| 1. |
- Bjornholm, Katrine Dahl, et al.
(author)
-
A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature
- 2023
-
In: CELL REPORTS METHODS. - : Elsevier. - 2667-2375. ; 3:3
-
Journal article (peer-reviewed)abstract
- Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain dis-eases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivas-cular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation proto-col suitable for different experimental modalities, such as single-cell analyses, western blotting, and pri-mary cell culture.
|
|
| 2. |
- Borgegard, Tomas, et al.
(author)
-
Alzheimers Disease: Presenilin 2-Sparing gamma-Secretase Inhibition Is a Tolerable A beta Peptide-Lowering Strategy
- 2012
-
In: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 32:48, s. 17297-17305
-
Journal article (peer-reviewed)abstract
- gamma-Secretase inhibition represents a major therapeutic strategy for lowering amyloid beta (A beta) peptide production in Alzheimers disease (AD). Progress toward clinical use of gamma-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The gamma-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between A beta production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1(PS1) over PS2 subclass of gamma-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain A beta levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious gamma-secretase targeting strategy for AD.
|
|
| 3. |
- Hedskog, Louise, et al.
(author)
-
Biochemical studies of poly t variants in the alzheimer's disease associated tomm40 gene
- 2012
-
In: Journal of Alzheimer's Disease. - 1387-2877 .- 1875-8908. ; 31:3, s. 527-536
-
Journal article (peer-reviewed)abstract
- The apolipoprotein E (APOE) gene remains the most strongly established risk factor for late onset Alzheimer's disease (LOAD). Recently the gene, TOMM40, which is in linkage disequilibrium with APOE, was identified to be associated with LOAD in genome-wide association studies. One of the identified polymorphisms in TOMM40 is rs10524523, which is located in intron 6 and composed of thymidine repeats varying between 14 to 36 base-pairs in length. Reported results are contradictory in regard to the very long poly-T variant that has been associated with both increased and decreased risk of LOAD. Our study aimed to elucidate the functional implication of rs10524523 in an in vitro model of human fibroblast cells obtained from cognitively healthy APOE epsilon 3/epsilon 4 carriers harboring very long or short poly-T variants coupled to their APOE epsilon 3 allele. We have studied (i) expression levels of TOM40 protein and mRNA, (ii) TOM40 mRNA splicing, and (iii) mitochondrial function and morphology; and we have found no significant differences in regards to very long or short poly-T variant.
|
|
| 4. |
- Oliveira, Daniel V., et al.
(author)
-
Molecular Chaperone BRICHOS Inhibits CADASIL-Mutated NOTCH3 Aggregation In Vitro
- 2022
-
In: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
-
Journal article (peer-reviewed)abstract
- CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common familial form of stroke, which is caused by mutations located in the epidermal growth factor (EGF)-like repeats of the NOTCH3 gene. Mutations cause the NOTCH3 (N3) protein to misfold and aggregate. These aggregates will be a component of granular osmiophilic material, which when accumulated around the arteries and arterioles is believed to cause the degradation of vascular smooth muscle cells (VSMC). VSMC degradation affects blood flow regulation and leads to white matter and neuronal death. Currently, there is no treatment for CADASIL. The dementia-relevant BRICHOS domain is a small multitalented protein with functions that include ATP-independent chaperone-like properties. BRICHOS has been shown to prevent the aggregation of both fibrillar and non-fibrillar structures. Therefore, the objective of this study is to investigate whether BRICHOS exhibits anti-aggregating properties on a recombinant CADASIL-mutated N3 protein consisting of the first five repeats of EGF (EGF(1-5)), harboring a cysteine instead of an arginine in the position 133, (R133C). We found that the N3 EGF(1-5) R133C mutant is more prone to aggregate, while the wildtype is more stable. Recombinant human Bri2 BRICHOS is able to interact and stabilize the R133C-mutated N3 protein in a dose-dependent manner. These results suggest an anti-aggregating impact of BRICHOS on the N3 EGF(1-5) R133C protein, which could be a potential treatment for CADASIL.
|
|
