A protocol for quantitative analysis of murine and human amyloid-β1-40 and 1-42

Illouz, Tomer; Madar, Ravit; Griffioen, Kathleen J.; Okun, Eitan

doi:10.1016/j.jneumeth.2017.07.022

Cited by 11 publications

(9 citation statements)

References 41 publications

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“…Next, cortical and hippocampal levels of Aβ 1–40 and Aβ 1–42 were quantified using sELISA as previously described ( Illouz et al, 2017 ). Cortical level of soluble Aβ 1–40 were higher in sham-vaccinated Ts65Dn mice compared with WT controls (6.13 ± 3.93 ng/ml and 0.93 ± 0.57 ng/ml, respectively, P < 0.05, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For biochemical analysis, cerebral cortex and hippocampi were separated, snap-froze on dry ice and stored at −80 °C until use. For Aβ quantification using ELISA, soluble and insoluble protein fractions were purified using our previously published protocol ( Illouz et al, 2017 ). Briefly, tissues were mechanically homogenized in 0.1% SDS-RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-base, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS in aqueous solution), 15 μl per 1 mg tissue, incubated on ice for 30 min followed by centrifugation for 90 min at 17,000g at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Soluble and insoluble levels Aβ 1–40 and Aβ 1–42 in the serum and brain-tissue were conducted using our previously published sandwich-ELISA protocol ( Illouz et al, 2017 ). In brief, 96-well polystyrene microplates (Microlon, 655061, Greinerbio-one, Monroe, NC) were covered with 50 μl of anti-mouse-N-terminus Aβ 1–16 (ab126873, Abcam) at a concentration of 5 μg/ml in carbonate-bicarbonate buffer (pH = 9.6) and incubated overnight at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

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Restoring microglial and astroglial homeostasis using DNA immunization in a Down Syndrome mouse model

Illouz

Madar

Biragyn³

et al. 2019

Brain, Behavior, and Immunity

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Down Syndrome (DS), the most common cause of genetic intellectual disability, is characterized by over-expression of the APP and DYRK1A genes, located on the triplicated chromosome 21. This chromosomal abnormality leads to a cognitive decline mediated by Amyloid-β (Aβ) overproduction and tau hyper-phosphorylation as early as the age of 40. In this study, we used the Ts65Dn mouse model of DS to evaluate the beneficial effect of a DNA vaccination against the Aβ1–11 fragment, in ameliorating Aβ-related neuropathology and rescue of cognitive and behavioral abilities. Anti-Aβ1–11 vaccination induced antibody production and facilitated clearance of soluble oligomers and small extracellular inclusions of Aβ from the hippocampus and cortex of Ts65Dn mice. This was correlated with reduced neurodegeneration and restoration of the homeostatic phenotype of microglial and astroglial cells. Vaccinated Ts65Dn mice performed better in spatial-learning tasks, exhibited reduced motor hyperactivity typical for this strain, and restored short-term memory abilities. Our findings support the hypothesis that DS individuals may benefit from active immunotherapy against Aβ from a young age by slowing the progression of dementia.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Restoring microglial and astroglial homeostasis using DNA immunization in a Down Syndrome mouse model

Illouz

Madar

Biragyn³

et al. 2019

Brain, Behavior, and Immunity

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“…1A). Cortical Aβ 40 and Aβ 42 levels were quantified using a modification of previously described sELISA (Illouz, Madar, Griffioen, & Okun, 2017) in soluble (TBST-soluble), pre-amyloid complexes (SDS-soluble) and insoluble-fibrillary (FA-soluble) Aβ extracts. M+/A- and M+/A+ mice exhibited reduced soluble Aβ 42 compared with both M-/A- and M-/A+ mice (0±0, 0±0, 465.4±191.5, and 143±112.3pg/mg, respectively, P<0.05, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Aβ 40 and Aβ 42 in the cortex were measured using a modification of a previously published sandwich-ELISA protocol (Illouz et al, 2017). The full protocol is found in the Supplementary Materials section.…”

Section: Methodsmentioning

confidence: 99%

Maternal antibodies facilitate Amyloid-β clearance by activating Fc-receptor-Syk-mediated phagocytosis

Illouz

Nicola

Ben-Shushan

et al. 2020

Preprint

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Down Syndrome (DS) features a life-long overexpression of the APP and DYRK1A genes, leading to a cognitive decline mediated by Amyloid-β (Aβ) overproduction and tau hyper-phosphorylation. As DS can be diagnosed in utero, maternally transferred anti-Aβ antibodies might promote removal of early accumulation of Aβ from the CNS. A DNA-vaccine expressing Aβ1-11 was delivered to wild-type female mice, followed by mating with 5xFAD males, which exhibit early Aβ plaque formation, similar to individuals with DS. Maternal Aβ-specific antibodies provided transgenic offspring with passive immunization against Aβ via the placental and subsequently lactation. Maternal antibodies reduced cortical Aβ levels 4 months after antibodies were undetectable, along with alleviating short-term memory deficits and activation of the Fc𝛾R1/Syk/Cofilin pathway in microglia. Sera from immunized dams facilitated Aβ clearance by microglia in a Syk-dependent manner. These data suggest that maternal anti-Aβ immunization is a potential strategy to alleviate cognitive decline in individuals with DS.

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Aging and brain free cholesterol concentration on amyloid‐β peptide accumulation in guinea pigs

Pang,

Peng,

et al. 2024

Biopharm & Drug Disp

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Alzheimer’s disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid‐β (Aβ40 and Aβ42) peptides, Mdr1 and APP (amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9–40 weeks old guinea pigs. Protein expression levels of P‐gp (Abcb1) and Cyp46a1 (24(S)‐hydroxylase) for Aβ40, and Aβ42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low‐density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aβ efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aβ peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aβ peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)‐hydroxycholesterol metabolite revealed a decreasing trend from 20 weeks old toward 40 weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aβ accumulation. The decreases in P‐gp and Lrp1 protein levels should further exacerbate the accumulation of Aβ peptides in guinea pig brain.

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A protocol for quantitative analysis of murine and human amyloid-β1-40 and 1-42

Cited by 11 publications

References 41 publications

Restoring microglial and astroglial homeostasis using DNA immunization in a Down Syndrome mouse model

Restoring microglial and astroglial homeostasis using DNA immunization in a Down Syndrome mouse model

Maternal antibodies facilitate Amyloid-β clearance by activating Fc-receptor-Syk-mediated phagocytosis

Aging and brain free cholesterol concentration on amyloid‐β peptide accumulation in guinea pigs

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