(B) Autophagic flux was analyzed in gba^+/+ and gba^−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.

(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. ^*p < 0.05.

(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.

(F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control.

(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba^+/+ and gba^−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.

(B) Sagittal brain stem sections from gba^+/+, gba^+/−, and gba^−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.

(A) Neurons and astrocytes from gba^+/+, gba^+/−, and gba^−/− mice were stained with TMRM. The mean florescence intensity in mitochondria was analyzed via confocal microscopy (n = 3, >32 cells analyzed/experiment).

(B) Neurons from gba^+/+ and gba^−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).

(C) Neurons from gba^+/+ and gba^−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).

(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, ^*p < 0.05. Neurons from gba^+/+ and gba^−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).

(A) Oxygen consumption rates of gba^+/+, gba^+/−, and gba^−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).

(B) Complex I-NADH: Ubiquinone reductase activity from gba^+/+, gba^+/−, and gba^−/− brains expressed as a ratio to citrate synthase (n = 3).

(C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba^+/+, gba^+/−, and gba^−/− brains expressed as a ratio to citrate synthase (n = 3).

(D) Complex IV activity from gba^+/+, gba^+/−, and gba^−/− brains expressed as a ratio to citrate synthase (n = 3).

(E) Neurons were treated with 3 nM decylTPP and MitoQ₁₀ for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).

(F) gba^+/+and gba^−/− cells treated with 3 nM decylTPP and MitoQ₁₀ for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, ^*p < 0.05.

(A) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst. Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes. "A" in nucleus denotes astrocytes. Scale bar, 20 μm.

(B) Mitochondrial morphology was blind counted from cells in (A) with neurons scored as MAP2 positive and astrocytes as GFAP positive. A "normal" morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild-type network. (n = 3; 100 cells counted/experiment).

(C) Levels of morphology proteins from gba^+/+, gba^+/−, and gba^−/− isolated mitochondria were analyzed via immunoblotting with the indicated antibodies.

(D) Midbrain gba^+/+, gba^+/−, and gba^−/− astrocytes expressing GFP-DRP1^K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy. Scale bar, 20 μm (n = 3, >15 cells/experiment). All data in this figure represent the mean ± SEM.

(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.

(B) Midbrain gba^+/+ and gba^−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.

(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).

(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba^+/+ and gba^−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.