Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats

Abstract

Alzheimer's disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25-35) oligomers (oAβ(25-35)) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ(25-35) and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ(25-35) was still present in the brain after 6 weeks. oAβ(25-35) injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ(25-35) injection resulted in increased APP expression, Aβ(1-42) generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.

Figure 1. Brain localization of Aβ_25–35 and particle characterization of Aβ_25–35 solutions A–I.

Localization within brain structures of oAβ_25–35-HLF, determined 6 weeks after its icv injection (10 µg/rat). oAβ_25–35-HLF was visualized in green, while the nucleus was counterstained with DAPI (blue labeling). Abbreviations: 3V: third ventricle; alv: alveus of the hippocampus; CA1: field CA1 of hippocampus; CA3: field CA3 of the hippocampus; cc: corpus callosum; D3V: dorsal third ventricle; ec: external capsule; fi: fimbria of the hippocampus; FrA: frontal association cortex; hf: hippocampal fissure; LV: lateral ventricle; MEE: median eminence, external part; MEI: median eminence, internal part; MHb: medial habenular nucleus; PVN: paraventricular hypothalamic nucleus; PVP: paraventricular thalamic nucleus, posterior part. Arrowhead: blood vessel. Scale bar  = 100 µm. J. Particle size distribution of the different fractions of Aβ_25–35 solution (1 µg/µl) was determined by PCS at 25°C. Samples were prepared as described in the materials and methods section. Black curve: Aβ_25–35 peptide dissolved in hexafluoroisopropanol (HFIP); Red curve: solution of aggregated Aβ_25–35 peptide; Green curve: supernatant of aggregated Aβ_25–35 peptide centrifuged at 1 000 g; Purple curve: re-suspended pellet of aggregated Aβ_25–35 peptide obtained after centrifugation at 1 000 g; Blue curve: supernatant of aggregated Aβ_25–35 peptide centrifuged at 16 000 g. Data were analyzed using a Zetasizer software 6.01 and expressed as size frequency distribution (%) in function of particles size (nm).

Figure 2. Physiological and behavioral effects of oAβ_25–35.

A. Body weight variations determined 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; scrambled group) or oAβ_25–35 (10 µg/rat; Aβ_25–35 group). The results are expressed as means ± SEM (with n = 6 per group). *p<0.05 vs. control value and +p<0.05 vs. scrambled value). B. Variations in locomotor activity and body temperature determined 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; scrambled group; n  = 7) or oAβ_25–35 (10 µg/rat; oAβ_25–35 group; n = 7). Locomotor activity and body temperature were monitored using telemetric sensors. The thick black line indicates the nocturnal phase (7:00 PM to 7:00 AM). The results are means/hour obtained the 6^th week following the icv injection. C. Effects of oAβ_25–35 icv injection (10 µg/rat) on the ability of rats to perform a spatial short-term memory task (T-maze). Six weeks after icv injection, animals were allowed to explore the T-maze, with one short arm closed (B), for 10 min. After a 1 h time interval, the pattern of exploration of the whole maze was recorded for 2 min. The icv injection of the scrambled Aβ_25–35 peptide (10 µg/rat) served as negative control. The results are expressed as means ± SEM. **p<0.01 vs. control un-injected rats, +p<0.05 and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns. D. Effects of oAβ_25–35 icv injection (10 µg/rat) on rat behavior in a spatial long-term memory test (Water-maze). Six weeks after icv injection, animals were allowed to swim for 90 s to find the training platform and 60 s without the platform for retention. The icv injection of the scrambled Aβ_25–35 peptide (10 µg/rat) served as negative control. The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control un-injected rats, +p<0.05 and ++p<0.01 vs. scrambled treated rats. E. The probe test was performed 4 h after the last training trial in a single 60 s-duration swimming without platform. The presence in the training quadrant was analyzed over the chance level (25%): # p<0.05 and ## p<0.01. The number of animals in each group is indicated within the columns. F. Variations in plasmatic corticosterone (CORT) levels determined in rats 6 weeks after icv injection of Aβ_25–35 scrambled peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). The values are means ± SEM. **p<0.01 vs. control un-injected rats (control group: C) and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns.

Figure 3. Oxidative stress.

Variations in lipid peroxidation levels in the frontal cortex, amygdala, hippocampus and hypothalamus, determined in rats 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). The results are expressed as means ± SEM. **p<0.01 vs. control un-injected rats (control group: C), +p<0.05 and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns.

Figure 4. Neurotrophic factor.

Variations in BDNF contents within the frontal cortex, amygdala, hippocampus and hypothalamus, determined in rats 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). The results are expressed as means ± SEM. **p<0.01 vs. control un-injected rats (control group: C), +p<0.05 and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns.

Figure 5. Mitochondrial stress.

Pro- and activated caspase-9 levels within the frontal cortex, amygdala, hippocampus and hypothalamus, determined in rats by western blot 6 weeks after oAβ_25–35 icv injection (10 µg/rat). Pro-caspase-9 (50 kDa) and activated caspase-9 (38 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with un-injected rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control group, +p<0.05 and ++p<0.01 vs. scrambled treated rats. Note that scrambled peptide injection (10 µg/rat) served as negative control and did not induce any modifications in pro- and activated caspase-9 levels. The number of animals in each group is indicated within the columns.

Figure 6. ER stress.

Variations in pro- and activated caspase-12 levels in the frontal cortex, amygdala, hippocampus and hypothalamus, determined in rats by western blot 6 weeks after oAβ_25–35 icv injection (10 µg/rat). Pro-caspase-12 (50 kDa) and activated caspase-12 (25 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with untreated rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control group, +p<0.05 and ++p<0.01 vs. scrambled treated rats. Note that scrambled peptide injection (10 µg/rat) served as negative control and did not induce any modifications in pro- and activated caspase-12 levels. The number of animals in each group is indicated within the columns.

Figure 7. Apoptosis.

Variations in pro- and activated caspase-3 levels in the frontal cortex, amygdala, hippocampus and hypothalamus, determined in rats by western blot 6 weeks after oAβ_25–35 icv injection (10 µg/rat). Pro-caspase-3 (35 kDa) and activated caspase-3 (19 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with untreated rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control group, +p<0.05 and ++p<0.01 vs. scrambled treated rats. Note that scrambled peptide injection (10 µg/rat) served as negative control and did not induce any modifications in pro- and activated caspase-3 levels. The number of animals in each group is indicated within the columns.

Figure 8. Astrocyte activation.

A. Variations in GFAP levels in the frontal cortex, amygdala, hippocampus and hypothalamus, determined in rats by western blot 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). GFAP (50 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with untreated rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control group, +p<0.05 and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns. B. Effects of oAβ_25–35 (10 µg/rat) icv injection on astrocyte reactions using GFAP immunolabeling into the frontal and parietal cortex, amygdala, hippocampus (CA1, CA2 & CA3 regions) and hypothalamus (periventricular nucleus: PeVN & paraventricular nucleus: PVN) determined in control (C) untreated rats and 6 weeks after Aβ_25–35 injection. The scrambled peptide injection (10 µg/rat) served as negative control and did not induce any modifications in the GFAP signal. 3v: third ventricle. brackets: hippocampus layer of granular cells layer. Scale bar  = 60 µm.

Figure 9. Microglial activation.

A. Effects of oAβ_25–35 (10 µg/rat) icv injection on microglial reaction using Iba-1 immunolabeling in the amygdala, frontal and parietal cortex, hypothalamus (paraventricular nucleus: PVN) and hippocampus (CA1 & CA3 regions) determined in control untreated rats and 6 weeks after Aβ_25–35 scrambled peptide (10 µg/rat; negative control) or Aβ_25–35 injection. Activated microglia was visualized with Alexafluor 488-labeled specific antibody against Iba-1 (green immunolabeling), while the nucleus was counterstained with DAPI (blue labeling). 3v: third ventricle. Scale bar  = 100 µm. B–E. Variations in Iba1 levels in the frontal cortex (B), amygdala (C), hippocampus (D) and hypothalamus (E), determined in rats by western blot 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). Iba1 (17 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with untreated rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control group, +p<0.05 and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns.

Figure 10. Cholinergic system.

Effects of oAβ_25–35 (10 µg/rat) icv injection on VAChT immunolabelling within the nucleus basalis of Meynert (A), mediobasal hypothalamus (B), parietal cortex (C) and hippocampus (D) determined in control untreated rats and 6 weeks after Aβ_25–35 injection. In (B): 3v: third ventricle. In (C): levels I to V cortical layers are indicated. In (D): brackets show the hippocampus granular cell layer. cc: corpus callosum. Scale bars  = 100 µm. Variations in VAChT levels in the hypothalamus (B) and hippocampus (D), determined in rats by western blot 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). VAChT (70 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with untreated rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control group, +p<0.05 and ++p<0.01 vs. scrambled treated rats. The number of animals in each group is indicated within the columns.

Figure 11. Hippocampus integrity.

Variations in hippocampus pyramidal cell numbers determined in rats 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). A. Representative microphotographs of coronal sections of Cresyl violet stained hippocampus CA1, CA2, CA3 and DG subfields, obtained in control untreated rats and after scrambled Aβ_25–35 peptide or Aβ_25–35 icv injection. Scale bar  = 300 and 100 µm. B. Average numbers of hippocampus pyramidal cells determined in untreated control rats (C) and 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). The results are expressed as means ± SEM (with n  = 4 per group). *p<0.05 and **p<0.01 vs. control rats, +p<0.05 and ++p<0.01 vs. respective scrambled peptide-treated rats. C. Effects of oAβ_25–35 (10 µg/rat) icv injection on hippocampus dendate gyrus (DG) neurogenesis using PSA-NCAM immunolabeling determined in untreated control rats and 6 weeks after Aβ_25–35 scrambled amyloid peptide (10 µg/rat; negative control) or oAβ_25–35 injection. Neurogenesis was visualized within coronal sections of the DG with Alexafluor555-labeled specific antibody against PSA-NCAM (red immunolabeling), while the nucleus was counterstained with DAPI (blue labeling). Scale bars = 200 µm.

Figure 12. APP processing.

Effects of oAβ_25–35 (10 µg/rat) icv injection on APP processing in the frontal cortex, amygdala, hippocampus and hypothalamus, determined by western blot in untreated control rats and 6 weeks after Aβ_25–35 scrambled amyloid peptide (10 µg/rat; negative control) or oAβ_25–35 injection. APP (125 KDa) and C99 (13 KDa) variations were normalized with β-tubulin (β-tub, 55 KDa) variations and compared with non-injected rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control non-injected rats (control group: C) and +p<0.05 and ++p<0.01 vs. respective scrambled peptide-treated rats. The number of animals in groups is indicated within the columns.

Figure 13. Tau phosphorylation.

Effects of oAβ_25–35 (10 µg/rat) icv injection on Tau phosphorylation in the frontal cortex, amygdala, hippocampus and hypothalamus, determined by western blot in untreated control rats and 6 weeks after Aβ_25–35 scrambled amyloid peptide (10 µg/rat; negative control) or oAβ_25–35 icv injection. The Tau hyperphosphorylation (AT8; 50 KDa) and the abnormal Tau phosphorylation (AT100; 50 KDa) variations were expressed in function of total Tau expression (50 KDa) and compared with non-injected rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control non-injected rats (control group: C) and +p<0.05 and ++p<0.01 vs. respective scrambled peptide-treated rats. The number of animals in groups is indicated within the columns.