Neurochemical classification of serotonin-immunoreactive neurons co-innervating motor endplates in the mouse esophagus.

Serotonin immunoreactivity was previously found in myenteric neurons co-innervating motor endplates in the mouse esophagus striated muscle and an involvement in motility control was suggested. However, it is not known if other neuroactive substances are present in these neurons and to what extent they co-localize. First, vasoactive intestinal peptide (VIP) was established as a bona fide marker for putative inhibitory myenteric neurons by evaluating co-localization with neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY). Then, co-localization of serotonin and VIP was tested in co-innervating axons on motor endplates, which were visualized with α-bungarotoxin (α-BT) by multilabel immunofluorescence. Myenteric ganglia were also surveyed for co-localization in neuronal perikarya and varicosities. nNOS, NPY, and VIP were completely co-localized in enteric co-innervating nerve terminals on motor endplates. After co-staining with VIP, we found (a) serotonin (5-HT)-positive nerve endings without VIP (44% of 5-HT-positively innervated endplates), (b) 5-HT- and VIP-positive endings without co-localization (35%), and (c) 5-HT- and VIP-positive endings with co-localization (21%). About one-fifth of nerve terminals on motor endplates containing 5-HT originate from putative inhibitory peptidegic nitrergic neurons. However, the majority represents a different population presumably subserving different functions.

Chemical Replacement of Noggin with Dorsomorphin Homolog 1 for Cost-Effective Direct Neuronal Conversion.

The direct conversion of adult human skin fibroblasts (FBs) into induced neurons (iNs) represents a useful technology to generate donor-specific adult-like human neurons. Disease modeling studies rely on the consistently efficient conversion of relatively large cohorts of FBs. Despite the identification of several small molecular enhancers, high-yield protocols still demand addition of recombinant Noggin. To identify a replacement to circumvent the technical and economic challenges associated with Noggin, we assessed dynamic gene expression trajectories of transforming growth factor-ß signaling during FB-to-iN conversion. We identified ALK2 (ACVR1) of the bone morphogenic protein branch to possess the highest initial transcript abundance in FBs and the steepest decline during successful neuronal conversion. We thus assessed the efficacy of dorsomorphin homolog 1 (DMH1), a highly selective ALK2-inhibitor, for its potential to replace Noggin. Conversion media containing DMH1 (+DMH1) indeed enhanced conversion efficiencies over basic SMAD inhibition (tSMADi), yielding similar ßIII-tubulin (TUBB3) purities as conversion media containing Noggin (+Noggin). Furthermore, +DMH1 induced high yields of iNs with clear neuronal morphologies that are positive for the mature neuronal marker NeuN. Validation of +DMH1 for iN conversion of FBs from 15 adult human donors further demonstrates that Noggin-free conversion consistently yields iN cultures that display high ßIII-tubulin numbers with synaptic structures and basic spontaneous neuronal activity at a third of the cost.

Direct Conversion of Human Fibroblasts to Induced Neurons.

Progressive aging is a physiological process that represents a central risk factor for the development of several human age-associated chronic diseases, including neurodegenerative diseases. A major focus in biomedical research is the pursuit for appropriate model systems to better model the biology of human aging and the interface between aging and disease mechanisms. Direct conversion of human fibroblasts into induced neurons (iNs) has emerged as a novel technology for the in vitro modeling of age-dependent neurological diseases. Similar to other cellular reprogramming techniques, e.g., iPSC-based cellular reprograming, direct conversion relies on the ectopic overexpression of transcription factors, typically including well-known pioneer factors. However, in contrast to alternative technologies to generate neurons, the entire process of direct conversion bypasses any proliferative or stem cell-like stage, which in fact renders it the unique aptitude of preserving age-associated hallmarks from the initial fibroblast source. In this chapter, we introduce direct conversion as a practical and easy-to-approach disease model for aging and neurodegenerative disease research. A focus here is to provide a stepwise protocol for the efficient and highly reproducible generation of iNs from adult dermal fibroblasts from human donors.

Age-dependent instability of mature neuronal fate in induced neurons from Alzheimer's patients.

Sporadic Alzheimer's disease (AD) exclusively affects elderly people. Using direct conversion of AD patient fibroblasts into induced neurons (iNs), we generated an age-equivalent neuronal model. AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways. Mapping iNs to longitudinal neuronal differentiation trajectory data demonstrated that AD iNs reflect a hypo-mature neuronal identity characterized by markers of stress, cell cycle, and de-differentiation. Epigenetic landscape profiling revealed an underlying aberrant neuronal state that shares similarities with malignant transformation and age-dependent epigenetic erosion. To probe for the involvement of aging, we generated rejuvenated iPSC-derived neurons that showed no significant disease-related transcriptome signatures, a feature that is consistent with epigenetic clock and brain ontogenesis mapping, which indicate that fibroblast-derived iNs more closely reflect old adult brain stages. Our findings identify AD-related neuronal changes as age-dependent cellular programs that impair neuronal identity.

Human neurons to model aging: A dish best served old.

With the advancing age of humans and with it, growing numbers of age-related diseases, aging has become a major focus in recent research. The lack of fitting aging models, especially in neurological diseases where access to human brain samples is limited, has highlighted direct conversion into induced neurons (iN) as an important method to overcome this challenge. Contrary to iPSC reprogramming and its corresponding cell rejuvenation, the generation of iNs enables us to retain aging signatures throughout the conversion process and beyond. In this review, we explore different cell reprogramming methods in light of age-associated neurodegenerative diseases and discuss different approaches, advances, and limitations.

Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects.

Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.