A Humanin Derivative Reduces Amyloid Beta Accumulation and Ameliorates Memory Deficit in Triple Transgenic Mice

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer\u27s disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN\u27s functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APPswe, tauP310L, and PS-1M146V that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD

Therapeutic versus neuroinflammatory effects of passive immunization is dependent on Abeta/amyloid burden in a transgenic mouse model of Alzheimer's disease

Abstract Background Passive immunization with antibodies directed to Aβ decreases brain Aβ/amyloid burden and preserves memory in transgenic mouse models of Alzheimer's disease (AD). This therapeutic strategy is under intense scrutiny in clinical studies, but its application is limited by neuroinflammatory side effects (autoimmune encephalitis and vasogenic edema). Methods We intravenously administered the monoclonal Aβ protofibril antibody PFA1 to aged (22 month) male and female 3 × tg AD mice with intermediate or advanced AD-like neuropathologies, respectively, and measured brain and serum Aβ and CNS cytokine levels. We also examined 17 month old 3 × tg AD female mice with intermediate pathology to determine the effect of amyloid burden on responses to passive immunization. Results The 22 month old male mice immunized with PFA1 had decreased brain Aβ, increased serum Aβ, and no change in CNS cytokine levels. In contrast, 22 month old immunized female mice revealed no change in brain Aβ, decreased serum Aβ, and increased CNS cytokine levels. Identical experiments in younger (17 month old) female 3 × tg AD mice with intermediate AD-like neuropathologies revealed a trend towards decreased brain Aβ and increased serum Aβ accompanied by a decrease in CNS MCP-1. Conclusions These data suggest that passive immunization with PFA1 in 3 × tg AD mice with intermediate disease burden, regardless of sex, is effective in mediating potentially therapeutic effects such as lowering brain Aβ. In contrast, passive immunization of mice with a more advanced amyloid burden may result in potentially adverse effects (encephalitis and vasogenic edema) mediated by certain proinflammatory cytokines.http://deepblue.lib.umich.edu/bitstream/2027.42/78261/1/1742-2094-7-57.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78261/2/1742-2094-7-57.pdfPeer Reviewe

SENP1 and SENP2 regulate SUMOylation of amyloid precursor protein

Amyloid β, a key molecule in the pathogenesis of Alzheimer's disease (AD), is produced from amyloid precursor protein (APP) by the cleavage of secretases. APP is SUMOylated near the cleavage site of β-secretase. SUMOylation of APP reduces amyloid β production, but its regulatory system is still unclear. SUMOylation, a modification at a lysine residue of a target protein, is mediated by activating, conjugating, and ligating enzymes and is reversed by a family of sentrin/SUMO-specific proteases (SENPs). Here, we found that both SENP1 and SENP2 induced de-SUMOylation of APP. Using quantitative PCR, we also found that expression of SENP1 but not SENP2 increased in an age-dependent manner only in female mice. The results of immunoblot analyses showed that the protein expression was consistent with the PCR results. Females, compared to males, have a higher incidence of AD in humans and show more aggressive amyloid pathology in AD mouse models. Our results provide a clue to understanding the role of SUMOylation in the sex difference in AD pathogenesis

SUMO3 Modification Accelerates the Aggregation of ALS-Linked SOD1 Mutants

<div><p>Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and aggregate to form intracellular inclusions. We report that both small ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant proteins at lysine 75 in a motoneuronal cell line, the cell type affected in ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9 of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather than by SUMO1, significantly increased the stability of the proteins and accelerated intracellular aggregate formation. These findings suggest the contribution of sumoylation, particularly by SUMO3, to the protein aggregation process underlying the pathogenesis of ALS.</p></div

SOD1 proteins are modified by SUMO1, SUMO2, and SUMO3.

<p>NSC34 cells were cotransfected with plasmids expressing FLAG-tagged SOD1 (either wild-type or mutant), HA-tagged SUMO 1/2/3, and myc-tagged Ubc9. The cell lysates were immunoprecipitated with an anti-FLAG M2 antibody, followed by immunoblotting with anti-HA antibody (upper panels) and anti-FLAG antibody (lower panels). <b>A.</b> SOD1 mutants (G93R, G85R, and N19S) were modified by both SUMO1 and SUMO2/3. A representative immunoblot result from four independent experiments is shown. <b>B.</b> The sumoylation of G93R-SOD1 by SUMO2/3 was markedly decreased by the K75R mutation but not by the K9R mutation. The presence (+) or absence (−) of mutations at K9 and K75 is indicated above the lane. A representative immunoblot result from three independent experiments is shown.</p

A familial ALS-linked SOD1 mutant is stabilized by SUMO3 modification.

<p>NSC34 cells were cotransfected with plasmids expressing FLAG-tagged G93R-SOD1, HA-tagged SUMO1, SUMO3, or the empty vector (vec), and myc-tagged Ubc9. After 16 h of transfection, the cells were treated with 50 µg/ml cycloheximide for 3 or 6 h or left untreated (time 0). The cell lysates were subjected to an immunoblot analysis with anti-FLAG antibody (<b>A</b> upper panel) and anti-β-actin antibody (<b>A</b> lower panel). <b>A.</b> A representative immunoblot result is shown. <b>B.</b> Quantitative analysis of the immunoblot. The intensity of each band was quantified using ImageJ and normalized to the arbitrary units of β-actin, and the means and SD (n = 4) were calculated. # indicates a statistically significant difference (p<0.01) among the three transfection conditions (vec, SUMO1, SUMO3) at time 0. Statistically significant differences (p<0.05) between time 0 of each condition are indicated by *. N.S. indicates not significant. <b>C.</b> Quantitative analysis of the immunoblot. The data in B are expressed relative to the 0 h value ( = 1).</p

SUMOs colocalize with familial ALS-linked SOD1 mutants in intracellular aggregates.

<p>CHO cells were cotransfected with plasmids expressing EGFP-fused SOD1 (either wild-type or mutant), HA-tagged SUMO 1/3, and myc-tagged Ubc9. The cells were fixed after 24 h of transfection and immunostained with anti-HA antibody and DyLight594-conjugated anti-mouse IgG antibody, followed by counterstaining with DAPI. Representative images from two independent experiments are shown. The bar in the upper left panel indicates 10 µm. Arrowheads and arrows indicate cells with and without intracellular SOD1 protein aggregates, respectively. In all triple transfections, all GFP-positive cells were DyLight594 (SUMO)-positive (detail in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101080#pone.0101080.s004" target="_blank">Table S1</a>).</p