The Tripeptide RER Mimics Secreted Amyloid Precursor Protein-Alpha in Upregulating LTP

Abstract

Secreted amyloid precursor protein-alpha (sAPPα), generated by enzymatic processing of the APP, possesses a range of neurotrophic and neuroprotective properties and plays a critical role in the molecular mechanisms of memory and learning. One of the key active regions of sAPPα is the central APP domain (E2) that contains within it the tripeptide sequence, RER. This sequence is exposed on the surface of a coiled coil substructure of E2. RER has by itself displayed memory-enhancing properties, and can protect newly formed engrams from interference in a manner similar to that displayed by sAPPα itself. In order to determine whether RER mimics other properties of sAPPα, we investigated the electrophysiological effects of the N-terminal protected acetylated RER (Ac-RER) and an isoform containing a chiral switch in the first amino acid from an l- to a d-orientation (Ac-rER), on synaptic plasticity. We found that, like sAPPα, exogenous perfusion with nanomolar concentrations of Ac-RER or Ac-rER enhanced the induction and stability of long-term potentiation (LTP) in area CA1 of rat and mouse hippocampal slices, in a protein synthesis- and trafficking-dependent manner. This effect did not occur with a control Ac-AAA or Ac-IFR tripeptide, nor with a full-length sAPPα protein where RER was substituted with AAA. Ac-rER also protected LTP against amyloid-beta (Aβ_{25 - 35})-induced LTP impairment. Our findings provide further evidence that the RER-containing region of sAPPα is functionally significant and by itself can produce effects similar to those displayed by full length sAPPα, suggesting that this tripeptide, like sAPPα, may have therapeutic potential.

Keywords: Alzheimer’s disease; amyloid precursor protein; amyloid-beta; field excitatory postsynaptic potential; hippocampus; long-term potentiation.

FIGURE 1

Schematic representation of the location of RER within the E2 domain of the sAPPα, while also showing the relative locations of Aβ_(1–42), Aβ_(25–35), sAPPα, and sAPPβ.

FIGURE 2

Acute administration of Ac-RER enhances LTP. (A) A mild TBS delivered to acute rat hippocampal slices produced a rapidly decaying LTP. Adding 1 nM Ac-RER to the solution for 30 min prior to delivery of the TBS enhanced the induction and persistence of the LTP produced. This enhancement was similar to the LTP facilitation caused by sAPPα. The plot shows average LTP obtained from slices pre-treated for 30 min under control (n = 9), 1 nM sAPPα (n = 8), or 1 nM Ac-RER (n = 7) conditions. The black bar indicates the period of perfusion with 1 nM Ac-RER or 1 nM sAPPα. The arrow indicates the delivery of the mild TBS (five bursts of five pulses). Example waveforms are averages of 10 sweeps from a single slice for each group: black, baseline; red, final LTP; scale bars: 2 mV, 5 ms. (B) Summary bar graph displaying the degree of LTP for each group. ^∗p < 0.05; ^∗∗p ≤ 0.01; Dunnett’s post hoc t-test. (C) Plotted responses for Ac-RER at various concentrations demonstrating that increases in the induction and persistence of LTP were concentration dependent. The arrow indicates the delivery of the half TBS (five bursts of five pulses). Example waveforms are averages of 10 sweeps from a single slice for each group: black, baseline; red, final LTP; scale bars: 2 mV, 5 ms. (D) Bar graph comparing LTP following perfusion with various concentrations of Ac-RER. The lowest concentration (0.3 nM RER; n = 6 slices) did not alter induction or persistence of LTP from controls, whereas higher concentrations (1 nM; n = 7 and 10 nM; n = 6) did increase LTP. ^∗p < 0.05; ^∗∗p ≤ 0.01. (E) Plot of responses following exposure to Ac-RER for 30–65 min of the protocol, but in the absence of TBS (n = 6). Basal responses to test pulses were not affected. Black bar indicates the period of perfusion with Ac-RER. Example waveforms are the average of 10 sweeps for pre- and post-Ac-RER treatment: black, pre-treatment; red, post-treatment; scale bars: 2 mV, 5 ms. (F) Paired-pulse facilitation, determined across a range of interpulse intervals, was not altered by superfusion with Ac-RER for 30 min prior to testing (1 nM, n = 9). (G) Presynaptic post-tetanic potentiation (PTP), measured 5 s after each of three TBS protocols delivered at 30 s intervals in the presence of D-AP5, was unchanged by administration of Ac-RER (1 nM, n = 9) for 30 min prior to the first TBS. All values in this and the following figures calculated as mean ± SEM.

FIGURE 3

Peptide sequence dependence of the LTP facilitation. (A) The full length sAPPα molecule, with the RER sequence replaced with AAA (sAPPα-AAA; n = 5) did not alter LTP in rat hippocampal slices. The control isolated tripeptide Ac-AAA (n = 7) also did not affect LTP. The black bar shows the period of perfusion, and the arrow the delivery of the TBS. Example waveforms are averages of 10 sweeps from a single slice for each group: black, baseline; red, final LTP; scale bars: 2 mV, 5 ms. (B) Summary bar graph shows that LTP obtained after exposure to these probe molecules was not altered from the control level. n.s., p > 0.05 compared to the control group.

FIGURE 4

Protein-synthesis and -trafficking inhibitors prevent Ac-RER enhancement of LTP in acute rat hippocampal slices. (A) BFA (35 μM) given 10 min prior to and during Ac-RER (1 nM) perfusion blocked the tripeptide enhancement of LTP induction and persistence. Solid line, BFA perfusion; perforated line, Ac-RER perfusion. Example waveforms are averages of 10 sweeps from a single slice for each group: black, baseline; red, final LTP; scale bars: 2 mV, 5 ms. (B) Bar graph comparing LTP in slices treated with BFA and Ac-RER (n = 6), to control (n = 5) and Ac-RER-treated slices (n = 7). The LTP enhancement was blocked by BFA. ^∗∗∗p < 0.001. (C) Plot of responses for control experiments (n = 5), BFA only perfusion (n = 4), and BFA with no TBS (far right bar, n = 5). Control LTP was not affected by BFA administration. In the absence of TBS, responses returned to basal levels following removal of BFA from the perfusion solution, indicating that the inhibitor did not persistently affect basal transmission. Example waveforms are averages of 10 sweeps from a single slice for each group: black, baseline; red, final LTP; scale bars: 2 mV, 5 ms. (D) Bar graph displaying the degree of response change for each group. n.s., p > 0.05 compared to the control group. (E) Sample blot showing the puromycin-containing proteins for each treatment condition. Tubulin was used as a loading control, as shown below. Incubation of peptides was at 1 nM, except for Ac-RER, where both 1 and 10 nM concentrations were assessed. (F) Bar graph displaying the relative puromycin optical density throughout the lanes, normalized to tubulin and compared to controls. There was a significant increase in newly synthesized protein in tissue treated with both 1 and 10 nM ac-RER, and with 1 nM sAPPα. ^∗∗p ≤ 0.01. (G) Inclusion of CHX for 10 min prior to and during Ac-RER (1 nM) perfusion reduced the effects of the tripeptide on LTP induction and persistence. Arrow indicates delivery of the TBS. Black line, CHX perfusion; perforated line, Ac-RER perfusion. Example waveforms are averages of 10 sweeps from a single slice for each group: black, baseline; red, final LTP; scale bars: 2 mV, 5 ms. (H) Bar graph comparing LTP between the different treatment groups in panel (G). CXH + Ac-RER (n = 7), control (n = 5), Ac-RER-treated slices (n = 7). ^∗p ≤ 0.05. (I) Plot of responses for controls (n = 5), CHX only (n = 5), and CHX with no TBS (n = 6). Responses returned to baseline levels following removal of CHX from the perfusion solution in the absence of a TBS. Control LTP was not affected by CHX perfusion. Example waveforms are averages of 10 sweeps from a single slice for each group: black = baseline; red = final LTP; scale bars: 2 mV, 5 ms. (J) Bar graph of the responses changes for the groups in panel (I). CHX by itself did not alter either basal responses or LTP. n.s., p > 0.05 compared to the control group.

FIGURE 5

Tripeptide effects in acute mouse slices. (A) The same Ac-RER tripeptide (n = 8) and a variant with the first arginine chirally switched to the d-oriented amino acid (Ac-rER; n = 10) were effective in enhancing LTP in adult C57/B6 mouse slices. An N-acetylated control tripeptide (Ac-IFR; n = 8) did not affect LTP. Example waveforms are averages of 10 sweeps from a single slice for each group: blue = sample EPSP 1 min before TBS; red = sample EPSP taken at 60 min post-TBS taken at 60 min post-TBS; scale bars: 1 mV, 10 ms. (B) Bar graph comparing the final level of LTP for the groups in panel (A). ^∗∗p ≤ 0.01; n.s., p > 0.05 compared to the control group.

FIGURE 6

Ac-rER prevents impairments in LTP following Aβ_(25–35) exposure in mouse slices. (A) Perfusion of mouse slices in aCSF containing 200 nM Aβ_(25–35) (Aβ; n = 5) for 28 min prior to and 2 min following the delivery of TBS (10 bursts) impaired LTP. Addition of 10 nM Ac-rER (n = 5) for 15 min prior to and during Aβ perfusion (n = 5) rescued the LTP. Solid line, treatment present. Arrow indicates delivery of TBS (10 bursts). Red = sample EPSP 1 min before TBS; blue = sample EPSP taken at 60 min post-TBS; scale bars: 1 mV, 10 ms. (B) Bar graph shows that perfusion with Aβ_(25–35) induced impaired LTP, which was rescued by pre-treatment with Ac-rER. ^∗p ≤ 0.05; n.s., p > 0.05 relative to the control group.