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Proteasomal Inhibition by α-Synuclein Filaments and Oligomers*

Open AccessPublished:January 07, 2004DOI:https://doi.org/10.1074/jbc.M306390200
      A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing α-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that α-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric α-synuclein. Proteasome binding to insoluble α-synuclein filaments and soluble α-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Aβ peptide aggregates. Endogenous ligands of aggregated α-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.
      The development of polyubiquitin-containing intracellular inclusions in cell bodies and dystrophic neurites of nerve cells is a key feature of several neurodegenerative disorders, e.g. in Alzheimer's disease, Parkinson's disease (PD),
      The abbreviations used are: PD, Parkinson's disease; AS, α-synuclein; DLB, dementia with Lewy bodies; GAPDH, glyceraldehyde-6-phosphate dehydrogenase; HSP70, heat shock protein-70; PBS, phosphate-buffered saline; SNpc, substantia nigra pars compacta; TBS, Tris-buffered saline; Z, benzyloxycarbonyl; MES, 4-morpholineethanesulfonic acid.
      1The abbreviations used are: PD, Parkinson's disease; AS, α-synuclein; DLB, dementia with Lewy bodies; GAPDH, glyceraldehyde-6-phosphate dehydrogenase; HSP70, heat shock protein-70; PBS, phosphate-buffered saline; SNpc, substantia nigra pars compacta; TBS, Tris-buffered saline; Z, benzyloxycarbonyl; MES, 4-morpholineethanesulfonic acid.
      dementia with Lewy bodies (DLB), and Huntington's disease (
      • Ellison D.
      • Love S.
      ). The polyubiquitination of proteins represents the result of the cellular system that recognizes misfolded or unwanted proteins and tags them for degradation by the proteasome via the sequential action of three enzymes (for a recent review on the ubiquitin-proteasomal system, see Ref.
      • Glickman M.H.
      • Ciechanover A.
      ). First, the ubiquitin-activating enzyme activates ubiquitin in an ATP-dependent process, after which the activated ubiquitin is passed onto the ubiquitin carrier protein class of ubiquitin conjugates. Then ubiquitin-protein isopeptide ligases recognize the misfolded proteins and mediate covalent binding of ubiquitin to them. Normally, polyubiquitinated proteins are recognized by the 19 S regulatory complex of the 26 S proteasome that unfolds the substrates and feeds them to the 20 S proteolytic core particle of the 26 S proteasome. The proteasome comprises three hydrolytic activities: the trypsin-like, the chymotrypsin-like, and the caspase-like hydrolytic activities that combined ensure the complete degradation of substrates. Accumulation of ubiquitinated proteins is an indicator for an imbalance between the production and ubiquitin labeling of misfolded proteins and the catabolic capacity of the proteasome.
      A group of neurodegenerative disorders, e.g. PD and DLB, is characterized by the filamentous Lewy body type of cytoplasmic inclusions in the degenerating cells (
      • Ellison D.
      • Love S.
      ). Aggregates of misfolded α-synuclein (AS) form the filamentous core of the Lewy bodies and Lewy neurites (
      • Goedert M.
      • Spillantini M.G.
      • Davies S.W.
      ), and the aggregates presumably play a role in the degeneration for more reasons. First, the association of missense mutations in AS with autosomal dominant PD shows a direct gain of toxic function for AS in the degenerative process where the dopaminergic neurons of the substantia nigra preferentially are lost (
      • Polymeropoulos M.H.
      • Lavedan C.
      • Leroy E.
      • Ide S.E.
      • Dehejia A.
      • Dutra A.
      • Pike B.
      • Root H.
      • Rubenstein J.
      • Boyer R.
      • Stenroos E.S.
      • Chandrasekharappa S.
      • Athanassiadou A.
      • Papapetropoulos T.
      • Johnson W.G.
      • Lazzarini A.M.
      • Duvoisin R.C.
      • Di Iorio G.
      • Golbe L.I.
      • Nussbaum R.L.
      ,
      • Krüger R.
      • Kuhn W.
      • Muller T.
      • Woitalla D.
      • Graeber M.
      • Kosel S.
      • Przuntek H.
      • Epplen J.T.
      • Schols L.
      • Riess O.
      ). Second, the pathogenic AS mutations, A30P and A53T increase AS propensity for aggregation (
      • Narhi L.
      • Wood S.J.
      • Steavenson S.
      • Jiang Y.
      • Wu G.M.
      • Anafi D.
      • Kaufman S.A.
      • Martin F.
      • Sitney K.
      • Denis P.
      • Louis J.C.
      • Wypych J.
      • Biere A.L.
      • Citron M.
      ,
      • Conway K.A.
      • Harper J.D.
      • Lansbury P.T.
      ). Likewise, aggregated insoluble AS has been demonstrated in tissue affected by sporadic PD, DLB, and multiple system atrophy (
      • Baba M.
      • Nakajo S.
      • Tu P.H.
      • Tomita T.
      • Nakaya K.
      • Lee V.M.
      • Trojanowski J.Q.
      • Iwatsubo T.
      ,
      • Dickson D.W.
      • Liu W.
      • Hardy J.
      • Farrer M.
      • Mehta N.
      • Uitti R.
      • Mark M.
      • Zimmerman T.
      • Golbe L.
      • Sage J.
      • Sima A.
      • D'Amato C.
      • Albin R.
      • Gilman S.
      • Yen S.H.
      ,
      • Kahle P.J.
      • Neumann M.
      • Ozmen L.
      • Muller V.
      • Odoy S.
      • Okamoto N.
      • Jacobsen H.
      • Iwatsubo T.
      • Trojanowski J.Q.
      • Takahashi H.
      • Wakabayashi K.
      • Bogdanovic N.
      • Riederer P.
      • Kretzschmar H.A.
      • Haass C.
      ). Third, the AS filaments in brain tissue resemble those formed in vitro from recombinant human AS that are rich in β-folded structures and both bind Congo Red and thioflavin S (
      • Serpell L.C.
      • Berriman J.
      • Jakes R.
      • Goedert M.
      • Crowther R.A.
      ,
      • Spillantini M.G.
      • Crowther R.A.
      • Jakes R.
      • Hasegawa M.
      • Goedert M.
      ,
      • Conway K.A.
      • Harper J.D.
      • Lansbury Jr., P.T.
      ). Accordingly, AS aggregates bear the characteristics of amyloid-type filaments accumulating in other diseases, e.g. formed from tau peptides in Alzheimer's disease and huntingtin in Huntington's disease (
      • Goedert M.
      • Spillantini M.G.
      • Davies S.W.
      ). The employment of animal models corroborates the involvement of aggregated AS in the neurodegenerative process where overexpression of AS leads to abnormal AS accumulation, misfolding, neuronal dysfunction, and overt degeneration (
      • Kahle P.J.
      • Neumann M.
      • Ozmen L.
      • Muller V.
      • Odoy S.
      • Okamoto N.
      • Jacobsen H.
      • Iwatsubo T.
      • Trojanowski J.Q.
      • Takahashi H.
      • Wakabayashi K.
      • Bogdanovic N.
      • Riederer P.
      • Kretzschmar H.A.
      • Haass C.
      ,
      • Masliah E.
      • Rockenstein E.
      • Veinbergs I.
      • Mallory M.
      • Hashimoto M.
      • Takeda A.
      • Sagara Y.
      • Sisk A.
      • Mucke L.
      ,
      • Feany M.B.
      • Bender W.W.
      ). A compromised proteasomal function in the substantia nigra in PD has been reported (
      • McNaught K.S.
      • Jenner P.
      ,
      • McNaught K.S.
      • Olanow C.W.
      • Halliwell B.
      • Isacson O.
      • Jenner P.
      ,
      • McNaught K.S.
      • Belizaire R.
      • Jenner P.
      • Olanow C.W.
      • Isacson O.
      ,
      • McNaught K.S.
      • Belizaire R.
      • Isacson O.
      • Jenner P.
      • Olanow C.W.
      ,
      • Tofaris G.K.
      • Razzaq A.
      • Ghetti B.
      • Lilley K.
      • Spillantini M.G.
      ), and this may be due to a direct effect of AS on the proteasome as transgenic cellular expression of AS inhibits the proteasome (
      • Stefanis L.
      • Larsen K.E.
      • Rideout H.J.
      • Sulzer D.
      • Greene L.A.
      ,
      • Tanaka Y.
      • Engelender S.
      • Igarashi S.
      • Rao R.K.
      • Wanner T.
      • Tanzi R.E.
      • Sawa A.L.
      • Dawson V.
      • Dawson T.M.
      • Ross C.A.
      ,
      • Petrucelli L.
      • O'Farrell C.
      • Lockhart P.J.
      • Baptista M.
      • Kehoe K.
      • Vink L.
      • Choi P.
      • Wolozin B.
      • Farrer M.
      • Hardy J.
      • Cookson M.R.
      ). Most important, aggregated AS displayed a strongly increased inhibitory activity toward the proteasome in vitro as compared with monomeric AS (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ). Such a proteasomal inhibition may have an impact on the dopaminergic neurons that seem to be particularly vulnerable to the stress of unfolded proteins (
      • Petrucelli L.
      • O'Farrell C.
      • Lockhart P.J.
      • Baptista M.
      • Kehoe K.
      • Vink L.
      • Choi P.
      • Wolozin B.
      • Farrer M.
      • Hardy J.
      • Cookson M.R.
      ).
      We demonstrate that AS, ubiquitin, and 20 S proteasomal components co-localize in Lewy bodies, and we show that aggregated but not monomeric AS binds efficiently to the 20 S proteasome part of the 26 S proteasome. The proteasome binding results in an efficient and selective non-competitive inhibition of the chymotrypsin-like proteasomal activity of the 20 S proteolytic particle. This inhibition, which is mimicked by Aβ amyloid filaments, is reverted by the amyloid targeting substances thioflavin S and Congo Red. Moreover, heat shock protein 70 (HSP70) that protects neurons toward AS-mediated toxicity in transgenic models (
      • Auluck P.K.
      • Chan H.Y.
      • Trojanowski J.Q.
      • Lee V.M.
      • Bonini N.M.
      ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bind to the AS filaments and abrogate their anti-proteasomal activity. Finally, subjecting cells to heat shock renders their cytosolic proteasomes less sensitive to the toxicity of aggregated AS thus demonstrating a potential for modulation of this toxicity by endogenous factors. The proteasomal inhibition by aggregated AS may represent a common neurodegenerative mechanism in diseases like PD, DLB, and multiple system atrophy where the degenerating cells accumulate AS aggregates. Abolition of the inhibition may open for new neuroprotective strategies.

      MATERIALS AND METHODS

      Miscellaneous and Proteins—All chemicals were of analytical grade if not otherwise stated. Thioflavin S and Congo Red were from Sigma. Human 20 S proteasome was isolated from human erythrocytes by affinity chromatography on immobilized MCP21 monoclonal antibody (
      • Hendil K.B.
      • Uerkvitz W.
      ). The specific activities of the 20 S proteasome are as follows: chymotrypsin-like activity, 4.0 nmol/min/mg protein using succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin; trypsin-like activity, 5.1 nmol/min/mg protein using Z-Ala-Ala-Arg-7-amido-4-methylcoumarine; caspase-like activity, 1.9 nmol/min/mg protein using Z-Leu-Leu-Glu-β-naphthylamide. The Aβ-(1-40) peptide was from (Schaefer-N., Copenhagen, Denmark). Recombinant human HSP70 was from Stressgen, (Victoria, British Columbia, Canada), and GAPDH was from Sigma. Both GAPDH and HSP70 were centrifuged for 30 min at 120,000 rpm at 4 °C in a TLA 120.1 rotor in a Optima TLX centrifuge (Beckman Instruments) to remove any aggregated materials prior to pull-down experiments. Recombinant human full-length AS-(1-140), C-terminally truncated AS-(1-95), and β-synuclein were expressed in Escherichia coli and purified as described previously (
      • Jensen P.H.
      • Højrup P.
      • Hager H.
      • Nielsen M.S.
      • Jakobsen L.
      • Olesen O.F.
      • Gliemann J.
      • Jakes R.
      ,
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ), followed by an additional reverse phase-high pressure liquid chromatography purification step on a Jupiter C18 column (Phenomenex) in 0.1% trifluoroacetic acid with an acetonitrile gradient. The proteins were subsequently aliquoted, lyophilized, and stored at -80 °C.
      Primary Antibodies—Sheep anti-AS was affinity-purified as described (
      • Gai W.P.
      • Power J.H.T.
      • Blumbergs P.C.
      • Jensen P.H.
      ) and obtained from Abcam, UK. Rabbit ASY-1 IgG was raised against full-length human recombinant AS (
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ). Rabbit FILA-1 IgG, specific for aggregated AS, was prepared by immunizing rabbits with sucrose density gradient-purified AS-(1-140) filaments. The immune serum was subsequently passed several times through a column with immobilized AS. This ensured the removal of all detectable immunoreactivity toward monomeric AS as determined by dot blotting. The serum lacking the monomer binding antibodies was then incubated with density gradient purified AS filaments, and the aggregate-binding antibodies were isolated by gradient co-sedimentation with the filaments. The antibodies were subsequently eluted from the filaments by incubation in 100 mm glycine, pH 2.5, followed by gradient centrifugation to separate the insoluble AS filaments from the FILA-1 IgG. The IgG was finally concentrated by protein A chromatography. The monoclonal antibodies, used to detect 20 S proteasome subunits, are as follows: MCP72 (α7 subunit); MCP196 (α5 subunit); MCP20 (α6 subunit); MCP231 (α2, α3, α6, α7 subunits); MCP421 (β1 subunit) (
      • Kristensen P.
      • Johnsen A.H.
      • Uerkvitz W.
      • Tanaka K.
      • Hendil K.B.
      ). The monoclonal antibodies to subunits of the 19 S regulatory proteasome complex are as follows: p31-38 (S14/Rpn12 subunit); S5a-18 (S5a/Rpn10 subunit); TBP1-19 (S6′/Rpt5 subunit) (
      • Hendil K.B.
      • Hartmann-Petersen R.
      • Tanaka K.
      ). A rabbit polyclonal antibody to S6′ (Affinity Bioreagents) was also used as were affinity-purified rabbit anti-ubiquitin (Dako, Denmark) and monoclonal antibodies against HSP70 (clone BRM-22) and β-actin (both from Sigma).
      Immunohistochemistry—Samples of substantia nigra from sporadic PD (n = 4), DLB (n = 3), and control subjects (n = 4) were obtained from the Netherlands Brain Bank (Amsterdam, The Netherlands) and fixed in 10% neutral buffered formalin and embedded in paraffin. Control samples were obtained among patients without neurological disease and with no pathological changes in sections of the substantia nigra pars compacta (SNpc). The diagnosis of PD in the human autopsies was based on loss of neuromelanin pigment and the excessive number of Lewy bodies in the remaining SNpc neurons in the eosin-stained section. The paraffin sections were cut at 2 μm and subjected to immunohistochemistry as described previously (
      • Mirza B.
      • Hadberg H.
      • Thomsen P.
      • Moos T.
      ). Complementary sections were demelanized using successive incubations with 0.25% potassium permanganate for 20 min followed by 1% oxalic acid and 1% potassium bisulfite for 4 min (
      • Leveugle B.
      • Faucheux B.A.
      • Bouras C.
      • Nillesse N.
      • Spik G.
      • Hirsch E.C.
      • Agid Y.
      • Hof P.R.
      ). The sections were incubated overnight at 4 °C with polyclonal rabbit anti-human ubiquitin diluted 1:500 (Dako, DK), sheep anti-human AS diluted 1:200 (Abcam, UK), or anti-human 20 S proteasome antibody MCP20. The sections were developed using 3,3-diaminobenzidine tetrahydrochloride as chromogen. To examine the extent of nonspecific binding, non-immune sera were substituted for the primary antibody. The relative ratio between the presence of AS and 20 S proteasome in Lewy bodies was estimated by staining four pairs of sections per autopsy for AS and 20 S proteasome, which were counted for their content of positive Lewy bodies.
      Immunolabeling of Isolated Lewy Bodies—Immunohistochemical staining was conducted in isolated Lewy bodies from fresh frozen brains of patients with DLB. The cingulate cortex from five DLB cases were analyzed. The isolation procedure was modified from Refs.
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ,
      • Gai W.P.
      • Power J.H.T.
      • Blumbergs P.C.
      • Jensen P.H.
      , and
      • Gai W.P.
      • Yuan H.X.
      • Li X.Q.
      • Power J.T.H.
      • Blumbergs P.C.
      • Jensen P.H.
      with the Lewy body smears prepared by washing the Lewy body enriched fraction three times in Tris-buffered saline (TBS, 0.1 m Tris-HCl, 0.9% NaCl, 5 mm EDTA, and protease inhibitors) followed by smearing on gelatin-coated glass slides and air-drying for 2 h at room temperature. The smears were fixed in 4% formaldehyde in TBS for 10 min and then incubated with 3% H2O2 in 50% methanol/TBS for 10 min to bleach endogenous peroxidase activity. Following three 5-min rinse in TBS, the smears were blocked with 20% normal horse serum for 30 min and then incubated for 60 min in primary antibody solution containing monoclonal antibody MCP72 (1:300) or MCP196 (1:300). To facilitate recognizing Lewy bodies, the primary antibody solution also included sheep anti-AS (1:300) or anti-ubiquitin (1:300). The smears were rinsed three times in TBS and then labeled for 60 min with Cy2-conjugated donkey anti-mouse IgG, in combination with Cy3-conjugated donkey anti-sheep IgG or Cy3-conjugated anti-rabbit IgG (all used 1:100 dilution, all from Jackson ImmunoResearch). The primary and secondary antibody dilutions used above were predetermined by serial titrations before formal experiments. Controls for antibody specificity included omitting primary or secondary antibodies and preabsorption the primary antibody with ubiquitin or AS. Lewy body staining was not detected in these control experiments. The smears were examined using a Bio-Rad confocal laser scanning microscope and software package (Bio-Rad MRC 1024) (
      • Gai W.P.
      • Yuan H.X.
      • Li X.Q.
      • Power J.T.H.
      • Blumbergs P.C.
      • Jensen P.H.
      ) with the scanned image representing an ∼1-μm thick slice through the center or equatorial plane of the Lewy body.
      Preparation of AS Aggregates—AS aggregates were made by resuspending lyophilized AS in 20 mm Tris, 150 mm NaCl, pH 7.5, 0.02% NaN3 at 7 mg/ml followed by ultracentrifugation to remove insoluble aggregates (
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ). Incubation of β-synuclein (5.5 mg/ml) and Aβ-(1-40) peptide (4 mg/ml) was performed by analogous procedures. The supernatant was incubated at 37 °C on a shaker for ∼14 days. Insoluble AS aggregates were isolated for proteasome activity assays and electron microscopy by sedimentation through a 40% sucrose cushion (
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ) followed by resuspension in the buffer of choice. Soluble AS aggregates were isolated by gel filtration of the soluble supernatant, obtained after ultracentrifugation of the aggregated AS sample, on a 24 × 1-cm Bio-Gel A 1.5M column (Bio-Rad) in 150 mm NaCl, 10 mm NaH2PO4, pH 7.4 (PBS), at 0.5 ml/min. The buffer was changed to 1 m Hepes, pH 8.0, when isolating soluble oligomers for the proteasome activity assay. The elution of the aggregated oligomers was determined by dot-blotting of the eluted fractions using the FILA-1 antibody, and the total AS was monitored by probing the membrane with the ASY-1 antibody.
      Electron Microscopy—Electron microscopy was used to analyze the insoluble AS filaments and to visualize the binding of purified 20 S proteasomes to the filaments. Filaments isolated from 30 μl of aggregated AS (7 mg/ml) were resuspended in 30 μl of PBS supplemented 20 mm NaN3 after pelleting through the sucrose cushion as described above. Purified 20 S proteasome was centrifuged for 10 min at 100,000 rpm at 4 °C in a TLA 120.1 rotor in an Optima TLX centrifuge (Beckman Instruments) to remove aggregated materials. Soluble 20 S proteasome (40 μl at 0.18 mg/ml) was incubated together with 5 μl of resuspended AS filaments (5 mg/ml) for 2 h at 25 °C. Non-bound proteasome was removed by sucrose gradient centrifugation of the filaments with bound 20 S proteasome. The sedimented filaments with attached 20 S proteasome was resuspended in 70 μl of distilled water. Filaments not incubated with 20 S proteasomes before the centrifugation and soluble 20 S proteasomes diluted to 0.16 mg/ml in PBS before direct application to the grid were used as control. All samples were pipetted onto carbon-coated nickel grids (3 μl for each grid) and allowed to stand for 2 min. The samples were stained with 1% aqueous uranyl acetate for 1 min. The grids were then air-dried and examined in a Morgagni 268, 80-kV electron microscope, and photographs were taken at 14,000, 18,000, or 22,000 times magnification.
      For immunogold labeling, grids with samples were blocked with blocking buffer (PBS with 0.05 m glycine and 0.1% milk protein) for 10 min and incubated with the primary antibody MCP72 (0.07 mg/ml) for 1 h. The grids were then washed three times for 5 min with blocking buffer and then incubated with goat anti-rabbit IgG conjugated to 5-nm diameter gold particles (Amersham Biosciences) diluted 1:100 in PBS with 1% fish gelatin, 0.06% polyethylene glycol, and 0.1% milk protein for 1 h. The grids were then washed twice for 5 min in PBS with 0.1% milk protein and then twice for 5 min in water. The grids were finally stained and examined as the non-immunolabeled samples.
      Filament Pull-down Assay—Cellular cytosol was prepared from primary cultures of normal human skin fibroblasts (
      • Rattan S.I.
      ) and from rat brain (
      • Jensen P.H.
      • Nielsen M.S.
      • Jakes R.
      • Dotti C.G.
      • Goedert M.
      ). Cells, at confluence, were trypsinized and collected by centrifugation (1000 × g, 10 min, 4 °C). The cells were sonicated in 50 mm NaCl, 10 mm Hepes, pH 8, 0.5 m sucrose, 1 mm EDTA, 0.2% (v/v) Triton X-100, 0.2 mm phenylmethanesulfonyl fluoride, 0.05% β-mercaptoethanol (5 ml for three 75-cm2 flasks) on ice. The supernatant was collected after centrifugation at 108,000 × g for 30 min at 4 °C giving a cytosol of 1 mg of protein/ml. To demonstrate the binding to AS filaments, 250 μl of cytosol were incubated with 70 μg of AS filaments for 1.5 h at 37 °C; 30 μg of GAPDH were incubated with 9 μg of AS filaments for 16 h at 4 °C; HSP70 (1.2 μg) were incubated with 21 μg of AS filaments for 2 h at 25 °C; purified 20 S (5.4 μg) were incubated with 70 μg of AS filaments for 16 h at 4 °C. All volumes were adjusted to 300 μl with 20 mm Tris and 150 mm NaCl, pH 7.4, except for the HSP70, where the buffer was 50 mm Tris, 100 mm NaCl, 1 mm dithioerythritol and 0.1 mm phenylmethanesulfonyl fluoride, pH 7.2. As negative controls cytosol, GAPDH, HSP70, 20 S proteasomes, or filaments were treated as above but without mixing the individual proteins. As positive controls, cytosol, GAPDH, HSP70, 20 S proteasome, or the filaments were loaded directly on the SDS-PAGE. Excess of purified monomeric AS was supplemented to the ligand-filament samples to determine whether this would inhibit the association of the ligands to the filaments or the interaction displayed a selectivity for the filaments as demonstrated by an unchanged amount of ligand in the pellet.
      The filament pull-down assay was performed by placing the protein samples on a cushion of 1.6 ml of 40% sucrose, 13 mm MES, and 1 mm EDTA, pH 7.0, followed by centrifugation at 55,000 rpm for 30 min in a TLS 55 swing out rotor at 4 °C in a Optima TLX centrifuge (Beckman Instruments). The pellets were either recovered for the proteasome assay by resuspension in 1 m Hepes, pH 8.0, or solubilized in 30 μl of 8 m urea with 4% SDS for 16 h in 37 °C. The latter was used to ensure complete depolymerization of the filaments. Non-bound proteins, remaining in the supernatant, were analyzed by the precipitation of 500 μl of the supernatant in trichloroacetic acid. The solubilized pellets and the precipitated supernatants were dissolved in dithioerythritol-containing SDS-PAGE loading buffer, heated 95 °C for 3 min, and subjected to SDS-PAGE followed by analysis with staining of the gel by silver or Coomassie Blue or by immunoblotting (
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ).
      Proteasome Assay—Approximately 80% confluent fibroblasts were scraped into 100 mm Tris-HCl buffer, pH 7.5 (1 ml per 75-cm2 flask). The cells were placed on ice for 15 min to allow lysis. The cytosolic extract was subsequently harvested as the supernatant after a centrifugation at 11,500 × g for 10 min at 4 °C. For the proteasome assay, the cytosol was used at a concentration of 100 μg of protein/ml and the purified human erythrocyte 20 S proteasome at a concentration of 2 μg of protein/ml. The hydrolytic activity for the chymotrypsin-like, trypsin-like, and caspase-like hydrolytic activities were determined as described previously (
      • Fonager J.
      • Beedholm R.
      • Clark B.F.
      • Rattan S.I.
      ). The coefficient of variation for the proteasome assay ranged from 0.8 to 6.3% in six independent experiments with the mean being 3%. Proteasome enzyme activities were calculated as the difference in activity measured in the absence and in the presence of the proteasome inhibitor, lactacystin, and in some experiments the peptide aldehyde Z-Leu-Leu-leucinal (MG132), both being potent inhibitors of primarily of the chymotrypsin-like activity. All the activity measurements were done in duplicate or triplicate from independent samples, as stated in the legends. Fluorescence of cleavage products from peptide substrate was measured on a Kontron SFM 25 spectrofluorometer. The effect of aggregated and non-aggregated AS on the proteasomal activity was determined by incubating proteasomes with AS for 60 min at 37 °C prior to the addition of the fluorogenic substrate.

      RESULTS

      Localization of 20 S Components in Lewy Bodies—Lewy bodies denote the characteristic inclusion bodies of PD and DLB (
      • McKeith I.G.
      • Galasko D.
      • Kosaka K.
      • Perry E.K.
      • Dickson D.W.
      • Hansen L.A.
      • Salmon D.P.
      • Lowe J.
      • Mirra S.S.
      • Byrne E.J.
      • Lennox G.
      • Quinn N.P.
      • Edwardson J.A.
      • Ince P.G.
      • Bergeron C.
      • Burns A.
      • Miller B.L.
      • Lovestone S.
      • Collerton D.
      • Jansen E.N.
      • Ballard C.
      • de Vos R.A.
      • Wilcock G.K.
      • Jellinger K.A.
      • Perry R.H.
      ); they contain abundant ubiquitinated material indicative of an insufficient proteasomal function (Fig. 1A). AS in a filamentous form comprise a main component of Lewy bodies (Fig. 1B) (
      • Spillantini M.G.
      • Schmidt M.L.
      • Lee V.M.
      • Trojanowski J.Q.
      • Jakes R.
      • Goedert M.
      ). 20 S proteasomes were observed in the cytoplasm of both control and brain disease cases, and the α6 subunit-specific antibody MCP20 also labeled Lewy bodies of both PD and DLB cases (Fig. 1C). Labeling of Lewy bodies by the anti-ubiquitin, anti-AS, and anti-proteasome 20 S antibodies was mainly confined to their peripheral zone (Fig. 1, A-C). Immunolabeling was not observed in nigral neurons when the primary antibody was omitted from the immunoreaction (Fig. 1D). The number of stained Lewy bodies was lower with 20 S proteasome antibodies than with AS.
      Figure thumbnail gr1
      Fig. 1Upper panel, ubiquitin, AS, and 20 S proteasome co-localize in Lewy bodies in SNpc neurons in PD and DLB. Lewy bodies are marked by arrowheads. A, ubiquitin is seen densely in the cytoplasm and in a Lewy body. The labeling of the peripheral zone of the Lewy body is higher than in the center. B, AS in Lewy body in DLB tissue. Note the predominant labeling of the peripheral zone of the Lewy body. C, 20 S proteasome in a neuron from a PD case obtained from a section that was subjected to demelanization before immunostaining. Note the labeling by the MCP20 antibody of the Lewy body with the main labeling of the peripheral zone. D, control section from a PD case that was subjected to demelanization before immunostaining but without incubation with the primary antibody. The bars represent 20 μm. Lower panel, co-localization of AS, ubiquitin, and 20 S proteasome in isolated Lewy bodies. Lewy bodies were isolated from cortical brain tissue affected by DLB. Lewy bodies were identified by AS staining, and the localization herein of the 20 S proteasomal α5 subunit by MCP196 was investigated by confocal laser scanning microscopy. The upper left panel show ubiquitin, and the lower left panel shows 20 S subunits. The middle column shows AS and the merged images are shown in the right column. The bar in lower middle image represents 10 μm and applies to all images in the lower panel. a-Syn, α-synuclein; Ub, ubiquitin; Ctl, control.
      We also used confocal laser scanning microscopy to analyze freshly isolated Lewy bodies from post-mortem brains of five patients with DLB. This technique is more sensitive and allows more detailed analysis of co-localization of AS and the 20 S proteasome components in Lewy bodies. Nearly all Lewy bodies (90-95%) were positive for ubiquitin. Approximately 70% of Lewy bodies (range 58-80%) were positively labeled by the antibodies MCP196 (Fig. 1, lower panel) and MCP72 (not shown) specific for 20 S proteasomal α5 and α7 subunits. There was no obvious difference in the number or intensity of the labeled Lewy bodies between the two antibodies. In general, both the ubiquitin and proteasome antibodies labeled the peripheral portion of Lewy bodies more intensely where the AS immunoreactivity was also most intense (Fig. 1, lower panel). Compared with that of ubiquitin, the labeling of proteasome subunits appeared punctate or granular and often extended further toward the central core of the Lewy bodies. Furthermore, while all Lewy neurites were also ubiquitin-positive, they were rarely labeled by the proteasome-specific antibodies (not shown). A similar co-localization was noted in AS-positive filamentous inclusions in astrocytes, isolated from brain tissue affected by multiple system atrophy (data not shown).
      Direct Interaction between AS Filaments and 20 S Proteasome Particles—The co-localization of 20 S proteasomes and AS in the Lewy bodies provides a topological frame for an interaction between the two molecular species. To examine a putative direct interaction between AS filaments and proteasome, we employed an AS filament binding assay based on the use of filaments formed from recombinant human AS (Fig. 2, A and D), which serve as bait for proteasomes in human fibroblasts cytosol. These filaments were readily depolymerized prior to SDS-PAGE by overnight incubation in 8 m urea, 4% SDS, as demonstrated in the lanes with pelleted filaments (Fig. 2A). This contrast to the AS aggregates previously used by Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) and demonstrates the aggregates are devoid of uncharacterized covalent modifications, e.g. by oxidative cross-links. Evidently, a range of cytosolic proteins was bound to the filaments as demonstrated by their presence in the pellet only when co-incubated with the filaments (Fig. 2A). The presence of putative proteasomal proteins was investigated by immunoblotting using monoclonal antibodies; the subunits S6′/Rpt5 and S14/Rpn12 from the 19 S regulatory complex were detected in the cytosol as were the subunits α2, α3, α6, α7, and β1 subunits from the 20 S proteasome core (Fig. 2B). Densitometric analysis of the blots showed that 5-10% of the subunits from 20 S proteasomes in the cytosolic input were bound to the filaments. In contrast, a negligible binding was found for the 19 S regulatory complex subunits S6′/Rpt5 and S14/Rpn12 although a faint band of both epitopes could be discerned. The S6′ antibody in Fig. 2B, top panel, was used by Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ). The 19 S regulatory complex can dissociate into two sub-complexes, the lid and the base (
      • Glickman M.H.
      • Rubin D.M.
      • Coux O.
      • Wefes I.
      • Pfeifer G.
      • Cjeka Z.
      • Baumeister W.
      • Fried V.A.
      • Finley D.
      ). S6′/Rpt5 is found in the base, whereas S14/Rpn12 is a component of the lid. Thus, neither lid nor base binds avidly to AS filaments.
      Figure thumbnail gr2
      Fig. 2AS filaments bind 20 S proteasomes. A, the binding of proteins from human fibroblast cytosol to AS filaments formed in vitro was investigated by co-sedimentation analysis and SDS-PAGE. Centrifugation of the filaments resulted in a pellet of the ∼20-kDa AS protein, whereas a small amount of protein was sedimented from the cytosol alone. However, co-incubation of the cytosol and filaments prior to centrifugation results in the detection of distinct proteins bands and a smear of proteins in the molecular range between 25 and 66 kDa. Electrophoresis of 8% of the input of cytosol and 25% of the input of filaments is shown, and the total pellets are displayed. Molecular size markers in kDa are indicated to the right. One of >3 experiments is presented. B, the binding of subunits of the 19 S regulatory complex (upper part) and 20 S proteasomes (lower part) from fibroblast cytosol to the AS filaments is demonstrated by immunoblotting. The total cytosolic input (C-T), the cytosolic pellet (C-P), and pellet formed by incubating cytosol and filaments (C+F-P) is shown to the left, middle, and right, and the antibodies used are indicated to the left. The subunit S6′/Rpt5 of the 19 S regulatory complex was probed by a commercial rabbit antibody S6′ and the TBP1-19 antibody with identical results. The S14/p31/Rpt5 subunit was detected with the p31-38 antibody. A faint filament-associated band is detectable by the TBP1-19 and the p31-38 antibodies. The 20 S proteasome subunits α7 and β1 were probed by MCP72 and MCP421 and the α-subunits 2, 3, 6, and 7 by MCP231, and all antibodies demonstrated filament-associated immunoreactivity corresponding to approximately the same proportion of the cytosolic input. One of three experiments is presented. C, the purity of purified human 20 S proteasomes is demonstrated by a Coomassie Blue staining of 8 μg of material resolved by reducing SDS-PAGE. Molecular size markers in kDa are indicated to the left. D, negative staining electron microscopy of purified AS filaments (panel 1) and filaments after incubation with purified 20 S proteasomes (panels 3-6). Note the smooth appearance of the native filaments (panel 1) and the presence of spherical, ∼11-nm particles, associated to the filaments upon incubation with the 20 S proteasomes (panels 3-6). Immunoelectron microscopy with the MCP72 antibody and 5-nm gold particles conjugated to the second antibody was performed to verify the presence of the 20 S proteasomes (panels 5 and 6). Panel 2 demonstrates the presence of approximately 11-nm electron dense particles labeled by the MCP72 antibody and shows that the particles are of similar diameter as those associated to the filaments (panels 3-6). Bars representing 25 nm are presented in panels 1-6. One of >3 independent analysis is presented. E, purified 20 S proteasomes bind preferentially to AS in its filamentous form. Lane 1 shows 30% of the input of 20 S proteasome subunit α7. Negligible amounts of the subunit were present in pellets upon sedimentation of the 20 S proteasome without filaments (lane 2) and in the presence of monomeric AS (lane 8). By contrast, proteasomes sedimented with filaments are shown in lanes 3-7. In lanes 4-7, increasing amounts of monomeric AS were added to the AS filaments. The molar ratio of monomeric to filamentous AS is indicated below the lanes. The absent effect of increasing the ratio of monomeric/filamentous (M/F) AS prior to incubation with the 20 S proteasome demonstrates selective binding of proteasomes to filaments. One of three similar experiments is presented.
      We next examined the direct interaction between AS filaments and human 20 S proteasome particles, purified from human erythrocytes, to further support the idea of a direct binding of the AS filament to the 20 S particles. The purity of the 20 S proteasome is demonstrated by Coomassie Blue staining of the characteristic subunits of about 25-30 kDa (Fig. 2C). Negative staining electron microscopy showed ∼11 nm structures on the AS filaments upon incubation with the 20 S proteasomes (Fig. 2D, panels 3 and 4). The 11 nm structures resembled 20 S proteasomes (Fig. 2D, panel 2). The identity of the filament-associated structures as 20 S proteasome was verified by immunogold labeling with the MCP72 antibody (Fig. 2D, panels 5 and 6).
      It was of pivotal importance to determine whether the interaction could represent a novel function of aggregated AS. Addition of up to an 8-fold excess of monomeric non-fibrillar AS had no detrimental effect on the co-sedimentation of purified 20 S proteasome particles with the filaments, thus supporting a filament selectivity of the binding (Fig. 2E). Similar data were obtained using proteasomes in cytosolic extracts (data not shown). Hence, proteasomal subunits co-localize with AS in Lewy bodies, and 20 S proteasomes bind directly to AS filaments without the need for auxiliary cytosolic components in this reaction, which represents an apparent novel function of AS developed upon aggregation.
      AS Filaments Selectively Inhibit the Chymotrypsin-like Activity of the 20 S Proteasome—The possible functional consequences of the interaction between AS filaments and the proteasome were determined by measuring the ubiquitin-independent hydrolytic activity of the proteasome in human fibroblasts cytosol and in purified 20 S proteasomes isolated from human erythrocytes. The assay uses small fluorogenic substrates for the individual hydrolytic activities of the 20 S proteasome: the caspase-like, the chymotrypsin-like, and the trypsin-like activity. The fluorescence in the presence of the proteasomal inhibitor lactacystin was subtracted as background in the assays shown in Fig. 3A. Similar background was obtained with the proteasome inhibitor MG132, and this inhibitor was used in subsequent analyses. Fig. 3A compares the effect on the individual hydrolytic activities of supplementing cytosolic proteasome and purified 20 S proteasome with 10 nm monomeric and filamentous AS and demonstrates a strong inhibition of the chymotrypsin-like activity by aggregated but not monomeric AS. Clearly, the effect on the individual activities in the cytosol and in the purified 20 S proteasome is highly similar with (i) a strong filament selective inhibition of the chymotrypsin-like activity, (ii) the trypsin-like activity being marginally inhibited by both monomeric and filamentous AS, and (iii) the caspase-like activity being barely affected. The molar concentration of the filaments cannot be expressed precisely due to their heterogeneous homopolymeric nature. We expressed their concentration by their monomeric AS building blocks, well aware that the actual concentration of the individual filaments is severalfold lower than the concentration of their individual subunits. By this approach, the IC50 value for the chymotrypsin-like activity with filaments was more than 103-fold lower than with monomeric AS and corresponded to 0.5 nm, expressed as the equivalent of monomeric AS (Fig. 3B). This means that the selectivity on a molar basis is even more pronounced and likely in the range 105-106, given that filaments are aggregates of 100-1000 monomers. The saturation curves for unpurified cytosolic proteasomes and purified 20 S proteasomes for the inhibition by monomeric and aggregated AS are nearly identical. The dose-response curve for the inhibition of the chymotrypsin-like activity by the proteasome inhibitor MG132 demonstrates an ∼10-fold lower potency as compared with the AS filaments, based on their monomeric concentration, and thus underscores the toxic potential of AS unleashed upon conversion into filaments (Fig. 3B). The mode of inhibition by AS filaments was investigated by measuring the rate of chymotrypsin-like catalysis at different substrate concentrations in the absence or presence of filaments. AS filaments inhibit the Vmax (Fig. 3C), thus demonstrating a non-competitive mode of inhibition. The reduction of the Vmax displayed a dose-dependent correlation to the concentration of the AS filaments (data not shown). The similar inhibitory pattern and saturation curves of the isolated 20 S proteasome and cytosolic proteasomes indicate a similar inhibitory mode by the aggregated AS that likely is mediated by a direct effect on the 20 S catalytic particle.
      Figure thumbnail gr3
      Fig. 3AS filaments inhibit the proteasomal chymotrypsin-like activity. A, the individual proteasomal hydrolytic activities, chymotrypsin-like (CL), trypsin-like (TL), and caspase-like (CAL) were measured in fibroblast cytosol and purified human 20 S proteasomes (hatched bars) by fluorogenic substrates in the absence (black column) and presence of 10 nm of monomeric (M) AS (gray columns) and filamentous AS (F) (white columns). The samples were incubated with the test substances for 60 min at 37 °C prior to addition of the fluorogenic substrate. The ordinate shows the ratio between the hydrolytic activity in the absence and presence of AS. The bars show the mean ± 1 S.D. of three replicates. One of three similar experiments is presented. The chymotrypsin-like activity was efficiently and selectively inhibited by the AS filaments, whereas the trypsin-like activity displayed some inhibition by both monomeric and filamentous AS. The asterisks demonstrate that only the chymotrypsin-like activity was significantly inhibited (p < 0.01, two sampled t test, unpaired data). B, concentration dependence of the inhibition of the chymotrypsin-like activity in fibroblast cytosol (filled circles and triangles) and purified 20 S proteasome (open circles and triangles) by monomeric (triangles) and filamentous AS (circles) and of cytosol in the presence of the proteasomal inhibitor MG132 (filled squares). The concentration of the inhibitors is displayed on the abscissa, and the ratio between the chymotrypsin-like activity at the indicated concentration of inhibitor and the chymotrypsin-like activity in the absence of inhibitor is displayed on the ordinate. The concentration of the filaments is indicated as the concentration of its monomeric constituents. One of three representative experiments is presented. C, non-competitive inhibition of the chymotrypsin-like activity by AS filaments. The substrate hydrolysis of increasing concentrations of substrate by cytosolic proteasomes was recorded in the absence (filled circles) and presence of 35 μg/ml purified AS filaments (open circles), and the initial rate of hydrolysis was measured. The abscissa shows the concentration of the fluorogenic substrate, and the ordinate shows the initial rate of hydrolysis in arbitrary units. Note that the filaments inhibit the maximal rate of hydrolysis, indicating a non-competitive mode of inhibition. One of three similar experiments is presented.
      Amyloid-like Characteristics Are Responsible for the 20 S Proteasomal Inhibition by AS Filaments—The structural characteristics of the aggregates responsible for the proteasomal inhibition were investigated by antibodies, filaments assembled from C-terminally truncated AS, and thioflavin S that targets amyloid structures (Fig. 4B). The FILA-1 antibody is specific for the filamentous form of AS by more than 103-fold as compared with monomeric AS (Fig. 4A). This antibody abrogated the inhibitory activity of the AS filaments just as potently as the ASY-1 antibody, raised against full-length AS, thus strengthening the requirement for a structural transition of the AS (Fig. 4B). The FILA-1 antibody bound only poorly to isolated Lewy bodies or to tissue investigated by immunohistochemistry (data not shown). This is likely due to the coating of the α-synuclein core fibrils by cytosolic proteins (
      • Gai W.P.
      • Pountney D.L.
      • Power J.H.
      • Li Q.X.
      • Culvenor J.G.
      • McLean C.A.
      • Jensen P.H.
      • Blumbergs P.C.
      ). The aggregation of AS depends on structural determinants within its N-terminal approximately 90 amino acids, and this allowed for the formation of filaments by the C-terminal truncated AS-(1-95) peptides. A similar potency was observed of filaments formed from full-length AS-(1-140) and AS-(1-95) (Fig. 4B). AS filaments exhibit characteristics of the β-pleated structures present in amyloid-type filaments that can be targeted by thioflavin S. Preincubation of the AS filaments with thioflavin S, prior to their density gradient purification, inhibited their chymotrypsin-like inhibitory activity by about 60% without solubilizing the aggregates (Fig. 4B). This underscores the requirement for amyloid-like structures in the proteasomal inhibition by AS filaments, which were further corroborated by the potent proteasomal inhibition by the model amyloid filaments assembled from synthetic Aβ-(1-40) peptide (Fig. 4B). Accordingly, the chymotrypsin-like inhibitory activity depends on amyloid-like structural elements that evolve during the development of AS filaments.
      Figure thumbnail gr4
      Fig. 4Characteristics of the filaments being responsible for the proteasomal inhibition. Requirements of the aggregated AS needed for inhibiting the proteasome were investigated by several approaches. A, first the filament specificity of the FILA-1 antibody was characterized by solid phase binding assay as described previously (
      • Jensen P.H.
      • Islam K.
      • Kenney J.
      • Nielsen M.S.
      • Power J.
      • Gai W.P.
      ) for MAP-1B. The percent binding of 125I-labeled FILA-1 IgG (50 pm) to immobilized filaments in the absence and presence of increasing concentrations of soluble filamentous (filled circles) and monomeric AS (open circles) is displayed on the ordinate. The abscissa displays the concentration of soluble filamentous and monomeric AS presented by their monomeric content. The points display the mean ± 1 S.D. of triplicates. One of three representative experiments is displayed. B, inhibition of the proteasomal chymotrypsin-like activity by amyloid filaments and reversal of the inhibition by antibodies and amyloid targeting agents. The proteasomal chymotrypsin-like activity in fibroblasts cytosol was measured in the presence of purified filaments formed from 170 nm full-length AS-(1-140), 170 nm C-terminally truncated AS-(1-95) and 170 nm Aβ-(1-40). AS-(1-140) aggregates was preincubated with non-immune (NI) rabbit IgG, ASY-1 IgG, FILA-1 IgG, in a 300-molar excess as compared with the AS, or 0.7% thioflavin S for 60 min at 20 °C prior to their isolation. The effect of the treatment on the proteasome assay was measured. The ordinate displays the difference between the total chymotrypsin-like activity and the activity measured in the presence of the inhibitors expressed as percent of the decrease in activity measured in the presence of AS-(1-140). The columns demonstrate the mean of duplicate samples from one of two similar experiments. The neutralization of the AS-(1-140) inhibitory activity marked by stars was significant (p < 0.01); two sampled t test, unpaired data.
      Soluble Amyloid-like AS Oligomers Inhibit the 20 S Proteasome—Strategies aiming at abrogating filament formation can increase the content of oligomeric AS (
      • Conway K.A.
      • Rochet J.C.
      • Bieganski R.M.
      • Lansbury Jr., P.T.
      ). Therefore, it is a question of considerable interest whether the detrimental effect of AS aggregates is due to the end state of insoluble filamentous forms or whether soluble oligomers can partake in the inhibitory process. AS oligomers were isolated by subjecting samples of aggregated AS to ultracentrifugation to remove insoluble aggregates, followed by the separation of the soluble AS species by gel filtration. Soluble oligomers, reacting with the filament-specific antibody FILA-1, eluted from the column corresponding to molecular size markers larger than 150 kDa when aggregated AS was used as starting material (Fig. 5A, upper panel). The neoepitope, formed when AS monomers polymerize, is therefore shared by insoluble filaments and soluble oligomers. No detectable FILA-1 but only ASY-1 immunoreactivity was present if non-aggregated AS was analyzed by gel filtration, although some of these ASY-1-positive structures likely represent AS multimers (Fig. 5A, lower panel). The soluble FILA-1-positive AS aggregates inhibited the proteasomal chymotrypsin-like activity, and this inhibition could be dose-dependently neutralized by FILA-1 IgG (Fig. 5B). These oligomers likely contain β-pleated amyloid-like characteristics as preincubation with 2.5 mm Congo Red abrogated their inhibitory activity (Fig. 5B). Congo Red is able to disrupt preformed amyloid-type oligomers (
      • Sanchez I.
      • Mahlke C.
      • Yuan J.
      ).
      Figure thumbnail gr5
      Fig. 5Soluble AS oligomers inhibit the chymotrypsin-like proteasome activity. A, AS oligomers were isolated by gel filtration of high speed supernatants to separate high molecular weight oligomers from monomeric AS. Non-aggregated AS was treated in parallel as control. Fractions were assayed by dot-blotting with a filament-specific antibody (FILA-1) and ASY-1, which reacts with both soluble and filamentous AS. Row NI shows the reactivity with a nonspecific antibody. The lower panel shows the immunoreactivity of the eluate from the monomeric sample analyzed by the same antibodies. The elution of molecular size markers in kDa is indicated above and the fraction numbers below. One of three similar experiments is presented. B, samples from the eluted fractions shown in A were assayed for the effect on the proteasomal chymotrypsin-like activity of fibroblast cytosol. Fractions from AS oligomers are indicated by filled circles and fractions from non-aggregated AS by filled squares. The abscissa shows the fraction number from the gel filtration, and the ordinate shows the chymotrypsin-like activity of the cytosol. The effect of supplementing the eluate with FILA-1 antibody (at 0.025 mg/ml (filled triangle) and 0.1 mg/ml (open circle)) and 2.5 mm Congo Red (open triangle) is demonstrated. The elution of molecular size markers in kDa is indicated above. One of three similar experiments is presented.
      The development of proteasomal inhibitory activity during the course of AS aggregation was compared with the development of insoluble aggregates to determine whether soluble AS oligomers could play a significant role. The initial rate of development of proteasome inhibitory activity was rapid with 50% of the maximal inhibition reached within 1-2 days and thereafter the rate declined, although the inhibition continued to increase during the 14-day period (Fig. 6A). This contrasted with the development of insoluble AS aggregates that are first detectable after 6 days on Coomassie Blue-stained gels (Fig. 6B). β-Synuclein was used as negative control for these experiments, because it is unable to form filaments due to the absence of an internal ∼11-amino acid peptide stretch (
      • Giasson B.I.
      • Murray I.V.
      • Trojanowski J.Q.
      • Lee V.M.
      ). Incubation of this control protein neither developed proteasome inhibitory activity nor insoluble aggregates (Fig. 6, A and B). The pathogenic AS mutations A30P and A53T have been reported to enhance AS filament formation, although the process at neutral pH and at the concentrations used in the present study takes several days (
      • Conway K.A.
      • Lee S.J.
      • Rochet J.C.
      • Ding T.T.
      • Williamson R.E.
      • Lansbury Jr., P.T.
      ). Of special interest was the observation that the A30P mutation specifically acted by increasing the formation of oligomers (
      • Conway K.A.
      • Lee S.J.
      • Rochet J.C.
      • Ding T.T.
      • Williamson R.E.
      • Lansbury Jr., P.T.
      ). Fig. 6C demonstrates the faster development of proteasomal inhibitory activity in samples of the A30P mutant as compared with the wild type and A53T mutant. Hence, the proteasome inhibitory activity of a given AS sample cannot simply be determined by assessing the amount of insoluble AS but may be more optimally reflected by the amount of FILA-1 immunoreactivity.
      Figure thumbnail gr6
      Fig. 6Development of chymotrypsin-like inhibitory activity. A, the temporal development of chymotrypsin-like inhibitory activity upon incubation of 0.5 mm AS (filled circles) and β-synuclein (open circles). The abscissa shows the days of incubation, and the ordinate shows the inhibition of the chymotrypsin-like activity by 7 μm AS samples with the activity at day 14 subtracted. The points demonstrate the mean of duplicate samples in one of two similar experiments. B, the development of insoluble aggregates in the AS and β-synuclein samples as demonstrated in A is shown by Coomassie Blue staining of the protein remaining soluble (S) or being pelleted (P) upon ultracentrifugation. Note the late development of insoluble AS aggregates as compared with the rapid development of chymotrypsin-like inhibitory activity in A. C, effect of AS mutations on the development of chymotrypsin-like inhibitory activity upon incubation of 0.5 mm wild type (triangles), A30P (filled circles), and A53T AS (open circles). The abscissa shows the hours of incubation, and the ordinate shows the inhibition of the chymotrypsin-like activity by 7 μm AS samples with the activity by A30P at 36 h subtracted. The points demonstrate the mean of duplicate samples in one of three similar experiments.
      HSP70 and GAPDH Attenuate the Proteasomal Toxicity of AS Filaments—HSP70 and GAPDH were discovered as AS filament-binding proteins in our search for brain proteins that selectively bind to aggregated AS (data not shown). The direct interaction of isolated GAPDH and HSP70 with insoluble AS filaments is demonstrated in Fig. 7, A and B. HSP70 is of special interest as it attenuates the toxicity of AS and the amyloid-like polyglutamine peptides in transgenic Drosophila models of PD and Huntington's disease (
      • Auluck P.K.
      • Chan H.Y.
      • Trojanowski J.Q.
      • Lee V.M.
      • Bonini N.M.
      ,
      • Chan H.Y.
      • Warrick J.M.
      • Gray-Board G.L.
      • Paulson H.L.
      • Bonini N.M.
      ). Recombinant HSP70 reverted the inhibitory effect of insoluble AS filaments on the chymotrypsin-like activity (Fig. 7C). In PD, GAPDH was reported to relocate from the cytoplasm to Lewy bodies and the nucleus (
      • Tatton W.G.
      • Chalmers-Redman R.M.
      • Elstner M.
      • Leesch W.
      • Jagodzinski F.B.
      • Stupak D.P.
      • Sugrue M.M.
      • Tatton N.A.
      ). Co-incubation of GAPDH with the AS aggregates completely reverted the inhibitory effects on the chymotrypsin-like activity (Fig. 7C). Accordingly, endogenous proteins carry the potential for modulating the toxic properties of aggregated AS.
      Figure thumbnail gr7
      Fig. 7HSP70 and GAPDH bind AS filaments and abrogate their chymotrypsin-like inhibitory activity. A, the binding of purified rabbit GAPDH to AS filaments was determined by the filament pull-down assay as described in . The panel shows a silver-stained SDS-PAGE gel where lane 1 demonstrates the input of 6 μg of GAPDH. Lanes 3-5 show pellets recovered after centrifugation of GAPDH alone (lane 3), AS filaments alone (lane 4), and GAPDH incubated with AS filaments (lane 5). The molecular size marker in kDa is shown to the left and the positions of AS and GAPDH to the right. One of 3 similar experiments is presented. B, the binding of recombinant human HSP70 to AS filaments was analyzed analogous to the above-described binding of GAPDH but visualized by immunoblotting, using a monoclonal anti-HSP70 antibody and enhanced chemiluminescence. Lane 1, input of 0.3 μg of recombinant HSP70. Lanes 3 and 5 show HSP70 sedimented in the absence and presence of AS filaments. One of three similar experiments is presented. C, reversal of the AS-dependent inhibition of the chymotrypsin-like activity of purified 20 S proteasome by HSP70 and GAPDH. The ordinate demonstrates the chymotrypsin-like activity in arbitrary units with the activity without filaments being 1 and in the presence of filaments being 0. The effects of HSP70 and GAPDH alone and in a 100-fold excess as compared with AS filaments are shown. The reversal of the inhibitory effects of the filaments by HSP70 and GAPDH are significant as marked by asterisk (p < 0.01) when measured by the two-sample t test of unpaired data. One of three similar experiments is presented. D, heat shock treatment of fibroblast increases the resistance of their cytosolic proteasome toward the inhibition by AS filaments. The ordinate shows the chymotrypsin-like activity in arbitrary units, and the columns show the chymotrypsin-like proteasomal activity in the cytosol from cells treated with or without heat shock, and absence (expressed as 1) or presence of AS filaments. The filamentous inhibition of the heat shock-treated cytosol differs significantly from untreated control (p < 0.05) as marked by asterisks when measured by the two-sample t test of unpaired data. The inset shows the presence of HSP70 and actin in cells treated with (HS) or without (C) heat shock as determined by immunoblotting. One of three similar experiments is presented.
      Heat shock treatment is known to induce the expression of various chaperones that assist the cell in combating unfolded protein stress (
      • Tatton W.G.
      • Chalmers-Redman R.M.
      • Elstner M.
      • Leesch W.
      • Jagodzinski F.B.
      • Stupak D.P.
      • Sugrue M.M.
      • Tatton N.A.
      ). Cytosolic proteasomes extracted from fibroblast cells after heat shock were more resistant to AS filaments than non-treated controls. HSP70 may participate in this protection as its expression was increased in this cytosol (Fig. 7D, inset). Accordingly, cells have evolved physiological systems for combating the detrimental effects of misfolded AS on the proteasome.

      DISCUSSION

      This report describes a mechanistic link between two hallmarks of the neurodegenerative disorders PD, DLB, Lewy body variant of PD, and multiple system atrophy, namely the presence of aggregated AS and the accumulation of polyubiquitinated proteins. The latter is likely due to an insufficient proteasomal activity (
      • McNaught K.S.
      • Olanow C.W.
      • Halliwell B.
      • Isacson O.
      • Jenner P.
      ). The functional link is the highly increased proteasome inhibitory activity that is developed upon aggregation of AS from its monomeric conformer. The increased inhibitory activity is selective toward the ubiquitin-independent proteasomal chymotrypsin-like activity in both human fibroblast cytosol and isolated human 20 S proteasomes. By contrast, there is only a minor effect of AS on the trypsin-like and caspase-like hydrolytic activities, and these effects do not depend on the aggregative state of AS. Most interesting, the chymotrypsin-like activity is far more important for degradation of proteins than are the trypsin-like and caspase-like activities (
      • Kisselev A.F.
      • Goldberg A.L.
      ). The chymotrypsin-like inhibition had an IC50 about 1 nm, so AS filaments are more potent than the proteasomal inhibitor MG132. This is in agreement with recent data on the AS inhibitory effect on HEK 293 cell extracts and the ubiquitin-dependent degradation by 26 S proteasomes in rabbit reticulocyte lysates (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ). The neutralization of the inhibition by the FILA-1 antibody, which selectively binds to aggregated AS, corroborates the need for an aggregation-dependent structural change. This inhibitory activity depends on structures related to amyloid-type aggregates, as demonstrated by the similar inhibition by Aβ-(1-40) aggregates. However, it does not require the aggregates to be in the insoluble filamentous state but merely requires a structural transition to a form present on both filaments and soluble oligomers, a transition that correlates to emergence of the epitopes recognized by the FILA-1 antibody. The inhibitory mode is non-competitive and thus resembles the inhibitory effect by HSP-90 (
      • Tsubuki S.
      • Saito Y.
      • Kawashima S.
      ). It likely represents a structure change by the binding of the aggregated AS to the exterior of the β5 subunit, responsible for the chymotrypsin-like activity, and relayed to its catalytic site on its interior surface of the proteasomal barrel. This would be analogous to the effect of binding of monoclonal antibodies to the 20 S proteasome (
      • Conconi M.
      • Djavadi-Ohaniance L.
      • Uerkvitz W.
      • Hendil K.B.
      • Friguet B.
      ). Alternatively, the effect might be allosteric by binding to another subunit that subsequently changes the β5 subunit, but this is less likely as the trypsin-like and caspase-like activities hardly were affected. Such interactions are corroborated by the electron micrographs showing direct binding of purified 20 S proteasomes to AS filaments.
      The proteasome exists in cells predominantly as 26 S proteasomes consisting of the 20 S proteolytic particles with 19 S regulatory complexes attached to the ends of the 20 S cylinder. However, naked 20 S proteasomes account for about one-third of the 20 S proteasomal particles in mammalian cells (
      • Tanahashi N.
      • Murakami Y.
      • Minami Y.
      • Shimbara N.
      • Hendil K.B.
      • Tanaka K.
      ), and hydrid proteasomes with 19 S particles attached to one end and the activator PA28 attached to the other end represent about 25% of 20 S proteasomes in HeLa cells (
      • Tanahashi N.
      • Murakami Y.
      • Minami Y.
      • Shimbara N.
      • Hendil K.B.
      • Tanaka K.
      ). All proteasomal hydrolysis is carried out within the 20 S proteasome particle, and this can take two forms. Ubiquitin-dependent protein degradation, representing the major fraction, relies solely on the 26 S proteasomes and the hybrid proteasomes. By contrast, ubiquitin-independent proteolysis of certain unfolded or modified proteins can be carried out by the single 20 S proteasome. Most interesting, AS belongs to the proteins, which can be degraded by the 20 S proteasome without participation of ubiquitin or the 19 S regulatory complex (
      • Tofaris G.K.
      • Razzaq A.
      • Ghetti B.
      • Lilley K.
      • Spillantini M.G.
      ).
      An important difference between the data presented by Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) and the present report relates to the effect of the aggregated AS on the 20 S proteasome. Our data are identical with that of Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) regarding the inhibitory mode toward the three hydrolytic activities when analyzing the effect of AS on the ubiquitin-independent proteasomal activity in cytosol. Moreover, the IC50 of ∼1 and 103 nm for aggregated and monomeric AS are similar for the cytosolic proteasomes. The discrepancy relates to the effect on the isolated 20 S proteasome, which we find behaves similar to the cytosolic proteasome. By contrast, Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) reports an IC50 of ∼1000 and 10,000 nm for the inhibition of the chymotrypsin-like activity of their 20 S preparation by aggregated and monomeric AS. More experimental details could account for this discrepancy. First, the purified 20 S proteasome (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) was from a commercial source and purified by ammonium sulfate precipitation and several chromatographic steps. In contrast, the 20 S proteasome preparation used in the present study was isolated by a milder procedure involving immunoaffinity chromatography (
      • Hendil K.B.
      • Uerkvitz W.
      ). We have experienced with one batch of purified 20 S proteasome stored for prolonged time at -20 °C that it lost the majority of its hydrolytic activity. However, the remaining lactacystin-inhibitable chymotrypsin-like activity was not well inhibited by aggregated AS (data not shown). In this context, it is also reassuring that we got similar results with purified proteasomes and cell extracts.
      Another reason for the discrepancy could be the different state of the aggregated AS. The material used in this study contains filaments, as demonstrated by electron microscopy (Fig. 2), and it can be readily depolymerized to the native monomeric AS as seen by SDS-PAGE (Fig. 2). By contrast, the AS used by Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) was a hexahistidine fusion protein incubated at an unspecified protein concentration and in an unspecified buffer, and the aggregates formed were very old and not depolymerized by SDS treatment. Hence, the latter material could contain uncharacterized modifications, e.g. oxidative cross-links. However, the differences of the aggregates are likely of less importance as similar effects of aggregated AS on the cytosolic proteasomal activity was measured in the two reports. Our data support the interpretation of a direct effect of aggregated AS on the 20 S proteasome particle because of the similar inhibitory profile on the individual proteasomal hydrolytic activities and the identical chymotrypsin-like inhibitory potency on purified 20 S proteasome and cytosolic proteasomes. This is further corroborated by the demonstration of a direct binding of isolated 20 S proteasomes to AS filaments.
      The inhibitory effect on the 20 S proteasome does not exclude an interaction with subunits in the 19 S regulatory complex as reported for the S6′/Rpt5 (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ). However, the reported binding of the S6′/Rpt5 subunit was not selective to AS in its aggregated state, and the efficiency of the binding was not reported in terms of how much of the input was bound to the aggregated AS, for example (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ). We performed filament pull-down experiments on fibroblast cytosol, and we compared the amount of the 19 S subunits S6′/Rpt5 and S14/Rpn12 with the amount of the 20 S proteasome subunits α2, α3, α6, α7, and β1 in the input with the filament-bound proteins using a panel of proteasome antibodies. By this approach ∼5-10% of all the 20 S proteasome subunits bound to the filaments. The equal binding of the individual subunits supports a binding of the 20 S particle in toto. The binding of the 19 S regulatory complex subunits was, by contrast, negligible, although the binding of the S6′/Rpt5 was verified in separate experiments optimized to detect low efficient binding. Accordingly, the effect of the interaction between aggregated AS and the 20 S proteasome particle is likely of primary significance as compared with the putative binding to 19 S regulatory complex subunits; especially because the 19 S regulatory complex is involved in the ubiquitin-dependent proteasomal functions, and we and Snyder et al. (
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ) demonstrate the filament-specific effect on the ubiquitin-independent hydrolysis in cell extracts.
      The recent demonstration of inhibition of proteasomal function by cellular expression of wild type AS or its mutant peptides may reflect different mechanisms (
      • Stefanis L.
      • Larsen K.E.
      • Rideout H.J.
      • Sulzer D.
      • Greene L.A.
      ,
      • Tanaka Y.
      • Engelender S.
      • Igarashi S.
      • Rao R.K.
      • Wanner T.
      • Tanzi R.E.
      • Sawa A.L.
      • Dawson V.
      • Dawson T.M.
      • Ross C.A.
      ,
      • Petrucelli L.
      • O'Farrell C.
      • Lockhart P.J.
      • Baptista M.
      • Kehoe K.
      • Vink L.
      • Choi P.
      • Wolozin B.
      • Farrer M.
      • Hardy J.
      • Cookson M.R.
      ,
      • Snyder H.
      • Mensah K.
      • Theisler C.
      • Lee J.
      • Matouschek A.
      • Wolozin B.
      ). Tanaka et al. (
      • Tanaka Y.
      • Engelender S.
      • Igarashi S.
      • Rao R.K.
      • Wanner T.
      • Tanzi R.E.
      • Sawa A.L.
      • Dawson V.
      • Dawson T.M.
      • Ross C.A.
      ) demonstrated an inhibition of the ubiquitin-independent hydrolysis of all the three hydrolytic activities by the A30P mutant but not the wild type peptide in cellular extracts. By contrast, Petrucelli et al. (
      • Petrucelli L.
      • O'Farrell C.
      • Lockhart P.J.
      • Baptista M.
      • Kehoe K.
      • Vink L.
      • Choi P.
      • Wolozin B.
      • Farrer M.
      • Hardy J.
      • Cookson M.R.
      ) showed in living M17 neuroblastoma cells a decreased ubiquitin-dependent degradation of an unstable degron fused to a green fluorescent protein. Stefanis et al. (
      • Stefanis L.
      • Larsen K.E.
      • Rideout H.J.
      • Sulzer D.
      • Greene L.A.
      ) demonstrated an accumulation of ubiquitinated proteins in the PC12 cells with the highest expression of the A53T mutant, which correlated to a significantly decreased chymotrypsin-like activity in cellular extracts. The interpretation of the relation of these experiments to the mechanism described in this report is not clear given the uncertainty of whether the cells had accumulated aggregated AS species. The use of antibodies like FILA-1 or a reversal of the inhibition by Congo Red (
      • Sanchez I.
      • Mahlke C.
      • Yuan J.
      ) or expression of HSP70, for example, may help to resolve this issue.
      The transition of AS monomers into insoluble aggregates denotes a nucleation-dependent process wherein soluble oligomers represent a step in the pathway. The oligomers have recently been characterized extensively by biophysical techniques (
      • Lashuel H.A.
      • Petre B.M.
      • Wall J.
      • Simon M.
      • Nowak R.J.
      • Walz T.
      • Lansbury Jr., P.T.
      ), and they have been proposed to represent pore-forming structures that may perturb the cellular ionic homeostasis (
      • Volles M.J.
      • Lee S.J.
      • Rochet J.C.
      • Shtilerman M.D.
      • Ding T.T.
      • Kessler J.C.
      • Lansbury Jr., P.T.
      ). We are the first to demonstrate that these structures, ∼200-800 kDa as demonstrated by gel filtration, share both structural and functional properties with the insoluble filaments represented by (i) the binding of the FILA-1 antibody, and (ii) the sensitivity of their inhibition of the proteasomal chymotrypsin-like activity to Congo Red. However, although the development of filaments is slow (
      • Conway K.A.
      • Lee S.J.
      • Rochet J.C.
      • Ding T.T.
      • Williamson R.E.
      • Lansbury Jr., P.T.
      ), the proteasome-inhibitory oligomers form within hours (Fig. 6A) (
      • Lashuel H.A.
      • Petre B.M.
      • Wall J.
      • Simon M.
      • Nowak R.J.
      • Walz T.
      • Lansbury Jr., P.T.
      ). The process of oligomer formation is not yet clear, but it is stimulated by the A30P mutation (
      • Conway K.A.
      • Lee S.J.
      • Rochet J.C.
      • Ding T.T.
      • Williamson R.E.
      • Lansbury Jr., P.T.
      ) and oxidative conjugation of dopamine to AS (
      • Conway K.A.
      • Rochet J.C.
      • Bieganski R.M.
      • Lansbury Jr., P.T.
      ). Our demonstration of a faster development of proteasome inhibitory activity in the A30P mutant (Fig. 6C), as compared with the A53T and the wild type AS, is thus compatible with biochemical experiments on aggregation (
      • Conway K.A.
      • Lee S.J.
      • Rochet J.C.
      • Ding T.T.
      • Williamson R.E.
      • Lansbury Jr., P.T.
      ). However, a comparison between different experiments on oligomer formation has to be exerted with great care, as the formation of these species is extremely sensitive to the protein concentration. Lashuel et al. (
      • Lashuel H.A.
      • Petre B.M.
      • Wall J.
      • Simon M.
      • Nowak R.J.
      • Walz T.
      • Lansbury Jr., P.T.
      ) demonstrated they can develop in less than 1 h at AS concentrations between 300 and 700 μm, which is in the range of the current experiments.
      Most important, we demonstrate that endogenous ligands carry the potential of attenuating the proteasomal inhibition by AS aggregates as shown for HSP70 and GAPDH. The HSP70-mediated rescue of proteasomal function from AS toxicity may be the reason for its cytoprotective effects in vivo when co-expressed with AS in Drosophila (
      • Auluck P.K.
      • Chan H.Y.
      • Trojanowski J.Q.
      • Lee V.M.
      • Bonini N.M.
      ) and likely forms the basis for the protective effect of heat shock treatment on the proteasomal sensitivity to AS aggregates (Fig. 7). GAPDH is considered as a household enzyme in the glycolytic pathway, but accumulating data indicate that it may participate in non-glycolytic processes (
      • Tatton W.G.
      • Chalmers-Redman R.M.
      • Elstner M.
      • Leesch W.
      • Jagodzinski F.B.
      • Stupak D.P.
      • Sugrue M.M.
      • Tatton N.A.
      ). Its relocalization into the nucleus and Lewy bodies in PD (
      • Tatton N.A.
      ) suggest it may be sequestered and rendered unable to form its protective function against aggregated AS species in the cytosol. However, the current data do not allow us to unambiguously state that the direct binding of GAPDH and HSP-70 to the AS aggregates causes the attenuation of the inhibition. A binding of GAPDH and HSP-70 to the 20 S proteasome could in principle also cause the effect. Solving this issue will require additional experimentation, e.g. with the identification of GAPDH mutants that fail to bind to the filaments. β-Synuclein may represent another factor that modulates the AS toxicity, because it is able to bind AS and inhibit its aggregation, and because the co-expression of AS and β-synuclein leads to an attenuation of the pathology observed in AS single transgenic mice (
      • Hashimoto M.
      • Rockenstein E.
      • Mante M.
      • Mallory M.
      • Masliah E.
      ).
      The amyloid-type characteristics are important for proteasomal inhibition, as demonstrated by the effects of Congo Red and thioflavin S on the inhibitory effect of AS aggregates, and the inhibitory effects of amyloid filaments of the Aβ peptide raise the following hypothesis. Diseases characterized by intracellular amyloid-type filaments are subject to an inhibition of the chymotrypsin-like proteasomal activity, which contributes to the accumulation of polyubiquitinated proteins and a general degeneration of the cellular homeostasis via an impaired protein catabolism. Such inhibition of the chymotrypsin-like activity has been reported in substantia nigral tissue affected by PD and DLB (
      • McNaught K.S.
      • Jenner P.
      ,
      • Tofaris G.K.
      • Razzaq A.
      • Ghetti B.
      • Lilley K.
      • Spillantini M.G.
      ). The cellular selectivity of the neurodegeneration displayed by specific aggregating proteins, as observed in striatal neurons in Huntington's disease (
      • Ellison D.
      • Love S.
      ), for example, contrast the often wider expression of the proteins. This may be due to a cell-specific expression of the attenuating proteins. The latter binds to protein-specific structures in the aggregating proteins that are different from the common amyloid structures and hereby revert the toxicity, as exemplified by HSP70 and GAPDH. This hypothesis is corroborated by the demonstration of in vivo proteasome inhibitory properties of aggregates of glutamine-expanded huntingtin peptides (
      • Bence N.F.
      • Sampat R.M.
      • Kopito R.R.
      ) that can be attenuated by HSP70 (
      • Chan H.Y.
      • Warrick J.M.
      • Gray-Board G.L.
      • Paulson H.L.
      • Bonini N.M.
      ) and the accumulation of ubiquitinated proteins in animal models expressing aggregation-prone AS and huntingtin (
      • Masliah E.
      • Rockenstein E.
      • Veinbergs I.
      • Mallory M.
      • Hashimoto M.
      • Takeda A.
      • Sagara Y.
      • Sisk A.
      • Mucke L.
      ,
      • Lee M.K.
      • Stirling W.
      • Xu Y.
      • Xu X.
      • Qui D.
      • Mandir A.S.
      • Dawson T.M.
      • Copeland N.G.
      • Jenkins N.A.
      • Price D.L.
      ,
      • Davies S.W.
      • Turmaine M.
      • Cozens B.A.
      • DiFiglia M.
      • Sharp A.H.
      • Ross C.A.
      • Scherzinger E.
      • Wanker E.E.
      • Mangiarini L.
      • Bates G.P.
      ).
      The linkage of AS aggregation to proteasomal inhibition represents a paradigm to be exploited for the protection against AS-mediated cellular degeneration. Structural insight into this interaction may allow therapeutic strategies based on the targeting of the aggregates by small molecules or endogenous proteins thereby preventing their binding to the proteasome. Such an approach may complement the ongoing attempts to design inhibitors of AS aggregation, and potentially it may have a wider applicability toward other intracellular amyloidoses.

      Acknowledgments

      We thank Prof. B. F. C. Clark for invaluable support, Prof. A. Maunsbach for valuable guidance on electron microscopy, and The Netherlands Brain Bank and the National Health and Medical Research Council Brain Bank for donating human brain tissue.

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