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. 2016 Oct 1;5(3):249-260.
doi: 10.3233/JHD-160211.

Autophagy Activation by Transcription Factor EB (TFEB) in Striatum of HDQ175/Q7 Mice

Petr Vodicka  1 , Kathryn Chase  2 , Maria Iuliano  1 , Adelaide Tousley  1 , Dana T Valentine  1 , Ellen Sapp  1 , Kimberly B Kegel-Gleason  1 , Miguel Sena-Esteves  3 , Neil Aronin  2 , Marian DiFiglia  1
Affiliations
Free PMC article

Autophagy Activation by Transcription Factor EB (TFEB) in Striatum of HDQ175/Q7 Mice

Petr Vodicka et al. J Huntingtons Dis. .
Free PMC article

Abstract

Background: Mutant huntingtin (mHTT) is encoded by the Huntington's disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy.

Objective: To examine if increasing TFEB protein levels in HD mouse striatum induces autophagy and influences mHTT levels.

Methods: We introduced cDNA encoding TFEB with an HA tag (TFEB-HA) under the control of neuron specific synapsin 1 promoter into the striatum of 3 month old HDQ175/Q7 mice using adeno-associated virus AAV2/9. The levels of exogenous TFEB were analyzed using qPCR and Western blot. Proteins involved in autophagy, levels of huntingtin, and striatal-enriched proteins were examined using biochemical and/or immunohistochemical methods.

Results: In HD mice expressing TFEB-HA, HA immunoreactivity distributed throughout the striatum in neuronal cell bodies and processes and preferentially in neuronal nuclei and overlapped with a loss of DARPP32 immunoreactivity. TFEB-HA mRNA and protein were detected in striatal lysates. There were increased levels of proteins involved with autophagosome/lysosome activity including LAMP-2A, LC3II, and cathepsin D and reduced levels of mutant HTT and the striatal enriched proteins DARPP32 and PDE10A. Compared to WT mice, HDQ175/Q7 mice had elevated levels of the ER stress protein GRP78/BiP and with TFEB-HA expression, increased levels of the astrocyte marker GFAP and pro-caspase 3.

Conclusion: These results suggest that TFEB expression in the striatum of HDQ175/Q7 mice stimulates autophagy and lysosome activity, and lowers mHTT, but may also increase a neuronal stress response.

Keywords: Adeno associated virus; GRP78/BiP; Huntington’s disease; LC3; TFEB; autophagy; huntingtin; neurodegeneration; striatum.

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Figures

Fig.1
Fig.1
Immunohistochemistry for TFEB-HA, DARPP32 and GFAP in mice injected with AAV hSyn1 TFEB-HA, AAV hSyn1 GFP or non-injected and examined 2 months post-injection: Immunoperoxidase labeling for HA (TFEB-HA) (A, B) or GFP (C) within the injected or control striatum. Images from corresponding sections stained with DARPP32 (D, E, F) and GFAP (G, H, I) show extent of labeling within the striatum. Numbered boxes correspond to areas displayed in the enlargements to the right. DARPP32 staining of cell bodies and processes is diminished in the TFEB-HA affected area of striatum (D). Note increased GFAP labeling in the area of striatum affected by TFEB-HA injection (G). N = 2 mice per treatment group.
Fig.2
Fig.2
TFEB mRNA quantification by qPCR and comparison to TFEB-HA protein levels. A) Relative quantification of TFEB mRNA in non-injected and AAV hSyn1 TFEB-HA injected HDQ175/Q7 mice. Data are presented as a fold-change over mean of control group (non-injected). Each point represents individual mouse and boxplots show overall data distribution (N = 4 mice per group). P-value from unpaired t-test. B) Comparison of TFEB mRNA levels in individual animals to TFEB-HA protein levels detected by HA antibody. Bar graphs show relative fold-change over mean of control group for mRNA and HA densitometry signal in crude homogenate normalized to GAPDH for protein. Kendall tau correlation coefficient and corresponding p-value for mRNA/ crude homogenate protein level is provided. C) Western blot for TFEB-HA protein levels as detected by HA antibody in P1 nuclear enriched fraction.
Fig.3
Fig.3
Activation of autophagy and reduction of mHTT in TFEB-HA treated striatum. A) Western blot analysis for LAMP-2A, LC3II, and procathepsin D (proCTSD) in crude homogenates (CH) of HDQ175/Q7 mice injected with AAV hSyn1 TFEB-HA or non-injected. B) Quantitative results of crude homogenate Western blots in A and Western blots in subcellular fractions (P1, S2 and P2). HSC70 and Rab7 in S2 fractions are included in the bar graphs along with the results for LAMP-2A. C) Western blots of HTT levels in crude homogenate (CH) and in membrane fractions P1 and P2 detected with anti-HTT aa1-100 antibody EPR5526. D) Bar-graphs represent the results of densitometry for mHTT and WT HTT on Western blots shown in C. Mutant HTT levels in the AAV hSyn1 TFEB-HA injected mice are reduced compared to the untreated mice. All mice are age matched and the injected mice were examined at 2 months post-injection. N = 4 per treatment group, p-values from unpaired t-test.
Fig.4
Fig.4
Western blot analysis of DARPP32, GFAP, PDE10A in striatal crude homogenates of mice injected with either AAV hSyn1 TFEB-HA or non-injected. A) Western blots for DARPP32, GFAP and PDE10A, GAPDH was used as a loading control. B) Bar graphs show results of densitometry for Western blots in A. All mice are age matched and the injected mice were examined at 2 months post-injection. Each column represents sample from a different mouse, N = 4 mice per treatment group, p-values from unpaired t-test.
Fig.5
Fig.5
Western blot analysis of apoptosis and ER stress markers in striatal crude homogenates of HDQ175/Q7 mice injected with AAV hSyn1 TFEB-HA or non-injected HDQ175/Q7 and WT mice. A) Western blots for GRP78/BiP and procaspase 3 in HDQ175/Q7 mice injected with AAV hSyn1 TFEB-HA and non-injected HDQ175/Q7 and WT mice. GAPDH was used as a loading control. B) Bar graphs show results of densitometry for Western blots in A. All mice are age matched and the injected mice were examined at 2 months post injection. Each column represents sample from a different mouse, N = 4 mice per treatment group, p-values from unpaired t-test.
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