Figure 10.
CA-ER-TRG mice enable spatiotemporal measurement of in vivo ER-phagy. (A) Generation of Cre-activated ER-TRG reporter mice (CA-ER-TRG) is illustrated. The gene targeting strategy (left) and the experimental procedure timeline (right) are depicted. CA-ER-TRG mice underwent AAV-Cre infection for 4 wk to induce reporter expression in specific cell types. (B) Confocal microscopy showcases GFP signals in the cerebellar and cerebral neurons of conditional ER-TRG (+/−) mice. Detailed images and 3D reconstructions reveal the ER morphology in Purkinje neurons of the cerebellum (left) and neurons in the brain (right). Infection with AAV-hSyn-Cre for 4 wk facilitated ER-TRG expression in mature neurons (n = 4 per group). Scale bar: 20 μm. (C) ER-phagy levels in mature forebrain neurons of CA-ER-TRG (+/−) mice were assessed. Expression of ER-TRG was induced in these neurons using AAV-hSyn-Cre. RFP-only signals were noted in the soma and axons of selected neurons. Top: Combined signals in the forebrain cortex. Bottom: Combined RFP and GFP signals in the CA1 region of the hippocampus (n = 4 per group). Scale bar: 20 μm. (D) Representative confocal images of RFP and GFP in CA-ER-TRG (+/−) mouse astrocytes. ER-TRG expression in astrocytes was induced using AAV-GFAP-Cre. ER-phagy levels varied among astrocytes in different brain regions. Top: High ER-phagy activity in cortical astrocytes (n = 4 per group). Bottom: Comparatively low activity in hippocampal astrocytes. Scale bar: 20 μm. (E and F) Expression of ssRFP-GFP-KDEL in various tissues of CA-ER-TRG (+/−) transgenic mice. Tissue homogenates from control and CA-ER-TRG (+/−) transgenic mice were analyzed via immunoblotting with specific antibodies. Activation of ssRFP-GFP-KDEL expression in the brain and liver was observed in mice treated with both AAV-GfaABC1D-Cre (E) and AAV-TBG-Cre (F). Full-length RFP-GFP, double red asterisk (**), and RFP fragments, single red asterisks (*), indicative of ER-phagy activity, were detected using an anti-RFP antibody. GAPDH and Tubulin served as internal controls. See also Fig. S5. Source data are available for this figure: SourceData F10.

CA-ER-TRG mice enable spatiotemporal measurement of in vivo ER-phagy. (A) Generation of Cre-activated ER-TRG reporter mice (CA-ER-TRG) is illustrated. The gene targeting strategy (left) and the experimental procedure timeline (right) are depicted. CA-ER-TRG mice underwent AAV-Cre infection for 4 wk to induce reporter expression in specific cell types. (B) Confocal microscopy showcases GFP signals in the cerebellar and cerebral neurons of conditional ER-TRG (+/−) mice. Detailed images and 3D reconstructions reveal the ER morphology in Purkinje neurons of the cerebellum (left) and neurons in the brain (right). Infection with AAV-hSyn-Cre for 4 wk facilitated ER-TRG expression in mature neurons (n = 4 per group). Scale bar: 20 μm. (C) ER-phagy levels in mature forebrain neurons of CA-ER-TRG (+/−) mice were assessed. Expression of ER-TRG was induced in these neurons using AAV-hSyn-Cre. RFP-only signals were noted in the soma and axons of selected neurons. Top: Combined signals in the forebrain cortex. Bottom: Combined RFP and GFP signals in the CA1 region of the hippocampus (n = 4 per group). Scale bar: 20 μm. (D) Representative confocal images of RFP and GFP in CA-ER-TRG (+/−) mouse astrocytes. ER-TRG expression in astrocytes was induced using AAV-GFAP-Cre. ER-phagy levels varied among astrocytes in different brain regions. Top: High ER-phagy activity in cortical astrocytes (n = 4 per group). Bottom: Comparatively low activity in hippocampal astrocytes. Scale bar: 20 μm. (E and F) Expression of ssRFP-GFP-KDEL in various tissues of CA-ER-TRG (+/−) transgenic mice. Tissue homogenates from control and CA-ER-TRG (+/−) transgenic mice were analyzed via immunoblotting with specific antibodies. Activation of ssRFP-GFP-KDEL expression in the brain and liver was observed in mice treated with both AAV-GfaABC1D-Cre (E) and AAV-TBG-Cre (F). Full-length RFP-GFP, double red asterisk (**), and RFP fragments, single red asterisks (*), indicative of ER-phagy activity, were detected using an anti-RFP antibody. GAPDH and Tubulin served as internal controls. See also Fig. S5. Source data are available for this figure: SourceData F10.

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