Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβx-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ1–38 occurs as Aβ1–42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders

Reduced evolutionary rate in reemerged Ebola virus transmission chains

On 29 June 2015, Liberia’s respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak (“flare-up”) of seven confirmed cases. We demonstrate that, similar to the March 2015 flare-up associated with sexual transmission, this new flare-up was a reemergence of a Liberian transmission chain originating from a persistently infected source rather than a reintroduction from a reservoir or a neighboring country with active transmission. Although distinct, Ebola virus (EBOV) genomes from both flare-ups exhibit significantly low genetic divergence, indicating a reduced rate of EBOV evolution during persistent infection. Using this rate of change as a signature, we identified two additional EVD clusters that possibly arose from persistently infected sources. These findings highlight the risk of EVD flare-ups even after an outbreak is declared over

Reovirus Infection or Ectopic Expression of Outer Capsid Protein μ1 Induces Apoptosis Independently of the Cellular Proapoptotic Proteins Bax and Bak ▿

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax−/−Bak−/− mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax−/−Bak−/− MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak

PADK selectively enhances cathepsin B levels in two transgenic mouse models.

<p>APP_SwInd and APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; n = 11−13) or vehicle (n = 10) for 9–11 days. Hippocampal homogenates were analyzed by immunoblot and mean immunoreactivities are shown for active cathepsin B (CB), neprilysin (nep), insulin-degrading enzyme (IDE), α-secretase (α-sec), and LAMP1.</p><p>***<i>P</i><0.0001, unpaired t-test.</p

Reduced intracellular Aβ_1–42 staining corresponds with enhanced cathepsin B.

<p>Fixed brain sections from vehicle-treated wildtype mice (wt) and from the APP-PS1 mice treated with vehicle (veh) or PADK were double-stained for Aβ_1–42 (green) and cathepsin B (red). Immunofluorescence images of CA1 pyramidal neurons (arrows) are shown, with view-field widths of 56 µm.</p

PADK has no inhibitory effect on β-secretase activity.

<p>Recombinant human β-secretase (10 ng/ml) was incubated with different concentrations of PADK (open triangles), CA074me (circles), and β-secretase inhibitor IV (closed triangles), and the enzyme activity was determined with the SensiZyme assay kit that uses the procaspase-3 variant containing the β-secretase cleavage sequence Gly-Ser-Ser-Glu-Ile-Ser-Tyr-Glu-Val-Glu-Phe-Arg-Glu-Phe (A). Activity was expressed in absorbance units (mean±SEM), and only β-secretase inhibitor IV elicited inhibition with an IC₅₀ of 19.8±2.4 nM. The three compounds were also tested against cathepsin B activity using the fluorogenic substrate Z-Arg-Arg AMC (mean fluorescence units±SEM plotted). β-secretase inhibitor IV had no effect on the cathepsin B activity, and PADK and CA074me resulted in IC₅₀ values of 9,200±1,030 and 120±13 nM, respectively (B).</p

PADK decreases 6E10 immunostaining in APP-PS1 mouse brain.

<p>APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; n = 11) or vehicle (n = 10) for 11 days. Fixed tissue was sectioned and stained with the 6E10 antibody. Image analysis for densitometric quantification of the immunostaining (mean integrated optical density±SEM) was conducted across view-fields of the hippocampal CA1 stratum pyramidale (sp). Area of deposit labeling above background was also measured for view-fields of the hippocampal stratum radiatum (sr) and piriform cortex (mean percent of total measured area±SEM). ANOVAs: <i>P</i><0.0001; Tukey's post hoc tests compared to APP−PS1+vehicle.</p><p>**<i>P</i><0.001.</p

Lysosomal modulation is associated with preservation of synaptic markers in APP_SwInd and APP-PS1 mice.

<p>Transgenic and wildtype (wt) mice were injected daily with PADK (+) or vehicle (–) for 9–11 days. Equal protein aliquots of hippocampal homogenates were analyzed by immunoblot for synaptic markers and actin, showing PADK-improved levels of GluA1 and synapsin II (syn II) in transgenic mice (A). The mean GluA1 immunoreactivities±SEM are shown for vehicle-treated wildtypes and for the vehicle- and PADK-treated transgenics (B). Post hoc tests compared to vehicle-treated transgenics: **<i>P</i><0.001.</p

PADK reduces behavioral deficits in APP_SwInd and APP-PS1 mice.

<p>In the first model, vehicle-treated wildtype mice (n = 11) were tested with groups of vehicle- (n = 10) and PADK-treated APP_SwInd mice (n = 13) across trials on the suspended rod test (A), and time maintained on the rod during the third trial was plotted (means±SEM). The animal groups were also tested across consecutive days in the same novel field, and the percent change±SEM in exploratory distance on the second day compared to the first was determined (B). In the second model, age-matched vehicle-treated wildtypes were tested with groups of vehicle- (n = 10) and PADK-treated APP-PS1 mice (n = 11) for spontaneous alternation behavior in a T-maze (C); data are plotted as percent of maximum alternations possible (mean±SEM). Open field mobility assessment confirmed no change in mean grid crossings±SEM across the three groups of mice (D). Post hoc tests compared to vehicle-treated transgenics: *<i>P</i>≤0.01, **<i>P</i><0.001.</p

PADK-mediated enhancement across brain regions of transgenic mouse models.

<p>APP_SwInd (10–11 months of age) and APPswe/PS1ΔE9 mice (APP-PS1; 20–22 months) were injected i.p. daily with PADK (20 mg/kg; n = 11−13) or vehicle (n = 10) for 9–11 days. Active cathepsin B in tissue homogenates was measured by immunoblot, and the mean levels were compared to the respective mean immunoreactivity in vehicle-injected transgenic samples to determine the fold increase across brain regions (±SEM).</p