Probing cell identity hierarchies by fate titration and collision during direct reprogramming.

Despite the therapeutic promise of direct reprogramming, basic principles concerning fate erasure and the mechanisms to resolve cell identity conflicts remain unclear. To tackle these fundamental questions, we established a single-cell protocol for the simultaneous analysis of multiple cell fate conversion events based on combinatorial and traceable reprogramming factor expression: Collide-seq. Collide-seq revealed the lack of a common mechanism through which fibroblast-specific gene expression loss is initiated. Moreover, we found that the transcriptome of converting cells abruptly changes when a critical level of each reprogramming factor is attained, with higher or lower levels not contributing to major changes. By simultaneously inducing multiple competing reprogramming factors, we also found a deterministic system, in which titration of fates against each other yields dominant or colliding fates. By investigating one collision in detail, we show that reprogramming factors can disturb cell identity programs independent of their ability to bind their target genes. Taken together, Collide-seq has shed light on several fundamental principles of fate conversion that may aid in improving current reprogramming paradigms.

Direct neuronal reprogramming: learning from and for development.

The key signalling pathways and transcriptional programmes that instruct neuronal diversity during development have largely been identified. In this Review, we discuss how this knowledge has been used to successfully reprogramme various cell types into an amazing array of distinct types of functional neurons. We further discuss the extent to which direct neuronal reprogramming recapitulates embryonic development, and examine the particular barriers to reprogramming that may exist given a cell's unique developmental history. We conclude with a recently proposed model for cell specification called the 'Cook Islands' model, and consider whether it is a fitting model for cell specification based on recent results from the direct reprogramming field.

Direct neuronal reprogramming of NDUFS4 patient cells identifies the unfolded protein response as a novel general reprogramming hurdle.

Mitochondria account for essential cellular pathways, from ATP production to nucleotide metabolism, and their deficits lead to neurological disorders and contribute to the onset of age-related diseases. Direct neuronal reprogramming aims at replacing neurons lost in such conditions, but very little is known about the impact of mitochondrial dysfunction on the direct reprogramming of human cells. Here, we explore the effects of mitochondrial dysfunction on the neuronal reprogramming of induced pluripotent stem cell (iPSC)-derived astrocytes carrying mutations in the NDUFS4 gene, important for Complex I and associated with Leigh syndrome. This led to the identification of the unfolded protein response as a major hurdle in the direct neuronal conversion of not only astrocytes and fibroblasts from patients but also control human astrocytes and fibroblasts. Its transient inhibition potently improves reprogramming by influencing the mitochondria-endoplasmic-reticulum-stress-mediated pathways. Taken together, disease modeling using patient cells unraveled novel general hurdles and ways to overcome these in human astrocyte-to-neuron reprogramming.

Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.

Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.

SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis.

Neural stem cells (NSCs) generate new hippocampal dentate granule neurons throughout adulthood. The genetic programs controlling neuronal differentiation of adult NSCs are only poorly understood. Here we show that, in the adult mouse hippocampus, expression of the SoxC transcription factors Sox4 and Sox11 is initiated around the time of neuronal commitment of adult NSCs and is maintained in immature neurons. Overexpression of Sox4 and Sox11 strongly promotes in vitro neurogenesis from adult NSCs, whereas ablation of Sox4/Sox11 prevents in vitro and in vivo neurogenesis from adult NSCs. Moreover, we demonstrate that SoxC transcription factors target the promoters of genes that are induced on neuronal differentiation of adult NSCs. Finally, we show that reprogramming of astroglia into neurons is dependent on the presence of SoxC factors. These data identify SoxC proteins as essential contributors to the genetic network controlling neuronal differentiation in adult neurogenesis and neuronal reprogramming of somatic cells.

Directing astroglia from the cerebral cortex into subtype specific functional neurons.

Astroglia from the postnatal cerebral cortex can be reprogrammed in vitro to generate neurons following forced expression of neurogenic transcription factors, thus opening new avenues towards a potential use of endogenous astroglia for brain repair. However, in previous attempts astroglia-derived neurons failed to establish functional synapses, a severe limitation towards functional neurogenesis. It remained therefore also unknown whether neurons derived from reprogrammed astroglia could be directed towards distinct neuronal subtype identities by selective expression of distinct neurogenic fate determinants. Here we show that strong and persistent expression of neurogenic fate determinants driven by silencing-resistant retroviral vectors instructs astroglia from the postnatal cortex in vitro to mature into fully functional, synapse-forming neurons. Importantly, the neurotransmitter fate choice of astroglia-derived neurons can be controlled by selective expression of distinct neurogenic transcription factors: forced expression of the dorsal telencephalic fate determinant neurogenin-2 (Neurog2) directs cortical astroglia to generate synapse-forming glutamatergic neurons; in contrast, the ventral telencephalic fate determinant Dlx2 induces a GABAergic identity, although the overall efficiency of Dlx2-mediated neuronal reprogramming is much lower compared to Neurog2, suggesting that cortical astroglia possess a higher competence to respond to the dorsal telencephalic fate determinant. Interestingly, however, reprogramming of astroglia towards the generation of GABAergic neurons was greatly facilitated when the astroglial cells were first expanded as neurosphere cells prior to transduction with Dlx2. Importantly, this approach of expansion under neurosphere conditions and subsequent reprogramming with distinct neurogenic transcription factors can also be extended to reactive astroglia isolated from the adult injured cerebral cortex, allowing for the selective generation of glutamatergic or GABAergic neurons. These data provide evidence that cortical astroglia can undergo a conversion across cell lineages by forced expression of a single neurogenic transcription factor, stably generating fully differentiated neurons. Moreover, neuronal reprogramming of astroglia is not restricted to postnatal stages but can also be achieved from terminally differentiated astroglia of the adult cerebral cortex following injury-induced reactivation.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Injury-specific factors in the cerebrospinal fluid regulate astrocyte plasticity in the human brain.

The glial environment influences neurological disease progression, yet much of our knowledge still relies on preclinical animal studies, especially regarding astrocyte heterogeneity. In murine models of traumatic brain injury, beneficial functions of proliferating reactive astrocytes on disease outcome have been unraveled, but little is known regarding if and when they are present in human brain pathology. Here we examined a broad spectrum of pathologies with and without intracerebral hemorrhage and found a striking correlation between lesions involving blood-brain barrier rupture and astrocyte proliferation that was further corroborated in an assay probing for neural stem cell potential. Most importantly, proteomic analysis unraveled a crucial signaling pathway regulating this astrocyte plasticity with GALECTIN3 as a novel marker for proliferating astrocytes and the GALECTIN3-binding protein LGALS3BP as a functional hub mediating astrocyte proliferation and neurosphere formation. Taken together, this work identifies a therapeutically relevant astrocyte response and their molecular regulators in different pathologies affecting the human cerebral cortex.

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Direct neuronal reprogramming: Fast forward from new concepts toward therapeutic approaches.

Differentiated cells have long been considered fixed in their identity. However, about 20 years ago, the first direct conversion of glial cells into neurons in vitro opened the field of "direct neuronal reprogramming." Since then, neuronal reprogramming has achieved the generation of fully functional, mature neurons with remarkable efficiency, even in diseased brain environments. Beyond their clinical implications, these discoveries provided basic insights into crucial mechanisms underlying conversion of specific cell types into neurons and maintenance of neuronal identity. Here we discuss such principles, including the importance of the starter cell for shaping the outcome of neuronal reprogramming. We further highlight technical concerns for in vivo reprogramming and propose a code of conduct to avoid artifacts and pitfalls. We end by pointing out next challenges for development of less invasive cell replacement therapies for humans.

Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons.

Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock-in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV-based strategy carrying intein-split-dCas9 in combination with activators (AAV-dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single-cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6-OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV-dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease.

ZFP423 coordinates Notch and bone morphogenetic protein signaling, selectively up-regulating Hes5 gene expression.

Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.

CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.

Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion.

A role for the transcription factor HEY1 in glioblastoma.

Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

Direct Neuronal Reprogramming: Achievements, Hurdles, and New Roads to Success.

The ability to directly reprogram mature cells to alternative fates challenges concepts of how cell identities are maintained, erased, and acquired. Recent advances in understanding and overcoming hurdles to direct neuronal conversion have provided new insights into mechanisms that maintain cell identity programs and have enabled high efficiency reprogramming in vivo. We discuss key cell-intrinsic molecular and metabolic constraints that influence the establishment of a new identity as well as environmental inputs from injured brains that favor or harm the conversion process. Finally, we outline the challenges ahead with a particular focus on direct neuronal reprogramming in vivo.

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming.

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo.

Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes.

Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

A critical period for experience-dependent remodeling of adult-born neuron connectivity.

Neurogenesis in the dentate gyrus (DG) of the adult hippocampus is a process regulated by experience. To understand whether experience also modifies the connectivity of new neurons, we systematically investigated changes in their innervation following environmental enrichment (EE). We found that EE exposure between 2-6 weeks following neuron birth, rather than merely increasing the number of new neurons, profoundly affected their pattern of monosynaptic inputs. Both local innervation by interneurons and to even greater degree long-distance innervation by cortical neurons were markedly enhanced. Furthermore, following EE, new neurons received inputs from CA3 and CA1 inhibitory neurons that were rarely observed under control conditions. While EE-induced changes in inhibitory innervation were largely transient, cortical innervation remained increased after returning animals to control conditions. Our findings demonstrate an unprecedented experience-dependent reorganization of connections impinging onto adult-born neurons, which is likely to have important impact on their contribution to hippocampal information processing.