Neurodegenerative diseases are defined by the propagation of neuroinflammation, driven in part by disease-associated microglia (DAM) that amplify inflammatory signaling and hasten neurodegeneration. Strategies to selectively eliminate DAM to attenuate disease progression remain elusive. Using existing datasets in combination with multiplexed immunofluorescence analysis of post-mortem ALS tissues, we identified the urokinase-type plasminogen activator receptor (uPAR) as a novel surface marker of DAM. uPAR protein is markedly elevated in IBA1⁺/CD68⁺ microglia within ALS-affected regions of both sporadic and familial cases, with negligible expression in unaffected areas or control tissues. These uPAR-high microglia drive neurite retraction in iPSC-derived neurons. To target these cells, we engineered 3rd-generation CAR-T cells expressing an anti-uPAR single-chain variable fragment, enabling specific recognition and elimination of uPAR-expressing microglia. Target specificity was assessed in human microglia C20 cells driven into a pathogenic state by poly(IC) or IFNγ stimulation, which resulted in robust surface uPAR expression alongside phagocytic (CD68⁺) and antigen-presenting (CD80⁺) markers. uPAR-CAR-T cells induced antigen-dependent cytolysis of uPAR-high microglia, reducing their viability by over 80% while sparing resting microglia and neurons in a mixed culture system. These results position uPAR-directed CAR-T cells as a viable immunotherapeutic approach to selectively disrupt disease-amplifying microglial subsets and modify the trajectories of neuroinflammatory diseases. ### Competing Interest Statement The authors have declared no competing interest. United States Department of Defense, https://ror.org/0447fe631, HT9425-23-1-0258

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. . While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/- +/- induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A-- haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 over- expression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination.

Adult neurogenic decline, inflammation, and neurodegeneration are phenotypic hallmarks of Alzheimer's disease (AD). Mobilization of transposable elements (TEs) in heterochromatic regions was recently reported in AD, but the underlying mechanisms are still underappreciated. Combining functional genomics with the differentiation of familial and sporadic AD patient derived-iPSCs into hippocampal progenitors, CA3 neurons, and cerebral organoids, we found that the upregulation of the AP-1 subunit, c-Jun, triggers decondensation of genomic regions containing TEs. This leads to the cytoplasmic accumulation of HERVK-derived RNA-DNA hybrids, the activation of the cGAS-STING cascade, and increased levels of cleaved caspase-3, suggesting the initiation of programmed cell death in AD progenitors and neurons. Notably, inhibiting c-Jun effectively blocks all these downstream molecular processes and rescues neuronal death and the impaired neurogenesis phenotype in AD progenitors. Our findings open new avenues for identifying therapeutic strategies and biomarkers to counteract disease progression and diagnose AD in the early, pre-symptomatic stages.

Subunit switches in the BAF chromatin remodeler are essential during development. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause neurodevelopmental disorders, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we leveraged ARID1B +/− Coffin-Siris patient-derived iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF). ARID1B-BAF regulates exit from pluripotency and lineage commitment by attenuating thousands of enhancers and genes of the NANOG and SOX2 networks. In iPSCs, these enhancers are maintained active by ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A- to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch, and maintain ARID1A-BAF at the pluripotency enhancers throughout all stages of CNCC formation. This leads to persistent NANOG/SOX2 activity which impairs CNCC formation. Despite showing the typical neural crest signature (TFAP2A/SOX9-positive), ARID1B -haploinsufficient CNCCs are also aberrantly NANOG-positive. These findings suggest a connection between ARID1B mutations, neuroectoderm specification and a pathogenic mechanism for Coffin-Siris syndrome.

Background: Adenosine to inosine (A-to-I) RNA editing is the most frequent editing event in humans. It converts adenosine to inosine in double-stranded RNA regions (in coding and non-coding RNAs) through the action of the adenosine deaminase acting on RNA (ADAR) enzymes. Long non-coding RNAs, particularly abundant in the brain, account for a large fraction of the human transcriptome, and their important regulatory role is becoming progressively evident in both normal and transformed cells. Results: Herein, we present a bioinformatic analysis to generate a comprehensive inosinome picture in long non-coding RNAs (lncRNAs), using an ad hoc index and searching for de novo editing events in the normal brain cortex as well as in glioblastoma, a highly aggressive human brain cancer. We discovered >10,000 new sites and 335 novel lncRNAs that undergo editing, never reported before. We found a generalized downregulation of editing at multiple lncRNA sites in glioblastoma samples when compared to the normal brain cortex. Conclusion: Overall, our study discloses a novel layer of complexity that controls lncRNAs in the brain and brain cancer.

MicroRNAs have emerged as regulators of brain development and function. Reduction of miR-101 expression has been reported in rodent hippocampus during ageing, in the brain of Alzheimer's disease (AD) patients and in AD animal models. In this study, we investigated the behavioral and molecular consequences of inhibition of endogenous miR-101 in 4–5-month-old C57BL/6J mice, infused with lentiviral particles expressing a miR-101 sponge (pLSyn-miR-101 sponge) in the CA1 field of the hippocampus. The sponge-infected mouse model showed cognitive impairment. The pLSyn-miR-101 sponge-infected mice were unable to discriminate either a novel object location or a novel object as assessed by object place recognition (OPR) and novel object recognition (NOR) tasks, respectively. Moreover, the sponge-infected mice evaluated for contextual memory in inhibitory avoidance task showed shorter retention latency compared to control pLSyn mice. These cognitive impairment features were associated with increased hippocampal expression of relevant miR-101 target genes, amyloid precursor protein (APP), RanBP9 and Rab5 and overproduction of amyloid beta (Aβ) 42 levels, the more toxic species of Aβ peptide. Notably, phosphorylation-dependent AMP-activated protein kinase (AMPK) hyperactivation is associated with AD pathology and age-dependent memory decline, and we found AMPK hyperphosphorylation in the hippocampus of pLSyn-miR-101 sponge mice. This study demonstrates that mimicking age-associated loss of miR-101 in hippocampal neurons induces cognitive decline and modulation of AD-related genes in mice.

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-β1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-β1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-β1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.

Glioblastoma (GBM) is the most aggressive, infiltrative, and lethal brain tumor in humans. Despite the extensive advancement in the knowledge about tumor progression and treatment over the last few years, the prognosis of GBM is still very poor due to the difficulty of targeting drugs or anticancer molecules to GBM cells. The major challenge in improving GBM treatment implicates the development of a targeted drug delivery system, capable of crossing the blood–brain barrier (BBB) and specifically targeting GBM cells. Aptamers possess many characteristics that make them ideal novel therapeutic agents for the treatment of GBM. They are short single-stranded nucleic acids (RNA or ssDNA) able to bind to a molecular target with high affinity and specificity. Several GBM-targeting aptamers have been developed for imaging, tumor cell isolation from biopsies, and drug/anticancer molecule delivery to the tumor cells. Due to their properties (low immunogenicity, long stability, and toxicity), a large number of aptamers have been selected against GBM biomarkers and tested in GBM cell lines, while only a few of them have also been tested in in vivo models of GBM. Herein, we specifically focus on aptamers tested in GBM in vivo models that can be considered as new diagnostic and/or therapeutic tools for GBM patients’ treatment.

Alterations of adult neurogenesis have been reported in several Alzheimer's disease (AD) animal models and human brains, while defects in this process at presymptomatic/early stages of AD have not been explored yet. To address this, we investigated potential neurogenesis defects in Tg2576 transgenic mice at 1.5 months of age, a prodromal asymptomatic age in terms of Aβ accumulation and neurodegeneration. We observe that Tg2576 resident and SVZ-derived adult neural stem cells (aNSCs) proliferate significantly less. Further, they fail to terminally differentiate into mature neurons due to pathological, tau-mediated, and microtubule hyperstabilization. Olfactory bulb neurogenesis is also strongly reduced, confirming the neurogenic defect in vivo. We find that this phenotype depends on the formation and accumulation of intracellular A-beta oligomers (AβOs) in aNSCs. Indeed, impaired neurogenesis of Tg2576 progenitors is remarkably rescued both in vitro and in vivo by the expression of a conformation-specific anti-AβOs intrabody (scFvA13-KDEL), which selectively interferes with the intracellular generation of AβOs in the endoplasmic reticulum (ER). Altogether, our results demonstrate that SVZ neurogenesis is impaired already at a presymptomatic stage of AD and is caused by endogenously generated intracellular AβOs in the ER of aNSCs. From a translational point of view, impaired SVZ neurogenesis may represent a novel biomarker for AD early diagnosis, in association to other biomarkers. Further, this study validates intracellular Aβ oligomers as a promising therapeutic target and prospects anti-AβOs scFvA13-KDEL intrabody as an effective tool for AD treatment.

In the subventricular zone (SVZ) of the adult brain, the neural stem cells (NSCs) ensure a continuous supply of new neurons to the olfactory bulb (OB), playing a key role in its plasticity and olfactory-related behavior. The activation and expansion of NSCs within the SVZ are finely regulated by environmental and intrinsic factors. Running represents one of the most powerful neurogenic stimuli, although is ineffective in enhancing SVZ neurogenesis. The cell cycle inhibitor p21 is an intrinsic inhibitor of NSCs’ expansion through the maintenance of their quiescence and the restrain of neural progenitor proliferation. In this work, we decided to test whether running unveils the intrinsic neurogenic potential of p21-lacking NSCs. To test this hypothesis, we examined the effect of three different paradigms of voluntary running (5, 12, and 21 days) on SVZ neurogenesis of p21 knockout (KO) male mice at two different stages of development, 2 and 12 months of age. In vivo and in vitro data clearly demonstrate that physical activity is consistent with the activation and expansion of NSCs and with the enhancement of SVZ neurogenesis in p21 KO mice. We also found that 12 days of running contribute to the increase in the number of new neurons functionally active within the OB, which associates with an improvement in olfactory performance strictly dependent on adult SVZ neurogenesis, i.e., the odor detection threshold and short-term olfactory memory. These data suggest that in the adult SVZ of p21 KO mice, NSCs retain a high neurogenic potential, triggered by physical activity, with long-term consequences in olfactory-related behavior.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. Statement of Significance In our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Adult neurogenesis occurs throughout life in the dentate gyrus (DG) and the subventricular zone (SVZ), where glia-like stem cells generate new neurons. Voluntary running is a powerful neurogenic stimulus triggering the proliferation of progenitor cells in the DG but, apparently, not in the SVZ. The antiproliferative gene Btg1 maintains the quiescence of DG and SVZ stem cells. Its ablation causes intense proliferation of DG and SVZ stem/progenitor cells in young mice, followed, during adulthood, by progressive decrease of the proliferative capacity. We have previously observed that running can rescue the deficit of DG Btg1-null neurogenesis. Here, we show that in adult Btg1-null SVZ stem and neuroblast cells, the reduction of proliferation is associated with a longer cell cycle and a more frequent entry into quiescence. Notably, running increases proliferation in Btg1-null SVZ stem cells highly above the levels of sedentary wild-type mice and restores normal values of cell cycle length and quiescence in stem and neuroblast cells, without affecting wild-type cells. Btg1-null SVZ neuroblasts show also increased migration throughout the rostral migratory stream and a deficiency of differentiated neurons in the olfactory bulb, possibly a consequence of premature exit from the cycle; running, however, normalizes migration and differentiation, increasing newborn neurons recruited to the olfactory circuitry. Furthermore, running increases the self-renewal of Btg1-null SVZ-derived neurospheres and, remarkably, in aged Btg1-null mice almost doubles the proliferating SVZ stem cells. Altogether, this reveals that SVZ stem cells are endowed with a hidden supply of self-renewal capacity, coupled to cell cycle acceleration and emerging after ablation of the quiescence-maintaining Btg1 gene and following exercise.

Amyloidβ oligomers (AβOs) are crucial neurotoxic species in Alzheimer’s Disease (AD) but they are still difficult targets for therapy. We demostrated that recombinant antibody fragments can be exploited as intracellular antibodies to block or modulate the toxicity of endogenous AβOs in living cells. In this way, we established a new experimental paradigm of subcellular-localized conformational-selective interference (CSI) (Meli et al., Nature Comm 2014). The intrabody-based CSI approach, previously established in mammalian fAD cell lines, was here developed through the use of new lentiviral systems, inducible or not, exploited in different murine and human primary cell systems. In detail, we investigated: i. primary neuronal stem cells (NSC) derived from neurogenic niches of the adult brain of AD mouse model Tg2576; ii. primary human fibroblasts from different AD patients (some of them carrying the V717I fAD mutation); iii. human iPS cells derived from primary fibroblasts. NSC were mantained in vitro as neurosphere cultures and differentiated in neurons or astrocytes. We demostrated that subcellular AβOs are responsible for alterations on growth and differentiation of NSC, and for dysfunctions of mitochondria and of homeostatic mechanisms in primary cells. These alterations and dysfunctions are functionally rescued by our CSI approach, in which the anti-AβOs intrabodies were targeted to the Endoplasmic Reticulum and the early secretory pathway. The subcellular localization of the intrabody-based CSI for AβOs in the Endoplasmic Reticulum and the early secretory pathway, strongly suggests the role of these subcellular compartments in determining the toxic activity of endogenously produced AβOs. In mammalian fAD cell lines we found an AD-relevant functional link between the ER and mitochondria, which seems to be confirmed in human primary fibroblasts. Thus, the intrabody-based CSI for AβOs is an useful approach to study AβO actions inside living cells and it gives a new perspective for in vivo immunotherapy. Supported by Alzheimer's Association NIRG-12-237751, Human Brain Project Neuroantibodies #604102.