Modulation of neuronal differentiation by CD40 isoforms

Abstract

Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40(-/-) deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40(-/-) mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may represent important therapeutic modalities for neurodegenerative and neurodevelopmental disorders, as well as, for enhancement of neurogenesis.

Figure 1

Characterization of anti-N-terminal CD40 antibody (N-CD40 Ab) and anti-C-terminal CD40 antibody (C-CD40 Ab) in CD40 deficient (CD40^−/−) mice. (A, B) Immunofluorescent staining analysis was performed on vibratome sections of CD40^−/− mouse (left) or wild-type mouse (CD40^+/+, right) brains by C-CD40 Ab or N-CD40 Ab as indicated. N-CD40 Ab positive cells (green) are neurons for both CD40^−/− and CD40^+/+ mice. However, C-CD40 Ab positive cells (red) are neurons for CD40^+/+ mouse, but not for CD40^−/−mouse. (C) CD40 isoform expression in brain homogenates from both CD40^−/− and CD40^+/+ mice by Western blotting analysis as indicated.

Figure 2

CD40 isoform expressions in situ on neurons. Immunofluorescent staining analysis was performed in situ on neurons from mice at 1 day old and 1 month of age by N-CD40 Ab (A) or C-CD40 Ab (B) as indicated. Western blotting analysis of CD40 isoform expression in brain homogenates from mice at 1 day old and 1 month of age by N-CD40 Ab (C) or C-CD40 Ab (D), with n = 6 for each group.

Figure 2

CD40 isoform expressions in situ on neurons. Immunofluorescent staining analysis was performed in situ on neurons from mice at 1 day old and 1 month of age by N-CD40 Ab (A) or C-CD40 Ab (B) as indicated. Western blotting analysis of CD40 isoform expression in brain homogenates from mice at 1 day old and 1 month of age by N-CD40 Ab (C) or C-CD40 Ab (D), with n = 6 for each group.

Figure 3

CD40 isoforms are expressed by neuron-like N2a cells. (A) Immunofluorescent staining by N-CD40 Ab or C-CD40 Ab was performed on cultured N2a cells prior to and after differentiation as indicated. N-CD40 Ab positive cells (green) are neuron-like cells for both differentiated N2a cells (Diff.) and undifferentiated N2a cells (Undiff.). However, C-CD40 Ab positive cells (red) are neuron-like cells for Diff. N2a cells, but not for Undiff. N2a cells. Western blotting analysis of CD40 isoform expression in N2a cells prior to and after differentiation by N-CD40 Ab (B) or C-CD40 Ab (B), with n = 4 for each condition presented.

Figure 3

CD40 isoforms are expressed by neuron-like N2a cells. (A) Immunofluorescent staining by N-CD40 Ab or C-CD40 Ab was performed on cultured N2a cells prior to and after differentiation as indicated. N-CD40 Ab positive cells (green) are neuron-like cells for both differentiated N2a cells (Diff.) and undifferentiated N2a cells (Undiff.). However, C-CD40 Ab positive cells (red) are neuron-like cells for Diff. N2a cells, but not for Undiff. N2a cells. Western blotting analysis of CD40 isoform expression in N2a cells prior to and after differentiation by N-CD40 Ab (B) or C-CD40 Ab (B), with n = 4 for each condition presented.

Figure 4

Anti-CD40 type I siRNA inhibits CD40L-induced neuronal differentiation. (A) Expression of CD40 type I and inhibition by transfection anti-CD40 type I siRNA, but not non-targeting control anti-GFP siRNA (Control), were confirmed by immunochemistry staining analysis in differentiated (48 h) cultures of N2a cells. (B) The efficiency for siRNA knockdown of CD40 type I is confirmed by Western blot analysis. Densitometry analysis shows the band density ratio of CD40 to actin as indicated in the panel to the right. One-way ANOVA revealed significant differences between anti-CD40 type I siRNA-transfected cells and non-targeting siRNA control treated cultures on expression of CD40 type I to actin (**P > 0.001). Similar data were obtained in three independent experiments. (C) In addition, N2a cells maintained in differentiation media for 48 h displayed low levels of neuron specific NeuN (upper left panel), however upon pretreatment with CD40L expression of NeuN increases dramatically (upper right panel). Expression of NeuN was decreased by transfection with anti-CD40 type I siRNA in N2a cells (lower left panel), not non-targeting control siRNA (lower right panel).

Figure 4

Anti-CD40 type I siRNA inhibits CD40L-induced neuronal differentiation. (A) Expression of CD40 type I and inhibition by transfection anti-CD40 type I siRNA, but not non-targeting control anti-GFP siRNA (Control), were confirmed by immunochemistry staining analysis in differentiated (48 h) cultures of N2a cells. (B) The efficiency for siRNA knockdown of CD40 type I is confirmed by Western blot analysis. Densitometry analysis shows the band density ratio of CD40 to actin as indicated in the panel to the right. One-way ANOVA revealed significant differences between anti-CD40 type I siRNA-transfected cells and non-targeting siRNA control treated cultures on expression of CD40 type I to actin (**P > 0.001). Similar data were obtained in three independent experiments. (C) In addition, N2a cells maintained in differentiation media for 48 h displayed low levels of neuron specific NeuN (upper left panel), however upon pretreatment with CD40L expression of NeuN increases dramatically (upper right panel). Expression of NeuN was decreased by transfection with anti-CD40 type I siRNA in N2a cells (lower left panel), not non-targeting control siRNA (lower right panel).