Direct Neuronal Reprogramming Research Articles

ONECUT transcription factors induce neuronal characteristics and remodel chromatin accessibility.

Remodeling of chromatin accessibility is necessary for successful reprogramming of fibroblasts to neurons. However, it is still not fully known which transcription factors can induce a neuronal chromatin accessibility profile when overexpressed in fibroblasts. To identify such transcription factors, we used ATAC-sequencing to generate differential chromatin accessibility profiles between human fibroblasts and iNeurons, an in vitro neuronal model system obtained by overexpression of Neurog2 in induced pluripotent stem cells (iPSCs). We found that the ONECUT transcription factor sequence motif was strongly associated with differential chromatin accessibility between iNeurons and fibroblasts. All three ONECUT transcription factors associated with this motif (ONECUT1, ONECUT2 and ONECUT3) induced a neuron-like morphology and expression of neuronal genes within two days of overexpression in fibroblasts. We observed widespread remodeling of chromatin accessibility; in particular, we found that chromatin regions that contain the ONECUT motif were in- or lowly accessible in fibroblasts and became accessible after the overexpression of ONECUT1, ONECUT2 or ONECUT3. There was substantial overlap with iNeurons, still, many regions that gained accessibility following ONECUT overexpression were not accessible in iNeurons. Our study highlights both the potential and challenges of ONECUT-based direct neuronal reprogramming.

Modeling Adult‐Onset Neurodegenerative Disorders with Human Neurons through Direct Neuronal Reprogramming

Studying late‐onset neurodegenerative disorders using human neurons remains a challenging task due to the inaccessibility of live neurons from the affected patients. Neurons from induced pluripotent stem cells (iPSCs) have limited potential to recapitulate cellular processes that occur in aged neurons as the induction of pluripotency resets the cellular age to an embryonic/fetal stage. The ability to generate human neurons that reflect the mature adult state will thus provide a powerful platform to investigate adult‐onset disorders. Brain‐enriched microRNAs (miRNAs), miR‐9/9* and miR‐124 (miR‐9/9*‐124) can function as potent cell fate regulators that can direct cell fate conversion (reprogramming) of non‐neural human somatic cells to neurons. When ectopically expressed in human adult fibroblasts, miR‐9/9*‐124 target multiple components of chromatin modifiers inducing an extensive reconfiguration of the chromatin landscape while erasing the fibroblast fate and promoting the neuronal identity. The miR‐9/9*‐124‐induced state becomes poised to receive inputs from additional subtype‐defining transcription factors (TFs), allowing the generation of specific neuronal subtypes including cortical, striatal, and spinal cord motor neurons. Importantly, for modeling adult‐onset disorders, we found that directly converted neurons preserve the age‐associated epigenetic memory reflecting the age signature of fibroblast donors. We have applied the neuronal conversion method specific for striatal medium spiny neurons (MSNs), the primary cell target that undergoes neurodegeneration in Huntington's disease (HD) in fibroblasts of HD patients (HD‐MSNs). We found that HD‐MSNs manifested hallmarks of adult‐onset HD including the aggregation of Huntingtin protein, increased DNA damage, mitochondrial dysfunction and spontaneous neuronal death in culture. Collectively, our studies demonstrate the potent activity of microRNAs as cell fate regulators and its utility to generate a patient‐derived cellular platform to model adult‐onset neurodegenerative diseases.Support or Funding InformationAndrew B and Virginia C. Craig Faculty Fellowship Endowment, Farrell Fund, Cure Alzheimer's Fund (CAF), Mallinckrodt Scholar Award, NIA (RF1AG056296), and NINDH (1R01NS107488)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct Neuronal Reprogramming Reveals Unknown Functions for Known Transcription Factors.

In recent years, the need to derive sources of specialized cell types to be employed for cell replacement therapies and modeling studies has triggered a fast acceleration of novel cell reprogramming methods. In particular, in neuroscience, a number of protocols for the efficient differentiation of somatic or pluripotent stem cells have been established to obtain a renewable source of different neuronal cell types. Alternatively, several neuronal populations have been generated through direct reprogramming/transdifferentiation, which concerns the conversion of fully differentiated somatic cells into induced neurons. This is achieved through the forced expression of selected transcription factors (TFs) in the donor cell population. The reprogramming cocktail is chosen after an accurate screening process involving lists of TFs enriched into desired cell lineages. In some instances, this type of studies has revealed the crucial role of TFs whose function in the differentiation of a given specific cell type had been neglected or underestimated. Herein, we will speculate on how the in vitro studies have served to better understand physiological mechanisms of neuronal development in vivo.

In vitro Methods to Cultivate Spiral Ganglion Cells, and Purification of Cellular Subtypes for Induced Neuronal Reprogramming.

Hearing loss can develop as a consequence of primary auditory neuron degeneration. These neurons are present within the spiral ganglion of the inner ear and co-exist with glial cells that assist in neuronal maintenance and function. There are limited interventions for individuals with hearing impairment, hence novel biological solutions must be explored. Regenerative strategies can benefit from in vitro methods to examine the long-term culture of purified cell populations. The culturing of neuronal, glial, and non-neuronal, non-glial cell types in both neonatal and adult mice is presented along with the whole-organ explant culture of the spiral ganglion. High yields of spiral ganglion glial and non-glial cells were cultured from both neonatal and adult mice. Dissociated spiral ganglion cells from Sox2-EGFP mice were sorted based on EGFP expression using fluorescence activated cell sorting. The EGFP+ fraction included purified glial populations, whereas the EGFP- fraction contained non-glial cells. Purified glial cells could be reprogrammed into induced neurons displaying neuronal markers and morphology at a higher efficiency than non-glial cells. Previous studies have only allowed for the short-term culturing of spiral ganglion cell populations and have placed emphasis on neonatal cells. There has also been a lack of methods able to cultivate pure cell populations. Here, the coupling of transgenic mouse lines, fluorescence activated cell sorting and advanced culture conditions allow cultivation and characterization of neuronal, glial and non-neuronal, non-glial cells from the spiral ganglion. These techniques are used to demonstrate that different spiral ganglion cell subtypes (glial vs. non-glial) display different competencies for direct neuronal reprogramming.

Regenerative Approaches in Huntington's Disease: From Mechanistic Insights to Therapeutic Protocols.

Huntington’s Disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the exon-1 of the IT15 gene encoding the protein Huntingtin. Expression of mutated Huntingtin in humans leads to dysfunction and ultimately degeneration of selected neuronal populations of the striatum and cerebral cortex. Current available HD therapy relies on drugs to treat chorea and control psychiatric symptoms, however, no therapy has been proven to slow down disease progression or prevent disease onset. Thus, although 24 years have passed since HD gene identification, HD remains a relentless progressive disease characterized by cognitive dysfunction and motor disability that leads to death of the majority of patients, on average 10–20 years after its onset. Up to now several molecular pathways have been implicated in the process of neurodegeneration involved in HD and have provided potential therapeutic targets. Based on these data, approaches currently under investigation for HD therapy aim on the one hand at getting insight into the mechanisms of disease progression in a human-based context and on the other hand at silencing mHTT expression by using antisense oligonucleotides. An innovative and still poorly investigated approach is to identify new factors that increase neurogenesis and/or induce reprogramming of endogenous neuroblasts and parenchymal astrocytes to generate new healthy neurons to replace lost ones and/or enforce neuroprotection of pre-existent striatal and cortical neurons. Here, we review studies that use human disease-in-a-dish models to recapitulate HD pathogenesis or are focused on promoting in vivo neurogenesis of endogenous striatal neuroblasts and direct neuronal reprogramming of parenchymal astrocytes, which combined with neuroprotective protocols bear the potential to re-establish brain homeostasis lost in HD.

Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons.

Biophysical cues can improve the direct reprogramming of fibroblasts into neurons that can be used for therapeutic purposes. However, the effects of a three-dimensional (3D) environment on direct neuronal reprogramming remain unexplored. Here, we show that brain extracellular matrix (BEM) decellularized from human brain tissue facilitates the plasmid-transfection-based direct conversion of primary mouse embryonic fibroblasts into induced neuronal (iN) cells. We first show that two-dimensional (2D) surfaces modified with BEM significantly increase the generation efficiency of iN cells and enhance neuronal transdifferentiation and maturation. Moreover, in an animal model of ischaemic stroke, iN cells generated on the BEM substrates and transplanted into the brain led to significant improvements in locomotive behaviours. We also show that compared with the 2D BEM substrates, 3D BEM hydrogels recapitulating brain-like microenvironments further promote neuronal conversion and potentiate the functional recovery of the animals. Our findings suggest that 3D microenvironments can boost nonviral direct reprogramming for the generation of therapeutic neuronal cells.

KMT2B Is Selectively Required for Direct Neuronal Reprogramming and Its Loss Exposes Dystonia-Relevant Targets

Direct reprogramming of mesoderm-derived fibroblasts into induced neurons (iNs) by neuronal-specific transcription factors Brn2, Myt1l and Ascl1 is a paradigmatic example of inter-lineage conversion across epigenetically distant cells. Despite tremendous progress on the transcriptional hierarchy underlying transdifferentiation, the concomitant epigenome resetting and its drivers remain elusive. Here we investigated the role of KMT2A and KMT2B, two histone H3 lysine 4 methylases with cardinal roles in development, through individual and compound inactivation. We find that Kmt2b, whose mutations cause human brain disorder dystonia, is selectively required for iN conversion through the suppression of the alternative myocyte program and the induction of neuronal maturation genes. The identification of KMT2B vulnerable targets that are specific to cell fate reassignment allowed in turn the identification of new dystonia-relevant variants and genes in a large cohort of patients, discovering 45 unique protein-impactful alterations in 39 KMT2B-targets that constitute promising candidates for KMT2B-related dystonia causation.

Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows for the possibility of generating patient-derived neurons. A unique feature of these so-called induced neurons (iNs) is the potential to maintain aging and epigenetic signatures of the donor, which is critical given that many diseases of the CNS are age related. Here, we review the published literature on the work that has been undertaken using iNs to model human brain disorders. Furthermore, as disease-modeling studies using this direct neuronal reprogramming approach are becoming more widely adopted, it is important to assess the criteria that are used to characterize the iNs, especially in relation to the extent to which they are mature adult neurons. In particular: i) what constitutes an iN cell, ii) which stages of conversion offer the earliest/optimal time to assess features that are specific to neurons and/or a disorder and iii) whether generating subtype-specific iNs is critical to the disease-related features that iNs express. Finally, we discuss the range of potential biomedical applications that can be explored using patient-specific models of neurological disorders with iNs, and the challenges that will need to be overcome in order to realize these applications.

Transient CREB-mediated transcription is key in direct neuronal reprogramming.

ABSTRACTCombinations of neuronal determinants and/or small-molecules such as Forskolin (Fk) can be used to convert different cell types into neurons. As Fk is known to activate cAMP-dependent pathways including CREB-activity, we aimed here to determine the role of CREB in reprogramming – including its temporal profile. We show that transient expression of the dominant-positive CREB-VP16 followed by its inactivation mediated by the dominant-negative ICER improves neuronal conversion of astrocytes mediated by the neurogenic determinant Ascl1. Contrarily, persistent over-activation by CREB-VP16 or persistent inhibition by ICER interferes with neuronal reprogramming, with the latter enhancing cell death. Taken together our work shows transient CREB activation as a key effector in neuronal reprogramming.

Reprogramming of somatic cells: iPS and iN cells.

Limited access to human neurons has posed a significant barrier to progress in biological and preclinical studies of the human nervous system. The advent of cell reprogramming technologies has widely disclosed unprecedented opportunities to generate renewable sources of human neural cells for disease modeling, drug discovery, and cell therapeutics. Both somatic reprogramming into induced pluripotent stem cells (iPSCs) and directly induced Neurons (iNeurons) rely on transcription factor-based cellular conversion processes. Nevertheless, they rely on very distinct mechanisms, biological barriers, technical limitations, different levels of efficiency, and generate neural cells with distinctive properties. Human iPSCs represent a long-term renewable source of neural cells, but over time genomic aberrations might erode the quality of the cultures and the in vitro differentiation process requires extensive time. Conversely, direct neuronal reprogramming ensures a fast and straightforward generation of iNeurons endowed with functional properties. However, in this last case, conversion efficiency is reduced when starting from adult human cells, and the molecular and functional fidelity of iNeurons with respect to their corresponding native neuronal subtype is yet to be fully ascertained in many cases. For any biomedical research application, it should be carefully pondered the reprogramming method to use for generating reprogrammed human neuronal subtypes that best fit with the following analysis considering the existing limitations and gap of knowledge still present in this young field of investigation.

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.

Stressed out? Healing Tips for Newly Reprogrammed Neurons

How do astrocyte-derived neurons deal with the sudden loss of their glial identity? Exciting new findings from Gascón et al. (2016) single out metabolic conversion as a critical checkpoint for direct neuronal reprogramming.

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.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. From the Clinical EditorIn the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons.

Environmental impact on direct neuronal reprogramming in vivo in the adult brain

Direct reprogramming of non-neuronal cells to generate new neurons is a promising approach to repair damaged brains. Impact of the in vivo environment on neuronal reprogramming, however, is poorly understood. Here we show that regional differences and injury conditions have significant influence on the efficacy of reprogramming and subsequent survival of newly generated neurons in the adult rodent brain. A combination of local exposure to growth factors and retrovirus-mediated overexpression of the neurogenic transcription factor Neurogenin2 (Neurog2) can induce new neurons from non-neuronal cells in the adult neocortex and striatum where neuronal turnover is otherwise very limited. These two regions respond to growth factors and Neurog2 differently and instruct new neurons to exhibit distinct molecular phenotypes. Moreover, ischemic insult differentially affects differentiation of new neurons in these regions. These results demonstrate strong environmental impact on direct neuronal reprogramming in vivo.