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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The BAF complex interacts with Pax6 in adult neural progenitors to establish a neurogenic cross-regulatory transcriptional network.

Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.

Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling.

The adult mouse subependymal zone (SEZ) harbours adult neural stem cells (aNSCs) that give rise to neuronal and oligodendroglial progeny. However it is not known whether the same aNSC can give rise to neuronal and oligodendroglial progeny or whether these distinct progenies constitute entirely separate lineages. Continuous live imaging and single-cell tracking of aNSCs and their progeny isolated from the mouse SEZ revealed that aNSCs exclusively generate oligodendroglia or neurons, but never both within a single lineage. Moreover, activation of canonical Wnt signalling selectively stimulated proliferation within the oligodendrogliogenic lineage, resulting in a massive increase in oligodendrogliogenesis without changing lineage choice or proliferation within neurogenic clones. In vivo activation or inhibition of canonical Wnt signalling respectively increased or decreased the number of Olig2 and PDGFR- α positive cells, suggesting that this pathway contributes to the fine tuning of oligodendrogliogenesis in the adult SEZ.

Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells.

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. A major challenge for the translation of neuronal reprogramming into therapy is whether the adult human brain contains cell populations amenable to direct somatic cell conversion. Here we show that cells from the adult human cerebral cortex expressing pericyte hallmarks can be reprogrammed into neuronal cells by retrovirus-mediated coexpression of the transcription factors Sox2 and Mash1. These induced neuronal cells acquire the ability of repetitive action potential firing and serve as synaptic targets for other neurons, indicating their capability of integrating into neural networks. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue-nonspecific alkaline phosphatase promoter corroborated the pericytic origin of the reprogrammed cells. Our results raise the possibility of functional conversion of endogenous cells in the adult human brain to induced neuronal fates.

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.

Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.

Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7-21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.