly, ELISA assays showed that the abilities of T cells generated from ATF4 cKO mice to produce the cytokines IFN-γ (Fig. 9c), IL-4 (Fig. 9d), and IL-17A (Fig. 9e) were not significantly changed compared to those from control mice. Thus, these data demonstrate that T cell priming is not altered in ATF4 cKO mice during EAE.