Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase

BACKGROUND: Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-beta (Abeta) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice. METHODOLOGY/PRINCIPAL FINDINGS: We found that while endogenous Abeta concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Abeta deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Abeta degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice. CONCLUSIONS/SIGNIFICANCE: Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases

Cortical Aβ deposition in brains of humans and mice.

<p>Representative examples of immunostaining for Aβ in brain sections obtained from 6-month-old (b) and 24-month-old NEP-deficient mice (d) and corresponding age-matched wildtype controls (a,c), and from post mortem human brain material of a 75-year-old Alzheimer patient (f) and a 31-year-old human control (e). Images shown in a–d represent the mouse somatosensory cortex (barrel field), while human brain sections (e,f) were obtained from the temporal cortex. All sections were immunostained under the same conditions and in the same experimental session with the biotinylated primary antiserum 4G8 which is known to react with both human and murine Aβ peptides. Scale bar represents 200 µm.</p

Behavioral studies in NEP-deficient mice and their wild-type controls.

<p>(a–c) Morris water maze: (a) Comparison of 6-month-old NEP-knockout mice (dotted line with open circles; n = 24) and their wild-type age-matched littermates (line with open quadrates; n = 19). Group means of escape latencies for the first trial per day to reach the platform ±s.e.m. in sec. Where standard variation is not visible it is within the symbols (b) Comparison of 21-month-old NEP-knockout mice (dotted line with filled circles; n = 12) and their wild-type age-matched littermates (line with filled quadrates; n = 9). Group means of escape latencies for the first trial per day to reach the platform ±s.e.m. in sec; statistical analyses by repeated measures of ANOVA (<i>P</i> = 0.016). (c) Comparison of time (in sec) animals needed to find the platform at both investigated time points; data were pooled for the three days of the experiment and plotted; statistical analysis by Student's t test (**<i>P</i><0.01). (d) Shuttle box: group means of the number of conditioned reactions ±s.e.m. of 21-month-old NEP-knockout mice (filled bars; n = 12) and their wild-type littermates (open bars; n = 10) during a training interval on five training days in this two-way active avoidance paradigm. Statistical analysis by repeated measures of ANOVA (**<i>P</i><0.01). (e) Marble burying test: Comparison of the number of marbles that have been buried by 21-month-old NEP-knockout mice (filled bars; n = 9) and their wild-type littermates (open bars; n = 11). Average±s.e.m., statistical analysis by Student's t test (*<i>P</i><0.05).</p

LTP studies in NEP-deficient mice and their wild-type controls.

<p>LTP in the hippocampus (CA1 region) and the amygdala (lateral nucleus of the amygdala (LA)) in adult (n = 7 mice) and aged NEP knockout mice (−/−; n = 7 mice) in comparison to wild-type (+/+) mice (n = 5 mice in each group). (a) 9-month-old mice: LTP magnitude (CA1: ○ +/+, n = 9 slices; • −/−, n = 11 slices; LA: ○ +/+, n = 10 slices, • −/−, n = 11 slices). Inserted representative records of PS potentials in the CA1 region before and after TBS and in the LA before and after HFS, above: obtained from −/−, below: obtained from +/+. (b) 24-month-old mice: LTP in both the CA1 region (○ +/+, n = 11 slices; • −/−, n = 16 slices) and the LA (○ +/+, n = 11 slices; • −/−, n = 10 slices). Data points in a and b represent mean amplitudes (mean±s.e.m.) normalized with respect to baseline values. Inserted representative records of PS potentials in the CA1 region before and after TBS and in the LA before and after HFS, above: obtained from −/−, below: obtained from +/+. (c) Bar histogram of data points shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004590#pone-0004590-g004" target="_blank">Figures 4a–b</a>, averaged 56 to 60 min after TBS/HFS and normalized with respect to baseline (mean±s.e.m.). *<i>P</i><0.05.</p