Deregulation of hypothalamic-pituitary-adrenal axis functions in an Alzheimer's disease rat model

International audienc

Alzheimer's Disease Related Markers, Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

International audienc

Time-Course and Regional Analyses of the Physiopathological Changes Induced after Cerebral Injection of an Amyloid β Fragment in Rats

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid β protein (Aβ) formed by pathological processing of the Aβ precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aβ fragment 25–35 (Aβ25-35) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aβ25-35 peptide was used as negative control. The aggregated forms of Aβ peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aβ25-35 decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aβ25-35, the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aβ25-35 injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD

Cholinergic system.

<p>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 <i>vs</i>. control group, +p<0.05 and ++p<0.01 <i>vs</i>. scrambled treated rats. The number of animals in each group is indicated within the columns.</p

Physiological and behavioral effects of oAβ_25–35.

<p><b>A.</b> 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 <i>vs</i>. control value and +p<0.05 vs. scrambled value). <b>B.</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. <b>C.</b> 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 <i>vs.</i> control un-injected rats, +p<0.05 and ++p<0.01 <i>vs.</i> scrambled treated rats. The number of animals in each group is indicated within the columns. <b>D.</b> 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 <i>vs.</i> scrambled treated rats. <b>E.</b> 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. <b>F.</b> 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 <i>vs</i>. control un-injected rats (control group: C) and ++p<0.01 <i>vs</i>. scrambled treated rats. The number of animals in each group is indicated within the columns.</p

Astrocyte activation.

<p><b>A.</b> 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 <i>vs</i>. control group, +p<0.05 and ++p<0.01 <i>vs</i>. scrambled treated rats. The number of animals in each group is indicated within the columns. <b>B.</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.</p

ER stress.

<p>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 <i>vs</i>. control group, +p<0.05 and ++p<0.01 <i>vs.</i> 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.</p

Apoptosis.

<p>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 <i>vs</i>. control group, +p<0.05 and ++p<0.01 <i>vs.</i> 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.</p

Neurotrophic factor.

<p>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 <i>vs</i>. control un-injected rats (control group: C), +p<0.05 and ++p<0.01 <i>vs.</i> scrambled treated rats. The number of animals in each group is indicated within the columns.</p

Hippocampus integrity.

<p>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). <b>A.</b> 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>B.</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. <b>C.</b> 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.</p