Brain-synthesized oestrogens regulate cortical migration in a sexually divergent manner.

Oestrogens play an important role in brain development where they have been implicated in controlling various cellular processes. Several lines of evidence have been presented showing that oestrogens can be synthesized locally within the brain. Studies have demonstrated that aromatase, the enzyme responsible for the conversion of androgens to oestrogens, is expressed during early development in both male and female cortices. Furthermore, 17ß-oestradiol has been measured in foetal brain tissue from multiple species. 17ß-oestradiol regulates neural progenitor proliferation as well as the development of early neuronal morphology. However, what role locally derived oestrogens play in regulating cortical migration and, moreover, whether these effects are the same in males and females are unknown. Here, we investigated the impact of knockdown expression of Cyp19a1, which encodes aromatase, between embryonic day (E) 14.5 and postnatal day 0 (P0) had on neural migration within the cortex. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 resulted in an increase in cells within the cortical plate, and a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. Interestingly, the opposite effect was observed in females, who displayed a significant reduction in cells migrating to the cortical plate. Together, these findings indicate that brain-derived oestrogens regulate radial migration through distinct mechanisms in males and females.

Glycosylated clusterin species facilitate Aß toxicity in human neurons.

Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer's disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU-AD mutations and amyloid-ß (Aß), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aß toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 -/- edited iPSCs and were incubated with aggregated Aß peptides. Aß induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aß. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 -/- and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aß25-35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2 -/- neurons. The latter also displayed partial protection against Aß-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 -/- neurons, potentially contributing to their reduced sensitivity to Aß toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aß toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aß toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 -/- and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 -/- neurons. This may contribute to the reduced sensitivity of these neurons to Aß, providing new mechanistic insights into Aß pathologies and therapeutic targets for AD.

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

Clusterin in Alzheimer's Disease: Mechanisms, Genetics, and Lessons From Other Pathologies.

Clusterin (CLU) or APOJ is a multifunctional glycoprotein that has been implicated in several physiological and pathological states, including Alzheimer's disease (AD). With a prominent extracellular chaperone function, additional roles have been discussed for clusterin, including lipid transport and immune modulation, and it is involved in pathways common to several diseases such as cell death and survival, oxidative stress, and proteotoxic stress. Although clusterin is normally a secreted protein, it has also been found intracellularly under certain stress conditions. Multiple hypotheses have been proposed regarding the origin of intracellular clusterin, including specific biogenic processes leading to alternative transcripts and protein isoforms, but these lines of research are incomplete and contradictory. Current consensus is that intracellular clusterin is most likely to have exited the secretory pathway at some point or to have re-entered the cell after secretion. Clusterin's relationship with amyloid beta (Aß) has been of great interest to the AD field, including clusterin's apparent role in altering Aß aggregation and/or clearance. Additionally, clusterin has been more recently identified as a mediator of Aß toxicity, as evidenced by the neuroprotective effect of CLU knockdown and knockout in rodent and human iPSC-derived neurons. CLU is also the third most significant genetic risk factor for late onset AD and several variants have been identified in CLU. Although the exact contribution of these variants to altered AD risk is unclear, some have been linked to altered CLU expression at both mRNA and protein levels, altered cognitive and memory function, and altered brain structure. The apparent complexity of clusterin's biogenesis, the lack of clarity over the origin of the intracellular clusterin species, and the number of pathophysiological functions attributed to clusterin have all contributed to the challenge of understanding the role of clusterin in AD pathophysiology. Here, we highlight clusterin's relevance to AD by discussing the evidence linking clusterin to AD, as well as drawing parallels on how the role of clusterin in other diseases and pathways may help us understand its biological function(s) in association with AD.

Clusterin Is Required for ß-Amyloid Toxicity in Human iPSC-Derived Neurons.

Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating ß-amyloid (Aß) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aß processing. Here we investigate how CLU mediates Aß-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate Aß-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aß using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with Aß25-35 peptides and Aß1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aß treatment. However, in CLU knockout neurons Aß exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to Aß in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aß and Dickkopf-1 (DKK1) proteins in an Aß-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in Aß treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aß compared to the wild type neurons supports the role of clusterin in Aß-mediated AD pathogenesis.