Driving mosaicism: somatic variants in reference population databases and effect on variant interpretation in rare genetic disease.

BACKGROUND: Genetic variation databases provide invaluable information on the presence and frequency of genetic variants in the 'untargeted' human population, aggregated with the primary goal to facilitate the interpretation of clinically important variants. The presence of somatic variants in such databases can affect variant assessment in undiagnosed rare disease (RD) patients. Previously, the impact of somatic mosaicism was only considered in relation to two Mendelian disease-associated genes. Here, we expand the analyses to identify additional mosaicism-prone genes in blood-derived reference population databases. RESULTS: To identify additional mosaicism-prone genes relevant to RDs, we focused on known/previously established ClinVar pathogenic and likely pathogenic single-nucleotide variants, residing in genes associated with early onset, severe autosomal dominant diseases. We asked whether any of these variants are present in a higher-than-expected frequency in the reference population databases and whether there is evidence of somatic origin (i.e., allelic imbalance) rather than germline heterozygosity (~ half of the reads supporting alternative allele). The mosaicism-prone genes identified were further categorized according to the processes they are involved in. Beyond the previously reported ASXL1 and DNMT3A, we identified 7 additional autosomal dominant RD-associated genes with known pathogenic single-nucleotide variants present in the reference population databases and good evidence of allelic imbalance: BRAF, CBL, FGFR3, IDH2, KRAS, PTPN11 and SETBP1. From this group of 9 genes, the majority (n = 7) was important for hematopoiesis. In addition, 4 of these genes were involved in cell proliferation. Further assessment of the known 156 hematopoietic genes led to identification of 48 genes (21 not yet associated with RDs) with at least some evidence of mosaicism detectable in reference population databases. CONCLUSIONS: These results stress the importance of considering genes involved in hematopoiesis and cell proliferation when interpreting the presence and frequency of genetic variants in blood-derived reference population databases, both public and private. This is especially important when considering new variants of uncertain significance in known hematopoietic/cell proliferation RD genes and future novel gene-disease associations involving this class of genes.

Importance of extracellular vesicle secretion at the blood-cerebrospinal fluid interface in the pathogenesis of Alzheimer's disease.

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathogenesis of Alzheimer's disease (AD). We previously reported that the blood-cerebrospinal fluid (CSF) interface, formed by the choroid plexus epithelial (CPE) cells, releases an increased amount of EVs into the CSF in response to peripheral inflammation. Here, we studied the importance of CP-mediated EV release in AD pathogenesis. We observed increased EV levels in the CSF of young transgenic APP/PS1 mice which correlated with high amyloid beta (Aß) CSF levels at this age. The intracerebroventricular (icv) injection of Aß oligomers (AßO) in wild-type mice revealed a significant increase of EVs in the CSF, signifying that the presence of CSF-AßO is sufficient to induce increased EV secretion. Using in vivo, in vitro and ex vivo approaches, we identified the CP as a major source of the CSF-EVs. Interestingly, AßO-induced, CP-derived EVs induced pro-inflammatory effects in mixed cortical cultures. Proteome analysis of these EVs revealed the presence of several pro-inflammatory proteins, including the complement protein C3. Strikingly, inhibition of EV production using GW4869 resulted in protection against acute AßO-induced cognitive decline. Further research into the underlying mechanisms of this EV secretion might open up novel therapeutic strategies to impact the pathogenesis and progression of AD.

Every-other-day feeding exacerbates inflammation and neuronal deficits in 5XFAD mouse model of Alzheimer's disease.

Food restriction has been widely associated with beneficial effects on brain aging and age-related neurodegenerative diseases such as Alzheimer's disease. However, previous studies on the effects of food restriction on aging- or pathology-related cognitive decline are controversial, emphasizing the importance of the type, onset and duration of food restriction. In the present study, we assessed the effects of preventive every-other-day (EOD) feeding regimen on neurodegenerative phenotype in 5XFAD transgenic mice, a commonly used mouse model of Alzheimer's disease. EOD feeding regimen was introduced to transgenic female mice at the age of 2 months and the effects on amyloid-ß (Aß) accumulation, gliosis, synaptic plasticity, and blood-brain barrier breakdown were analyzed in cortical tissue of 6-month-old animals. Surprisingly, significant increase of inflammation in the cortex of 5XFAD fed EOD mice was observed, reflected by the expression of microglial and astrocytic markers. This increase in reactivity and/or proliferation of glial cells was accompanied by an increase in proinflammatory cytokine TNF-α, p38 MAPK and EAAT2, and a decrease in GAD67. NMDA receptor subunit 2B, related to glutamate excitotoxicity, was increased in the cortex of 5XFAD-EOD mice indicating additional alterations in glutamatergic signaling. Furthermore, 4 months of EOD feeding regimen had led to synaptic plasticity proteins reduction and neuronal injury in 5XFAD mice. However, EOD feeding regimen did not affect Aß load and blood-brain barrier permeability in the cortex of 5XFAD mice. Our results demonstrate that EOD feeding regimen exacerbates Alzheimer's disease-like neurodegenerative and neuroinflammatory changes irrespective of Aß pathology in 5XFAD mice, suggesting that caution should be paid when using food restrictions in the prodromal phase of this neurodegenerative disease.

Short-Term Fish Oil Treatment Changes the Composition of Phospholipids While Not Affecting the Expression of Mfsd2a Omega-3 Transporter in the Brain and Liver of the 5xFAD Mouse Model of Alzheimer's Disease.

Long-term fish oil (FO) supplementation is able to improve Alzheimer's disease (AD) pathology. We aimed to determine the impact of short-term fish oil (FO) intake on phospholipids composition and plaque pathology in 5xFAD mice, a widely used animal model of AD. A 3-week-long FO supplementation administered at 3 months of age decreased the number of dense core plaques in the 5xFAD cortex and changed phospholipids in the livers and brains of wild-type (Wt) and 5xFAD mice. Livers of both genotypes responded by increase of n-3 and reciprocal decrease of n-6 fatty acids. In Wt brains, FO supplementation induced elevation of n-3 fatty acids and subsequent enhancement of n-6/n-3 ratio. However, in 5xFAD brains the improved n-6/n-3 ratio was mainly due to FO-induced decrease in arachidonic and adrenic n-6 fatty acids. Also, brain and liver abundance of n-3 fatty acids were strongly correlated in Wts, oppositely to 5xFADs where significant brain-liver correlation exists only for n-6 fatty acids. Expression of omega-3 transporter Mfs2a remained unchanged after FO supplementation. We have demonstrated that even a short-term FO intake improves the phospholipid composition and has a significant effect on plaque burden in 5xFAD brains when applied in early stages of AD pathology.

Counteracting the effects of TNF receptor-1 has therapeutic potential in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, and neuroinflammation is an important hallmark of the pathogenesis. Tumor necrosis factor (TNF) might be detrimental in AD, though the results coming from clinical trials on anti-TNF inhibitors are inconclusive. TNFR1, one of the TNF signaling receptors, contributes to the pathogenesis of AD by mediating neuronal cell death. The blood-cerebrospinal fluid (CSF) barrier consists of a monolayer of choroid plexus epithelial (CPE) cells, and AD is associated with changes in CPE cell morphology. Here, we report that TNF is the main inflammatory upstream mediator in choroid plexus tissue in AD patients. This was confirmed in two murine AD models: transgenic APP/PS1 mice and intracerebroventricular (icv) AßO injection. TNFR1 contributes to the morphological damage of CPE cells in AD, and TNFR1 abrogation reduces brain inflammation and prevents blood-CSF barrier impairment. In APP/PS1 transgenic mice, TNFR1 deficiency ameliorated amyloidosis. Ultimately, genetic and pharmacological blockage of TNFR1 rescued from the induced cognitive impairments. Our data indicate that TNFR1 is a promising therapeutic target for AD treatment.

Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles.

Here, we identified release of extracellular vesicles (EVs) by the choroid plexus epithelium (CPE) as a new mechanism of blood-brain communication. Systemic inflammation induced an increase in EVs and associated pro-inflammatory miRNAs, including miR-146a and miR-155, in the CSF Interestingly, this was associated with an increase in amount of multivesicular bodies (MVBs) and exosomes per MVB in the CPE cells. Additionally, we could mimic this using LPS-stimulated primary CPE cells and choroid plexus explants. These choroid plexus-derived EVs can enter the brain parenchyma and are taken up by astrocytes and microglia, inducing miRNA target repression and inflammatory gene up-regulation. Interestingly, this could be blocked in vivo by intracerebroventricular (icv) injection of an inhibitor of exosome production. Our data show that CPE cells sense and transmit information about the peripheral inflammatory status to the central nervous system (CNS) via the release of EVs into the CSF, which transfer this pro-inflammatory message to recipient brain cells. Additionally, we revealed that blockage of EV secretion decreases brain inflammation, which opens up new avenues to treat systemic inflammatory diseases such as sepsis.

The choroid plexus-cerebrospinal fluid interface in Alzheimer's disease: more than just a barrier.

The choroid plexus is a complex structure which hangs inside the ventricles of the brain and consists mainly of choroid plexus epithelial (CPE) cells surrounding fenestrated capillaries. These CPE cells not only form an anatomical barrier, called the blood-cerebrospinal fluid barrier (BCSFB), but also present an active interface between blood and cerebrospinal fluid (CSF). CPE cells perform indispensable functions for the development, maintenance and functioning of the brain. Indeed, the primary role of the choroid plexus in the brain is to maintain homeostasis by secreting CSF which contains different molecules, such as nutrients, neurotrophins, and growth factors, as well as by clearing toxic and undesirable molecules from CSF. The choroid plexus also acts as a selective entry gate for leukocytes into the brain. Recent findings have revealed distinct changes in CPE cells that are associated with aging and Alzheimer's disease. In this review, we review some recent findings that highlight the importance of the CPE-CSF system in Alzheimer's disease and we summarize the recent advances in the regeneration of brain tissue through use of CPE cells as a new therapeutic strategy.

Dietary restriction suppresses apoptotic cell death, promotes Bcl-2 and Bcl-xl mRNA expression and increases the Bcl-2/Bax protein ratio in the rat cortex after cortical injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability in humans. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. Using an animal model of TBI we examined the effect of dietary restriction (DR) on the neuroapoptosis and Bcl-2 family genes as the main regulators of the intrinsic apoptotic pathway. Bcl-2, Bcl-xl and Bax mRNA and protein expression in the ipsilateral cortex of adult Wistar rats exposed to DR before TBI were studied from 2 to 28 days post injury. Our results showed that DR suppressed neuroapoptosis and promoted significant upregulation of antiapoptotic Bcl-2 and Bcl-xl mRNAs in the ipsilateral cortex following injury. Expression of the proapoptotic Bax gene increased in ad libitum (AL) fed rats but remained unchanged in rats exposed to DR. Although the expression of Bcl-2, Bcl-xl and Bax proteins was changed in a similar manner in both experimental groups, DR promoted a continuous increase in the Bcl-2:Bax protein ratio throughout the recovery period. Together with our previous finding that DR mediates inhibition of the extrinsic apoptotic pathway the present work reveals that modulation of the intrinsic pathway contributes to the beneficial effect of DR in brain injury. These findings provide new insight into the effects of DR on pro-survival signaling after injury, lending further support to its neuroprotective effect.

Friends or Foes: Matrix Metalloproteinases and Their Multifaceted Roles in Neurodegenerative Diseases.

Neurodegeneration is a chronic progressive loss of neuronal cells leading to deterioration of central nervous system (CNS) functionality. It has been shown that neuroinflammation precedes neurodegeneration in various neurodegenerative diseases. Matrix metalloproteinases (MMPs), a protein family of zinc-containing endopeptidases, are essential in (neuro)inflammation and might be involved in neurodegeneration. Although MMPs are indispensable for physiological development and functioning of the organism, they are often referred to as double-edged swords due to their ability to also inflict substantial damage in various pathological conditions. MMP activity is strictly controlled, and its dysregulation leads to a variety of pathologies. Investigation of their potential use as therapeutic targets requires a better understanding of their contributions to the development of neurodegenerative diseases. Here, we review MMPs and their roles in neurodegenerative diseases: Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and multiple sclerosis (MS). We also discuss MMP inhibition as a possible therapeutic strategy to treat neurodegenerative diseases.

Amyloid ß Oligomers Disrupt Blood-CSF Barrier Integrity by Activating Matrix Metalloproteinases.

The blood-CSF barrier (BCSFB) consists of a monolayer of choroid plexus epithelial (CPE) cells that maintain CNS homeostasis by producing CSF and restricting the passage of undesirable molecules and pathogens into the brain. Alzheimer's disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid ß (Aß) plaques and neurofibrillary tangles in the brain. Recent research shows that Alzheimer's disease is associated with morphological changes in CPE cells and compromised production of CSF. Here, we studied the direct effects of Aß on the functionality of the BCSFB. Intracerebroventricular injection of Aß1-42 oligomers into the cerebral ventricles of mice, a validated Alzheimer's disease model, caused induction of a cascade of detrimental events, including increased inflammatory gene expression in CPE cells and increased levels of proinflammatory cytokines and chemokines in the CSF. It also rapidly affected CPE cell morphology and tight junction protein levels. These changes were associated with loss of BCSFB integrity, as shown by an increase in BCSFB leakage. Aß1-42 oligomers also increased matrix metalloproteinase (MMP) gene expression in the CPE and its activity in CSF. Interestingly, BCSFB disruption induced by Aß1-42 oligomers did not occur in the presence of a broad-spectrum MMP inhibitor or in MMP3-deficient mice. These data provide evidence that MMPs are essential for the BCSFB leakage induced by Aß1-42 oligomers. Our results reveal that Alzheimer's disease-associated soluble Aß1-42 oligomers induce BCSFB dysfunction and suggest MMPs as a possible therapeutic target. SIGNIFICANCE STATEMENT: No treatments are yet available to cure Alzheimer's disease; however, soluble Aß oligomers are believed to play a crucial role in the neuroinflammation that is observed in this disease. Here, we studied the effect of Aß oligomers on the often neglected barrier between blood and brain, called the blood-CSF barrier (BCSFB). This BCSFB is formed by the choroid plexus epithelial cells and is important in maintaining brain homeostasis. We observed Aß oligomer-induced changes in morphology and loss of BCSFB integrity that might play a role in Alzheimer's disease progression. Strikingly, both inhibition of matrix metalloproteinase (MMP) activity and MMP3 deficiency could protect against the detrimental effects of Aß oligomer. Clearly, our results suggest that MMP inhibition might have therapeutic potential.

Long-term dietary restriction differentially affects the expression of BDNF and its receptors in the cortex and hippocampus of middle-aged and aged male rats.

Dietary restriction (DR) exerts significant beneficial effects in terms of aging and age-related diseases in many organisms including humans. The present study aimed to examine the influence of long-term DR on the BDNF system at the transcriptional and translational levels in the cortex and hippocampus of middle-aged (12-month-old) and aged (24-month-old) male Wistar rats. The obtained results revealed that the DR upregulated the expression of exon-specific BDNF transcripts in both regions, followed by elevated levels of mBDNF only in the cortex in middle-aged animals. In aged animals, DR modulated BDNF protein levels by increasing proBDNF and by declining mBDNF levels. Additionally, elevated levels of the full-length TrkB accompanied by a decreased level of the less-glycosylated TrkB protein were observed in middle-aged rats following DR, while in aged rats, DR amplified only the expression of the less-glycosylated form of TrkB. The levels of phosphorylated TrkB(Y816) were stable during aging regardless of feeding. Reduced levels of p75(NTR) were detected in both regions of middle-aged DR-fed animals, while a significant increase was measured in the cortex of aged DR-fed rats. These findings shed additional light on DR as a modulator of BDNF system revealing its disparate effects in middle-aged and aged animals. Given the importance of the proBDNF/BDNF circuit-level expression in different brain functions and various aspects of behavior, it is necessary to further elucidate the optimal duration of the applied dietary regimen with regard to the animal age in order to achieve its most favorable effects.

Morphofunctional characteristics of ACTH cells in middle-aged male rats after treatment with genistein.

The soybean phytoestrogen, genistein, is increasingly consumed as an alternative therapeutic for age-related diseases. The aim of this study was to examine the morphofunctional characteristics of adrenocorticotrophic (ACTH) cells and blood concentrations of ACTH in sham-operated, orchidectomized and genistein-treated orchidectomized, 16-month-old Wistar male rats. Genistein (10 mg/kg/day) was administered subcutaneously for three weeks, while the control groups received the vehicle alone. Orchidectomy and genistein treatment decreased the volume density of ACTH cells and reduced (p < 0.05) circulating ACTH concentrations in comparison with control groups. In conclusion, genistein modulated the morphofunctional features of ACTH cells and decreased blood ACTH levels.