Neuronal Differentiation
Trevor Morey, Student #: 998595177


Neuronal differentiation is the process through which pluripotent neuronal stem cells adopt specific cell fates as different functional neurons. The process begins when cellular morphogens, such as WNT, Sonic Hedgehog (SHH), Retinoic Acid (RA) or Fibroblast Growth Factor (FGF) are deposited in gradients within the developing organism. Specific concentrations of these growth factors determine what type of neuron a stem cell will become. The type of neurotransmitter they secrete and the area of the brain in which they reside can be used to distinguish different groups of neuronal cells. Each group of neuronal cells has a different differentiation process involving different morphogens. Current scientific research is making an effort into being able to fully differentiate different types neurons in vitro to treat neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis (ALS).(1)



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A timeline of neuronal development showing the morphogens and the gene expression patterns that form different groups of fully differentiated neurons. (1)



















Cortical Glutaminergic Neurons



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A schematic of the development process for glutaminergic neurons. (1)

Cortical Glutaminergic Neurons are the primary excitatory neurons within the brain.
They release the transmitter glutamate, which is a non essential amino acid. Glutamate binds to AMPA and NMDA receptors on post- synaptic neurons causing positively charged sodium ions to diffuse through the neuronal cell membrane and depolarize the cell creating an excitatory post-synaptic potential, which can lead to the triggering of action potentials.(2) Glutamate receptors are thought to be very important in the process of long-term potentiation, which is involved with post-synaptic plasticity. These cortical neurons can be further modified during cortical patterning to create different functional regions of the cerebral cortex.







WNT_SHH.jpg
A depiction of the SHH and WNT morphogen gradients in the developing coretx (Left), as well as the genes that are expressed due to these gradients (right). (1)

Differentiation Process

Current research suggests that glutaminergic neurons arise as a result of the default pathway that pluripotent stem cells take to become neuronal stem cells. They differentiate out of the Dorsal
Pallium of the Anterior Neural Tube, which is created by a high concentration of Wingless-Type MMTV Integration Site Family (WNT) and Bone Morphogenic Proteins (BMP) and a low concentration of Sonic HedgeHog (SHH).(3) This specificmorphogen gradient leads to the expression of the Pax6 gene and repression of the Nkx2.1 gene.(4) These homeodomain transcription factors then begin transcribing genes that ultimately lead to the characteristics of glutaminergic neurons such as vesicular glutamate transporters.(5)











GABAergic Projection Neurons


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An illustration of a GABAergic synapse. (22)

GABAergic Projection Neurons are one of the most important sets of neurons located in the brain. They project from various brain stem nuclei to regions of the cortex, helping to regulate cortical and subcortical activity.(1) These neurons release the neurotransmitter Gamma aminobutyric acid (GABA), which binds to GABAa ionotropic (6) or GABAb metabotropic (7) receptors on the post-synaptic membrane. When GABA binds to its receptor it acts to either bring chloride ions into the cell or potassium ions out of the cell, leading to an inhibitory post-synaptic potential. This makes the post-synaptic cell more negative and thus more difficult to create action potentials.(6,7)












Differentiation Process

This subset of neurons is created from the Lateral Ganglionic Emminence (LGE). This particular region has a low concentration of SHH and a low concentration of WNT and BMP. (8)
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A schematic of the development process for GABAergic neurons. (1)

This leads to a high expression of the Gsx2 and Pax6 genes and a low expression of the Nkx2.1 gene. (1) These transcription factors act on genes that help to synthesize the transmitter GABA and the other characteristics of these GABAergic neurons. (9)






Basal Forebrain Cholinergic Neurons



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Cholinergic neurons plated in vitro. The enzyme ChAT has been tagged with GFP causing only cholinergic neurons to fluoresce. (11)

As one of the key nuclei for cortical activation, basal forebrain cholinergic neurons play a crucial role in the cognitive function of human beings. They are important for awareness, attention and other executive functions. These neurons produce the neurotransmitter acetylcholine, which acts on nicotinic (ionotropic) and muscarinic (metabotropic) receptors on post-synaptic neurons.When acetylcholine binds to its receptor it promotes the flow of sodium and potassium ions into the cell causing a depolarizing excitatory post-synaptic potential, which promotes action potential production. (10)



Differentiation Process


This group of neurons arises from the
medial ganglionic eminence (MGE).
In this particular area there is a relatively
high concentration of the morphogen SHH
and little to no WNT or BMP. (11) This promotes the expression of the Nkx2.1
BFCN.jpg
A schematic of the development process for basal forebrain cholinergic neurons. (1)

gene and repression of the Pax6 gene.(12) These transcription factors act to promote the expression of genes such as Choline aceytltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are involved in acetylcholine production and release. (11)




Alzheimer’s Disease Treatment


Alzheimer’s disease is characterized by post mortem amyloid beta plaques and tau fibrillary tangles. These pathologicalsubstances are thought to primarily damage the basal forebrain cholinergic neurons, as well as, hippocampal neurons. Tuszynski, M.H. et al.,have recently performed an experiment where they genetically engineered human fibroblasts to express nerve growth factor (NGF) and then implanted these cells into basal forebrain of human Alzheimer’s disease patients. It was found that with increased concentration of NGF the patients showed improved cognitive and memory functions. Post-mortem specimens also showed substantial neuronal growth of cholinergic neurons in the forebrain. (13) Another study by Bissonette, C.J., et al., showed that basal forebrain cholinergic neurons could be grown in vitro by applying WNT inhibitors as well as SHH to the growth medium. These results suggest future treatments may involve the implantation of neurons grown in vitro to replace those that are lost in Alzheimer’s patients. (11)











Spinal Cord Motor Neurons


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A schematic of the development process for spinal cord motor neurons. (1)

Similar to the basal forebrain cholinergic neurons, spinal cord motor neurons use the neurotransmitter acetylcholine to transfer signals to their targets. However, instead of cortical neuronal targets these neurons innervate skeletal muscle. The acetylcholinebinds to nicotinic receptors at the neuromuscular junction, which leads to the opening of sodium and potassium channels on the muscle membrane causing a depolarization of the muscle fiber. This ultimately leads to an inflow of calcium resulting in the muscle contraction. (14) After being generated the spinal cord motor neurons must undergo axonal pathfinding to reach their appropriate effector targets.



Differentiation Process

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A representation of the Retinoic Acid (RA) gradient in the Hindbrain (HB) and Spinal Cord (SC) of a developing organism. (1)

Spinal cord motor neurons are one of the most caudally extending subsets of neurons that arise during the development of an organism. A gradient of the morphogen retinoic acid (RA) is found along the rostral-caudal axis of the neural tube during development. The presence of RA along with SHH (15) is thought to promote the development of cervical spinal cord motor neurons by promoting the expression of the Olig2 transcription factor gene. (16)









ALS Disease Treatment

Amyotrophic Lateral Sclerosis (ALS) is a condition that arises from the degeneration of spinal cord motor neurons. (14) Lee, H., et al., and their team

devised a protocol for differentiating spinal motor neurons in vitro from embryonic stem cells. They placed the stem cells on a MS5 stromal feeder and applied RA and SHH to the medium. These neurons were then grafted into the spinal cords of adult rats and developing chick embryos. After implantation the axons of these neurons were found to be choline acetyltransferase positive, suggesting that they had differentiated into spinal cord motor neurons. This provides the groundwork for studies that could be conducted on humans to treat conditions where spinal cord motor neurons are lost such as in ALS. (17)















Midbrain Dopaminergic Neurons



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Dopaminergic neurons grown from embryonic stem cells in vitro. The enzyme Tyrosine Hydroxylase has been tagged by RFP and fluoresces red. (19)

Dopamine has many different functions within the brain; it is thought to be involved in reward, cortical activation and regulating motor function. Midbrain dopaminergic neurons are found primarily in two areas, the ventral tegmental area for reward and the substantia nigra for motor regulation. Dopamine’s effect depends on the receptor that it binds to. For example, D1 dopaminergic post-synaptic receptors will respond to the binding of dopamine by opening sodium channels leading to an excitatory post-synaptic potential. Whereas, if dopamine binds to a D2 receptor it will open chloride ion channels leading to an inhibitory post-synaptic potential. (18) Dopaminergic neurons are also very important in the developmental processes that characterize the teenage brain, as well as the pathology of alcohol and social addictions.




Differentiation Process

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A schematic of the development process for midbrain dopaminergic neurons. (1)

Midbrain Dopaminergic neurons arise from a group of neuroepithelial cells that originate in the ventral midbrain. These neural stem cells receive high concentrations of the morphogens Fibroblast Growth Factor 8 (FGF8) and SHH during the development of the midbrain. These morphogens activate genes such as FoxA2 and EN1, which encode transcription factors that increase the production of proteins specific to dopaminergic neurons such as tyrosine hydroxylase, which creates dopamine from tyrosine. (19) It has also been found that adding dopaminergic neuroprotective chemicals such as Glial line Derived Neurotrophic Factor (GDNF) to with medium with differentiating neuronal stem cells that the yield of dopaminergic neurons becomes much higher and more appealing for clinical uses. (20)




Parkinson’s Disease Treatment


Current research into the process of neuronal differentiation by Swistowski et al.shows the potential clinical applications of being able to fully differentiate neurons in vitro. This group of scientists treated induced pluripotent stem cells derived from adult somatic cells with a combination of Fibroblast Growth Factor and Sonic Hedgehog to induce their differentiation into midbrain dopaminergic neurons in vitro. They then implanted these differentiated neurons into Parkinsonian rat models, which showed a functional motor recovery after the treatment. This research suggests that the implantation of neuronal cells differentiated in vitro may be a viable treatment option for neurodegenerative disorders, such as Parkinson's Disease. (21)















References



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2. Li, Q., et al. Modulation of NMDA and AMPA-Mediated Synaptic Transmission by CB1 Receptors in Frontal Cortical Pyramidal Cells. Brain Res. 1342: 127-137 (2010).

3. Gunhaga, L., et al. Specification of Dorsal Telencephalic Character by Sequential WNT and FGF signaling. Nature Neuroscience. 6(7): 701- 707 (2003).

4. Zhang, X., et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell. 7(1): 90-100 (2010).

5. Winpenny, E., et al. Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells. Neural Development. 6(12): 1-18 (2011).

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11. Bissonnette, C.J., et al. The Controlled Generation of Functional Basal Forebrain Cholinergic Neurons from Human Embryonic Stem Cells. Stem Cells. 29: 802-811 (2011).

12. Goulburn, A.L., et al. A Targeted NKX2.1 Human Embryonic Stem Cell Reporter Line Enables Identification of Human Basal Forebrain Derivatives. Stem Cells. 29: 462-473 (2011).

13. Tuszynski, M.H., et al. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nature Medicine. 11(5): 551-555 (2005).

14. Dimos, J.T., et al. Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons. Science. 321: 1218- 1221 (2008).

15. Li, X.J., et al. Directed Differentiation of Ventral Spinal Progenitors and Motor Neurons from Human Embryonic Stem Cells by Small Molecules. Stem Cells. 26(4): 886-893 (2008).

16. Li, H., et al. Phosphorylation Regulates OLIG2 Cofactor Choice and the Motor Neuron-Oligodendrocyte Fate Switch. Neuron. 69(5): 918-929 (2011).

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18. Simon, N. W., et al. Dopaminergic Modulation of Risky Decision-Making. Journal of Neuroscience. 31(48): 17460-17470 (2011).

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