PMC

Extracellular field excitatory postsynaptic potential (fEPSP) was measured as described in . Arrows indicate tetanus application. The horizontal bar indicates the period during which Aβ and/or LM11A-31 were added to the bath solution. Aβ did not affect baseline transmission. Application of LM11A-31 (100 nM) rescued Aβ-induced impairment of LTP without affecting baseline transmission. The values for the last data point in the graph are as follows: vehicle (veh, n = 8) 252.4±27.6%; Aβ (n = 7) 134.0±9.3%; LM11A-31 (n = 7) 230.2±24.3%.; Aβ+LM11A-31 (n = 7) 233.6±17.6%. Vehicle vs. Aβ, p = 0.02; Aβ vs. Aβ+LM11A-31, p = 0.0006; vehicle vs. LM11A-31, p = 0.8131.

Organotypic slice cultures were prepared from PND-8 rat brain and allowed to mature in vitro for 11–19 days. Pyramidal neuron death was detected by propidium iodide (PI) staining, and all experimental conditions were compared to Aβ treatment alone. Upper row, at baseline only trace levels of PI staining were detected. Middle row, PI staining after a 24 hour exposure to either culture medium (CM), Aβ or Aβ+LM11A-31 at 100 nM demonstrates readily apparent Aβ-induced pyramidal neuron death that is inhibited in the presence of p75^NTR ligand. Bottom row, PI staining shows maximum neuronal death after 24 hour treatment with NMDA. In the lower panel, quantitative analysis of PI staining demonstrates a significant reduction in Aβ-induced neuronal death (n = 56–59 brain slices derived from 4 independent studies).

(A–C) 6–7 DIV hippocampal neurons were treated with: (A) CM alone; (B) 5 µM Aβ; (C) 5 µM Aβ+100 nM LM11A-31 for 72 hours and then stained with TUNEL/DAPI. Many neurons exposed to Aβ were TUNEL-positive (green), indicative of death; whereas the majority of neurons co-treated with Aβ and LM11A-31 were TUNEL-negative. (D) Quantitation of the percentage of TUNEL-positive neurons demonstrated that LM11A-31 alone had no effect on baseline death while NGF (100 ng/ml) was associated with a significant increase in death. Aβ caused an approximate 2.0-fold increase in death that was significantly inhibited by LM11A-31 but not NGF. In the presence of NGF, LM11A-31 failed to prevent Aβ-induced death (n = 98–131 fields derived from a total of 6 separate experiments). (E) In 6–7 DIV hippocampal neuronal cultures derived from C57Bl/6 p75^NTR+/+ and −/− mice, Aβ triggered a 2.0-fold increase in death in p75^NTR+/+ cultures that was significantly inhibited by LM11A-31. In p75^NTR−/− cultures, Aβ triggered a significant 1.5-fold increase in death, a degree of increase significantly less than that found in +/+ cultures. In p75^NTR−/− cultures, LM11A-31 demonstrated no protective effect (n = 69–140 fields per condition, derived from a total of 4 separate p75^NTR−/− cultures and 6 separate p75^NTR+/+ cultures).

21–22 DIV hippocampal neurons were exposed to the following conditions: (A) culture medium (CM); (B) 5 µM oligomeric-NaOH-derived Aβ; or (C) 5 µM oligomeric-NaOH-derived Aβ+100 nM LM11A-31. After 48 hours, cultures were fixed and immunostained for MAP2 to visualize dendrites. In the presence of CM alone, dystrophic changes including beading and tortuosity were rare. In contrast, Aβ induced beading (arrowheads) and increased tortuosity (brackets), and each of these changes was markedly reduced with co-administration of LM11A-31. (D) Dystrophic neurites, defined as neurites exhibiting beading and/or multiple abrupt turns (i.e., tortuosity) were measured by a blinded observer. Data is expressed as average numbers of dystrophic neurites per field. All conditions were compared to Aβ alone (n = 8 randomly chosen fields from 3 independent experiments). (E) Mean differential curvature (MDC) analysis in randomly selected fields demonstrated that oligomeric-NaOH-derived Aβ induced a significant increase in MDC which was prevented by LM11A-31 (n = 7–9 fields per condition). (F) Distribution analysis of MDC values (X axis) demonstrates a rightward shift in the presence of oligomeric-NaOH-derived Aβ that was mitigated in the presence of LM11A-31. (G–H) The same assays and analyses were performed using 5 µM oligomeric-HFIP-derived Aβ which demonstrated similar findings (n = 9 fields per condition derived from 3 independent experiments).

6–7 DIV hippocampal, cortical and septal neurons were exposed to: culture medium alone (CM); CM containing 10 µM Aβ(S), a control peptide containing Aβ(1-42) residues in a scrambled sequence; 10 µM Aβ alone or with NGF (100 ng/ml , ∼4 nM), LM11A-24, -31, or -36 (100 nM). After 72 hours, cultures were fixed and photographed under phase contrast microscopy. (A) In CM alone, neurons had round, phase bright cell bodies with outgrowth of intact neurites (normal morphology). (B) In the presence of Aβ, many neurons exhibited the degenerative findings of cell body shrinkage (arrowheads), vacuolated cytoplasm, and neurite beading and fragmentation (arrows). (C, D) Neurons co-treated with Aβ and LM11A-24 (C) or LM11A-31 (D) exhibited normal morphology. (E–J) Neuronal survival was quantitated using morphological criteria (see ) and survival in each experimental condition was statistically compared to Aβ alone. In cultures of hippocampal (E) cortical (F) and septal (G) neurons, Aβ significantly decreased survival while Aβ (S) had no effect. LM11A-24 and -31 inhibited Aβ-induced death while NGF and LM11A-36 negative control compound demonstrated no protective effects with the exception of NGF exhibiting a small protective effect in hippocampal and septal cultures (for hippocampal cultures, n = 45–146 fields counted over 5–16 separate experiments; for cortical cultures, n = 25–40 fields counted over 5 separate experiments; for septal cultures, n = 50–60 fields counted over 6 separate experiments). (H, I) 6–7 DIV hippocampal neurons were exposed to CM alone or Aβ in the presence or absence of LM11A-24 (H) or -31 (I) at the indicated concentrations. LM11A-24 and -31 were protective against Aβ in a dose-dependent manner, with an EC₅₀ of approximately 20 nM (LM11A-24: n = 20–146 fields counted over 3–16 separate experiments. LM11A-31: n = 43–146 fields counted over 5–16 separate experiments). (J) 6–7 DIV hippocampal neurons were exposed to CM alone or 10 µM fibrillar Aβ±LM11A-31. LM11A-31 inhibited fibril-induced neuronal death (n = 30 fields counted over 3 separate experiments).

For each study, LM11A-24 and -31 were present at 100 nM and NGF at 50 or 100 ng/ml. All conditions were compared to Aβ treatment alone, unless indicated otherwise (e.g. Fig. 5B). For the majority of protein preparations, each derived from separate experiments; two independent Western analyses were conducted. (A) DIV 21–22 hippocampal neurons were treated with CM alone, CM+Aβ, or Aβ in the presence of LM11A-24, -31 or NGF and examined at the 4 hour time point. AKT activation was significantly decreased by Aβ, while LM11A-24, -31 and NGF prevented this decrease. The ability of NGF to prevent Aβ-induced AKT inhibition was diminished relative to that of the small molecules (n = 8–14 separate Western blots from 9 independent protein preparations). (B) Neurons were treated with CM alone or with the indicated combinations of the PI3K inhibitor LY294002, Aβ and LM11A-24 or -31. After 72 hours, cultures were stained with Hoechst 33258 for cell death quantitation. In CM, LY294002 increased cell death by approximately 1.7-fold without reaching significance. In the presence of Aβ, LY294002 blocked the protective effect of p75^NTR ligands and resulted in a > 2.0-fold increase in cell death (n = 35–45 fields derived from a total of 4–5 experiments for each condition). (C–E) DIV 21–22 hippocampal neurons were treated with CM or Aβ in the presence of LM11A-24, -31 or NGF for 4 hours. (C) Addition of Aβ resulted in increased calpain-induced cleavage of full-length α-fodrin (250 kDa) to its 150 and 145 kDa fragments. Co-treatment with LM11A-24, -31 or NGF, significantly reduced Aβ-induced calpain activation (n = 4–7 separate Western blots from 4 independent protein preparations). (D) Addition of Aβ induced cdk5 activity as revealed by the increased ratio of the cleaved (p25) to uncleaved (p35) regulatory subunit. Activation was inhibited by LM11A-24, -31 or NGF (n = 8–10 separate Western blots from 7 independent protein preparations). (E) Addition of Aβ induced GSK3β activation as revealed by a decrease in the ratio of p-GSK3β^Ser9 signal to total GSK3β. Activation was significantly inhibited by LM11A-24 and 31, but not NGF (n = 8–14 separate Western blots from 7 independent protein preparations). (F) In DIV 6–7 neuronal cultures examined at the 12 hour time point, Aβ induced a 1.8-fold increase in the proportion of phospho-c-Jun positive nuclei, a measure of JNK/c-Jun activation. Activation was significantly inhibited by LM11A-24 and 31, but not NGF (n = 139–210 fields from 6 individual experiments). (G) In DIV 21–22 hippocampal neuronal cultures examined at the 4 hour time point, Aβ induced a 3.5-fold increase in Tau^Ser202 phosphorylation which was significantly inhibited by LM11A-24 and -31 (n = 10 separate Western blots from 5 independent protein preparations). (H) In DIV 21–22 hippocampal neuronal cultures examined at the 3 hour time point, Aβ significantly decreased CREB phosphorylation by 43%, while this decrease was prevented by LM11A-24 and -31 (n = 10 separate Western blots from 5 independent protein preparations).