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STIM2 protects hippocampal mushroom spines from amyloid synaptotoxicity.

Popugaeva E, Pchitskaya E, Speshilova A, Alexandrov S, Zhang H, Vlasova O, Bezprozvanny I - Mol Neurodegener (2015)

Bottom Line: Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD.We discovered that application of Aβ42 oligomers to hippocampal cultures or injection of Aβ42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII).Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neurodegeneration, Department of Medical Physics, Peter the Great St.Petersburg Polytechnic University, St. Petersburg, Russian Federation. lena.popugaeva@gmail.com.

ABSTRACT

Background: Alzheimer disease (AD) is a disease of lost memories. Mushroom postsynaptic spines play a key role in memory storage, and loss of mushroom spines has been proposed to be linked to memory loss in AD. Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD. It is important to evaluate effects of amyloid on stability of mushroom spines.

Results: In this study we used in vitro and in vivo models of amyloid synaptotoxicity to investigate effects of amyloid peptides on hippocampal mushroom spines. We discovered that application of Aβ42 oligomers to hippocampal cultures or injection of Aβ42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII). We further discovered that expression of STIM2 protein rescued CaMKII activity and protected mushroom spines from amyloid toxicity in vitro and in vivo.

Conclusions: Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.

No MeSH data available.


Related in: MedlinePlus

Synaptotoxic effects of amyloid oligomers in primary neuronal cultures. a Primary hippocampal neuronal cultures exposed to Aβ40, Aβ42 or vehicle treated (Ctrl). The cultures were fixed and stained for synaptic marker Synapsin I (green) and neuronal marker MAP2 (red). Scale bar corresponds to 20 μm. b Mean fluorescent intensities of synapsin I staining were divided by mean fluorescent intensities of MAP2 staining for hippocampal cultures treated with Aβ40, Aβ42 or vehicle treated (Ctrl). Average results from three independent experiments are shown. Values are shown as mean ± SEM. *: p < 0.05 by ANOVA one-way test
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Fig1: Synaptotoxic effects of amyloid oligomers in primary neuronal cultures. a Primary hippocampal neuronal cultures exposed to Aβ40, Aβ42 or vehicle treated (Ctrl). The cultures were fixed and stained for synaptic marker Synapsin I (green) and neuronal marker MAP2 (red). Scale bar corresponds to 20 μm. b Mean fluorescent intensities of synapsin I staining were divided by mean fluorescent intensities of MAP2 staining for hippocampal cultures treated with Aβ40, Aβ42 or vehicle treated (Ctrl). Average results from three independent experiments are shown. Values are shown as mean ± SEM. *: p < 0.05 by ANOVA one-way test

Mentions: In our experiments we set out to establish neuronal culture model of amyloid toxicity. In these studies we focused on soluble amyloid oligomers, which have been demonstrated to exert synaptotoxic effects in AD [1, 12–17]. Soluble amyloid oligomers were prepared from synthetic Aβ42 peptides by following previously described procedures [18] (for details see Materials and Methods section). The oligomeric state of resulting Aβ42 preparation was confirmed by AFM analyses as well as by SDS gel Western blotting experiments (Additional file 1: Figure S1). According to AFM data Aβ42 oligomers appear as globular structures with 1–2 nm height and around 10 nm width (Additional file 1: Figure S1A). Western blot experiments showed that Aβ42 preparation is mainly in oligomeric form and its molecular weight is around 26 kDa (Additional file 1: Figure S1B) that corresponds to pentamers or hexamers [19]. To mimic physiological situation more closely, we utilized low concentrations (less than 100 nM as calculated based on initial amount of peptides utilized for preparation of oligomers) of Aβ42 oligomers in our experiments. We also evaluated effects of Aβ40 oligomers. Aβ40 peptides were processed the same way as Aβ42 peptides (Additional file 1: Figure S1A). Equivalent amounts of Aβ42 and Aβ40 peptides were used in our experiments. Generated oligomers of Aβ42 and Aβ40 were added to DIV11 primary hippocampal neuronal cultures. In control experiments neuronal cultures were treated with vehicle (equivalent amount of growth media). At DIV14 these cultures were fixed, permeabilized and stained for neuronal marker MAP2 and synaptic marker synapsin I (Fig. 1a). To evaluate synaptogenesis state in studied cultures, mean synapsin signal intensity was divided by the mean of MAP2 intensity. Consistent with previous observations [15, 16, 20], we observed significant loss of synapsin staining in cultures exposed to Aβ42 oligomers (Fig. 1a, 1b). There was also a trend towards synaptic loss in cultures exposed to Aβ40 oligomers, but these changes have not reached a level of significance when compared to control cultures (Fig. 1a, 1b).Fig. 1


STIM2 protects hippocampal mushroom spines from amyloid synaptotoxicity.

Popugaeva E, Pchitskaya E, Speshilova A, Alexandrov S, Zhang H, Vlasova O, Bezprozvanny I - Mol Neurodegener (2015)

Synaptotoxic effects of amyloid oligomers in primary neuronal cultures. a Primary hippocampal neuronal cultures exposed to Aβ40, Aβ42 or vehicle treated (Ctrl). The cultures were fixed and stained for synaptic marker Synapsin I (green) and neuronal marker MAP2 (red). Scale bar corresponds to 20 μm. b Mean fluorescent intensities of synapsin I staining were divided by mean fluorescent intensities of MAP2 staining for hippocampal cultures treated with Aβ40, Aβ42 or vehicle treated (Ctrl). Average results from three independent experiments are shown. Values are shown as mean ± SEM. *: p < 0.05 by ANOVA one-way test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4536802&req=5

Fig1: Synaptotoxic effects of amyloid oligomers in primary neuronal cultures. a Primary hippocampal neuronal cultures exposed to Aβ40, Aβ42 or vehicle treated (Ctrl). The cultures were fixed and stained for synaptic marker Synapsin I (green) and neuronal marker MAP2 (red). Scale bar corresponds to 20 μm. b Mean fluorescent intensities of synapsin I staining were divided by mean fluorescent intensities of MAP2 staining for hippocampal cultures treated with Aβ40, Aβ42 or vehicle treated (Ctrl). Average results from three independent experiments are shown. Values are shown as mean ± SEM. *: p < 0.05 by ANOVA one-way test
Mentions: In our experiments we set out to establish neuronal culture model of amyloid toxicity. In these studies we focused on soluble amyloid oligomers, which have been demonstrated to exert synaptotoxic effects in AD [1, 12–17]. Soluble amyloid oligomers were prepared from synthetic Aβ42 peptides by following previously described procedures [18] (for details see Materials and Methods section). The oligomeric state of resulting Aβ42 preparation was confirmed by AFM analyses as well as by SDS gel Western blotting experiments (Additional file 1: Figure S1). According to AFM data Aβ42 oligomers appear as globular structures with 1–2 nm height and around 10 nm width (Additional file 1: Figure S1A). Western blot experiments showed that Aβ42 preparation is mainly in oligomeric form and its molecular weight is around 26 kDa (Additional file 1: Figure S1B) that corresponds to pentamers or hexamers [19]. To mimic physiological situation more closely, we utilized low concentrations (less than 100 nM as calculated based on initial amount of peptides utilized for preparation of oligomers) of Aβ42 oligomers in our experiments. We also evaluated effects of Aβ40 oligomers. Aβ40 peptides were processed the same way as Aβ42 peptides (Additional file 1: Figure S1A). Equivalent amounts of Aβ42 and Aβ40 peptides were used in our experiments. Generated oligomers of Aβ42 and Aβ40 were added to DIV11 primary hippocampal neuronal cultures. In control experiments neuronal cultures were treated with vehicle (equivalent amount of growth media). At DIV14 these cultures were fixed, permeabilized and stained for neuronal marker MAP2 and synaptic marker synapsin I (Fig. 1a). To evaluate synaptogenesis state in studied cultures, mean synapsin signal intensity was divided by the mean of MAP2 intensity. Consistent with previous observations [15, 16, 20], we observed significant loss of synapsin staining in cultures exposed to Aβ42 oligomers (Fig. 1a, 1b). There was also a trend towards synaptic loss in cultures exposed to Aβ40 oligomers, but these changes have not reached a level of significance when compared to control cultures (Fig. 1a, 1b).Fig. 1

Bottom Line: Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD.We discovered that application of Aβ42 oligomers to hippocampal cultures or injection of Aβ42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII).Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neurodegeneration, Department of Medical Physics, Peter the Great St.Petersburg Polytechnic University, St. Petersburg, Russian Federation. lena.popugaeva@gmail.com.

ABSTRACT

Background: Alzheimer disease (AD) is a disease of lost memories. Mushroom postsynaptic spines play a key role in memory storage, and loss of mushroom spines has been proposed to be linked to memory loss in AD. Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD. It is important to evaluate effects of amyloid on stability of mushroom spines.

Results: In this study we used in vitro and in vivo models of amyloid synaptotoxicity to investigate effects of amyloid peptides on hippocampal mushroom spines. We discovered that application of Aβ42 oligomers to hippocampal cultures or injection of Aβ42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII). We further discovered that expression of STIM2 protein rescued CaMKII activity and protected mushroom spines from amyloid toxicity in vitro and in vivo.

Conclusions: Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.

No MeSH data available.


Related in: MedlinePlus