Modulating Golgi Stress Signaling Ameliorates Cell Morphological Phenotypes Induced by CHMP2B with Frontotemporal Dementia-Associated p.Asp148Tyr

Abstract

Some charged multivesicular body protein 2B (CHMP2B) mutations are associated with autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 7 (FTDALS7). The main aim of this study is to clarify the relationship between the expression of mutated CHMP2B protein displaying FTD symptoms and defective neuronal differentiation. First, we illustrate that the expression of CHMP2B with the Asp148Tyr (D148Y) mutation, which preferentially displays FTD phenotypes, blunts neurite process elongation in rat primary cortical neurons. Similar results were observed in the N1E-115 cell line, a model that undergoes neurite elongation. Second, these effects were also accompanied by changes in neuronal differentiation marker protein expression. Third, wild-type CHMP2B protein was indeed localized in the endosomal sorting complexes required to transport (ESCRT)-like structures throughout the cytoplasm. In contrast, CHMP2B with the D148Y mutation exhibited aggregation-like structures and accumulated in the Golgi body. Fourth, among currently known Golgi stress regulators, the expression levels of Hsp47, which has protective effects on the Golgi body, were decreased in cells expressing CHMP2B with the D148Y mutation. Fifth, Arf4, another Golgi stress-signaling molecule, was increased in mutant-expressing cells. Finally, when transfecting Hsp47 or knocking down Arf4 with small interfering (si)RNA, cellular phenotypes in mutant-expressing cells were recovered. These results suggest that CHMP2B with the D148Y mutation, acting through Golgi stress signaling, is negatively involved in the regulation of neuronal cell morphological differentiation, providing evidence that a molecule controlling Golgi stress may be one of the potential FTD therapeutic targets at the molecular and cellular levels.

Keywords: Arf4; CHMP2B; Golgi stress; Hsp47; differentiation.

Figure 1

Wild-type CHMP2B is present in MVB-like structures, whereas CHMP2B with the D148Y mutation forms aggregate-like structures. (A) COS-7 cells were transiently transfected with the plasmid encoding wild-type CHMP2B tagged with EGFP (a–c) at its C-terminus or EGFP-tagged CHMP2B with the D148Y mutation (d–f). Successfully transfected cells with CHMP2B (green, (a,d)) were also stained with DAPI to detect nuclear positions (blue, (b,e)). Merged images are also depicted in (c,f). It is possible that the nuclei of COS-7 cells have some amplified chromosomes and/or nuclei arrested in the middle of cell division. (B) Cells with MVB-like structures were statistically depicted in the graph (** p < 0.01; n = 3 fields). (C) N1E-115 cells were transiently transfected with the plasmid encoding wild-type CHMP2B tagged with EGFP (a–c) at its C-terminus or EGFP-tagged CHMP2B with the D148Y mutation (d–f). Successfully transfected cells with CHMP2B (green, (a,d)) were also stained with DAPI to detect nuclear positions (blue, (b,e)). Merged images are also depicted in (c,f). (D) Cells with MVB-like structures are statistically depicted in the graph (** p < 0.01; n = 3 fields).

Figure 2

CHMP2B with the D148Y mutation alters the expression levels of molecules associated with Golgi stress. (A) N1E-115 cells were transfected with the plasmid encoding wild-type CHMP2B tagged with EGFP or EGFP-tagged CHMP2B with the D148Y mutation. The lysates of transfected cells were immunoblotted with the respective Golgi stress molecule antibodies against Hsp47 (a), Arf4 (b), GM130 (c), or control actin (d). In the Hsp47 blot, the asterisk (*) indicates probable non-specific immunoreactive bands. The protein bands slightly above 45 kDa correspond to the immunoreactive ones of Hsp47. (B) The immunoreactive bands were scanned, and each band was statistically analyzed ((a) Hsp47; (b) Arf4; (c) GM130; (d) actin) with the control band at 100% for (a,d) and with the band in the D148Y mutation at 100% for (b,c) (** p < 0.01; n = 3 blots).

Figure 3

CHMP2B with the T148N mutation inhibits neuronal morphological differentiation with decreased expression levels of differentiation markers. (A) Cells harboring wild-type CHMP2B (a,b) or CHMP2B with the D148Y mutation (c,d) were allowed to differentiate for 0 (a,c) or 2 (b,d) days. Cells with processes with more than two cell body lengths were counted as differentiated cells at 0 (e) or 2 (f) days. Their cells are statistically depicted in the graph (** p < 0.01; n = 3 fields). (B) The lysates were immunoblotted with their respective differentiation marker antibodies against GAP43 (a), Tau (b), and actin (c). The immunoreactive bands were scanned, and each band was statistically analyzed with the control band (d–f) as 100% (** p < 0.01; n = 3 blots).

Figure 4

Modulating the expression levels of Golgi stress molecules recovers phenotypes of cells with CHMP2B with the T148N mutation with improved expression levels of differentiation markers. (A) Cells harboring CHMP2B with the D148Y mutation were transfected with mock (a) or Hsp47 (b) and allowed to differentiate for 2 days. Differentiated cells in cell images are statistically depicted (c) in the graph (** p < 0.01; n = 3 fields). The lysates were immunoblotted with an antibody against GAP43 (d), Tau (e), and actin (f). The immunoreactive bands were scanned, and each band was statistically analyzed with the control band at 100% for (f) and with the band in Hsp47 at 100% for (d,e) (** p < 0.01; n = 3 blots). (B) Cells harboring CHMP2B with the D148Y mutation were transfected with control siRNA (a) or Arf4 siRNA (b) and allowed to differentiate for 2 days. Differentiated cells are statistically depicted (c) in the graph (** p < 0.01; n = 3 fields). The lysates were immunoblotted with an antibody against GAP43 (d), Tau (e), and actin (f). The immunoreactive bands were scanned, and each band was statistically analyzed with the control band at 100% for (f) and with the band in the Arf4 siRNA at 100% for (d,e) (** p < 0.01; n = 3 blots).