Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5

Delineation of the Mdm20 protein domains affecting polyQ aggregate formation: Independence from the Nat5-interaction.

<div><p>A. Schematic drawings of Mdm20 deletion constructs. The tri-peptide repeat (TRP) domain and a nuclear localization sequence (NLS) are indicated as black and gray squares, respectively. Amino acid residue numbers are given for each deletion.</p> <p>B. Western blots showing the expression efficiency of each Mdm20-deletion construct and the association with Nat5 in HEK293 cells. Forty-eight hours post-transfection, the cells were immunoprecipitated (IP) with an anti-Flag antibody and then immunoblotted with anti-Flag and anti-Nat5 antibodies. Note that only the full-length Mdm20 interacts with Nat5, and all the other partial deletion constructs fail to interact with Nat5.</p> <p>C. Comparison of the polyQ aggregate forming efficacy among the Mdm20-deletion constructs. The relative levels of polyQ-bearing cells are compared with the mock transfection without any Mdm20 constructs and only with the polyQ81 and GFP plasmids. The data represent the mean +/- S.D. (n=3) **P<0.001. ***P<0.01.</p></div

Mdm20 co-localizes with aggresome, wrapping the polyQ aggregates at the peri-nuclear region.

<div><p>A. Immunohistochemistry showing the cellular localization of Mdm20 (upper panels: a-c) and Nat5 (lower panels: d-f) following transfection with Flag-tagged Q79 containing plasmids into HEK293 cells. The cells were fixed, and the images were captured 48 hr post-transfection. The arrows indicate the polyQ aggregates at the perinuclear region. (Scale bar: 10 μm).</p> <p>B. Mdm20 co-localizes with polyQ aggregates, wrapping the aggregates in rat primary cultured hippocampal neurons. Immunohistchemistry shows the cellular localization of Mdm20 (b) following transfection with GFP-polyQ81 (a) into rat primary cultured hippocampal neurons. High-magnification images of GFP-polyQ81 and Mdm20 staining (area as indicated with a dot-lined square) are shown within each panels. The arrow indicates polyQ aggregates. (Scale bar: 20 μm).</p> <p>C. Mdm20 accumulates in areas surrounding polyQ aggregates. Immunostaining of Mdm20, vimentin and polyQ aggregates following transfection with a Flag-tagged polyQ79-containing plasmid is shown. The images in d,h,l are high-magnification images of areas in a,e,i. The arrows indicate polyQ aggregates at the periphery of nucleus. (Scale bar: 10 μm (c, g, k), 3 μm (d, h, l)).</p></div

Immunocytochemical staining and cellular biochemical fractionation reveal distinct localizations of Mdm20 and Nat5.

<div><p>A. Distribution of NatB complex in HEK293 cells. Left: Immunocytochemistry of Mdm20 and Nat5 in HEK293 cells. After fixing HEK293 cells with paraformaldehyde, the cells were immunostained with anti-Mdm20 (green) and anti-Nat5 (red) antibodies. Shown are low magnification views (a,c,e) and partial-zoom views (b,d,f) of the area indicated (dot lined squares). (Scale bar: 20 μm (a, c, e), 3 μm (b, d, f)) Right: Cellular fractionation experiments in HEK293 cells. Western blots of Mdm20 and Nat5 together with other sub-cellular marker proteins are shown to reveal the efficiency of the cell fractionation process (g). F1, cytosolic fraction; F2, membranes and membrane organelles; F3, nuclear proteins; F4, components of cytoskeletal proteins. Marker proteins examined: Rpl3 and Rps3 (ribosomal proteins of large and small subunits, respectively), Lamp2 (marker of membrane fraction), Myc (marker of nuclear fraction), Histone3 (marker of nuclear chromatin fraction), vimentin (marker of cytoskeletal fraction), and actin (a general marker).</p> <p>B. Distribution of NatB complex in primary cultured rat hippocampal neurons. Left: Immunohistochemistry of Mdm20 and Nat5 in primary cultured rat hippocampal neurons at 7 DIV. Immunofluorescence analysis was performed for Mdm20 (a), Nat5 (e), and MAP2 (b,f) (a specific marker of neuronal dendrites). High-magnification images of Mdm20 and Nat5 staining (area as indicated with a dot-lined square) are shown (c,g). Merged images are also shown (d,h). (Scale bar: 20 μm (d, h), 5 μm (c, g)) (i): Cellular fractionation experiments of rat hippocampus at E18.5. Biochemical fractionations were performed as in A-(g) using brain regions from the hippocampi of embryos at E18.5. N-Shc was used as a neuron-specific marker protein. The relative intensities of the Mdm20 and Nat5 bands were quantified by densitometry and indicated below each blot.</p></div

Mdm20 stimulates polyQ aggregate formation in HEK293 cells.

<div><p>A. The polyQ aggregate-bearing cells increase in Mdm20-OE HEK293 cells, but not in Nat5-OE cells. Upper panels (a) indicate the GFP-polyQ 81 transfected HEK293 cells with mock, Flag-Mdm20 or mCherry-Nat5. Arrows show the polyQ aggregate-positive cells. (Scale bar: 100 μm) Western blots of Mdm20, Nat5, and actin in transfected and non-transfected cells are shown in (b). The arrow indicates a correct Mdm20 or mCherry-Nat5 band. An asterisk denotes a cross-reacting band. As in (c), the polyQ aggregate-positive cells were counted and evaluated. The relative ratio of polyQ-bearing cells to GFP-positive cells was determined in Mdm20, Nat5, and mock transfected cells. **P<0.001.</p> <p>B. Mdm20-KD by siRNA reduces the number of polyQ-bearing cells. No difference was found between two sets of Nat5-KD cells and either Nat5-1 or Nat5-2 siRNAs. (a): GFP-polyQ aggregates formed HEK293 cells. (Scale bar: 100 μm) (b): A western blot showing the effectiveness of the siRNAs. Evaluation of polyQ aggregates by cell counting as in (c). **P<0.001.</p></div

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO)mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity

Mdm20 affects the polyQ aggregate formation by the regulation of pAkt^Ser473 level.

<div><p>A. A phospho-mimic mutant of Akt (Akt-S473D) increases polyQ aggregate formation. Left: Shown are western blots for Akt and phospho-Akt (at Ser473 and Thr308). HEK293 cells were co-transfected with GFP-polyQ81 and Flag-tagged wild type Akt (F-Akt) or various phospho- and none-phospho-mimic Akt mutants as indicated. Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. Experiments were performed in naïve HEK293 cells (green bars) and Mdm20-KD cells (red bars). The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of mock transfection. The data represent the mean +/- S.D. (n=3) ***P<0.01.</p> <p>B. Mdm20-KD cells also reduce the phosphorylation level of Akt and polyQ aggregate formation. Left: The effects of Mdm20-KD on the phosphorylation level of Akt in the presense of various chemical treatment (DMSO, rapamycin (100 nM), Akt-inhibitor-VIII (5 μM) or 3MA (1 mM)). Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of control transfection. The data represent the mean +/- S.D. (n=3) **P<0.001. ***P<0.01.</p> <p>C. Mdm20-OE cells also increase the phosphorylation level of Akt and polyQ aggregate formation. Left: The effects of Mdm20-OE on the phosphorylation level of Akt in the presense of various chemical treatment same as B. The arrow indicates a correct mCherry-Nat5 band. An asterisk denotes a cross-reacting band. Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of mock transfection. The data represent the mean +/- S.D. (n=3) **P<0.001. ***P<0.01.</p></div

Mdm20 regulates the phosphorylation status of Akt.

<div><p>A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods.</p> <p>B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and PP1. The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. </p> <p>C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot. </p></div