Increased G3BP2-Tau interaction in tauopathies is a natural defense against Tau aggregation.

Many RNA-binding proteins (RBPs), particularly those associated with RNA granules, promote pathological protein aggregation in neurodegenerative diseases. Here, we demonstrate that G3BP2, a core component of stress granules, directly interacts with Tau and inhibits Tau aggregation. In the human brain, the interaction of G3BP2 and Tau is dramatically increased in multiple tauopathies, and it is independent of neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). Surprisingly, Tau pathology is significantly elevated upon loss of G3BP2 in human neurons and brain organoids. Moreover, we found that G3BP2 masks the microtubule-binding region (MTBR) of Tau, thereby inhibiting Tau aggregation. Our study defines a novel role for RBPs as a line of defense against Tau aggregation in tauopathies.

Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies.

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3' UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

Functional imaging of brain organoids using high-density microelectrode arrays.

Abstract: Studies have provided evidence that human cerebral organoids (hCOs) recapitulate fundamental milestones of early brain development, but many important questions regarding their functionality and electrophysiological properties persist. High-density microelectrode arrays (HD-MEAs) represent an attractive analysis platform to perform functional studies of neuronal networks at the cellular and network scale. Here, we use HD-MEAs to derive large-scale electrophysiological recordings from sliced hCOs. We record the activity of hCO slices over several weeks and probe observed neuronal dynamics pharmacologically. Moreover, we present results on how the obtained recordings can be spike-sorted and subsequently studied across scales. For example, we show how to track single neurons across several days on the HD-MEA and how to infer axonal action potential velocities. We also infer putative functional connectivity from hCO recordings. The introduced methodology will contribute to a better understanding of developing neuronal networks in brain organoids and provide new means for their functional characterization. Impact statement: Human cerebral organoids (hCOs) represent an attractive in vitro model system to study key physiological mechanisms underlying early neuronal network formation in tissue with healthy or disease-related genetic backgrounds. Despite remarkable advances in the generation of brain organoids, knowledge on the functionality of their neuronal circuits is still scarce. Here, we used complementary metal-oxide-semiconductor (CMOS)-based high-density microelectrode arrays (HD-MEAs) to perform large-scale recordings from sliced hCOs over several weeks and quantified their activity across scales. Using single-cell and network metrics, we were able to probe aspects of hCO neurophysiology that are more difficult to obtain with other techniques, such as patch clamping (lower yield) and calcium imaging (lower temporal resolution). These metrics included, for example, extracellular action potential (AP) waveform features and axonal AP velocity at the cellular level, as well as functional connectivity at the network level. Analysis was enabled by the large sensing area and the high spatiotemporal resolution provided by HD-MEAs, which allowed recordings from hundreds of neurons and spike sorting of their activity. Our results demonstrate that HD-MEAs provide a multi-purpose platform for the functional characterization of hCOs, which will be key in improving our understanding of this model system and assessing its relevance for translational research. Supplementary Information: The online version contains supplementary material available at 10.1557/s43577-022-00282-w.

A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community ( https://www.angelman-proteome-project.org/ ).

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation.

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3'UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities.

Pluripotent Stem Cells for Brain Repair: Protocols and Preclinical Applications in Cortical and Hippocampal Pathologies.

Brain injuries causing chronic sensory or motor deficit, such as stroke, are among the leading causes of disability worldwide, according to the World Health Organization; furthermore, they carry heavy social and economic burdens due to decreased quality of life and need of assistance. Given the limited effectiveness of rehabilitation, novel therapeutic strategies are required to enhance functional recovery. Since cell-based approaches have emerged as an intriguing and promising strategy to promote brain repair, many efforts have been made to study the functional integration of neurons derived from pluripotent stem cells (PSCs), or fetal neurons, after grafting into the damaged host tissue. PSCs hold great promises for their clinical applications, such as cellular replacement of damaged neural tissues with autologous neurons. They also offer the possibility to create in vitro models to assess the efficacy of drugs and therapies. Notwithstanding these potential applications, PSC-derived transplanted neurons have to match the precise sub-type, positional and functional identity of the lesioned neural tissue. Thus, the requirement of highly specific and efficient differentiation protocols of PSCs in neurons with appropriate neural identity constitutes the main challenge limiting the clinical use of stem cells in the near future. In this Review, we discuss the recent advances in the derivation of telencephalic (cortical and hippocampal) neurons from PSCs, assessing specificity and efficiency of the differentiation protocols, with particular emphasis on the genetic and molecular characterization of PSC-derived neurons. Second, we address the remaining challenges for cellular replacement therapies in cortical brain injuries, focusing on electrophysiological properties, functional integration and therapeutic effects of the transplanted neurons.

The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation.

The morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3'UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3'UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping.

Neurons Generated by Mouse ESCs with Hippocampal or Cortical Identity Display Distinct Projection Patterns When Co-transplanted in the Adult Brain.

The capability of generating neural precursor cells with distinct types of regional identity in vitro has recently opened new opportunities for cell replacement in animal models of neurodegenerative diseases. By manipulating Wnt and BMP signaling, we steered the differentiation of mouse embryonic stem cells (ESCs) toward isocortical or hippocampal molecular identity. These two types of cells showed different degrees of axonal outgrowth and targeted different regions when co-transplanted in healthy or lesioned isocortex or in hippocampus. In hippocampus, only precursor cells with hippocampal molecular identity were able to extend projections, contacting CA3. Conversely, isocortical-like cells were capable of extending long-range axonal projections only when transplanted in motor cortex, sending fibers toward both intra- and extra-cortical targets. Ischemic damage induced by photothrombosis greatly enhanced the capability of isocortical-like cells to extend far-reaching projections. Our results indicate that neural precursors generated by ESCs carry intrinsic signals specifying axonal extension in different environments.

Assessment of antibody library diversity through next generation sequencing and technical error compensation.

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging.

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.