Apolipoprotein E binds to and potentiates the biological activity of ciliary neurotrophic factor

Abstract

Expression of apolipoprotein E (apoE) and ciliary neurotrophic factor (CNTF), a pleiotropic neuron survival factor, increases in the CNS in response to injury. Although CNTF is believed to act as a survival factor after injury in the CNS, the functions of apoE in the CNS remain mainly unknown. Similarities between apoE and CNTF, including coinciding patterns of postinjury expression, extracellular localization, homologous tertiary structure, and ability to form homodimers led us to examine the possibility that apoE and CNTF directly associate and thereby facilitate the neurotrophic activity of CNTF. We identified two binding interactions between apoE and CNTF: (1) reversible binding of both the apoE3 and apoE4 isoforms to CNTF under nondenaturing conditions, and (2) a higher avidity, SDS-stable binding of apoE3 with CNTF. Purified lipid-free apoE, as well as apoE in cerebrospinal fluid, binds CNTF. We demonstrate here that the survival-promoting activity of CNTF on cultured hippocampal neurons is potentiated by apoE. In the absence of apoE, survival of hippocampal neurons with 1 ng/ml CNTF was 20% above control survival values. In contrast, in the presence of apoE, survival of hippocampal neurons with 1 ng/ml CNTF was 40% above control survival values. These data, which indicate a novel function for apoE in the nervous system, support the hypothesis that apoE secreted locally at sites of injury can facilitate neural repair by promoting the activity of certain growth factors, in particular CNTF.

Fig. 1.

ApoE3 and apoE4 bind CNTF. Western blot of retained CNTF in the SCIB assay shows that CNTF binds to apoE3- or apoE4-coated microfuge tubes. CNTF was incubated in tubes precoated with apoE3 (lane 1) or apoE4 (lane 3) or in tubes with no apoE (lane 2). All tubes were blocked with BSA before CNTF was added. Unbound or weakly associated protein was removed by washing with 1 m NaCl; then bound proteins were stripped from the tubes with nonreducing Laemmli buffer, and the proteins were separated on a 10% SDS-polyacrylamide gel (nonreducing). The Western blot was probed with anti-CNTF antibody (A) and then stripped and reprobed with anti-apoE antibody (B). The positions of molecular weight standards are shown (right). Note that these were nonreduced samples, and protein mobility does not correlate precisely with molecular weights. The positions of the apoE monomer, apoE3 dimer, and CNTF monomer and dimer are shown (right, arrows).

Fig. 2.

Gel-shift assays demonstrate the SDS-stable apoE3/CNTF complex. A, CNTF (250 ng) and apoE3 or apoE4 (375 ng; equal molar amounts) were incubated alone or together, as indicated, in 50 μl of TBS at 37°C for 3 hr. Proteins were separated on a 7.5% SDS-polyacrylamide separating gel (nonreducing). The left three lanes of this Western blot were probed with an anti-apoE antibody while the right three lanes were probed with anti-CNTF antibody. The apoE3/CNTF complex is recognized by both antibodies, whereas apoE monomer and dimer are immunoreactive only with the anti-apoE antibody, and the CNTF multimers are recognized only by the anti-CNTF antibody. The samples were run on a 7.5% gel to resolve the apoE3/CNTF complex from the CNTF trimer. On a 7.5% gel the CNTF monomer is not resolved. The positions of molecular weight standards are indicated (right). Note that, under nonreducing conditions, protein mobility on SDS-PAGE may not correlate precisely with the molecular weights of proteins.B, Shown here, 500 ng of purified and lipid-free apoE3 or apoE4 and 500 ng of recombinant CNTF were incubated alone or in combination, as indicated. These samples were separated on a 10% SDS-polyacrylamide nonreducing gel. Western blots were probed with anti-apoE antibody. ApoE3, but not apoE4, forms a high-avidity complex with CNTF that resists denaturing.

Fig. 3.

Time course and specificity of apoE3/CNTF complex formation. ApoE3 (50 ng) and CNTF, LIF, NT3, or bFGF (50 ng) were incubated together in 40 μl of TBS for up to 4 hr. After the incubations were terminated at the indicated times, the samples were separated on a 10% SDS-polyacrylamide gel (nonreducing), and Western blots were probed with an anti-apoE antibody as described in Materials and Methods. ApoE/CNTF complex was detectable within 30 min and reached a maximum by 4 hr. In contrast, no complex was detected between apoE and the growth factors LIF, NT3, or bFGF.

Fig. 4.

ApoE3 in CSF forms a complex with CNTF. CSF samples with no detectable CNTF or apoE3 immunoreactivity at the molecular weight of the apoE/CNTF complex were used. A representative sample of CSF is shown. Ten microliters of CSF were incubated alone or with 500 ng of exogenous CNTF at 37°C for 3 hr. Gel-shift assay with anti-apoE showed the formation of an apoE3/CNTF complex. Note that the apoE3/CNTF complex is resolved as a doublet. This suggests that apoE3/CNTF complex formation is not affected by differences in post-translational sialation of apoE3.

Fig. 5.

Dose–response effect of CNTF on survival of hippocampal neurons in the presence and absence of either apoE3 or apoE4. A, The 96-well plates were preplated with 1 ng/ml of apoE3 or apoE4. Control plates had no apoE. Dissociated hippocampal cells were cultured in the presence or absence of increasing concentrations of CNTF (0.004–25 ng/ml) for 3 d. Survival was assayed at the end of the culture period by the MTS viability assay. The results from four independent experiments were pooled and are presented as the mean percentage of absorbance as compared with controls ± SEM (n = 4–5/experiment). The difference in maximum survival between apoE and no apoE conditions is significant at p < 0.0005, using the Student’st test for the differences between the means.B, C, Phase-contrast photomicrographs of hippocampal cultures after 3 d cultured in 40 pg/ml CNTF in the presence (B) or absence (C) of 1 ng/ml apoE3. Calibration bar, 50 μm.