User:AAR3643/Epigenetic regulation of neurogenesis

Sandbox belonging to Andre Ribeiro, Amulya Cherian, and Andrew Spires.

Embryonic neurogenesis

Histone modifications

Neural stem cells are involved in the development of the cortex in a precise "inside out" manner with carefully controlled timing mechanisms. Early born neurons form deep layers in the cortex while newer born neurons form the upper layers. This timing program is seen in vitro as well as in vivo. Mutant analysis has shown that histone methylation modulates the production of deep layer and upper layer neurons through epigenetic regulation. Specifically, deletion of a portion of the PRC2 complex, Ezh2, encoding histone methyltransferase, led to a twofold reduction of POU3F2/BRN2-expressing and SATB2-expressing upper layer neurons without affecting the number of neurons in layers V and VI . In mouse embryonic stem cell-derived neural progenitors, increased histone acetylation induced by the histone deacetylase (HDAC) inhibitor valproic acid not only induced neuronal differentiation, but also selectively enriched the upper layer neuronal population. Therefore, it has been proposed that HDAC inhibition promotes the progression of neuronal differentiation, leading to a fate-switch from deep-layer producing progenitors into upper-layer progenitors. However, the reasons behind this selective differentiation and timing control as a result of HDAC inhibition are not yet fully understood.

Astrocyte reprogramming

Astrocytes are specialized glial cells that vastly outnumber neurons in the adult brain, due to their ability to multiply as needed to sustain proper levels for brain function through multiple functions, including blood brain barrier control, supporting synapses as well as axon pathfinding. Unlike neurons, these specialized glial cells are able to alter their cell fate prior to reaching full maturation and “dedifferentiate,” in large part due to epigenetic factors. This dedifferentiation allows astrocytes to potentially reach a different cell fate entirely, so long as this dedifferentiation occurs before complete maturation occurs, and can lead to their consequent differentiation and conversion from glial cells into neurons in the adult brain. Prior to complete maturation while dedifferentiation is still possible, the expression of genes Mash1, NeuroG1 and NeuroG2 can allow for reprogramming of astrocytes into neurons. In addition to the expression of these genes in brain cells, multiple epigenetic factors play a role in these gene’s expression patterns. An up-regulation of acetylation at H3K9 and H3K14 residues adjacent to both NeuroG1 and NeuroG2 genes has been shown to accompany astrocyte dedifferentiation, over-expression and/or forced expression of these genes can directly induce the differentiation of astrocytes.

In addition, silencing of methylation mechanisms, specifically the silencing of many classes of DNA methyltransferases, which themselves are involved in silencing expression, inhibits the progenitor cells of astrocytes from differentiating back to their original fate as glial cells. Despite this knowledge of the mechanism of methylation repression, the identity of these silenced genes is not yet fully known. While this overall repression of methylation is necessary to prevent expression of specific genes needed to allow an astrocyte to fully mature and reach an astrocytic cell fate, it was found that the over-expression of one specific histone methyltransferase, Ezh2, which catalyzes the tri-methylation of H3K27, represses genes needed for astrocyte maintenance, thus allowing the cell to retain its neural stem cell morphology. This demonstrates that differential methylation by distinct methyltransferases and their consequent repression or over-expression have differing roles in the dedifferentiation of astrocytes to form neurons. Furthermore, while not sufficient to induce astrocyte dedifferentiation alone, Ezh2 is necessary for astrocytes to dedifferentiate as they are inhibited from reaching complete maturity into their original cell fate. Once in this inhibited stage, expression of the gene NeuroD4 in these specified glial cells has been shown to lead to neuronal formation, and thus neurogenesis, from the dedifferentiated astrocytes in adult mammalian brains.

Histone Modifications via Acetylation

Histone acetylation, deacetylation, as well as the inhibition of histone deacetylation mechanisms also play large roles in the proliferation and self-renewal of post-natal neural stem cells. DNA, which encodes genes within the genome, including those involved in adult neurogenesis, is packaged into chromatin. Chromatin itself is made up of nucleosome subunits, each consisting of two copies each of histone proteins H2A, H2B, H3 and H4. One of the primary roles acetylation plays in the regulation of gene expression is through the inhibition of adjacent nucleosome interactions. When the H4 histones are not acetylated, they are basic in nature, and insert into the acidic pocket of the H2A-H2B protein dimers in adjacent nucleosomes, leading to tight association between nucleosomes and further packing of the chromatin. Thus, acetylation causes the H4 histone to lose its basicity, and prevents nucleosome cross-linking. This acetylation of histone tails additionally increase the affinity of chromatin remodeling enzyme complexes such as SWI–SNF and ISWI, which utilize ATP to produce nucleosome-free regions at promoter and enhancer sites. This allows for greater ability of recognition of these sites by transcription factors, particularly TFIID which is the major transcription factor involved in transcription initiation. Furthermore, the acetylation of lysine residues on histone tails can be recognized by the TAF1 component of TFIID, and when bound, TAF1 becomes a histone acetyltransferase (HAT), further acetylating adjacent H3 and H4 histones and recruiting more HATs in the process. DNA wraps around the histones of chromatin, and the acetylation of these histone tails leads to reduction of the positive charge associated with the histones. This results in the negatively-charged DNA to lose affinity for the histone, allowing for more space for transcription factors to bind promoter regions and further facilitate expression.

Eventually, the processes of histone acetylation and consequent chromatin remodeling allow for greater expression of target genes, including those involved in adult neurogenesis. The most prominently studied and well-understood regulators of chromatin remodeling, which play an important role in adult neurogenesis are histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs add acetyl groups to nucleosomes, while HDACs remove them. The acetylation of histones leads to decreased condensation of the nucleosomes to target DNA, and increases the likelihood that gene expression may occur by freeing up the DNA targets to bind to their respective transcriptional factors. This process is involved in neural proliferation regulation, as different neuronal cell genes are expressed and repressed. Deacetylation of histones leads to the reverse, and increases the likelihood for the repression of gene expression.

Role of Histone Modifications in Neurogenesis

HDAC inhibitors (HDACi), such as valproic acid (VPA) and trichostatin A can promote proliferation of adult neurogenesis through the inhibition of HDAC activity, inducing differentiation of adult progenitor cells. Neural-expressed HDACs interact with Tlx, an essential neural stem cell regulator, to suppress TLX target genes. This includes the cyclin-dependent kinase inhibitor P21 and the tumor suppressor gene Pten to promote neural stem cell proliferation. Inhibition of HDACs by the antiepileptic drug valproic acid induces neuronal differentiation as in embryonic neurogenesis, but also inhibits glial cell differentiation of adult neural stem cells. This is likely mediated through upregulation of neuronal specific genes such as the neurogeneic basic helix-loop-helix transcription factors NEUROD, NEUROGENENIN1, and Math1. Conditional loss of HDAC1 and HDAC2 in neural progenitor cells prevented them from differentiating into neurons and their loss in oligodendrytic progenitor cells disrupted oligodendrocyte formations, suggesting that histone deacetlyation plays important but varying roles in different stages of neuronal development.

miRNAs

MicroRNAs (miRNAs) are small noncoding RNAs that play a significant role in eukaryotic epigenetic regulation. miRNAs function to modulate protein expression levels of their mRNA targets without affecting the sequences of the genes of interest. While miRNAs play a large role in the modulation of epigenetic mechanisms, they are also modified and regulated by other epigenetic factors including DNA methylation, histone modifications and other RNA modifications. Together, miRNAs create an epigenetic feedback loop with other epigenetic factors to affect the expression levels of specific genes. A number of specific miRNAs have been implicated as agents of epigenetic regulation in adult neurogenesis. miR-9 targets the nuclear receptor TLX in adult neurogenesis to promote neural differentiation and inhibit neural stem cell proliferation. It also influences neuronal subtype specification and regulates axonal growth, branching, and targeting in the central nervous system through interactions with HES1, a neural stem cell homeostasis molecule. miR-124 promotes cell cycle exit and neuronal differentiation in adult neurogenesis. Mouse studies have shown that ectopic expression of miR-124 showed premature neural progenitor cell differentiation and exhaustion in the subventricular zone.

In addition to miR-9 and miR-124, other miRNAs play essential roles in regulation of adult neurogenesis. miR-137, miR-184 and miR-195 regulate adult neural stem cell proliferation, with their over-expression leading to up-regulated proliferation while their down-regulation leads to a decrease in neuronal proliferation. Methyl-CpG binding protein 1 (MBD1) represses miR-184, which is a microRNA responsible for proliferation of adult neural stem/progenitor cells (aNSCs) along with the inhibition of differentiating these cells. miR-184 regulates embryonic brain development by binding to the mRNA for the Numblike (Numbl) protein and altering its expression. MBD1, Numbl, and miR-184 all work together to regulate the proliferation and differentiation of aNSCs. In addition, miR-195 works closely with MBD1 to regulate aNSC proliferation and differentiation. mIR-194 and MBD1 form a negative regulatory loop in aNSCs and work to repress the expression of each other. Inhibition of miR-195 promotes aNSC differentiation. Once differentiation has occurred, levels of miR-195 decreases.

Epigenetic dysregulation and neurological disorders

Epigenetic dysregulation, or alterations in epigenomic machinery, can cause DNA methylation and histone acetylation processes to go rogue. The epigenetic machinery influences neural differentiation regulation (i.e. neurogenesis) and are also involved in processes related to memory consolidation and learning in healthy individuals. Increasing age can produce various epigenetic changes such as reduced global heterochromatin, nucleosome remodeling, altered histone marks, and changes in DNA methylation. For instance, nucleosome loss occurs due to aging because core histone proteins are lost and less protein synthesis occurs. As aging is the main risk for many neurological disorders, epigenetic dysregulation can in turn lead to alterations on the transcriptional level of genes involved in the pathogenesis of neural degenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, and bipolar disease.

Huntington’s disease

Histone acetylation has received increasing support over the years as a proposed mechanism through which epigenetic dysregulation leads changes in gene expression that contribut to HD. Studies that look at mice with HD versus the wild type (WT) have shown that specific gene loci (Drd2, Penk1, Actb, and Grin1) decrease in histone acetylation levels, suggesting that a mutation of the Huntington (HTT) gene and its overexpression may be the cause of this epigenetic dysregulation.

It has been thought that HDAC inhibitors (HDACi's) could partially reverse the low acetylation levels seen in patients with HD. Preclinical studies have been performed using various HDACi’s [such as suberoxylanilide hydroxamic acid (SAHA), Trichostatin A (TSA), phenylbutyrate, and sodium butyrate (NaB)] that target HDACI and HDACII. Although these inhibitors improve some phenotypes of HD in mice, such as neuropathology and motor function, these beneficial effects do not lead towards a conclusion for the definitive need for increasing acetylation levels in HD patients. However, inactivation of a target of SAHA, Hdac 4, alleviates neurodegenerative complications in mice with HD through a transcription-independent mechanism which acts upon mutant Htt aggregation processes-which may indicate that there is a mechanism involving non-histone proteins. The proposed mechanism through which SAHA is speculated to act is through a RANBP2-mediated proteasome degradation model-which likely comes about as a general outcome of HDAC inhibitor actions. In this mechanism, SAHA is shown to down-regulate Hdac 4 through an increase in sumoylation, which is then followed up with the activation of degradation through a proteasomal pathway. This mechanism reveals the connectivity between acetylation, deacetylation, and sumoylation processes.

As of 2014, HDACi treatment has not been shown to restore normal expression of neuronal-identity genes. However clinical studies using HDACi are currently ongoing and the results are pending, with the Phase II studies showing promise for safe and tolerable use of several compounds such as phenylbutyrate.

Non-histone-mediated beneficial effects of HDACi have also been documented in models of Parkinson disease, suggesting common mechanisms between several neurodegenerative diseases.

Parkinson’s disease

DNA methylation analysis showed that there is significant dysregulation of methylation on CpG islands in patients with PD when compared to healthy individuals. Although this was genome-wide, this also occurred on many PD risk genes.

Mitochondrial DNA methylation of cytosine has also been shown to fluctuate over time due to age variance, as there is a growing body of literature linking mtDNA methylation to aging and oxidative stress. A study from 2015 by Hashizume et al. showed that SHMT2 mRNA levels are significantly reduced in the fibroblasts of old people when compared to younger individuals. The study also further indicated that decreased GCAT and SHMT2 levels of gene expression via shRNA and siRNA, respectively, in the fibroblasts of young patients led to a respiratory chain dysfunction typical for senile individuals-suggesting that an epigenetic mechanism may be the cause for the phenotypic change. As mitochondria plays a role in the development of the PD, further research into the area will help uncover any implications that mitochondrial DNA methylation plays in the pathogenesis of PD.

The use of dopaminergic neurons that have been isolated from the PD patients indicated that there were increases in acetylation (at H2A, H3 and H4) when compared to the age-control group. Another study involving MPP+ (a compound that can cause a disease state resembling mammals and humans with PD)-treated cells and (MPP+)-treated mouse brains showed decreased HDAC levels, as well as in midbrain samples from patients with PD. This is seen potentially due to how MPP+ promotes the breakdown of HDAC1 and HDAC2 via autophagy, a bodily process of cycling out old cells to make room for newer, healthier cells. These results point towards the stress of histone modifications in regards to chromatin remodeling and its implication in the pathogenesis of PD.

miRNAs are also emerging as relevant contributors to neurodegeneration in PD. In particular, the frontal cortex PD patients have shown higher levels of LRRK2 and lower levels of miR-205 when compared to healthy individuals. Connecting this to the findings of miR-205’s ability to bind to the 3′ UTR of LRRK2 mRNA and suppress expression, as well as miR-205’s prevention of defects after introduction to a R1441G LRRK2 mutation, these results point towards miR-205 and its regulatory role in LRRK2 expression-which in turn suggest a regulatory role in the pathogenesis of PD.

In another study in which increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 showed prevention of the association of mutant LRRK2 with microtubules, inhibition of deacetylases HDAC6 and Sirt2 through knockdown processes rescued both axonal transport and locomotor behavior. This further connects to the common mechanisms involving HDACi in various neurodegenerative diseases.

Bipolar Disorder

Bipolar disorders are both highly complex and heritable, which makes it an interesting disorder to examine for epigenetic modifications. DNA methylation, DNA hydroxymethylation, and histone modifications are all capable of contributing to the formation of bipolar disorder.  

For example, studies of monozygotic twins revealed that individuals with bipolar disorder had lower methylation of the peptidylprolyl isomerase E-like (PPIEL) gene, which can be attributed to the dopamine transmission. The studies indicated that hypermethylation of SLC6A4, a serotinin transporter gene, is also involved with bipolar disorder. Greater expression of DNA methyltransferase 1 in cortical GABAergic interneurons may enable hypermethylation. Hypermethylation may prompt hydroxymethylation to occur in order to overcompensate for the repressive effects of hypermethylation. The methylation of CpG regions are relevant to bipolar disorders. Patients with bipolar disorder showed lower methylation levels for the CpG region of the KCNQ3 gene, which is responsible for the voltage-gated K+ channel. Childhood maltreatment contributed to the methylation status of CpG2 III of 5-hydroxytryptamine 3A, which alters how maltreatment affects bipolar disorder.

Moreover, therapeutic interventions such as engineered transcription factors could modify chromatin structure to address the epigenetic changes found in those with bipolar disorder. DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors could possibly reverse epigenetic modifications in order to therapeutically address bipolar disorder. DNMT inhibitors and HDAC often produces antidepressant-like effects.