TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity

Abstract

The aggregation of α-synuclein plays a major role in Parkinson disease (PD) pathogenesis. Recent evidence suggests that defects in the autophagy-mediated clearance of α-synuclein contribute to the progressive loss of nigral dopamine neurons. Using an in vivo model of α-synuclein toxicity, we show that the PD-like neurodegenerative changes induced by excess cellular levels of α-synuclein in nigral dopamine neurons are closely linked to a progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of transcription factor EB (TFEB), a major transcriptional regulator of the autophagy-lysosome pathway. The changes in lysosomal function, observed in the rat model as well as in human PD midbrain, were reversed by overexpression of TFEB, which afforded robust neuroprotection via the clearance of α-synuclein oligomers, and were aggravated by microRNA-128-mediated repression of TFEB in both A9 and A10 dopamine neurons. Delayed activation of TFEB function through inhibition of mammalian target of rapamycin blocked α-synuclein induced neurodegeneration and further disease progression. The results provide a mechanistic link between α-synuclein toxicity and impaired TFEB function, and highlight TFEB as a key player in the induction of α-synuclein-induced toxicity and PD pathogenesis, thus identifying TFEB as a promising target for therapies aimed at neuroprotection and disease modification in PD.

Keywords: Beclin; adeno-associated virus; aggregates; synucleinopathy.

Fig. 1.

α-Synuclein impairs autophagy by sequestrating TFEB. (A) qPCR analysis showing mRNA expression of ALP markers (Beclin-1, Cathepsin B, Cathepsin D, Lamp-1, and TFEB) and dopaminergic markers (TH and Aldh1) in the ventral midbrain 10 d, 3 wk, and 8 wk after intranigral injection of AAV-GFP (white bar) or AAV–α-syn vectors at high (2.1 × 10¹² gc/mL) (black bar) and low concentration (3.8 × 10¹¹ gc/mL) (gray bar). *P < 0.05 compared with AAV-GFP group (Student t test) (n = 5 per group). (B) Protein expression of Beclin-1, Lamp-1, Cathepsin D, LC3I/II, and TH in the midbrain 10 d (presymptomatic stage), and 3 wk (initiation of neurodegeneration) after overexpression of GFP or α-syn. (C–E) Fluorescent immunohistochemistry showing the colocalization of human α-syn with Beclin-1 (C), Cathepsin D (D), and p62 (E) 3 wk after intranigral injection of AAV–α-syn. (Scale bars, 15 μm.) (F) Fluorescent immunohistochemistry illustrating the accumulation of dense ubiquitin-positive structures inside DA neurons overexpressing α-syn 8 wk after AAV–α-syn injection. (Scale bar, 15 μm.) (G) Western blot showed an important accumulation of the autophagy substrate p62 8 wk after AAV–α-syn injection in the SN compared with the GFP and control group. (H) Western blot analysis illustrating the progressive accumulation of high molecular weight (HMW) α-syn oligomers in the striatum and p62 in midbrain extracts at 10 d, 3 wk, and 8 wk after AAV–α-syn injection (high titer). (I) Western blot analysis showed an increased nuclear expression of TFEB at 10 d, and a reduced nuclear localization of TFEB at 3 wk after AAV–α-syn injection at high titer. Shift in the molecular weight corresponds to the phosphorylated and dephosphorylated forms of TFEB in the cytoplasm and nucleus, respectively. Control of nuclear extraction was performed by analysis of the nuclear marker HDAC1. (J) Coimmunoprecipitation (IP) of TFEB with α-syn and 14-3-3 in α-syn–overexpressing midbrain tissue (3 wk postinjection). Lysates were subjected to IP for TFEB or control IgG, and immunoblotted for α-syn and 14-3-3 (Inp: input, no I.P).

Fig. 2.

Changes in TFEB localization in human PD brains. (A and B) TH immunohistochemistry showing the DA neurons in control and PD human midbrain. Note the reduced density of TH⁺ neurons in the PD brain consistent with the clinical diagnostic. Red and blue squares in A indicate the regions analyzed to study the expression of TFEB in nigral (shown in C–E) and VTA neurons (shown in F and G), respectively. (Scale bar, 500 μm.) (C and D) Double-immunofluorescence staining of human postmortem midbrain showed that TFEB is well expressed in neuromelanin-containing nigral DA neurons (C). (Scale bar, 60 μm.) High-power magnification revealed that TFEB is localized both in the cytoplasmic and nuclear compartments of A9 neurons (D). (Scale bar, 12 μm.). (E) In PD brains, although the expression level did not seem to be altered, TFEB was not observed in the nuclear compartment and appeared clustered in the cytoplasm (arrowhead). In addition, TFEB colocalized with α-syn in Lewy bodies containing nigral neurons (arrow). Identity of the DA neurons was further confirmed by the expression of neuromelanin (light microscopy). (Scale bar, 12 μm.). Immunofluorescence staining of TFEB from control (F) and PD (G) human midbrain showing that its pattern of expression is unchanged in VTA DA neurons. (Scale bars, 30 μm.) (H) Percentage of nigral and VTA DA neurons presenting TFEB immunoreactivity in the nucleus. A total of 200 neuromelanin-containing cells from five different brains were examined in both the VTA and SN in control and PD human brains.

Fig. 3.

Gene transfer-mediated stimulation of autophagy protects DA neurons against α-syn toxicity. (A–C) Motor function was assessed at 8 wk in rats overexpressing α-syn (high titer: 2.1 × 10¹² gc/mL) together with GFP (white bar), TFEB (black bar), or Beclin-1 (gray bar), using the cylinder test (A), stepping test (B), and amphetamine-induced rotation test (C). Overexpression of Beclin-1 and TFEB prevented the development of behavioral deficits in these three tests compared with the GFP group. *P < 0.05 compared with AAV-GFP group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (D and E) Midbrain sections were stained for TH (D) and stereological estimation of the number of TH⁺ nigral neurons was performed 8 wk after vector injection (E). Counts showed that Beclin-1 and TFEB overexpression afforded robust neuroprotection of nigral DA neurons against α-syn–induced toxicity. *P < 0.05 compared with AAV-GFP group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (Scale bar, 2 mm.) Asterisk indicates injection site. (F and G) Forebrain sections were stained for TH (F) and assessment of striatal TH⁺ innervation density was performed by optical densitometry 8 wk after vector injection (G). Measurements showed significant survival of striatal TH⁺ terminals in the Beclin-1 and TFEB-overexpressing groups compared with GFP control animals. *P < 0.05 compared with AAV-GFP group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (Scale bar, 3 mm.) (H) HPLC measurement revealed that striatal DA concentration was significantly higher in rats overexpressing Beclin-1 or TFEB compared with the GFP control group 8 wk after vector injection. *P < 0.05 compared with AAV-GFP group (one-way ANOVA, Bonferroni post hoc test; n = 5 per group). (I and J) Representative traces of in vivo real-time chronoamperometry measurements recorded simultaneously from the left (control side) and right (injected side: AAV–α-syn+AAV-GFP or AAV–α-syn+AAV-TFEB and AAV–α-syn+AAV-Beclin-1) striatum after delivery of a KCl pulse (arrow). Quantitative analysis of repeated measurements showed that TFEB or Beclin-1 overexpression significantly preserved DA release (peak amplitude and linear rise rate) and reuptake, as well as the total amount of DA released (AUC, area under the curve) (P). *P < 0.05 compared with GFP group (Student t test; n = 5 per group). (K) Western blot analysis showed that Beclin-1 and TFEB-mediated neuroprotection is associated with reduced accumulation of α-syn oligomers (HMW, high molecular weight), and reduced levels of p62 (n = 5 per group). (L) Computer-based 3D reconstruction of z-stacked pictures allowed the detection and quantification of large-sized (>20 μm³) α-syn⁺ axonal swellings in the striatum of rats overexpressing α-syn at high titer together with GFP, Beclin-1, or TFEB. Beclin-1 of TFEB overexpression prevented the accumulation of large α-syn⁺ structures. *P < 0.05 compared with GFP group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (M) The load of α-syn was estimated by calculating the ratio of the number of striatal α-syn axonal swellings (Fig. S3 A–D) over the number of surviving nigral DA neurons (F). Beclin-1 or TFEB overexpression markedly reduced the abundance of α-syn swellings. *P < 0.05 compared with AAV-GFP group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group).

Fig. 4.

miR-128-induced inhibition of autophagy aggravates α-syn toxicity. (A) Midbrain expression levels of ALP and dopaminergic markers, analyzed by qPCR 3 wk after intranigral injection of AAV-GFP (open bars) or AAV–miR-128 (eGFP) (low titer, 1.2 × 10¹⁰ gc/mL, black bars). *P < 0.05 compared with GFP group (Student t test; n = 5 per group). (B and C) Triple-immunofluorescence staining showing the colocalization of TH (red), GFP (green in B), miR-128 (green, visualized by the GFP reporter in C, low titer 1.2 × 10¹⁰ gc/mL), and α-syn (low titer: 3.8 × 10¹¹ gc/mL, blue) in nigral DA neurons 3 wk after vector injection. (Scale bar, 100 μm.) (D–F) Motor function was assessed at 8 wk in rats overexpressing α-syn at low titer together with GFP or miR-128 using the cylinder test (D), stepping test (E), and amphetamine-induced rotation test (F). miR-128 overexpression (low titer 1.2 × 10¹⁰ gc/mL) together with α-syn promoted the development of behavioral deficits on the side controlateral to the vector injection in these three tests compared with the GFP-overexpressing animals. *P < 0.05 compared with α-syn+GFP group (Student t test; n = 8 per group). (G–I) Brain sections were stained for TH (I) and survival of nigral DA neurons (G) and loss of striatal innervation (H) was determined at 8 wk by stereological counting and optical densitometry, respectively. Measurements showed that miR-128 overexpression at low titer aggravated α-syn toxicity (low titer: 3.8 × 10¹¹ gc/mL) and triggered a significant loss of nigral TH⁺ neurons and striatal TH⁺ terminals compared with GFP-overexpressing rats. *P < 0.05 compared with α-syn+GFP group (Student t test; n = 8 per group). (Scale bar, 1.5 mm.) Asterisk indicates the injection site. (J) HPLC measurement showed that striatal DA concentration was significantly lower in rats coexpressing α-syn+miR-128 compared with the GFP controls. *P < 0.05 compared with α-syn+GFP group (Student t test; n = 5 per group). (K) Western blot analysis showed that miR-128–mediated inhibition of autophagy enhanced the accumulation of HMW striatal α-syn oligomers (n = 5 per group, both vectors delivered at low titer). (L) The load of α-syn was estimated by calculating the ratio of the number of striatal α-syn accumulated axonal swellings over the number of surviving nigral TH⁺ neurons (G). miR-128 overexpression (low titer) significantly increased the burden of α-syn aggregation in these structures. *P < 0.05 compared with α-syn+GFP group (Student t test; n = 8 per group). Data in J–L were all obtained at 8-wk survival.

Fig. 5.

miR-128-induced knock-down of TFEB renders the A10 neurons more vulnerable to α-syn toxicity. (A and B) Double-immunofluorescence staining showing the expression of GFP (A) and miR-128 (high titer: 6.4 × 10¹¹ gc/mL) (B) in green (visualized by the GFP reporter) together with α-syn (high titer: 2.1 × 10¹² gc/mL) (in blue) in VTA DA neurons (TH in red), 3 wk after vector injection. (Scale bar, 120 μm.) (C and D) Midbrain sections from animals overexpressing α-syn at high titer together with GFP or miR-128 (given in high titer) were stained for TH (C) and the number of VTA TH⁺ neurons was estimated by stereological method (D). Co-overexpression of miR-128 made the A10 neurons more sensitive to α-syn–induced toxicity as assessed 8 wk after vector injection. (Scale bar, 500 μm.) *P < 0.05 compared with α-syn+GFP group (Student t test; n = 5 per group). Asterisk indicates the injection site. (E and F) Forebrain sections were stained for TH (E), and TH⁺ innervation density was determined by optical densitometry in the nucleus accumbens (F). miR-128 overexpression induced a significant loss of TH⁺ DA terminals in this structure. (Scale bar, 600 μm.) *P < 0.05 compared with α-syn+GFP group (Student t test; n = 5 per group). (G) Western blot analysis showed that coexpression of miR-128, together with α-syn, in VTA neurons promoted the formation of HMW oligomers compared with the α-syn+GFP injected control animals, 8 wk survival (n = 5 per group).

Fig. 6.

Delayed mTOR inhibition slows the progression of α-syn–induced neurodegeneration. AAV–α-syn injected rats were treated with CCI-779 (20 mg/kg, three times per week) for 5 wk, starting 3 wk after vector injection. (A–C) Motor function was assessed at 3 wk in rats overexpressing α-syn (high titer: 2.1 × 10¹² gc/mL) (white bar), and at 8 wk in rats treated with CCI-779 (black bar) or vehicle (gray bar) during the last 5 wk, using the cylinder test (A), stepping test (B), and amphetamine-induced rotation test (C). Drug treatment significantly prevented the development of behavioral deficits in these three tests compared with the vehicle-treated animals. *P < 0.05 compared with the 3-wk α-syn group; ^#P < 0.05 compared with α-syn+vehicle group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (D) HPLC measurement revealed that striatal DA concentration was significantly higher in α-syn–overexpressing rats treated with the mTOR inhibitor compared with the vehicle-treated animals. *P < 0.05 compared with the 3-wk α-syn group; ^#P < 0.05 compared with α-syn+vehicle group (one-way ANOVA, Bonferroni post hoc test; n = 5 per group). (E and F) Midbrain sections were stained for TH (E) and stereological estimation of the number of TH⁺ nigral neurons was performed at 3 wk (i.e., before initiation of treatments) or 8 wk after vector injection (i.e., after 5 wk of treatment) (F). Counts showed that delayed CCI-779 treatment, starting at 3 wk, slowed down the neurodegeneration and afforded robust neuroprotection of nigral DA neurons against α-syn–induced toxicity compared with vehicle-treated animals. *P < 0.05 compared with the 3-wk α-syn group; ^#P < 0.05 compared with α-syn+vehicle group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (Scale bar, 1.2 mm.) Asterisk indicates injection site. (G and H) Forebrain sections were stained for TH (G) and the density of striatal TH⁺ innervation was assessed by optical densitometry at 3 or 8 wk after vector injection (G). Measurements showed significant survival of striatal TH⁺ terminals in the drug-treated groups compared with control animals. *P < 0.05 compared with the 3-wk α-syn group; ^#P < 0.05 compared with α-syn+vehicle group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (Scale bar, 2 mm.) (I) The load of α-syn was estimated by calculating the ratio of the number of striatal α-syn accumulated axonal swellings (Fig. S3 A–D) over the number of surviving nigral TH⁺ neurons (F). CCI-779 treatment significantly reduced the abundance of α-syn accumulated swellings. *P < 0.05 compared with the 3-wk α-syn group; ^#P < 0.05 compared with α-syn+vehicle group (one-way ANOVA, Bonferroni post hoc test; n = 8 per group). (J) Western blot analysis showed that treatment with the mTOR inhibitor reduced the accumulation of HMW striatal α-syn oligomers (n = 5 per group). (K and L) Western blot analysis demonstrated that the neuroprotective effect of CCI-779 was associated with a reduced activation of mTOR (K) and alleviation of the sequestration of TFEB in the cytoplasm (L). Cellular fractionation was confirmed by the expression of GAPDH and HDAC1 in the cytoplasmic and nuclear fractions, respectively (n = 5 per group).