and Differentiation of Neural Progenitors and Adult Neural Stem Cells
he importance of glial cells in developmental neurobiology and clinical neuroscience is becoming increasingly clear. Emerging evidence indicates that glial cells are dynamic entities, whose ability to proliferate, migrate and differentiate is critical for brain development and for response to injury. Glial cells also express electrophysiological properties and coupling, thereby modulating neuronal function. Glial cells outnumber neurons as the brain mass increases in phylogenetically higher organisms, thus implicating these cells in the evolution of complex behavior. In broad terms, I am interested in defining the molecular events regulating development and pathology of a specialized glial cell type: the oligodendrocyte. These are the myelin-forming cells of the central nervous system. Previous studies suggested that they originate from immunophenotypically defined lineage restricted progenitors. However, several reports have challenged this theory and proposed an origin from multipotential progenitors, with the ability to generate neurons. This scientific debate has raised the need for conceptually innovative models of oligodendrocyte differentiation and for a redefinition of the cellular identity of these cells.
From a clinical perspective, oligodendrocyte differentiation occurs during the last gestational weeks of human development and, therefore, is severely compromised in preterm infants and in several conditions affecting this critical period, including deficient maternal diet (i.e. low iron), hormonal state (i.e. low thyroid hormone) or exposure to toxins (i.e. alcohol). Therefore, the study of mechanisms of oligodendrocyte differentiation is critical for the neurological development of the fetus and the neonate and for therapeutic intervention in a great number of clinical conditions associated with defective or severely delayed myelination and long-term neurological disabilities.
In the adult brain, progenitors retain the ability to differentiate into myelinating oligodendrocytes. This is a highly desirable property for repair of demyelinating lesions consequent to toxic, inflammatory, metabolic or ischemic damage. Remyelination in the adult brain is also essential for recovery of function after spinal cord injury or traumatic brain injury and in demyelinating disorders, such as multiple sclerosis. Defective differentiation of adult progenitors, in contrast, could lead to tumor formation when associated with mutations providing a proliferative advantage. Finally, recent evidence of white matter abnormalities and altered myelin gene expression in autism and schizophrenia has led to new interest in the role of these cells in complex neurological and psychiatric disorders.
My laboratory has a long-standing interest in the definition of the events at the transition between cell cycle exit and initiation of a developmental program of oligodendrocyte differentiation. Together with other laboratories, we have contributed to the definition of an obligate relationship between cell cycle exit and oligodendrocyte differentiation. The characterization of oligodendrocyte differentiation in mice, with targeted deletion of genes encoding for cell cycle inhibitors performed by our laboratory and others, reinforced this concept and suggested that cell cycle exit was the driving force for differentiation. However, over-expression of specific cell cycle inhibitors in proliferating progenitors stopped proliferation, but was not sufficient to also induce the progression to myelinating phenotype.
Because the proliferative state of a cell is associated with changes of chromatin components, we hypothesized that secondary modifications of nucleosomal histones, the basic unit of chromatin, would be the necessary element linking cell cycle exit to the activation of the oligodendrocyte differentiation program. Our laboratory was the first one to demonstrate this concept in the myelin field and it has now received independent validation by several other groups.
We suggested that the differentiation of progenitors into oligodendrocytes involves epigenetic repression of specific genes, and is associated with specific histone modifications. These changes in chromatin components favor the repression of inhibitory molecules that keep the progenitors unable to synthesize myelin genes, while repressing alternative fates. These studies were conducted by Marin-Husstege and Aixiao Liu, and later followed by graduate student Siming Shen, who graduated in May 2007, earned the Best Thesis Award 2007 from the Graduate School of Biomedical Sciences, received the Marian Kies Award and was invited to organize a symposium at the ASN Meeting in March 2008.
Dr. Liu’s work focused on two critical steps in oligodendrocyte differentiation: branching and myelin protein synthesis. His studies indicated both processes are essential for developmental myelination or remyelination in demyelinating disorders. His work demonstrated that specific cytoskeletal proteins, like stathmin, are crucial for the morphological change of oligodendrocytes. He also thoroughly characterized the mechanisms of action of the transcription factor Hes5, in modulating myelin gene expression. Most recently, Dr. Liu addressed the critical question of whether oligodendrocyte progenitors retain the plasticity to revert to neural stem cells and differentiate into other lineages. This question, in an era of translational research, is critical for its important therapeutic implications. For a long time, it was believed that oligodendrocytes originate from “committed” progenitor cells (OPCs). However, it was reported by the group of M.C. Raff and colleagues that these progenitor cells have the ability to revert to a stem-like cell state. Using pharmacological manipulation of histone acetylation, Dr. Liu demonstrated that OPCs can be reverted into stem-like cells and these “reverted” stem-like cells could acquire a different fate and become neurons and astrocytes both in vitro and in vivo. These findings may provide some understanding of the underlying mechanism controlling the lineage choice of stem cells, and were recently highlighted for their relevance to neural development in Nature Neuroscience. Studies are being carried out in our laboratory to further define the underlying mechanisms and their potential for clinical applications.
Another important contribution of our laboratory to the field of oligodendrocyte differentiation was made by graduate student, Ye He, who noticed that one quarter of the genes regulated by histone deacetylation during oligdendrocyte differentiation have the ability to bind a protein called Yin Yang 1 (YY1). She demonstrated that YY1 is expressed in the oligodendrocyte lineage, and that conditional knockout mice for this gene have a very severe clinical phenotype, characterized by tremor, ataxia, and head wobbling, and progressively deteriorate to paralysis. Additional ultrastructural and immunohistochemical studies revealed severe hypomyelination in the central nervous system of these mice. Ye He then identified the mechanisms responsible for this phenotype and found that deletion of yy1 blocked the differentiation of oligodendrocyte progenitors due to persistent histone acetylation and high levels of transcriptional inhibitors. These innovative findings were recently published in the prestigious journal Neuron. She then continued to characterize the role of YY1 in the peripheral nervous system and discovered that YY1 can modulate the ability of Schwann cells to myelinate axons, and is testing the hypothesis that these findings might provide benefits for treatments aimed at remyelination from spinal cord injury.
Collectively, the work of our laboratory has shown that the activity of enzymes catalyzing the removal of the acetyl group from nucleosomal histones (HDAC) was necessary for oligodendrocyte differentiation in vitro and for developmental myelination in vivo. We have also shown that systemic administration of HDAC inhibitors in neonatal rats, and even in zebrafish, results in complete inhibition of myelination in the developing brain. We don’t yet know, however, whether the same events that regulate developmental myelination also modulate remyelination in pathological conditions. To define our work’s translational relevance, we have recently started to investigate the role of HDAC activity and HDAC inhibitors in the adult brain by characterizing the mechanisms involved in adult progenitor differentiation and new myelin formation in the demyelinated brain. The long-term goal is to identify “cell-specific” pathways affected by HDAC in order to design therapies targeted at promoting repair for demyelinating conditions, including multiple sclerosis.
Our laboratory’s second area of study is the mechanism of control of proliferation of adult neural stem cells. We’re interested in discovering the molecules controlling the decision of neural stem cells to either proliferate and/or differentiate. When proliferation is “under control” in the developing brain, it is crucial to generate the appropriate number of cells. In the adult brain, “controlled proliferation” is also beneficial because it allows “new” cells to replace those that were lost after an injury, stroke, or infections. However, when proliferation is “out of control,” it can be very harmful and lead to brain tumors. Typically, a biochemical approach is used to identify the molecules responsible for a specific event (e.g. proliferation). We then ask if this molecule is necessary or “dispensable” to obtain the desired response (either proliferation or growth arrest) using a loss of function approach in vitro (by gene transfer of dominant negative molecules) and in vivo (by phenotypic analysis of knockout animals). Finally, we ask whether the identified molecule by itself is sufficient to induce the desired response, using a gain of function approach. Although stem cell therapy has been proposed for repair, still relatively little is known about the behavior of these cells in the adult injured CNS. Our laboratory is attempting to define how mitogens affect the proliferative potential of adult SVZ cells and how dysregulation of cell cycle molecules and tumor suppressor genes leads to glioblastomas, a very common and devastating brain tumor. Based on the proteomic and gene profiling studies, we are attempting to define molecular mechanisms and downstream signaling networks that modulate lineage determination and survival.
Patrizia Casaccia-Bonnefil earned an MD from UCSC (Catholic University), Rome, Italy, and a PhD in cell biology from SUNY-Health Sciences Center at Brooklyn (HSCB). Her postgraduate training included a residency in neurology at UCSC, a research fellowship at the Institute for Basic Research, Staten Island, NY, and a post-doctoral fellowship at Cornell University Medical College. She was a research associate at the Skirball Institute for Biomolecular Medicine and is currently an associate professor in the Department of Neuroscience and Cell Biology at UMDNJ-Robert Wood Johnson Medical School and a member of the Cancer Institute of NJ. Her research is supported by the National Institutes of Health, the National Multiple Sclerosis Society, The Christopher and Dana Reeves Foundation, the Multiple Sclerosis Research Foundation and the New Jersey Commission on Traumatic Brain Injury. A grant from the New Jersey Commission on Science and Technology supports a focus on the critical areas of gene delivery and differentiation of human embryonic stem cells.