Rare A360T Mutation Alters GSK3ß(Ser9) Binding in the Cytosolic Loop of Presenilin 1, Influencing ß-Catenin Nuclear Localization and Pro-Death Gene Expression in Alzheimer's Disease Case.

Presenilin 1 (PS1) forms, via its large cytosolic loop, a trimeric complex with N-cadherin and ß-catenin, which is a key component of Wnt signaling. PS1 undergoes phosphorylation at 353 and 357 serines upon enhanced activity and elevated levels of the GSK3ß isoform. PS1 mutations surrounding these serines may alter the stability of the ß-catenin complex. Such mutations are found in some cases of familial early-onset Alzheimer's disease (fEOAD), but their functional impact remains obscure. One of such variants of PS1, the A360T substitution, is located close to GSK3ß-targeted serine residues. This variant was recently demonstrated in the French population, but more detail is needed to understand its biological effects. To assess the significance of this variant, we employed functional studies using a fibroblast cell line from an Alzheimer's disease case (a female proband) carrying the A360T mutation. Based on functional transcriptomic, cellular, and biochemical assays, we demonstrated atypically impaired ß-catenin/GSK3ß signaling in the A360T patient's fibroblasts. In detail, this was characterized by a decreased level of active cytosolic ß-catenin and bound by PS1, an increased level of nuclear ß-catenin, an increased level of inhibited GSK3ß phosphorylated on Ser9, and enhanced interaction of GSK3ß(Ser9) with PS1. Based on the transcriptomic profile of the A360T fibroblasts, we proposed a dysregulated transcriptional activity of ß-catenin, exemplified by increased expression of various cyclin-dependent kinases and cyclins, such as cyclin D1, potentially inducing neurons' cell cycle re-entry followed by apoptosis. The A360T cells did not exhibit significant amyloid pathology. Therefore, cell death in this PS1 cytosolic loop mutation may be attributed to impaired ß-catenin/GSK3ß signaling rather than amyloid deposition per se. We further estimated the biological and clinical relevance of the A360T variant by whole exome sequencing (WES). WES was performed on DNA from the blood of an A360T female proband, as well as an unrelated male patient carrying the A360T mutation and his mutation-free daughter (both unavailable for the derivation of the fibroblast cell lines). WES confirmed the highest-priority AD causality of the A360T variant in PS1 and also profiled the pathways and processes involved in the A360T case, highlighting the greatest importance of altered Wnt signaling.

mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.

BACKGROUND: Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. METHODS: The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. RESULTS: We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. CONCLUSIONS: Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.

Oxidative DNA Damage Signalling in Neural Stem Cells in Alzheimer's Disease.

The main pathological symptoms of Alzheimer's disease (AD) are ß-amyloid (Aß) lesions and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. Unfortunately, existing symptomatic therapies targeting Aß and tau remain ineffective. In addition to these pathogenic factors, oxidative DNA damage is one of the major threats to newborn neurons. It is necessary to consider in detail what causes neurons to be extremely susceptible to oxidative damage, especially in the early stages of development. Accordingly, the regulation of redox status is crucial for the functioning of neural stem cells (NSCs). The redox-dependent balance, of NSC proliferation and differentiation and thus the neurogenesis process, is controlled by a series of signalling pathways. One of the most important signalling pathways activated after oxidative stress is the DNA damage response (DDR). Unfortunately, our understanding of adult neurogenesis in AD is still limited due to the research material used (animal models or post-mortem tissue), providing inconsistent data. Now, thanks to the advances in cellular reprogramming providing patient NSCs, it is possible to fill this gap, which becomes urgent in the light of the potential of their therapeutic use. Therefore, a decent review of redox signalling in NSCs under physiological and pathological conditions is required. At this moment, we attempt to integrate knowledge on the influence of oxidative stress and DDR signalling in NSCs on adult neurogenesis in Alzheimer's disease.

Wild-type p53 oligomerizes more efficiently than p53 hot-spot mutants and overcomes mutant p53 gain-of-function via a "dominant-positive" mechanism.

Human p53 protein acts as a transcription factor predominantly in a tetrameric form. Single residue changes, caused by hot-spot mutations of the TP53 gene in human cancer, transform wild-type (wt) p53 tumor suppressor proteins into potent oncoproteins - with gain-of-function, tumor-promoting activity. Oligomerization of p53 allows for a direct interplay between wt and mutant p53 proteins if both are present in the same cells - where a mutant p53's dominant-negative effect known to inactivate wt p53, co-exists with an opposite mechanism - a "dominant-positive" suppression of the mutant p53's gain-of-function activity by wt p53. In this study we determine the oligomerization efficiency of wt and mutant p53 in living cells using FRET-based assays and describe wt p53 to be more efficient than mutant p53 in entering p53 oligomers. The biased p53 oligomerization helps to interpret earlier reports of a low efficiency of the wt p53 inactivation via the dominant-negative effect, while it also implies that the "dominant-positive" effect may be more pronounced. Indeed, we show that at similar wt:mutant p53 concentrations in cells - the mutant p53 gain-of-function stimulation of gene transcription and cell migration is more efficiently inhibited than the wt p53's tumor-suppressive transactivation and suppression of cell migration. These results suggest that the frequent mutant p53 accumulation in human tumor cells does not only directly strengthen its gain-of-function activity, but also protects the oncogenic p53 mutants from the functional dominance of wt p53.

Functional characterization of a novel progranulin mutation in a patient with progressive nonfluent aphasia.

Loss-of-function mutations in progranulin (PGRN) gene cause frontotemporal lobar degeneration. Here, we report a case of a 63-year-old woman with a 2-year history of speech impairment, diagnosed with a nonfluent variant of primary progressive aphasia, a subtype of frontotemporal lobar degeneration. In this patient, a novel heterozygous frameshift mutation, c.77delG, in exon 2 of PGRN gene, introducing premature stop codon, p.(C26SfsX28), has been identified. Cultured fibroblasts derived from the patient and her asymptomatic first-degree relative with c.77delG mutation had decreased levels of PGRN messenger RNA (mRNA) and protein. However, PGRN mRNA levels did not recover upon incubation with inhibitors of nonsense-mediated mRNA decay (cycloheximide or puromycin), suggesting involvement of other mRNA degradation pathways. In addition, we observed upregulated wingless-type mouse mammary tumor virus integration site (WNT) signaling pathway gene, WNT3A, in fibroblasts of the patient and her asymptomatic first-degree relative with c.77delG mutation. As reported previously, this is an early hallmark of PGRN deficiency.

Hypermethylation of TRIM59 and KLF14 Influences Cell Death Signaling in Familial Alzheimer's Disease.

Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.

Overactive BRCA1 Affects Presenilin 1 in Induced Pluripotent Stem Cell-Derived Neurons in Alzheimer's Disease.

The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.

What factors determine phenotype of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)? Considerations in the context of a novel pathogenic R110C mutation in the NOTCH3 gene.

We report patients from a Polish family with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) who possess a novel heterozygous R110C mutation in exon 3 of the NOTCH3 gene leading to stereotypical cysteine loss. The proband had only seizure attacks and her magnetic resonance imaging (MRI) showed very numerous hyperintense foci in the cerebral white matter in a location characteristic of CADASIL. Distinctive ultrastructural assessment of vessels from skin-muscle biopsy revealed only mild degenerative changes but relatively numerous homogeneous deposits of granular osmiophilic material (GOM). In the other symptomatic family members with the same mutation ischaemic strokes were present but not epilepsy. In the proband's affected brother at a similar age, the brain MRI was normal but vessels showed pronounced degenerative changes and irregular GOM deposits. The present report not only extends the list of known pathogenic mutations responsible for CADASIL but also emphasizes clinical and morphologic variability among family members with the same NOTCH3 mutation, suggesting that probably additional factors, not only mutations, may influence the disease phenotype..