Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-β (Aβ) peptide that deposits as plaques in the Alzheimer’s disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aβ42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1^M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aβ42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1^M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.

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.

Background: Although Alzheimer’s disease (AD) is the leading cause of dementia worldwide, there are currently no treatments available that limit the neurodegeneration or slow down the disease progression. Hence, innovative therapeutic approaches are clearly required. The role of astrocytes in AD has recently received much attention, due to their central function in brain homeostasis and synaptic function. Accumulating evidence indicates that astrocytes may lose the ability to fulfil some of their physiological tasks when shifting towards an inflammatory state. Our previous data demonstrate that astrocytes can ingest large amounts of aggregated amyloid-beta (Aβ), but then store, rather than degrade, the ingested material. This incomplete degradation results in severe cellular stress, which could be of relevance for AD progression.

Methods: In this study, we aimed to investigate how inclusions of aggregated Aβ in astrocytes affect their interplay with neurons, focusing on cellular viability and synaptic function. For this purpose, human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Αβ42 fibrils and their impact on hiPSC-derived neurons was analyzed by performing neuron-astrocyte co-cultures as well as additions of conditioned media or extracellular vesicles to pure neuronal cultures.

Results: In the co-culture setup, the presence of Aβ inclusions led to an elevated clearance of dead cells by the astrocytes, indicating increased glial reactivity. In contrast, conditioned media from control, but not from Aβ-exposed astrocytes, benefited the wellbeing of neuronal monocultures. Furthermore, electrophysiological recordings showed a significant decrease in the frequency of excitatory post synaptic current (sEPSCs) in neurons co-cultured with Aβ-astrocytes compared to control astrocytes, while conditioned media from Aβ-exposed astrocytes had the opposite effect.

Conclusions: Taken together, our results demonstrate that inclusions of aggregated Aβ affect the reactivity state of astrocytes, as well as their ability to support neuronal function.

Background: Alzheimer's disease (AD) is a slowly progressive condition and the most common cause of dementia worldwide. The pathological changes associated with the disease are estimated to occur decades before the first clinical symptoms emerge. Although neurons have been the main research focus, astrocytes have recently gained attention because of their participation in several key cellular functions. Our previous data suggest that astrocytes engulf large amounts of aggregated amyloid-beta (Aβ), but are unable to successfully degrade the material. The intracellular accumulation is, at least initially, very stressful for the astrocytes and long-term Aβ deposits may be detrimental for their functionality. However, if the astrocytes can handle the Aβ storage it could instead be beneficial in a longer perspective, by minimizing the spread of potentially pathogenic Aβ species to neighboring cells.

Methods: We aimed to evaluate the effects of long-term Aβ inclusions in astrocytes. Mature human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils and then further cultured for one week or ten weeks in Aβ-free medium. Cells from both time points were analyzed for lysosomal proteins and astrocyte reactivity markers and the medium was screened for inflammatory cytokines. In addition, the overall health of cytoplasmic organelles was investigated by immunocytochemistry and electron microscopy.

Results: Long-term astrocytes displayed frequent Aβ inclusions that were contained within LAMP1 positive organelles and sustained markers associated with reactivity. Furthermore, increased levels of CCL2/MCP-1 were detected in the media from Aβ-exposed long-term cultures. Finally, Aβ accumulation induced endoplasmic reticulum and mitochondrial swelling as well as formation of pathological lipid structures in astrocytes.

Conclusions: This study provides valuable information about how intracellular Aβ deposits affect astrocytes over time, and thereby contribute to the understanding of astrocytes role in AD progression.