Evangelos Konstantinidis: CRISPR-Cas9-based therapies and the role of astrocytes in Alzheimer’s disease and Parkinson’s disease
- Date: 30 September 2022, 09:00
- Location: Rudbecksalen, Rudbecklaboratoriet, Dag Hammarsköljds väg 20, Uppsala
- Type: Thesis defence
- Thesis author: Evangelos Konstantinidis
- External reviewer: Harald Steiner
- Supervisors: Martin Ingelsson, Anna Erlandsson, Vilmantas Giedraitis, Magnus Essand
- DiVA
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.