Reprogramming the proteostasis network to prevent the accumulation of alpha-synuclein aggregates

Protein misfolding and aggregation characterizes the development of a number of neurodegenerative diseases, such as Parkinson’s, Alzheimer’s and Huntington’s disease. The hallmark of Parkinson’s disease is the formation of proteinaceous inclusions, which consist primarily of α-synuclein (α-syn), a natively unstructured protein with propensity to misfold and aggregate. Cells have evolved sophisticated systems of protein quality control to prevent accumulation of non-native proteins and maintain protein homeostasis. However, the load of misfolded α-syn typically exceeds the capacity of the quality control system. Aberrant accumulation of misfolded α-syn leads to proteotoxic stress, eventually resulting in neurodegeneration. The objective of this project is to investigate chemical and genetic approaches to modulate the protein quality control system and reduce the accumulation of aberrant α-syn species. Studying α-syn aggregation in cells presents a number of challenges mainly due to the limited availability of tools to quantitatively distinguish between different α-syn conformational species within the cellular environment. To address this need, we engineered an in vitro model system based on neuroglioma cells that accumulate α-syn aggregates and developed a set of analytical tools based on the use of aggregation responsive probes to quantify α-syn aggregation in cells. To test whether modulating the protein quality control system affects the accumulation of α-syn aggregates, we investigated a series of complementary approaches aimed at i) enhancing the innate cellular chaperone machinery, which promotes folding and prevents aggregation, and ii) upregulating the autophagy pathway, which mediates clearance of aggregated proteins. We demonstrated that chemical modulation of Hsp70, a ubiquitously expressed molecular chaperone, affects the accumulation of α-syn aggregates. Particularly, the Hsp70 upregulator carbenoxolone was found to reduce α-syn aggregation and prevent α-syn-induced cytotoxicity via activation of the heat shock response. We also found that activation of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, results in enhanced autophagic clearance of α-syn aggregates. We demonstrated that cell treatment with 2-hydroxypropyl-β-cyclodextrin reduces the accumulation of aggregated α-syn specifically by upregulating TFEB-mediated autophagic clearance. These findings lay the foundation for the development of pharmacological strategies to reduce the accumulation of misfolded and aggregated α-syn for the treatment of Parkinson’s disease

Quantitative Analysis of a-Synuclein Solubility in Living Cells Using Split GFP Complementation

Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 60 years of age. The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions (Lewy bodies) in surviving neurons. Lewy bodies are mainly composed of the pre-synaptic protein alpha-synuclein (αsyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble αsyn and monitoring αsyn aggregation in living cells is fundamental to study the molecular mechanisms of αsyn-induced cytotoxicity and develop therapeutic strategies to prevent αsyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify αsyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed αsyn variants and showed that this method can be used to study how αsyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on αsyn solubility. The results presented underscore the utility of the split GFP assay to quantify αsyn solubility in living cells

HPβCD-mediated clearance of α-syn aggregates does not depend on the ability of HPβCD to alter cholesterol levels.

<p><b>a)</b> Immunofluorescence microscopy analyses of TFEB subcellular localization in H4/α-syn-GFP cells untreated or treated with HPβCD (1 mM) or HPβCD–cholesterol complexes (1 mM) for 24 h. TFEB nuclear localization was monitored using a FLAG-specific antibody and DAPI nuclear stain. Colocalization of DAPI (blue, column 1) and TFEB (red, column 2) is shown in purple (column 3). Representative images are reported. Scale bar represents 10 μm. <b>b)</b> Fluorescence microscopy analyses of H4/α-syn-GFP cells untreated or treated with HPβCD (1 mM) or HPβCD–cholesterol complex (1 mM) for 24 h. Images of α-syn-GFP fluorescence (green, column 1) and aggregates, detected using the ProteoStat dye (red, column 2), were merged (column 3) and analyzed using NIH ImageJ software. Representative images are reported. Scale bar represents 20 μm.</p

TFEB overexpression results in reduced accumulation of α-syn aggregates.

<p><b>a)</b> Fluorescence microscopy analyses of H4/α-syn-GFP cells transduced to express TFEB-3xFLAG or S142A TFEB-3xFLAG. Images of α-syn-GFP fluorescence (green, column 1) and aggregates, detected using the ProteoStat dye (red, column 2), were merged (column 3) and analyzed using NIH ImageJ software. Representative images are reported. Scale bar represents 20 μm. <b>b)</b> Total protein aggregation in H4/α-syn-GFP cells transduced to express TFEB-3xFLAG or S142A TFEB-3xFLAG. Total protein aggregation was quantified by measuring binding of the ProteoStat dye by flow cytometry. The aggregation propensity factor (APF) was calculated as described in Methods and normalized to TFEB mRNA expression. Data are reported as mean ± SD (n ≥ 3; p < 0.01). <b>c)</b> Fluorescence microscopy analyses of H4/α-syn-GFP cells treated with control siRNA or <i>TFEB</i> siRNA. Images were analyzed as described in (a). Representative images are reported. Scale bar represents 20 μm. <b>d)</b> Total protein aggregation in H4/α-syn-GFP treated with control siRNA or <i>TFEB</i> siRNA. Total protein aggregation was quantified as described in (b). Data are reported as mean ± SD (n ≥ 3; p < 0.01). e) Immunofluorescence microscopy analyses of TFEB subcellular localization in H4/α-syn-GFP cells transduced to express TFEB-3xFLAG or S142A TFEB-3xFLAG. TFEB nuclear localization was monitored using a FLAG-specific antibody and DAPI nuclear stain. Colocalization of DAPI (blue, column 1) and TFEB-3xFLAG (red, column 2) is shown in purple (column 3). Representative images are reported. Scale bar represents 10 μm. <b>f)</b> Percentage of cells transduced as described in (e) presenting TFEB nuclear localization. Representative fields containing 50–100 cells were analyzed (p < 0.05).</p

Genetic and Chemical Activation of TFEB Mediates Clearance of Aggregated α-Synuclein

<div><p>Aggregation of α-synuclein (α-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson’s disease (PD). The formation of α-syn aggregates results from aberrant accumulation of misfolded α-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of α-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated α-syn by inducing autophagic clearance of α-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-β-cyclodextrin promotes autophagic clearance of aggregated α-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of α-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of α-syn aggregates.</p></div

HPβCD treatment induces activation of TFEB in H4/α-syn-GFP cells.

<p><b>a-b)</b> Immunofluorescence microscopy analysis of TFEB subcellular localization in H4/α-syn-GFP cells treated with HPβCD (1 mM). TFEB nuclear localization was monitored using a TFEB-specific antibody and DAPI nuclear stain. Colocalization of DAPI (blue, row 1) and TFEB (red, row 2) is shown in purple (row 3). Scale bar represents 10 μm. <b>c)</b> Percentage of HPβCD-treated cells presenting TFEB nuclear localization. Representative fields containing 50–100 cells were analyzed (p < 0.05). <b>d)</b> Relative mRNA expression levels of representative CLEAR network genes in H4/α-syn-GFP cells treated with HPβCD (1 mM) for 24 h. <i>GBA</i>, <i>HEXA</i>, and <i>LAMP1</i> mRNA expression levels were obtained by qRT-PCR and calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120819#pone.0120819.g002" target="_blank">Fig. 2C</a>. Data are reported as mean ± SD (n ≥ 3; p < 0.01).</p

TFEB mediates reduction of α-syn aggregates by inducing autophagic clearance.

<p>a) Immunofluorescence microscopy analyses of LC3 and LAMP2 in H4/α-syn-GFP cells transduced to express TFEB-3xFLAG. Colocalization of LC3 (red, column 1) and LAMP2 (blue, column 2) is shown in purple (column 3). Representative images are reported. Scale bar represents 20 μm. <b>b)</b> Quantification of LC3- LAMP2 colocalization was calculated using randomly selected images containing 30–50 cells obtained from three independent experiments (p < 0.001). <b>c)</b> Relative mRNA expression levels of representative genes involved in the autophagy pathway in H4/α-syn-GFP cells transduced to overexpress TFEB. <i>MAPLC3</i>, <i>SQSTM1</i>, <i>BECN1</i>, and <i>UVRAG</i> mRNA expression levels were obtained by qRT-PCR, corrected for the expression of the housekeeping genes <i>GAPDH</i> and <i>ACTB</i>, and normalized to those of untreated cells (dashed line). Data are reported as mean ± SD (n ≥ 3; p < 0.05, *p < 0.01). <b>d)</b> Total protein aggregation in H4/α-syn-GFP cells transduced to express TFEB-3xFLAG and treated with bafilomycin (100 nM). Total protein aggregation was quantified by measuring binding of the ProteoStat dye by flow cytometry. The APF was calculated as described in the Methods. Data are reported as mean ± SD (n ≥ 3; p < 0.05).</p

HPβCD treatment enhances autophagic clearance of α-syn aggregates in H4/α-syn-GFP cells.

<p><b>a)</b> Relative mRNA expression levels of representative genes of the autophagy pathway in H4/α-syn-GFP cells treated with HPβCD (1 mM) for 24 h. <i>MAPLC3</i>, <i>SQSTM1</i>, <i>BECN1</i>, and <i>UVRAG</i> mRNA expression levels were obtained by qRT-PCR and calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120819#pone.0120819.g002" target="_blank">Fig. 2C</a> (p < 0.05). <b>b)</b> Western blot analyses of LC3 isoforms and GAPDH (used as loading control) in H4/α-syn-GFP cells treated with HPβCD (1 mM) for 24 h and quantification of LC3-II bands. Band intensities were quantified with NIH ImageJ software and corrected by GAPDH band intensities (p < 0.05) <b>c)</b> Immunofluorescence microscopy analysis of LC3 and LAMP2 in H4/α-syn-GFP cells treated with HPβCD (1 mM) for 24 h. Colocalization of LC3 (red, column 1) and LAMP2 (blue, column 2) is shown in purple (column 3). Representative images are reported. Scale bars represent 20 μm. <b>d)</b> Quantification of LC3-LAMP2 colocalization was calculated using randomly selected images containing 30–50 cells obtained from three independent experiments (p < 0.001). <b>e)</b> Fluorescence microscopy analyses of H4/α-syn-GFP cells untreated or treated with HPβCD (1 mM) and/or bafilomycin (100 nM) for 24 h. Images of α-syn-GFP fluorescence (green, column 1) and aggregates, detected using the ProteoStat dye (red, column 2), were merged (column 3) and analyzed using NIH ImageJ software. Representative images are reported. Scale bar represents 20 μm. f) Total protein aggregation in H4/α-syn-GFP cells untreated or treated with HPβCD (1 mM) and/or bafilomycin (100 nM) for 24 h. Total protein aggregation was quantified by measuring binding of the ProteoStat aggregation dye by flow cytometry. The APF was calculated as described in the Methods. Data are reported as mean ± SD (n ≥ 3; p < 0.05).</p

ProteoStat® co-localization assay.

a<p>Low co-localization – 35 to 60, yellow pixels.</p>b<p>High co-localization – 0 to 35, red pixels.</p

Inhibition of proteasomal degradation lowers αsyn solubility.

<p>(<b>A</b>) Quantitative analysis of GFP fluorescence of cells expressing αsyn-GFP₁₁+ GFP_1–10 (blue) and TP αsyn-GFP₁₁+ GFP_1–10 (red). Relative fluorescence was calculated by normalizing the fluorescence of cells 12 and 24 hrs post transfection to the fluorescence measured at 0 hr. *<i>p</i><0.005; **<i>p</i><0.05. (<b>B</b>) Relative fluorescence of cells expressing αsyn-GFP₁₁ and GFP_1–10 (blue) and TP αsyn-GFP₁₁+ GFP_1–10 (red), 24 hrs post transfection. Cells were incubated for 24 hrs with increasing concentrations of lactacystin (0–5 µM). Relative fluorescence was evaluated by normalizing the fluorescence of treated cells to the fluorescence of untreated cells. *<i>p</i><0.01, **<i>p</i><0.05. Data points are reported as mean ± S.E.M. (n = 3) (<b>C</b>) Representative western blot of cells expressing αsyn-GFP₁₁, treated with lactacystin (5 µM) for 24 hrs, using αsyn-specific antibody. (<b>D</b>) Western blots band quantification of cells expressing αsyn-GFP₁₁. Bands were quantified by NIH ImageJ analysis software. GAPDH was used as loading control.</p