Bidirectional interplay of HSF1 degradation and UPR activation promotes tau hyperphosphorylation
Fig 5
HSF1 degradation caused by overexpressed TauRD ΔK280 and further facilitated by eIF2α-CHOP activation.
(A-C) HSF1 degradation and CHOP activation in N2a-TauRD ΔK280 stable cell line. (A) Western blot analysis on N2a neuroblastoma cells transiently overexpressing GFP-tagged repeated domain (RD) tau constructs (TauRD WT, TauRD P301L, TauRD ΔK280) and empty vector (control). (B) A graph indicates relative protein levels of HSF1 and CHOP normalized to β-actin in N2a and N2a-TauRD ΔK280 stable cell line (means ± SEM, *P <0.05, **P <0.01, comparing to control, n = 4). (C) HSF1 degradation through both autophagy-lysosome and proteasome in N2a-TauRD ΔK280. N2a-TauRD ΔK280 were treated with either autophagy-lysosomal (Baf A1, NH4Cl, CQ) or proteasomal blocker (MG 132, 20 μM) for 6 hr at different concentrations indicated. Left: Control N2a treated with either MG132 or NH4Cl. Right (short exposure of film): Baf A1: baflomycin A1, CQ: chloroquine. (D) N2a-TauRD ΔK280 were transfected with either HSF1 WT or HSF1 S303A (constitutively active form) or empty vector as control. Cleaved-caspase 3 as apoptotic marker. A right graph represents reduced relative CHOP protein expression levels normalized to β-actin in N2a-TauRD ΔK280 overexpressing HSF1 WT (means ± SEM, ***P < 0.001, comparing to N2a-TauRD ΔK280 transfected with empty vector, n = 4). (E) N2a-TauRD ΔK280 were treated with either thapsigargin (TG, 1μM) for 6 hr or salubrinal (20 μM) for 12 hr or both of them. Their protein lysates were subjected to western blot (left). A graph on quantitative measurement of the relative HSF1 protein expression (right, means ± SEM, *P < 0.05, n = 4, N.S., non-significant). (F) Treatment of thapsigargin (1 μM), rapamycin (0.5 μM) and NH4Cl (5 mM) in N2a-TauRD ΔK280. (G) The change on expression levels of HSF1 was assessed in N2a-TauRD ΔK280 transfected with CHOP siRNA and later subjected to either thapsigargin or tunicamycin (1 μM) treatment for different duration time as indicated. Quantitative measurement of relative HSF1 protein levels normalized to β-actin (bottom, arrow indicates dramatica increase in HSF1 protein by siCHOP expression.) (H) A graph indicates relative HSF1 protein levels in N2a-TauRD ΔK280 transfected with CHOP siRNA in the presence or absence of thapsigargin (means ± SEM, *P < 0.05, N.S., non-significant, n = 3).