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Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

Izzo NJ, Xu J, Zeng C, Kirk MJ, Mozzoni K, Silky C, Rehak C, Yurko R, Look G, Rishton G, Safferstein H, Cruchaga C, Goate A, Cahill MA, Arancio O, Mach RH, Craven R, Head E, LeVine H, Spires-Jones TL, Catalano SM - PLoS ONE (2014)

Bottom Line: We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo.These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models.We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Cognition Therapeutics Inc., Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.

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C-terminal antibodies directed against the C-terminus of PGRMC1 prevent (A–D) and displace (E–H) Abeta oligomer binding to neurons and glia.Abeta oligomers bind to a subset of neurons and glia in mature hippocampal primary neurons 21DIV (A, E, red bar in I) compared to vehicle-treated (no Abeta) cultures (B, F, blue bar in I). Graphs in I are average of 3 experiments (avg. intensity of Abeta oligomer puncta + S.E.M., expressed as a percentage of Abeta oligomer-treated condition, difference in binding intensity vs. Abeta oligomer condition *p<0.05, Student's t-test). Abeta oligomer binding to cultured neurons is significantly reduced in the presence of C-terminal antibody to sigma-2/PGRMC1 regardless of whether it is added before (D, green bar in I [prevention], 58% reduction) or after (H, green hatched bar in I [treatment], 26% reduction) oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (C, G and maroon bars in I) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition. J Effects of antibodies on membrane trafficking rate in the presence or absence of Abeta oligomers (expressed as a percentage of vehicle-treated in the absence of Abeta, difference in trafficking rate vs. Abeta oligomer- or vehicle-treated condition *p<0.05, Student's t-test). The C-terminal antibody directed against amino acids 185–195 in sigma-2/PGRMC1 does not rescue oligomer-induced deficits, but induces trafficking deficits on its own in the absence of Abeta oligomers, pointing to a critical role of this protein in normal membrane trafficking.
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pone-0111899-g006: C-terminal antibodies directed against the C-terminus of PGRMC1 prevent (A–D) and displace (E–H) Abeta oligomer binding to neurons and glia.Abeta oligomers bind to a subset of neurons and glia in mature hippocampal primary neurons 21DIV (A, E, red bar in I) compared to vehicle-treated (no Abeta) cultures (B, F, blue bar in I). Graphs in I are average of 3 experiments (avg. intensity of Abeta oligomer puncta + S.E.M., expressed as a percentage of Abeta oligomer-treated condition, difference in binding intensity vs. Abeta oligomer condition *p<0.05, Student's t-test). Abeta oligomer binding to cultured neurons is significantly reduced in the presence of C-terminal antibody to sigma-2/PGRMC1 regardless of whether it is added before (D, green bar in I [prevention], 58% reduction) or after (H, green hatched bar in I [treatment], 26% reduction) oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (C, G and maroon bars in I) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition. J Effects of antibodies on membrane trafficking rate in the presence or absence of Abeta oligomers (expressed as a percentage of vehicle-treated in the absence of Abeta, difference in trafficking rate vs. Abeta oligomer- or vehicle-treated condition *p<0.05, Student's t-test). The C-terminal antibody directed against amino acids 185–195 in sigma-2/PGRMC1 does not rescue oligomer-induced deficits, but induces trafficking deficits on its own in the absence of Abeta oligomers, pointing to a critical role of this protein in normal membrane trafficking.

Mentions: We have shown that our sigma-2 selective small molecules are capable of preventing and displacing Abeta oligomer binding to mature primary hippocampal and cortical cultures 21DIV [30]. To confirm the role of the sigma-2/PGRMC1 protein in mediating the binding of Abeta 1-42 oligomers, we treated cultures with Abeta oligomers for 30 minutes, then added a C-terminal specific PGRMC1 antibody to live cultures for 30 minutes, or vice-versa. The cells were then fixed and immunofluorescently labeled to detect Abeta binding (Fig. 6A–H). This antibody, which is directed against the C-terminal amino acids 185–195 of sigma-2/PGRMC1, significantly reduced Abeta oligomer binding to synaptic terminals on hippocampal and cortical neurites regardless of whether it was added before (Fig.6D, green bar in I [prevention], 58% reduction) or after (Fig. 6H, green hatched bar in I [treatment], 26% reduction) the oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (Fig. 6C, G and maroon bars in I) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition.


Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

Izzo NJ, Xu J, Zeng C, Kirk MJ, Mozzoni K, Silky C, Rehak C, Yurko R, Look G, Rishton G, Safferstein H, Cruchaga C, Goate A, Cahill MA, Arancio O, Mach RH, Craven R, Head E, LeVine H, Spires-Jones TL, Catalano SM - PLoS ONE (2014)

C-terminal antibodies directed against the C-terminus of PGRMC1 prevent (A–D) and displace (E–H) Abeta oligomer binding to neurons and glia.Abeta oligomers bind to a subset of neurons and glia in mature hippocampal primary neurons 21DIV (A, E, red bar in I) compared to vehicle-treated (no Abeta) cultures (B, F, blue bar in I). Graphs in I are average of 3 experiments (avg. intensity of Abeta oligomer puncta + S.E.M., expressed as a percentage of Abeta oligomer-treated condition, difference in binding intensity vs. Abeta oligomer condition *p<0.05, Student's t-test). Abeta oligomer binding to cultured neurons is significantly reduced in the presence of C-terminal antibody to sigma-2/PGRMC1 regardless of whether it is added before (D, green bar in I [prevention], 58% reduction) or after (H, green hatched bar in I [treatment], 26% reduction) oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (C, G and maroon bars in I) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition. J Effects of antibodies on membrane trafficking rate in the presence or absence of Abeta oligomers (expressed as a percentage of vehicle-treated in the absence of Abeta, difference in trafficking rate vs. Abeta oligomer- or vehicle-treated condition *p<0.05, Student's t-test). The C-terminal antibody directed against amino acids 185–195 in sigma-2/PGRMC1 does not rescue oligomer-induced deficits, but induces trafficking deficits on its own in the absence of Abeta oligomers, pointing to a critical role of this protein in normal membrane trafficking.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4229119&req=5

pone-0111899-g006: C-terminal antibodies directed against the C-terminus of PGRMC1 prevent (A–D) and displace (E–H) Abeta oligomer binding to neurons and glia.Abeta oligomers bind to a subset of neurons and glia in mature hippocampal primary neurons 21DIV (A, E, red bar in I) compared to vehicle-treated (no Abeta) cultures (B, F, blue bar in I). Graphs in I are average of 3 experiments (avg. intensity of Abeta oligomer puncta + S.E.M., expressed as a percentage of Abeta oligomer-treated condition, difference in binding intensity vs. Abeta oligomer condition *p<0.05, Student's t-test). Abeta oligomer binding to cultured neurons is significantly reduced in the presence of C-terminal antibody to sigma-2/PGRMC1 regardless of whether it is added before (D, green bar in I [prevention], 58% reduction) or after (H, green hatched bar in I [treatment], 26% reduction) oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (C, G and maroon bars in I) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition. J Effects of antibodies on membrane trafficking rate in the presence or absence of Abeta oligomers (expressed as a percentage of vehicle-treated in the absence of Abeta, difference in trafficking rate vs. Abeta oligomer- or vehicle-treated condition *p<0.05, Student's t-test). The C-terminal antibody directed against amino acids 185–195 in sigma-2/PGRMC1 does not rescue oligomer-induced deficits, but induces trafficking deficits on its own in the absence of Abeta oligomers, pointing to a critical role of this protein in normal membrane trafficking.
Mentions: We have shown that our sigma-2 selective small molecules are capable of preventing and displacing Abeta oligomer binding to mature primary hippocampal and cortical cultures 21DIV [30]. To confirm the role of the sigma-2/PGRMC1 protein in mediating the binding of Abeta 1-42 oligomers, we treated cultures with Abeta oligomers for 30 minutes, then added a C-terminal specific PGRMC1 antibody to live cultures for 30 minutes, or vice-versa. The cells were then fixed and immunofluorescently labeled to detect Abeta binding (Fig. 6A–H). This antibody, which is directed against the C-terminal amino acids 185–195 of sigma-2/PGRMC1, significantly reduced Abeta oligomer binding to synaptic terminals on hippocampal and cortical neurites regardless of whether it was added before (Fig.6D, green bar in I [prevention], 58% reduction) or after (Fig. 6H, green hatched bar in I [treatment], 26% reduction) the oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (Fig. 6C, G and maroon bars in I) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition.

Bottom Line: We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo.These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models.We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Cognition Therapeutics Inc., Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.

Show MeSH
Related in: MedlinePlus