2020 Volume 43 Issue 12 Pages 1975-1978
The pathological significance of amyloid-β1–42 (Aβ1–42) dynamics is poorly understood in the brain extracellular compartment. Here we test which of the concentration or the retention is critical for Aβ1–42 toxicity after injection of equal dose into dentate granule cell layer of freely moving rats. The toxicity of Aβ1–42 (25 µM) was compared between injections at the rate of 0.25 µL/min for 4 min (fast injection) and 0.025 µL/min for 40 min (slow injection). Dentate gyrus long-term potentiation (LTP) was affected 1 and 2 h after the fast injection, but not 4 h. In contrast, LTP was affected even 72 h after the slow injection. Aβ1–42 staining 5 min after finish of the slow injection was more intense in the dentate granule cell layer than of the fast injection. The present study indicates that the retention of Aβ1–42 in the extracellular fluid is correlated with neuronal Aβ1–42 uptake and plays a key role in Aβ1–42 neurotoxicity. In the extracellular fluid of the dentate gyrus, the retention period of Aβ1–42 is much more critical for Aβ1–42 toxicity than Aβ1–42 concentration. It is likely that Aβ1–42 toxicity is accelerated by the disturbance of Aβ1–42 metabolism in the dentate gyrus.
In familial Alzheimer’s disease (AD), more than 200 mutations have been identified in the three genes encording the amyloid precursor protein (APP), presenilin 1 and presenilin 2 that are involved in amyloid β (Aβ) production. All the mutations increase Aβ1–42 as a primary pathogenic agent in the AD development. Sporadic AD, the etiology of which is poorly understood, accounts for most AD patients.1) However, sporadic AD is essentially indistinguishable from familial AD in both pathological and neurological terms. Thus, it is important to understand why Aβ accumulates in the sporadic AD brain and this understanding contributes to preventing the AD development.
Transsynaptic progression of Aβ-induced neuronal dysfunction is observed in the entorhinal-hippocampal network.2) The findings indicate that Aβ release from the perforant pathway is closely linked with the damages of the postsynaptic dentate granule cells. On the other hand, Neprilysin is a rate-limiting peptidase involved in the physiological degradation of Aβ in the brain3) and is most abundant in the stratum lacunosum-moleculare of the CA1–CA3 field and the molecular layer of the dentate gyrus in the mouse hippocampus.4)
However, Aβ1–42 dynamics is poorly understood in the extracellular compartment of the dentate gyrus. Here we test which of the concentration or the retention period in the extracellular compartment is critical for Aβ1–42 neurotoxicity after injection of equal dose into dentate granule cell layer of freely moving rats.
Male Wistar rats (<20 weeks of age, Japan SLC, Hamamatsu, Japan) were caged under the standard conditions with a diurnal 12-h light cycle. The room temperature and relative humidity were controlled at 23 ± 1 °C and 55 ± 5%, respectively. The rats were allowed free access to a standard laboratory food and water. The experiments were done in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka that refer to the American Association for Laboratory Animals Science and the guidelines laid down by the NIH in the U.S.A. (NIH Guide for the Care and Use of Laboratory Animals). This work has been approved by the Ethics Committee for Experimental Animals in the University of Shizuoka.
Synthetic human Aβ1–42 (ChinaPeptides, Shanghai, China) was dissolved in saline before the experiments and used immediately.
In Vivo Long-Term Potentiation (LTP) RecordingLTP was recorded at the perforant pathway-dentate granule cell synapses in freely moving and unanesthetized rats as reported previously.5) To monitor population spike (PS) amplitude, test stimuli (0.05 Hz) were delivered at 20 s intervals and PS amplitudes were recorded for 10 min for the baseline recording in freely moving and unanesthetized rats. LTP was induced by delivering high-frequency stimulation (HFS; 10 trains of 20 pulses at 200 Hz separated by 1 s) and under the same condition. PS amplitudes, which were measured for 10 min, were recorded 24–72 h (the baseline recording) and immediately (0 h) before high-frequency stimulation, and 1 h after HFS stimulation, averaged, and expressed as percentages of the mean PS amplitude recorded during the 10-min baseline period prior to LTP induction, which was expressed as 100%.
In Vivo Aβ ImmunostainingAβ immunostaining was done as reported previously.6) Saline (1 µL) and Aβ in saline (25 µM, 1 µL) were bilaterally injected into the dentate granule cell layer of freely moving rats via injection cannulae at the rate of 0.25 µL/min for 4 min and the rate of 0.025 µL/min for 40 min. Five minutes later, hippocampal slices were prepared, incubated at 4 °C with Aβ monoclonal antibody, 4G8 (COVANCE), and then incubated with Alexa Fluor 633 goat anti-mouse immunoglobulin G (IgG) secondary antibody. Images for immunostaining were captured using a confocal laser-scanning microscopic system and Alexa Fluor 633 florescence intensity was analyzed using NIH Image J to assess the level of Aβ uptake.
Data AnalysisData were expressed as means ± standard error. Differences among groups were analyzed by one-way ANOVA followed by post hoc test using the Tukey’s test for multiple comparisons (the statistical software, GraphPad Prism 5). A value of p < 0.05 was considered significant.
LTP is affected 1 h after the local injection of Aβ1–42 (25 µM, 1 µL) into the dentate gyrus of anesthetized rats at the rate of 0.25 µL/min for 4 min, consistent with memory deficit, but not after injection of Aβ1–42 (12.5 µM, 1 µL) at the rate of 0.25 µL/min for 4 min.7) In the present study, LTP was affected 1 h after injection of Aβ1–42 (25 µM, 1 µL) into the dentate gyrus of unanesthetized rats at the rate of 0.25 µL/min for 4 min (Fig. 1). LTP was also affected 2 h after the injection, but not 4 h.
Saline and Aβ in saline (25 µM, 1 µL) were injected into the dentate granule cell layer via an injection cannula attached to a recording electrode 1 (n = 10), 2 (n = 3), and 4 h (n = 4) before LTP induction at the rate of 0.25 µL/min for 4 min as shown by the gray bars. High-frequency stimulation (HFS) was delivered as shown by the arrow (upper-left). Magnitude of LTP 1 h after HFS ***, p < 0.001, **, p < 0.01, vs. saline (n = 8) (upper-right). Representative field excitatory postsynaptic potential (fEPSP) recordings at the time −24 h (black line), −5–0 min (grey line) and 50–60 min (red line) are shown (lower). (Color figure can be accessed in the online version.)
In the case of injection at the slow rate of 0.025 µL/min for 40 min, LTP was affected 4 h after the injection and even 72 h (Fig. 2). As shown as the PS amplitudes just before LTP induction, the baseline of synaptic neurotransmission was not significantly modified after Aβ1–42 injection (Fig. 2), suggesting that the slow injection of Aβ1–42 has persistently toxic effect and acts on the mechanism of LTP induction.
Aβ in saline (25 µM, 1 µL) was injected into the dentate granule cell layer via an injection cannula attached to a recording electrode 72 (n = 4) and 4 h (n = 4) before LTP induction at the rate of 0.025 µL/min for 40 min as shown by the gray bars. Saline (1 µL) was injected 1 h before LTP induction at the rate of 0.25 µL/min for 4 min as shown by the gray bar. High-frequency stimulation (HFS) was delivered as shown by the arrow (upper-left). Magnitude of LTP 1 h after HFS **, p < 0.01, vs. saline (n = 8) (upper-right). Representative fEPSP recordings at the time −72 h (black line, Aβ/−72 h), −24 h (black line, Aβ/ − 4 h and saline), −5–0 min (grey line) and 50–60 min (red line) are shown (lower). (Color figure can be accessed in the online version.)
Aβ1–42 staining was more intensely observed in the dentate granule cell layer after the slow injection than the fast injection (Fig. 3).
Aβ staining in the dentate granule cell layer (GCL) was determined with Alexa 633 fluorescence, which is represented by the ratio to the control (saline, n = 6) expressed as 100%. *, p < 0.05, Aβ (4 min, n = 6) vs. Aβ (40 min, n = 6) (right). Bar, 50 µm (left). (Color figure can be accessed in the online version.)
The toxicity of Aβ1–42 (25 µM) was compared between injections at the rate of 0.25 µL/min for 4 min (fast injection) and 0.025 µL/min for 40 min (slow injection). LTP was affected 1 and 2 h after the fast injection, but not 4 h. In contrast, LTP was affected even 72 h after the slow injection. Aβ1–42 staining 5 min after finish of the slow injection was more intense in the dentate granule cell layer than of the fast injection. Aβ1–42 staining is observed around the nuclei of dentate granule cells in an extracellular Zn2+-dependent manner after the fast injection,6) suggesting that a portion of Zn-Aβ1–42 formed in the extracellular compartment is taken up into dentate granule cells in addition to the interaction with the plasma membranes.8) The present study indicates that the retention of Aβ1–42 in the extracellular fluid is correlated with neuronal Aβ1–42 uptake and plays a key role in Aβ1–42 neurotoxicity. In the extracellular fluid of the dentate gyrus, the retention period of Aβ1–42 is more critical for Aβ1–42 toxicity than Aβ1–42 concentration.
Neuronal Aβ1–42 accumulation in the human brain occurs from approx. 40 years old and the Aβ1–42 level in normal brain are close to that of clinically AD-affected patients with progression of aging.9) Thus, aging is regarded as the most potent risk factor for sporadic AD. The levels of Neprilysin are characteristically reduced by 20–40% in the outer molecular layer and polymorphic layer of the dentate gyrus of aged mice, indicating that neprilysin is selectively decreased at the terminal zones and on axons of the lateral perforant pathway.10) It is likely that Aβ1–42 toxicity is accelerated by the disturbance of Aβ1–42 metabolism in the dentate gyrus.
The authors declare no conflict of interest.
