Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes.

Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-ß and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher (~two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

Contrasting patterns of somatic mutations in neurons and glia reveal differential predisposition to disease in the aging human brain.

Characterizing the mechanisms of somatic mutations in the brain is important for understanding aging and disease, but little is known about the mutational patterns of different cell types. We performed whole-genome sequencing of 71 oligodendrocytes and 51 neurons from neurotypical individuals (0.4 to 104 years old) and identified >67,000 somatic single nucleotide variants (sSNVs) and small insertions and deletions (indels). While both cell types accumulate mutations with age, oligodendrocytes accumulate sSNVs 69% faster than neurons (27/year versus 16/year) whereas indels accumulate 42% slower (1.8/year versus 3.1/year). Correlation with single-cell RNA and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These patterns highlight differences in the mutagenic processes in glia and neurons and suggest cell type-specific, age-related contributions to neurodegeneration and oncogenesis.

Rates and Patterns of Clonal Oncogenic Mutations in the Normal Human Brain.

Although oncogenic mutations have been found in nondiseased, proliferative nonneural tissues, their prevalence in the human brain is unknown. Targeted sequencing of genes implicated in brain tumors in 418 samples derived from 110 individuals of varying ages, without tumor diagnoses, detected oncogenic somatic single-nucleotide variants (sSNV) in 5.4% of the brains, including IDH1 R132H. These mutations were largely present in subcortical white matter and enriched in glial cells and, surprisingly, were less common in older individuals. A depletion of high-allele frequency sSNVs representing macroscopic clones with age was replicated by analysis of bulk RNA sequencing data from 1,816 nondiseased brain samples ranging from fetal to old age. We also describe large clonal copy number variants and that sSNVs show mutational signatures resembling those found in gliomas, suggesting that mutational processes of the normal brain drive early glial oncogenesis. This study helps understand the origin and early evolution of brain tumors. SIGNIFICANCE: In the nondiseased brain, clonal oncogenic mutations are enriched in white matter and are less common in older individuals. We revealed early steps in acquiring oncogenic variants, which are essential to understanding brain tumor origins and building new mutational baselines for diagnostics.This article is highlighted in the In This Issue feature, p. 1.

Comprehensive identification of somatic nucleotide variants in human brain tissue.

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations.

BACKGROUND: Mosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation. METHODS: To overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples. RESULTS: The Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions. CONCLUSIONS: MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.

Mesenchymal Stem Cell Transplantation Promotes Neurogenesis and Ameliorates Autism Related Behaviors in BTBR Mice.

Autism spectrum disorders (ASD) are characterized by social communication deficits, cognitive rigidity, and repetitive stereotyped behaviors. Mesenchymal stem cells (MSC) have a paracrine regenerative effect, and were speculated to be a potential therapy for ASD. The BTBR inbred mouse strain is a commonly used model of ASD as it demonstrates robust behavioral deficits consistent with the diagnostic criteria for ASD. BTBR mice also exhibit decreased brain-derived neurotrophic factor (BDNF) signaling and reduced hippocampal neurogenesis. In the current study, we evaluated the behavioral and molecular effects of intracerebroventricular MSC transplantation in BTBR mice. Transplantation of MSC resulted in a reduction of stereotypical behaviors, a decrease in cognitive rigidity and an improvement in social behavior. Tissue analysis revealed elevated BDNF protein levels in the hippocampus accompanied by increased hippocampal neurogenesis in the MSC-transplanted mice compared with sham treated mice. This might indicate a possible mechanism underpinning the behavioral improvement. Our study suggests a novel therapeutic approach which may be translatable to ASD patients in the future.

Dopaminergic-like neurons derived from oral mucosa stem cells by developmental cues improve symptoms in the hemi-parkinsonian rat model.

Achieving safe and readily accessible sources for cell replacement therapy in Parkinson's disease (PD) is still a challenging unresolved issue. Recently, a primitive neural crest stem cell population (hOMSC) was isolated from the adult human oral mucosa and characterized in vitro and in vivo. In this study we assessed hOMSC ability to differentiate into dopamine-secreting cells with a neuronal-dopaminergic phenotype in vitro in response to dopaminergic developmental cues and tested their therapeutic potential in the hemi-Parkinsonian rat model. We found that hOMSC express constitutively a repertoire of neuronal and dopaminergic markers and pivotal transcription factors. Soluble developmental factors induced a reproducible neuronal-like morphology in the majority of hOMSC, downregulated stem cells markers, upregulated the expression of the neuronal and dopaminergic markers that resulted in dopamine release capabilities. Transplantation of these dopaminergic-induced hOMSC into the striatum of hemi-Parkinsonian rats improved their behavioral deficits as determined by amphetamine-induced rotational behavior, motor asymmetry and motor coordination tests. Human TH expressing cells and increased levels of dopamine in the transplanted hemispheres were observed 10 weeks after transplantation. These results demonstrate for the first time that soluble factors involved in the development of DA neurons, induced a DA phenotype in hOMSC in vitro that significantly improved the motor function of hemiparkinsonian rats. Based on their neural-related origin, their niche accessibility by minimal-invasive procedures and their propensity for DA differentiation, hOMSC emerge as an attractive tool for autologous cell replacement therapy in PD.

Astrocyte-like cells derived from human oral mucosa stem cells provide neuroprotection in vitro and in vivo.

Human oral mucosa stem cells (hOMSC) are a recently described neural crest-derived stem cell population. Therapeutic quantities of potent hOMSC can be generated from small biopsies obtained by minimally invasive procedures. Our objective was to evaluate the potential of hOMSC to differentiate into astrocyte-like cells and provide peripheral neuroprotection. We induced hOMSC differentiation into cells showing an astrocyte-like morphology that expressed characteristic astrocyte markers as glial fibrillary acidic protein, S100ß, and the excitatory amino acid transporter 1 and secreted neurotrophic factors (NTF) such as brain-derived neurotrophic factor, vascular endothelial growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1. Conditioned medium of the induced cells rescued motor neurons from hypoxia or oxidative stress in vitro, suggesting a neuroprotective effect mediated by soluble factors. Given the neuronal support (NS) ability of the cells, the differentiated cells were termed hOMSC-NS. Rats subjected to sciatic nerve injury and transplanted with hOMSC-NS showed improved motor function after transplantation. At the graft site we found the transplanted cells, increased levels of NTF, and a significant preservation of functional neuromuscular junctions, as evidenced by colocalization of α-bungarotoxin and synaptophysin. Our findings show for the first time that hOMSC-NS generated from oral mucosa exhibit neuroprotective effects in vitro and in vivo and point to their future therapeutic use in neural disorders.

Mesenchymal stem cells protect from sub-chronic phencyclidine insult in vivo and counteract changes in astrocyte gene expression in vitro.

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine strategies in brain diseases. Experimental studies have shown that repeated administration of phencyclidine (PCP) leads to schizophrenia-like behavioral changes in mice. The aim of the present study was to explore the effectiveness of MSC transplantation into the hippocampus in attenuating PCP-induced social behavior deficits. PCP was administered subcutaneously to C57bl mice (10mg/kg daily) for 2 weeks. On the first day of PCP administration, adult human MSCs were transplanted into the hippocampus. A week after the last PCP dose, the mice underwent social preference testing. MSC transplantation was associated with a significant reduction in the adverse social behavior induced by PCP. Immunohistochemical analysis revealed that the stem cells survived in the mouse brain, and hippocampal Western blot analysis revealed a statistical trend towards a decrease in cleaved caspase 3 protein levels in the stem cell treated group. Upon in vitro co-culture of astrocytes and MSCs, the MSCs, in the presence of PCP, positively regulated astrocyte expression of genes involved in glutamate metabolism and antioxidant defenses. These findings suggest that MSC transplantation into the hippocampus may serve as a novel neuroprotective tool for the treatment of the PCP-induced schizophrenia-like social endophenotype. The mechanism underlying the beneficial behavioral effect may involve modulation of host astrocyte functioning, including glutamate processing and antioxidant capacity.

Cell replacement therapy for Parkinson's disease: how close are we to the clinic?

Cell replacement therapy (CRT) offers great promise as the future of regenerative medicine in Parkinson´s disease (PD). Three decades of experiments have accumulated a wealth of knowledge regarding the replacement of dying neurons by new and healthy dopaminergic neurons transplanted into the brains of animal models and affected patients. The first clinical trials provided the proof of principle for CRT in PD. In these experiments, intrastriatal transplantation of human embryonic mesencephalic tissue reinnervated the striatum, restored dopamine levels and showed motor improvements. Sequential controlled studies highlighted several problems that should be addressed prior to the wide application of CRT for PD patients. Moreover, owing to ethical and practical problems, embryonic stem cells require replacement by better-suited stem cells. Several obstacles remain to be surpassed, including identifying the best source of stem cells for A9 dopaminergic neuron generation, eliminating the risk of tumor formation and the development of graft-induced dyskinesias, and standardizing dopaminergic cell production in order to enable clinical application. In this article, we present an update on CRT for PD, reviewing the research milestones, various stem cells used and tailored differentiation methods, and analyze the information gained from the clinical trials.

Nanoparticulate architecture of protein-based artificial viruses is supported by protein-DNA interactions.

UNLABELLED: AIM & METHODS: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine-glycine-aspartic acid cell-binding domains and promote significant transgene expression levels. RESULTS & CONCLUSION: The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of α-helix and the absence of cross-molecular, ß-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA-protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy.