Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Abstract Background Alzheimer’s disease (AD), the most common cause of dementia, is characterized by the progressive deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Mouse models of Aβ amyloidosis generated by knock-in (KI) of a humanized Aβ sequence provide distinct advantages over traditional transgenic models that rely on overexpression of amyloid precursor protein (APP). In App-KI mice, three familial AD-associated mutations were introduced into the endogenous mouse App locus to recapitulate Aβ pathology observed in AD: the Swedish (NL) mutation, which elevates total Aβ production; the Beyreuther/Iberian (F) mutation, which increases the Aβ42/Aβ40 ratio; and the Arctic (G) mutation, which promotes Aβ aggregation. App NL-G-F mice harbor all three mutations and develop progressive Aβ amyloidosis and neuroinflammatory response in broader brain areas, whereas App NL mice carrying only the Swedish mutation exhibit no overt AD-related pathological changes. To identify behavioral alterations associated with Aβ pathology, we assessed emotional and cognitive domains of App NL-G-F and App NL mice at different time points, using the elevated plus maze, contextual fear conditioning, and Barnes maze tasks. Results Assessments of emotional domains revealed that, in comparison with wild-type (WT) C57BL/6J mice, App NL-G-F/NL-G-F mice exhibited anxiolytic-like behavior that was detectable from 6 months of age. By contrast, App NL/NL mice exhibited anxiogenic-like behavior from 15 months of age. In the contextual fear conditioning task, both App NL/NL and App NL-G-F/NL-G-F mice exhibited intact learning and memory up to 15–18 months of age, whereas App NL-G-F/NL-G-F mice exhibited hyper-reactivity to painful stimuli. In the Barnes maze task, App NL-G-F/NL-G-F mice exhibited a subtle decline in spatial learning ability at 8 months of age, but retained normal memory functions. Conclusion App NL/NL and App NL-G-F/NL-G-F mice exhibit behavioral changes associated with non-cognitive, emotional domains before the onset of definitive cognitive deficits. Our observations consistently indicate that App NL-G-F/NL-G-F mice represent a model for preclinical AD. These mice are useful for the study of AD prevention rather than treatment after neurodegeneration

Amyloid-β plaque formation and reactive gliosis are required for induction of cognitive deficits in App knock-in mouse models of Alzheimer’s disease

Abstract Background Knock-in (KI) mouse models of Alzheimer’s disease (AD) that endogenously overproduce Aβ without non-physiological overexpression of amyloid precursor protein (APP) provide important insights into the pathogenic mechanisms of AD. Previously, we reported that App NL-G-F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited emotional alterations before the onset of definitive cognitive deficits. To determine whether these mice exhibit deficits in learning and memory at more advanced ages, we compared the Morris water maze performance of App NL-G-F and App NL mice, which harbor only the Swedish mutation, with that of wild-type (WT) C57BL/6J mice at the age of 24 months. To correlate cognitive deficits and neuroinflammation, we also examined Aβ plaque formation and reactive gliosis in these mice. Results In the Morris water maze, a spatial task, 24-month-old App NL-G-F/NL-G-F mice exhibited significantly poorer spatial learning than WT mice during the hidden training sessions, but similarly to WT mice during the visible training sessions. Not surprisingly, App NL-G-F/NL-G-F mice also exhibited spatial memory deficits both 1 and 7 days after the last training session. By contrast, 24-month-old App NL/NL mice had intact spatial learning and memory relative to WT mice. Immunohistochemical analyses revealed that 24-month-old App NL-G-F/NL-G-F mice developed massive Aβ plaques and reactive gliosis (microgliosis and astrocytosis) throughout the brain, including the cortex and hippocampus. By contrast, we observed no detectable brain pathology in App NL/NL mice despite overproduction of human Aβ40 and Aβ42 in their brains. Conclusions Aβ plaque formation, followed by sustained neuroinflammation, is necessary for the induction of definitive cognitive deficits in App-KI mouse models of AD. Our data also indicate that introduction of the Swedish mutation alone in endogenous APP is not sufficient to produce either AD-related brain pathology or cognitive deficits in mice

eNOS-dependent antisenscence effect of a calcium channel blocker in human endothelial cells.

Senescence of vascular endothelial cells is an important contributor to the pathogenesis of age-associated vascular disorders such as atherosclerosis. We investigated the effects of antihypertensive agents on high glucose-induced cellular senescence in human umbilical venous endothelial cells (HUVECs). Exposure of HUVECs to high glucose (22 mM) for 3 days increased senescence-associated- β-galactosidase (SA-β-gal) activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. The calcium channel blocker nifedipine, but not the β1-adrenergic blocking agent atenolol or the angiotensin-converting enzyme inhibitor perindopril, reduced SA-β-gal positive cells and prevented a decrease in telomerase activity in a high-glucose environment. This beneficial effect of nifedipine was associated with reduced reactive oxygen species (ROS) and increased endothelial nitric oxide synthase (eNOS) activity. Thus, nifedipine prevented high glucose-induced ROS generation and increased basal eNOS phosphorylation level at Ser-1177. Treatment with N (G)-nitro-L-arginine (L-NAME) and transfection of small interfering RNA (siRNA) targeting eNOS eliminated the anti-senscence effect of nifedipine. These results demonstrate that nifedipine can prevent endothelial cell senescence in an eNOS-dependent manner. The anti-senescence action of nifedipine may represent a novel mechanism by which it protects against atherosclerosis

MOESM3 of Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Additional file 3: Fig. S3. Locomotor activity in AppNL-G-F/NL-G-F and AppNL/NL mice during the pre-shock period in the contextual fear conditioning task. The distance travelled during the pre-shock period (3-min period just prior to the first footshock) in conditioning was compared among genotypes at both 6–9 (a and b) and 15–18 (c and d) months of age. Representative images of movement tracks during the pre-shock period in each genotype at 6–9 (a) and 15–18 (c) months of age were shown. At 6–9 months of age, AppNL-G-F/NL-G-F mice exhibited a slight decrease in distance travelled during the pre-shock period in comparison with WT mice (b). At 15–18 months of age, AppNL/NL mice exhibited a significant decrease in distance travelled during the pre-shock period in comparison with WT mice (d). Locomotor activity in AppNL-G-F/NL-G-F mice was also slightly decreased in comparison with WT mice. 6–9 month-old; n = 6 WT (B6 J), n = 6 AppNL/NL, n = 9 AppNL-G-F/NL-G-F. 15–18 month-old; n = 8 WT (B6 J), n = 7 AppNL/NL, n = 7 AppNL-G-F/NL-G-F. *p < 0.05 versus WT (B6J)

Knockdown of <i>wfs1</i>, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in <i>Drosophila</i>

<div><p>Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (<i>WFS1</i>), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. <i>WFS1</i> encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic β-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by <i>WFS1</i> deficiency remain elusive. Here, we investigated the role of <i>WFS1</i> in the maintenance of neuronal integrity <i>in vivo</i> by knocking down the expression of <i>wfs1</i>, the <i>Drosophila</i> homolog of <i>WFS1</i>, in the central nervous system. Neuronal knockdown of <i>wfs1</i> caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of <i>wfs1</i> in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that <i>wfs1</i> has important functions in both cell types. Although <i>wfs1</i> knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of <i>wfs1</i>. This study highlights the protective role of <i>wfs1</i> against age-associated neurodegeneration and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases.</p></div

MOESM2 of Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Additional file 2: Fig. S2. Locomotor activity of AppNL-G-F/NL-G-F and AppNL/NL mice during the first and second trials in the elevated plus maze task. The distance travelled during the 10-min test of the first and second trials in the elevated plus maze task was compared among genotypes at both 6–9 (a–d) and 15–18 (e–h) months of age. Representative images of movement tracks during the first and second trials for each genotype at 6–9 (a and c) and 15–18 (e and g) months of age were shown (closed arms are indicated by shaded areas). At 6–9 months of age, AppNL-G-F/NL-G-F mice exhibited slight increases in distance travelled during the first (b) and second (d) trials in comparison with WT mice. By contrast, locomotor activity in AppNL/NL mice was comparable with WT mice in the two trials. At 15–18 months of age, AppNL-G-F/NL-G-F mice exhibited a slight increase in movement compared to WT mice during the first (f) and second (g) trials. AppNL/NL mice moved at similar levels compared with WT mice in the two trials. 6–9 month-old; n = 8 WT (B6J), n = 8 AppNL/NL, n = 8 AppNL-G-F/NL-G-F. 15–18 month-old; n = 12 WT (B6J), n = 10 AppNL/NL, n = 11 AppNL-G-F/NL-G-F. †p < 0.05 versus AppNL/NL

Developmental alterations in anxiety and cognitive behavior in serotonin transporter mutant mice

RATIONALE: A promoter variant of the serotonin transporter (SERT) gene is known to affect emotional and cognitive regulation. In particular, the "short" allelic variant is implicated in the etiology of multiple neuropsychiatric disorders. Heterozygous (SERT(+/-)) and homozygous (SERT(-/-)) SERT mutant mice are valuable tools for understanding the mechanisms of altered SERT levels. Although these genetic effects are well investigated in adulthood, the developmental trajectory of altered SERT levels for behavior has not been investigated. OBJECTIVES: We assessed anxiety-like and cognitive behaviors in SERT mutant mice in early adolescence and adulthood to examine the developmental consequences of reduced SERT levels. Spine density of pyramidal neurons was also measured in corticolimbic brain regions. RESULTS: Adult SERT(-/-) mice exhibited increased anxiety-like behavior, but these differences were not observed in early adolescent SERT(-/-) mice. Conversely, SERT(+/-) and SERT(-/-) mice did display higher spontaneous alternation during early adolescence and adulthood. SERT(+/-) and SERT(-/-) also exhibited greater neuronal spine densities in the orbitofrontal but not the medial prefrontal cortices. Adult SERT(-/-) mice also showed an increased spine density in the basolateral amygdala. CONCLUSIONS: Developmental alterations of the serotonergic system caused by genetic inactivation of SERT can have different influences on anxiety-like and cognitive behaviors through early adolescence into adulthood, which may be associated with changes of spine density in the prefrontal cortex and amygdala. The altered maturation of serotonergic systems may lead to specific age-related vulnerabilities to psychopathologies that develop during adolescence

Heterozygous mutation in <i>Eaat1</i>, a glial high-affinity glutamate transporter, significantly worsens behavioral deficits caused by <i>wfs1</i> deficiency.

<p>(<b>A</b>) Flies with neuronal (<i>elav</i>), neuronal and glial (<i>elav-Repo</i>) knockdown of <i>wfs1</i> or <b>(B</b>) <i>wfs1</i> mutant with PiggyBac insertion (<i>wfs1</i>^{<i>LL07290/LL07290</i>}) required longer time to complete righting reflex than control flies. n = 10–26, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Mann-Whitney <i>U</i>-test. (<b>C</b>) The mRNA levels of genes related to synaptic activities with focus on GABAergic as well as glutamatergic systems in fly brains with neuronal and glial knockdown of <i>wfs1</i> were determined by qRT-PCR. n = 4, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>D</b>) The mRNA level of <i>Eaat1</i> in fly brains with neuronal knockdown of <i>wfs1</i> was determined by qRT-PCR. n = 4, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>E</b>) Heterozygous mutation in <i>Eaat1</i> did not induce locomotor defects in aged flies (left panel). In the <i>wfs1</i> knockdown background, heterozygous mutation in <i>Eaat1</i> caused locomotor deficits (right panel) as revealed by climbing assay. Average percentages of flies that climbed to the top (white), climbed to the middle (light gray), or stayed at the bottom (dark gray) of the vials. Percentage of flies that stayed at the bottom were subjected to statistical analyses. n = 4 independent experiments. n = 5, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>F</b>) A heterozygous mutation in <i>Eaat1</i> induced ER stress responses in <i>wfs1</i> knockdown background, but not in control background. The mRNA levels of <i>Xbp1-RB</i>, <i>PEK</i> and <i>Hsc70-3</i> in fly brains were determined by qRT-PCR. n = 4, *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>G</b>) mRNA levels of genes related to oxidative stress responses in fly heads carrying a heterozygous mutation in <i>Eaat1</i> were prominently elevated in <i>wfs1</i> knockdown background compared to control background, as determined by qRT-PCR. n = 4, *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>H</b>-<b>I</b>) Riluzole increased the survival time in flies with neuronal and glial knockdown of <i>wfs1</i> (<b>H</b>) and in flies carrying homozygous mutations of <i>wfs1</i> with PiggyBac insertion (<i>wfs1</i>^{<i>LL07290/LL07290</i>}) (<b>I</b>). The survival rates were determined by Kaplan-Meier survival analysis with log-rank test, and Holm-Sidak method was used for multiple comparison analysis (n = 138, <i>mcherry</i> RNAi with Riluzole 0 mM, n = 128, <i>mcherry</i> RNAi with Riluzole 5 mM, n = 102, <i>wfs1</i> RNAi with Riluzole 0 mM, n = 101, <i>wfs1</i> RNAi with Riluzole 5 mM, n = 82, <i>wfs1</i>^{LL07290/LL07290} with Riluzole 0 mM, n = 85, <i>wfs1</i>^{LL07290/LL07290} with Riluzole 5 mM). The statistical significance was indicated in the figure. Genotypes and ages of flies are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007196#pgen.1007196.s001" target="_blank">S1 Table</a>.</p

EDEM Function in ERAD Protects against Chronic ER Proteinopathy and Age-Related Physiological Decline in Drosophila

Summary The unfolded protein response (UPR), which protects cells against accumulation of misfolded proteins in the ER, is induced in several age-associated degenerative diseases. However, sustained UPR activation has negative effects on cellular functions and may worsen disease symptoms. It remains unknown whether and how UPR components can be utilized to counteract chronic ER proteinopathies. We found that promotion of ER-associated degradation (ERAD) through upregulation of ERAD-enhancing α-mannosidase-like proteins (EDEMs) protected against chronic ER proteinopathy without inducing toxicity in a Drosophila model. ERAD activity in the brain decreased with aging, and upregulation of EDEMs suppressed age-dependent behavioral decline and extended the lifespan without affecting the UPR gene expression network. Intriguingly, EDEM mannosidase activity was dispensable for these protective effects. Therefore, upregulation of EDEM function in the ERAD protects against ER proteinopathy in vivo and thus represents a potential therapeutic target for chronic diseases

Knockdown of <i>wfs1</i> alone does not induce ER stress in the fly brains.

<p><b>(A)</b> Neuronal (<i>elav</i>), <b>(B)</b> neuronal and glial (<i>elav-Repo</i>) knockdown of <i>wfs1</i> or <b>(C</b>) <i>wfs1</i> mutant with MiMIC insertion (<i>wfs1</i>^{<i>MI14041/MI14041</i>}) did not increase mRNA levels of <i>Xbp1-RB</i>, <i>PEK</i> and <i>Hsc70-3</i> in fly brains, as determined by qRT-PCR. n = 4, *<i>p</i> < 0.05 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. Genotypes and ages of flies are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007196#pgen.1007196.s001" target="_blank">S1 Table</a>.</p