Nicotinamide restricts neural precursor proliferation to enhance catecholaminergic neuronal subtype differentiation from mouse embryonic stem cells

Emerging evidence indicates that a strong relationship exists between brain regenerative therapies and nutrition. Early life nutrition plays an important role during embryonic brain development, and there are clear consequences to an imbalance in nutritional factors on both the production and survival of mature neuronal populations and the infant’s risk of diseases in later life. Our research and that of others suggest that vitamins play a fundamental role in the formation of neurons and their survival. There is a growing body of evidence that nicotinamide, the water-soluble amide form of vitamin B3, is implicated in the conversion of pluripotent stem cells to clinically relevant cells for regenerative therapies. This study investigated the ability of nicotinamide to promote the development of mature catecholaminergic neuronal populations (associated with Parkinson’s disease) from mouse embryonic stem cells, as well as investigating the underlying mechanisms of nicotinamide’s action. Nicotinamide selectively enhanced the production of tyrosine hydroxylase-expressing neurons and serotonergic neurons from mouse embryonic stem cell cultures (Sox1GFP knock-in 46C cell line). A 5-Ethynyl-2´-deoxyuridine (EdU) assay ascertained that nicotinamide, when added in the initial phase, reduced cell proliferation. Nicotinamide drove tyrosine hydroxylase-expressing neuron differentiation as effectively as an established cocktail of signalling factors, reducing the proliferation of neural progenitors and accelerating neuronal maturation, neurite outgrowth and neurotransmitter expression. These novel findings show that nicotinamide enhanced and enriched catecholaminergic differentiation and inhibited cell proliferation by directing cell cycle arrest in mouse embryonic stem cell cultures, thus driving a critical neural proliferation-to-differentiation switch from neural progenitors to neurons. Further research into the role of vitamin metabolites in embryogenesis will significantly advance cell-based regenerative medicine, and help realize their role as crucial developmental signalling molecules in brain development

Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations.

Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington's disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons. METHODS: Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods. RESULTS: Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons. CONCLUSIONS: Our results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons

One Giant Leap from Mouse to Man: The Microbiota–Gut–Brain Axis in Mood Disorders and Translational Challenges Moving towards Human Clinical Trials

The microbiota–gut–brain axis is a bidirectional communication pathway that enables the gut microbiota to communicate with the brain through direct and indirect signaling pathways to influence brain physiology, function, and even behavior. Research has shown that probiotics can improve several aspects of health by changing the environment within the gut, and several lines of evidence now indicate a beneficial effect of probiotics on mental and brain health. Such evidence has prompted the arrival of a new term to the world of biotics research: psychobiotics, defined as any exogenous influence whose effect on mental health is bacterially mediated. Several taxonomic changes in the gut microbiota have been reported in neurodevelopmental disorders, mood disorders such as anxiety and depression, and neurodegenerative disorders such as Alzheimer’s disease. While clinical evidence supporting the role of the gut microbiota in mental and brain health, and indeed demonstrating the beneficial effects of probiotics is rapidly accumulating, most of the evidence to date has emerged from preclinical studies employing different animal models. The purpose of this review is to focus on the role of probiotics and the microbiota–gut–brain axis in relation to mood disorders and to review the current translational challenges from preclinical to clinical research

The Influence of Nicotinamide on Health and Disease in the Central Nervous System

Nicotinamide, the amide form of vitamin B 3 (niacin), has long been associated with neuronal development, survival, and function in the central nervous system (CNS), being implicated in both neuronal death and neuroprotection. Here, we summarise a body of research investigating the role of nicotinamide in neuronal health within the CNS, with a focus on studies that have shown a neuroprotective effect. Nicotinamide appears to play a role in protecting neurons from traumatic injury, ischaemia, and stroke, as well as being implicated in 3 key neurodegenerative conditions: Alzheimer’s, Parkinson’s, and Huntington’s diseases. A key factor is the bioavailability of nicotinamide, with low concentrations leading to neurological deficits and dementia and high levels potentially causing neurotoxicity. Finally, nicotinamide’s potential mechanisms of action are discussed, including the general maintenance of cellular energy levels and the more specific inhibition of molecules such as the nicotinamide adenine dinucleotide-dependent deacetylase, sirtuin 1 (SIRT1)

Early nicotinamide administration accelerates the conversion of neural progenitor populations to mature cell types.

<p>(A-B) Early nicotinamide treatment (days 0–7) induced a significant decrease in the <i>Sox1</i>GFP⁺ population in comparison to untreated cells. (A,C) Nicotinamide added between days 7 and 14 did not affect the population of <i>Sox1</i>GFP-expressing cells. (D) Histogram showing the proportion of cells with different levels of <i>Sox1</i>GFP expression. Larger numbers of cells displayed weak <i>Sox1</i>GFP expression in cultures that were exposed to 10 mM nicotinamide between days 0 and 7 (red bars), compared with controls. (E-F) Addition of nicotinamide increased the population of GABAergic neurons from mESCs. Nicotinamide (10 mM) added to media generated a significant increase in the percentage of the total GABAergic neuron population at day 14, compared against the control untreated group. Some cells showed intense GABA immunoreactivity (yellow arrows) and some less strong GABA expression (white arrow). Scale bar 50 μm applies to all high magnification images. ***p<0.001, **p<0.01.</p

Nicotinamide enhanced neuronal maturation in GABAergic populations.

<p>(A and E) The proportion of 46C-derived cells displaying “short” primary processes was significantly decreased in cultures exposed to 10 mM nicotinamide between days 0 and 7, concomitant with an obvious increased trend for “longer” neurite processes, compared with controls (F). (B) Total length of neurites was significantly increased in the presence of nicotinamide. (C) There was no difference in the number of primary neurites per neurons between controls and nicotinamide-stimulated conditions. (D) An upward trend in the proportion of mESC-derived cells displaying “strong” levels of GABA expression was demonstrated in cultures exposed to 10 mM nicotinamide, which correlated with a downward trend in “weaker”-expressing GABAergic cells, compared with control conditions. ***p<0.001, **p<0.01, *p<0.05. Micrographs show morphologies of GABA+ neurons differentiated in control conditions (E), or cultures treated with 10 mM nicotinamide (F). Scale bar = 100 μm.</p

Schematic diagram of time-course analysis of neural and neuronal differentiation in the presence of nicotinamide.

<p>Undifferentiated cells were cultured as monolayers in N2B27 medium for different durations to investigate whether nicotinamide would influence the conversion of Oct4⁺undifferentiated stem cells (scale bar = 100 μm) to <i>Sox1</i>GFP neural progenitors (scale bar = 50 μm) and thus drive differentiation toward βIII-tubulin⁺ neurons (scale bar = 50 μm). The potential of nicotinamide was assessed using immunocytochemistry at different culture periods from days 2–14.</p