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The glymphatic system is a fluid-transport system that accesses all regions of the brain. It facilitates the exchange of cerebrospinal fluid and interstitial fluid and clears waste from the metabolically active brain. Astrocytic endfeet and their dense expression of the aquaporin-4 water channels promote fluid exchange between the perivascular spaces and the neuropil. Cerebrospinal and interstitial fluids are together transported back to the vascular compartment by meningeal and cervical lymphatic vessels. Multiple lines of work show that neurological diseases in general impair glymphatic fluid transport. Insofar as the glymphatic system plays a pseudo-lymphatic role in the central nervous system, it is poised to play a role in neuroinflammation. In this review, we discuss how the association of the glymphatic system with the meningeal lymphatic vessel calls for a renewal of established concepts on the CNS as an immune-privileged site. We also discuss potential approaches to target the glymphatic system to combat neuroinflammation.

Parkinson’s disease (PD) is characterized by a drastic loss of dopaminergic neurons already at diagnosis. As this loss of neurons starts decades before diagnosis, understanding the prodromal stages of the disease might offer novel strategies to curb its progression. While the precise pathogenic mechanisms underlying PD remain incompletely understood, growing evidence suggests that neuroinflammation and immune dysregulation play a central role in the development and progression of the disease. Here, we delve into the emerging roles of microglia, the resident immune cells of the central nervous system, in the pathogenesis of prodromal and early-stage PD. We emphasize that microglia contribute to neuroinflammation, protein aggregation and neurodegeneration, although the underlying mechanisms are not yet known. Neuroimaging studies have provided valuable insights into the patterns of microglial activation detected in individuals with prodromal PD and at the time of clinical diagnosis. Furthermore, we highlight the complex interplay between immune dysregulation and neurodegeneration along PD development, including alterations in the peripheral immune system, brain-gut interactions and brain-immune interfaces. Lastly, we outline existing models for investigating microglial involvement in prodromal PD, along with the impact of anti-inflammatory therapies and strategies to modify risk factors. In conclusion, targeting microglial activation and immune dysfunctions in individuals at risk of PD could represent a promising preventive measure and may offer novel therapeutic strategies for early intervention and disease modification.

Background Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson’s disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7 /DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. Methods Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7 /DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives, we conducted corresponding analyses in human PARK7 /DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). Results By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7 /DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7 /DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7 /DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7 /DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 h after acute inflammation, as also observed in BMDMs. Conclusions Taken together, our results show that, under inflammatory conditions, PARK7 /DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7 /DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.

Microglia are the immune effector cells of the brain playing critical roles in immune surveillance and neuroprotection in healthy conditions, while they can sustain neuroinflammatory and neurotoxic processes in neurodegenerative diseases, including Parkinson’s disease (PD). Although the precise triggers of PD remain obscure, causative genetic mutations, which aid in the identification of molecular pathways underlying the pathogenesis of idiopathic forms, represent 10% of the patients. Among the inherited forms, loss of function of  PARK7 , which encodes the protein DJ-1, results in autosomal recessive early-onset PD. Yet, although protection against oxidative stress is the most prominent task ascribed to DJ-1, the underlying mechanisms linking DJ-1 deficiency to the onset of PD are a current matter of investigation. This review provides an overview of the role of DJ-1 in neuroinflammation, with a special focus on its functions in microglia genetic programs and immunological traits. Furthermore, it discusses the relevance of targeting dysregulated pathways in microglia under DJ-1 deficiency and their importance as therapeutic targets in PD. Lastly, it addresses the prospect to consider DJ-1, detected in its oxidized form in idiopathic PD, as a biomarker and to take into account DJ-1-enhancing compounds as therapeutics dampening oxidative stress and neuroinflammation.

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.

With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T‐cell phenotype (T‐ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T‐ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA‐sequencing analysis on patient‐derived samples followed by splice isoform‐specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T‐ALL. Moreover, patients with T‐ALL displayed pro‐survival splice isoform switching favoring pro‐survival isoforms compared to normal healthy stem cells. Cumulatively, pro‐survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T‐ALL proliferation and survival, thus serving as a potential therapeutic option.

The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.

The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002-2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Even very poor quality embryos with few cells in the ICM can give origin to hESC lines.

Key Points Methylation of RNA at N 6 -methyladenosine (m 6 A) has been identified in humans, viruses and mice and has been linked to several diseases. m 6 A plays an important role in pluripotency and differentiation and has therefore been associated with cancer development; it can promote the translation of several oncogenes. Malignant adenosine-to-inosine RNA editing controls the self-renewal of cancer stem cells (CSCs), and raises the possibility that targeting this pathway may provide a new strategy for eliminating CSCs. RNA splicing disruption promotes generation of aberrant splice isoforms in pre-malignant and malignant haematopoietic disorders and is a key therapeutic vulnerability in a growing number of human malignancies. Therapeutic splicing modulation has the potential to target bulk tumour cells as well as self-renewing CSCs that contribute to disease progression and relapse. This Review discusses the roles of deregulated RNA processing, including RNA methylation, RNA editing, RNA splicing and RNA binding protein activity, in cancer stem cells and highlights the potential of these events as biomarkers and therapeutic targets. Cancer stem cells (CSCs) can regenerate all facets of a tumour as a result of their stem cell-like capacity to self-renew, survive and become dormant in protective microenvironments. CSCs evolve during tumour progression in a manner that conforms to Charles Darwin's principle of natural selection. Although somatic DNA mutations and epigenetic alterations promote evolution, post-transcriptional RNA modifications together with RNA binding protein activity (the 'epitranscriptome') might also contribute to clonal evolution through dynamic determination of RNA function and gene expression diversity in response to environmental stimuli. Deregulation of these epitranscriptomic events contributes to CSC generation and maintenance, which governs cancer progression and drug resistance. In this Review, we discuss the role of malignant RNA processing in CSC generation and maintenance, including mechanisms of RNA methylation, RNA editing and RNA splicing, and the functional consequences of their aberrant regulation in human malignancies. Finally, we highlight the potential of these events as novel CSC biomarkers as well as therapeutic targets.