19 research outputs found
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 Ī² peptide (AĪ²) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including AĪ²1ā42. 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 AĪ² pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ĪE9 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 AĪ² immunostaining as well as AĪ²x-42 sandwich ELISA measures in APPSwInd mice of 10ā11 months. More extensive AĪ² deposition in 20-22-month APPswe/PS1ĪE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic AĪ²1ā38 occurs as AĪ²1ā42 levels decrease in the mouse models, indicating that PADK treatment leads to AĪ² truncation. Associated with AĪ² 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 AĪ²1ā42 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 AĪ² 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, Liberiaās respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak (āflare-upā) 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 Ī¼1 Induces Apoptosis Independently of the Cellular Proapoptotic Proteins Bax and Bak āæ
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 Ī¼1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed Ī¼1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of Ī¼1 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 Ī¼1-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 Ī¼1 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 Ī¼1, indicating that caspases were not required. Additionally, Ī¼1- or reovirus-induced release of cytochrome c occurred efficiently in Baxā/āBakā/ā mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Baxā/āBakā/ā 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<sub>SwInd</sub> and APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; nā=ā11ā13) or vehicle (nā=ā10) for 9ā11 days. Hippocampal homogenates were analyzed by immunoblot and mean immunoreactivities are shown for active cathepsin B (CB), neprilysin (nep), insulin-degrading enzyme (IDE), Ī±-secretase (Ī±-sec), and LAMP1.</p><p>***<i>P</i><0.0001, unpaired t-test.</p
Reduced intracellular AĪ²<sub>1ā42</sub> 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 AĪ²<sub>1ā42</sub> (green) and cathepsin B (red). Immunofluorescence images of CA1 pyramidal neurons (arrows) are shown, with view-field widths of 56 Āµm.</p
PADK has no inhibitory effect on Ī²-secretase activity.
<p>Recombinant human Ī²-secretase (10 ng/ml) was incubated with different concentrations of PADK (open triangles), CA074me (circles), and Ī²-secretase inhibitor IV (closed triangles), and the enzyme activity was determined with the SensiZyme assay kit that uses the procaspase-3 variant containing the Ī²-secretase cleavage sequence Gly-Ser-Ser-Glu-Ile-Ser-Tyr-Glu-Val-Glu-Phe-Arg-Glu-Phe (A). Activity was expressed in absorbance units (meanĀ±SEM), and only Ī²-secretase inhibitor IV elicited inhibition with an IC<sub>50</sub> of 19.8Ā±2.4 nM. The three compounds were also tested against cathepsin B activity using the fluorogenic substrate Z-Arg-Arg AMC (mean fluorescence unitsĀ±SEM plotted). Ī²-secretase inhibitor IV had no effect on the cathepsin B activity, and PADK and CA074me resulted in IC<sub>50</sub> values of 9,200Ā±1,030 and 120Ā±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; nā=ā11) or vehicle (nā=ā10) for 11 days. Fixed tissue was sectioned and stained with the 6E10 antibody. Image analysis for densitometric quantification of the immunostaining (mean integrated optical densityĀ±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 areaĀ±SEM). ANOVAs: <i>P</i><0.0001; Tukey's post hoc tests compared to APPāPS1+vehicle.</p><p>**<i>P</i><0.001.</p
Lysosomal modulation is associated with preservation of synaptic markers in APP<sub>SwInd</sub> and APP-PS1 mice.
<p>Transgenic and wildtype (wt) mice were injected daily with PADK (+) or vehicle (ā) for 9ā11 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 immunoreactivitiesĀ±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<sub>SwInd</sub> and APP-PS1 mice.
<p>In the first model, vehicle-treated wildtype mice (nā=ā11) were tested with groups of vehicle- (nā=ā10) and PADK-treated APP<sub>SwInd</sub> mice (nā=ā13) across trials on the suspended rod test (A), and time maintained on the rod during the third trial was plotted (meansĀ±SEM). The animal groups were also tested across consecutive days in the same novel field, and the percent changeĀ±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- (nā=ā10) and PADK-treated APP-PS1 mice (nā=ā11) for spontaneous alternation behavior in a T-maze (C); data are plotted as percent of maximum alternations possible (meanĀ±SEM). Open field mobility assessment confirmed no change in mean grid crossingsĀ±SEM across the three groups of mice (D). Post hoc tests compared to vehicle-treated transgenics: *<i>P</i>ā¤0.01, **<i>P</i><0.001.</p
PADK-mediated enhancement across brain regions of transgenic mouse models.
<p>APP<sub>SwInd</sub> (10ā11 months of age) and APPswe/PS1ĪE9 mice (APP-PS1; 20ā22 months) were injected i.p. daily with PADK (20 mg/kg; nā=ā11ā13) or vehicle (nā=ā10) for 9ā11 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 (Ā±SEM).</p