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. 2012 Feb;23(4):553-66.
doi: 10.1091/mbc.E11-06-0573. Epub 2011 Dec 21.

Neuronal Dystonin Isoform 2 Is a Mediator of Endoplasmic Reticulum Structure and Function

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Free PMC article

Neuronal Dystonin Isoform 2 Is a Mediator of Endoplasmic Reticulum Structure and Function

Scott D Ryan et al. Mol Biol Cell. .
Free PMC article

Abstract

Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum (dt) mice results in hereditary sensory neuropathy. Although loss of expression of neuronal dystonin isoforms (dystonin-a1/dystonin-a2) is sufficient to cause dt pathogenesis, the diverging function of each isoform and what pathological mechanisms are activated upon their loss remains unclear. Here we show that dt(27) mice manifest ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration through induction of a proapoptotic caspase cascade. dt sensory neurons display neurodegenerative pathologies, including Ca(2+) dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism of dt neuropathology and proposes a role for dystonin-a2 as a mediator of normal ER structure and function.

Figures

FIGURE 1:
FIGURE 1:
Dystonin deficiency leads to degeneration of sensory neurons in mice. DRGs from P4 (A, B) and P15 (C, D) WT and dt27 mice were stained with Fluoro-Jade B to identify degenerating neurons. No degeneration of sensory neurons is evident prephenotype (A, B), whereas Fluoro-Jade–positive degenerating neurons were evident at P15 in phenotypic dt27 animals (C, D). Analysis of TUNEL labeling from P4 (E, F) and P15 (G, H) WT and dt27 DRGs yielded no difference in apoptotic index prephenotype (E, F). At P15, a significant increase in percentage TUNEL-positive cells was observed in phenotypic dt27 animals relative to WT controls (G, H). Scale bars, 50 μm; Student's t test, **p < 0.01, n = 5.
FIGURE 2:
FIGURE 2:
Isoform-specific loss of dystonin-a2 promotes neuronal cell death. (A) Efficiency of siRNA-mediated knockdown of exogenously expressed dyst-a1–YFP and dyst-a2–YFP protein was evaluated in COS-1 cells by epifluorescence. (B) Efficiency of siRNA-mediated knockdown of endogenously expressed dystonin-a1 and dystonin-a2 mRNA was measured in F11 neuronal cells by RT-PCR. Knockdown of dystonin-a2 resulted in an increase in death as assessed by ethidium homodimer incorporation within 24 h (C) and an increase in TUNEL labeling within 48 h of silencing relative to scrambled control. Loss of dystonin-a1 has no impact on cell death (C, D). Scale bars, 10 μm; ANOVA, post hoc Dunnett's t test, *p < 0.05, **p < 0.01, n = 5.
FIGURE 3:
FIGURE 3:
Loss of neuronal dystonin-a2 results in activation of a caspase 2–dependent caspase cascade in F11 cells. F11 neuronal cells were screened for caspase activation using FLICA assays at multiple time points following knockdown of either dystonin-a1 or dystonin-a2. (A) No induction of death receptor–associated caspase 8 was observed at any time point. (B) A modest increase in caspase 12 activation was measured following 48 h of dystonin-a2 depletion. A pronounced increase in both caspase 2 (C) and caspase 3 (D) activity was observed following 24 and 48 h of dystonin-a2 depletion relative to scrambled siRNA control cells. Loss of dystonin-a1 had no effect on caspase activation (A–D). ANOVA, post hoc Dunnett's t test, *p < 0.05, **p < 0.01, n = 5.
FIGURE 4:
FIGURE 4:
Loss of dystonin results in activation of an ER-associated caspase cascade in DRGs. (A) A postnatal time course of caspase 3 activity was conducted by immunofluorescence analysis of cleaved caspase 3 expression at P4, P10, and P15 in WT and dt27 DRG sections; scale bars, 50 μm. (B) Survival of cultured primary sensory neurons was then assessed by TUNEL labeling of WT and dt27 DRGNs at P4 and P15; scale bars, 10 μm. DNase-treated sensory neurons were processed as a positive control. Fold change in TUNEL reactivity was quantified at (C) P4 and (D) P15. Data are expressed as fold increase in TUNEL-positive dt27 neurons relative to WT; Student's t test, n = 3–6. (E) WT and dt27 primary sensory neurons were screened for caspase activation following 48 h in culture using FLICA assays. Although no induction of death receptor–associated caspase 8 was measured, an increase in activity of executioner caspases 2 and 3 was recorded; ANOVA, post hoc Dunnett's t test, *p < 0.05, n = 9. (F) Caspase 2 activity was confirmed by Western analysis of pro–caspase 2 cleavage in both P4 and P15 WT and dt27 DRGs. Caspase 2 cleavage in (G) P4 and (H) P15 WT and dt27 DRGs was quantified by densitometry and normalized to tubulin standard; Student's t test, *p < 0.05, n = 5.
FIGURE 5:
FIGURE 5:
Caspase 2 is ER associated in F11 neuronal cells. F11 cells overexpressing either calreticulin-YFP (A, B) or Golgi-YFP (C, D) were antigenically labeled for caspase 2 following 48-h treatment with either (A, C) control or (B, D) dystonin-a2 siRNAs (scale bars, 10 μm). Colocalization of caspase 2 with either the ER (calreticulin) or Golgi was subsequently determined and colocalization masks generated. Quantification of the percentage colocalized area between caspase 2 and (E) the ER or caspase 2 and (F) the Golgi was subsequently performed; Student's t test, n = 12. Loss of dystonin has no effect on localization. (G) ER macrostructure was visualized by antigenic labeling of endogenous calreticulin (scale bars, 10 μm). Loss of dystonin-a2 had little effect on calreticulin subcellular organization.
FIGURE 6:
FIGURE 6:
Loss of dystonin results in ER stress–mediated induction of the unfolded protein response. (A, B) Antigenic labeling of CHOP by immunofluorescence confirmed an induction of CHOP expression in whole DRGs from dt27 animals relative to WT littermate controls (scale bars, 50 μm). (C) Phenotypic WT and dt27 DRGs were analyzed by Western blot for induction of UPR proteins BiP and CHOP. A significant induction of both BiP and CHOP was observed in P15 dt27 DRGs (each lane represents pooled DRGs from three animals). (D) Induction of BiP expression following loss of dystonin-a2 was confirmed by immunofluorescence antigenic labeling (scale bars, 10 μm). (E–I) Isoform-specific depletion of dystonin was performed in F11 neuronal cells and the effect on UPR induction determined. (E, F) Although Western analysis did not reveal a change in BiP expression following dystonin-a1 silencing, an increase in (G, H) BiP and (G, I) CHOP expression was detected within 48 h of dystonin-a2 silencing; ANOVA, post hoc Dunnett's t test, *p < 0.05, **p < 0.01, n = 3–7). (J, K) UPR-mediated activation of XBP1 splicing was then determined by total cell counts of XBP1-GFP expression. A 48-h dystonin-a1 depletion resulted in a modest induction of XBP1 splicing, whereas a dramatic increase in splicing was observed following loss of dystonin-a2 (scale bars, 10 μm); ANOVA, post hoc Dunnett's t test, *p < 0.05, **p < 0.01, n = 3–7.
FIGURE 7:
FIGURE 7:
ER Ca2+ homeostasis is perturbed in the sensory neurons of dt27 mice. Electron microscopy of WT and dt27 DRGs at (A) P5, (B) P10, and (C) P15. Normal striated patterning of ER can be observed in WT DRGs (A–C top, arrows). In contrast, the ER in dt27 DRGs is dilated (A–C, bottom arrows), leading to vacuole formation in some instances (C, bottom, arrowhead; scale bars, 500 nm). (D) Quantification of ER dilation shows a significant increase in ER dilation at P10 and P15 in dt27 DRGs relative to WT; one-way Student's t test, *p < 0.05, **p < 0.01, n = 3. Ca2+ mobilization from the ER of (E) P15 WT and (F) dt27 primary sensory neurons was visualized and recorded using Fura-2AM dye, and a representative graph is depicted. Neurons were perfused with PSS for 100 s prior to Ca2+ depletion in tyrode (Ty) buffer for 30 s. Caffeine was then administered for 30 s in tyrode buffer [Ty(+)] to stimulate Ca2+ efflux from the ER. Following Ca2+ efflux, neurons were then reperfused in PSS to replenish Ca2+ stores and the sequence was repeated. Ca2+ efflux from the ER is indicated by a shift in the 340/380-nm ratio of Fura-2 fluorescence emittance. (F, G) dt27 sensory neurons showed reduced ER calcium efflux relative to WT sensory neurons as indicated by a reduction in maximum peak amplitude following caffeine administration; Student's t test, **p < 0.01, n = 61–63. Ca2+ reuptake was normal in both WT and dt27 sensory neurons as indicated by the second peak response. The experimental paradigm in E was repeated in (H) WT and (I) dt27 sensory neurons, with the exception that between the first and second sequences the neurons were perfused with thapsigargin (Thaps) for 180 s to block Ca2+ reuptake by SERCA pumps. Following SERCA inhibition, caffeine stimulation did not result in Ca2+ mobilization, confirming that caffeine was indeed stimulating efflux from ER stores alone and that all Ca2+ in the ER was effluxed following stimulation with caffeine.
FIGURE 8:
FIGURE 8:
Inhibition of ER stress and the associated caspase cascade rescues dystonin-a2–deficient neurons from death. (A) F11 neuronal cells were screened for caspase 3 activation following knockdown of dystonin-a2 in the presence of various caspase inhibitors using the FLICA assay. Pan-caspase inhibition or inhibition of caspase 2 but not caspase 12 prevented downstream caspase 3 activation in F11 neuronal cells; ANOVA, post hoc Dunnett's t test, *p < 0.05. (B) In P15 primary sensory neurons from dt27 mice subjected to the same assay, pan-caspase inhibition in addition to inhibition of either caspase 2 or caspase 12 prevented downstream caspase 3 activation; ANOVA, post hoc Dunnett's t test, *p < 0.05, n = 9. (C) Western analysis of F11 neuronal cells treated with various concentrations of salubrinal confirmed that salubrinal inhibits downstream UPR signaling by preventing dephosphorylation of eIF2α. Fold change in ethidium homodimer incorporation showed that whereas dystonin-a2 depletion causes a significant increase in death (ANOVA, post hoc Tukey, **p < 0.01, n = 9), salubrinal treatment at 15 and 25 μM significantly reduces death resulting from depletion of dystonin-A2 in F11 neuronal cells relative to vehicle (DMSO)-treated cells (D, E) (scale bars, 10 μm; ANOVA, post hoc Tukey, #p < 0.05). (F) Measurements of fold change in ethidium homodimer incorporation indicate that 25 μM salubrinal treatment rescues dt27 primary sensory neurons from death; Student's t test, **p < 0.01, n = 18–20.
FIGURE 9:
FIGURE 9:
Proposed model of apoptotic signaling initiated by depletion of dystonin-a2 in sensory neurons. 1) Schematic representation of the events that occur following depletion of dystonin-a2 in sensory neurons. 2) Depletion of dystonin-a2 results in a loss of cytoskeletal integrity that culminates in dilation of the ER. 3) Perturbed ER homeostasis leads to a decrease in the steady-state levels of Ca2+ in the ER. 4) The associated rise in free intracellular Ca2+ likely results in activation of an ER-dependent caspase cascade, inhibition of which can maintain cell viability. 5) Blockade of downstream eIF2α signaling with salubrinal partially rescues neurodegeneration following dystonin-a2 depletion, implicating PERK-mediated eIF2α signal transduction in the apoptotic cascade. 6) Enhanced XBP1 splicing likely coupled with activation of ATF-6 and IRE1 promotes expression of protein chaperones (7) BiP and CHOP in an attempt to maintain ER function. 8) The inability to rescue ER structural integrity culminates in DNA cleavage and programmed cell death.

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