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- Forsberg, Lars A., 1974-, et al.
(författare)
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Mosaic loss of chromosome Y in leukocytes matters
- 2019
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 51:1, s. 4-7
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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- Giedraitis, Vilmantas
(författare)
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Candidate gene analyses and genome-wide screens in multiple sclerosis
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Specific disease causing mutations have been identified for many disorders. Most of these disorders are of Mendelian origin and there have been very few successful attempts in the genetic dissection of complex traits. Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease of the central nervous system with clear influence of both environmental and genetic factors. It is genetically complex, however to date the only genetic region clearly demonstrated to be involved in MS is the major histocompatibility complex. In our studies we have analyzed two NIS candidate genes as well as tried to identify chromosomal regions harboring MS susceptibility genes by genome-wide screens. Alteration in interferon-gamma (IFN-gamma) production has been found in several diseases including multiple sclerosis. Such alterations could theoretically be caused by polymorphisms in the gene. We screened the IFN-gamma gene promoter and part of the first intron for possible mutations by sequencing. We identified a C to T substitution in the IFN-gamma promoter at position -333. Screening for this mutation by sequence specific PCR in 214 MS patients and 164 controls identified two patients, both heterozygous, but no controls with this mutation. The IFN-gamma gene seems to be rather conserved and most probably changes in IFN-gamma expression are due to variations in transcription factor activity, rather than gene polymorphisms. One of the factors influencing IFN-gamma production is interleukin-18 (IL-18). We cloned the human IL-18 promoter and screened it for possible polymorphisms. Five single nucleotide polymorphisms were identified. Three combinations of these polymorphisms were observed in the Swedish population and were analyzed for activity. All alleles had clear promoter activity, which increased after stimulation with PMA/ionomicin. There were no significant differences in promoter activity between alleles before stimulation, but after stimulation, one of the alleles had clearly lower activity than the others. Measurement of IL-18 and IFN-gamma production by RT-PCR showed slightly higher expression of IL-18 in individuals homozygous for the most frequent allele. Two polymorphisms, identifying promoter alleles, were analyzed by sequence specific PCR in MS patients and healthy controls, however no significant differences were found. In the next tree studies we have used isolated and localized Swedish populations to screen the genome for NIS susceptibility regions. By haplotype sharing analysis we studied a genetically isolated population from Överkalix in Northern Sweden, where a high incidence of MS was previously reported. Genealogical analysis had shown that 19 MS patients available for our study, originated from a single common ancestral couple in the eighteenth century. Five affected individuals were selected for an initial genomic screen with 390 micro-satellite markers. Shared haplotypes identified in these patients were analyzed in other MS patients and healthy relatives. A conserved haplotype spanning 10 cM was identified on 17p11. Surprisingly, DR-typing revealed no significant sharing of the 14LA region. In the next study we extended the analysis of the Överkalix MS pedigrees and performed a genomewide screen in all available families. We analyzed data by haplotype based transmission disequilibrium test (TDT) and likelihood-ratio test for linkage disequilibrium. Four regions showed significant p-values in both tests. By a haplotype-based TDT analysis, several additional regions were identified. The two most interesting loci were in chromosomes 10p13 and 17p11, being identified by both approaches and corresponding to loci identified in other studies. We also performed a genome-wide screen with 834 microsatellite marker in a family material collected in Värmland county of Sweden, consisting of 54 MS patients and 114 healthy family members. A group of families were possible to track back to common ancestors in the 17th century. We used single marker and haplotype based TDT analysis and nonparametric linkage analysis to analyze data. Regions on chromosomes 2q21-33, 6p25-23, 6q25-27, 14q24-31 and 17q22 were found to be in transmission disequilibrium with MS. Most interesting region was 14q24-31, where several dimarker haplotypes were in transmission disequilibrium in the affected individuals. One marker in this region was also positive in single marker TDT.
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- Konstantinidis, Evangelos, 1990-
(författare)
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CRISPR/Cas9-based therapies and the role of astrocytes in Alzheimer’s disease and Parkinson’s disease
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative disorders. Whereas the AD brain features plaques of amyloid-beta (Aβ) and neurofibrillary tangles of tau, the PD brain is characterized by Lewy bodies and Lewy neurites containing α-synuclein (αSyn). Rare familial disease forms have illustrated a central involvement of these proteins in the respective pathogeneses. Mutations in the genes for the presenilins (PSEN1, PSEN2) result in AD by an increased generation of the more aggregation prone Aβ42 peptide, whereas mutations in the αSyn gene (SNCA) cause PD by affecting aggregation of αSyn.This thesis has investigated the gene editing tool CRISPR/Cas9 as a potential treatment strategy against AD and PD. When targeting PSEN1 M146L in patient fibroblasts, the increased Aβ42/Aβ40 ratio was partially restored and the treatment typically normalized the mutation-induced conformation of presenilin 1. Moreover, the treatment did not cause any major off-target effects across the genome. For SNCA, both the wild-type form and the A53T mutant were targeted. Lentivirus-mediated delivery of CRISPR/Cas9 to patient fibroblasts and HEK293T cells led to a targeting efficiency of up to 87%. However, treatment of A53T mutant patient fibroblasts only resulted in low and inconsistent targeting efficiencies.During the course of AD, progressive cellular dysfunction and degeneration cause widespread neuronal death. Apart from neurons, also glial cells are affected by the disease process. Astrocytes, the most abundant glial cell type, play a key role in maintaining brain homeostasis. However, in a neurodegenerative environment, astrocytes enter a reactive and inflammatory state that can potentially harm nearby neurons.To further investigate the role of astrocytes in AD, we generated a co-culture system of human induced pluripotent stem cell-derived neurons and astrocytes. We observed a differential effect of direct and remote astrocytic control on neuronal viability and functionality. Physical astrocytic contact combined with the presence of Aβ resulted in increased phagocytosis and clearance of dead cells as well as a reduced neuronal activity. However, indirect contact via conditioned media from control astrocytes improved the viability of neurons, whereas addition of Aβ led to hyperactivity. Analyses of long-term astrocytic cultures revealed a persistent reactive state accompanied by a limited Aβ degradation capacity and severe cellular stress.Overall, this thesis has explored novel gene therapeutic strategies for AD and PD as well as contributed with knowledge regarding the role of astrocytes in AD progression.
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- Molisak, Agnieszka, et al.
(författare)
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CRISPR/Cas9 as a tool to disrupt wild-type and A53T SNCA in sporadic and familial Parkinson’s disease
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Parkinson’s Disease (PD) is characterized by pathological accumulation of α-synuclein (αSyn) as Lewy bodies and Lewy neurites in the brain. Current treatment strategies can only alleviate the symptoms but do not interfere with the disease progression. With the discovery of the CRISPR/Cas9 gene editing tool, it has become possible to target the generation of pathological protein aggregates at the DNA level. Disrupting the αSyn gene (SNCA) could prevent the formation of Lewy-related pathologies. Here, we have designed two CRISPR/Cas9-based approaches by using guide RNAs (gRNAs) that are targeting either wild-type (WT) SNCA (pan-SNCA) or mutant A53T SNCA that causes early-onset familial PD. We could demonstrate that plasmid vector-mediated transfection of the pan-SNCA gRNA led to robust allelic disruption in HEK293T cells and human fibroblasts and that the editing efficiency was further increased with the use of a lentiviral transduction system. In addition, the SNCA A53T gRNA was specific towards the mutation site, but resulted in low and inconsistent targeting efficiencies in human patient fibroblasts carrying the SNCA A53T mutation. Our results indicate that SNCA can be targeted by CRISPR/Cas9, although the system efficiency varies across different cell types. In the future, systemically administered gene-editing treatments based on CRISPR/Cas9 could provide a valid therapeutic approach for PD patients.
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| 7. |
- Nowak, Christoph, et al.
(författare)
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Metabolite profiles during an oral glucose tolerance test reveal new associations with clamp-measured insulin sensitivity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Impaired insulin sensitivity (IS) is a major risk factor for cardiovascular disease and type 2 diabetes. Metabolomic profiling during an oral glucose tolerance test (OGTT) can reveal early pathogenic alterations in healthy individuals. Our aim was to identify IS biomarkers and gain new pathophysiologic insights by applying untargeted metabolomics to repeated OGTT plasma samples in association with a hyperinsulinemic-euglycemic clamp assessment. We studied 192 metabolites identified by non-targeted liquid chromatography/mass spectrometry in plasma samples taken at 0, 30, and 120 min during an OGTT in 470 non-diabetic 71-yr-old men. Insulin sensitivity was associated with 35 metabolites at one or more time points in multivariable-adjusted linear regression. The trajectories of nine metabolites during the OGTT were related to IS, six of which (oleic and palmitoleic acid, decanoyl- and dodecanoylcarnitine, deoxycholate-glycine and hexose) showed no associations with IS in the baseline fasting state. The strongest effects were detected for medium-chain acylcarnitines, which increased between 30-120 min in insulin-resistant individuals compared to those with normal IS. In this large community sample, we identified novel associations between clamp-measured IS and metabolite profiles that became apparent only after an oral glucose challenge. Associations of differential medium-chain acylcarnitine and monounsaturated fatty acid trajectories with IS provide new insights into the pathogenesis of insulin resistance.
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| 8. |
- Pagnon de la Vega, María, 1994-
(författare)
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Characterization of the novel “Uppsala mutation” causing a familial form of early onset Alzheimer’s disease
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Alzheimer’s disease (AD) brain displays extracellular plaques of amyloid-β (Aβ), neurofibrillary tangles of tau and neuronal loss. The 40-42 amino acid Aβ peptide is formed from the amyloid precursor protein (APP) by β-secretase and γ-secretase, while α-secretase prevents Aβ generation. According to the amyloid cascade hypothesis, AD is initiated by increased brain levels of toxic Aβ species. Our laboratory has previously identified two APP mutations, causing early onset AD. Whereas the Swedish mutation results in increased β-secretase cleavage leading to higher Aβ levels, the Arctic mutation leads to a conformational Aβ change that promotes formation of toxic Aβ protofibrils.In this thesis, we have performed a screen for novel disease-causing mutations in 102 patients with early onset dementia disorders, who underwent investigation at the Memory clinic at Uppsala University Hospital. Mainly, we found a new APP mutation, which causes familial dominantly inherited AD with age at symptom onset in the early forties. This Uppsala APP mutation, consists of an intra-Aβ deletion of six consecutive amino acids, which results in Aβ with 34-36 amino acids (AβUppΔ19-24).Affected mutation-carriers develop symptoms typical of AD. As for biomarkers, the patients display expected changes although brain Aβ imaging by [11C]PIB-PET is only slightly pathological and Aβ42-analysis of cerebrospinal fluid yields normal results. By investigating neuropathological, biochemical and structural properties of AβUppΔ19-24 in patient samples, on synthetic peptides and in cell culture models we found evidence that Uppsala APP is pathogenic via three mechanisms: increased β-secretase cleavage, altered α-secretase cleavage and rapid formation of Aβ fibrils into unique polymorphs.To allow for in vivo studies of molecular mechanisms related to the Uppsala APP mutation we generated transgenic mice, expressing human APP with this mutation together with Swedish APP (to increase Aβ levels). In the brain of tg-UppSwe mice, we observed diffuse aggregates of mainly AβUpp42Δ19-24, which, given their normal γ-secretase activity, distinguishes these mice from most transgenic mouse models. In order to study if AβUppΔ19-24 co-aggregates with wild-type Aβ (Aβwt), we crossed tg-UppSwe with tg-Swe. Analyses of brains from such mice indicated that AβUppΔ19-24 may act as seeds for Aβwt by changing its aggregation behavior and thereby increasing its deposition in brain.Taken together, our studies of the Uppsala APP mutation have provided new knowledge of pathogenic molecular mechanisms in AD and of basic Aβ biology. Such insights may in a longer perspective help us to develop new diagnostics and therapeutics for this disorder.
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