Research Overview:

Within the context of our work on glial progenitor cells, we are now focusing on the different stages of astrocytic differentiation and the role of astrocytes as critical modulators in response to injury or stress. The importance of understanding this process is emphasized by our recent discovery that the generation of mature astrocytes may be impaired in Vanishing White Matter leukodystrophy (Nat Med. 2005 Mar;11(3):277-83.). The ability to study astrocyte development in normal and pathological conditions, provides a unique opportunity to test the utility of glial precursor cells and their astrocytic progeny for cell transplantation therapy in genetic CNS disease paradigms, such as Vanishing White Matter leukodystrophy.

A comprehensive approach to CNS precursor biology

The potential of stem cells for cell replacement therapies has placed them at the center of what many believe will be the next biomedical revolution. During normal development, stem cells sequentially proliferate and differentiate, thereby giving rise to the various differentiated cells that make up adult tissues. Even though this ability to generate new cells, and the potential to replace diseased cell populations have received the greatest attention, other properties of these precursor cells are equally important for the development of effective therapies.

An increasing number of neurological diseases are thought to be caused or exacerbated by defects in glial cells. Consequently, we have focused on the study of glial precursor cells. In collaboration with the laboratories of Dr. Noble and Dr. Mayer-Pröschel, who through their pioneering work were instrumental in identifying the two best characterized glial-restricted progenitor cells known to date, we have established an experimental system of early glial differentiation. This system has not only allowed us to identify distinct classes of glial progenitors within the context of normal development, but also provides an essential tool-set to study the role of precursor cells in disease processes.

1) Lineage restriction and the astroglial cell fate
Although the exact mechanisms of progressive lineage restriction of stem cells and progenitor cells in the CNS remain largely unknown, it is clear that lineage-restricted progenitor cells are the real work-horses of organogenesis and tissue building. Stem cells in contrast, function as a source of the lineage-restricted progenitor cells and only in the earliest stages of development do they constitute a numerically significant population in any tissue. Consequently understanding the signals that control the proliferation and lineage decisions of restricted progenitors, will be essential to understanding tissue formation and repair.

Based on our studies of Vanishing White Matter leukodystrophy and collaborative studies of spinal cord injury paradigms (see below), we have become increasingly interested in the importance and heterogeneity of astrocytes. Astrocytes have been studied in specific developmental and disease paradigms, and while GFAP continues to be the most frequently employed defining marker of astrocytes, it is clear that phenotypes can vary dramatically between astrocytes found during development, in acute lesions and in glial scar tissue.

My laboratory has taken a two-pronged approach towards studying the generation of astrocytes: (a) the micro-array based analysis of astrocyte differentiation and (b) the systematic development of a library of markers that can be used to identify and purify distinct neural cell populations, including astrocyte progenitors and different astrocytic sub-populations. In both cases we have taken advantage of our ability to generate enriched populations of two distinct types of astrocytes from glial restricted progenitor (GRP) cells. These astrocyte populations, referred to as GRP-Derived Astrocyte type 1 (GDA1) and type-2 (GDA2) astrocytes, differ in multiple properties including their morphology, marker expression and in their ability to support neurite outgrowth. Some have likened GDA1 astrocytes to astrocytes found during normal development, while GDA2 have been suggested to resemble reactive astrocytes formed in response to numerous CNS insults.

2) Precursor cells and disease.
The complexity and plasticity of the mammalian nervous system have been major obstacles in identifying the cell biological processes that result in the manifestation of a disease. This is particularly true for genetic diseases, in which the causative mutations may have been identified, but the consequences at the cellular or tissue level remain unclear, thereby dramatically limiting our ability to devise effective therapies.

My laboratory is particularly interested in the effects of genetic mutation on the function of human glial precursor cells. The successful isolation and continued propagation of human neural precursors has only recently been accomplished and the realization that genetic diseases may have their origin in the disruption of lineage development is a very new concept. The research conducted in my lab has provided the first example of a devastating genetic disease of the CNS from whom the precursor cells of an afflicted patient have been isolated, a cellular phenotype was identified and recreated by targeting the appropriate protein in normal human neural precursors. These studies were conducted on one of the heritable leukodystrophies for which identification of mutations per se has not provided insights into cellular pathophysiology. The autosomal recessive Childhood Ataxia with Diffuse CNS Hypomyelination (CACH)/Vanishing White Matter Disease (VWM) is known to be associated with mutations in the subunits of translation initiation factor 2B (eIF2B), yet the lack of a suitable experimental system in which to directly test the effect of eIF2B mutations has been a major obstacle in understanding the etiology of CACH/VWM disease.

We have discovered that neural precursors from the brain of a CACH/VWM disease patient with known mutations in the e subunit of eIF2B (EIF2B5) are specifically compromised in their ability to generate astrocytes. Analysis of early cultures derived from the CNS of a VWM disease patient, using lineage specific markers revealed the presence of neurons, oligodendrocytes and astrocytes. In light of the clinical pathology, we initially focused on the oligodendrocyte compartment. Contrary to our expectations, oligodendrocytes derived from the patient’s brain appeared normal by morphological criteria and progressive maturation of oligodendroglial lineage cells could be observed in culture. Surprisingly, however, only few glial fibrillary acidic protein (GFAP) expressing cells were present and most of these cells exhibited an atypical morphology. In addition, generation of GFAP+ astrocytes from precursor cells was severely impaired, even under conditions known to promote astrocyte differentiation.

3) Precursor cells and tissue repair paradigms.
The third approach, and more conventional application of precursor cells in disease paradigms, is the transplantation of these cells into models of genetic disease or injury of the CNS. Within this context we are studying the usefulness of early glial progenitors in different paradigms: (a) as a means of replacing injured cells, (b) for the stimulation of endogenous repair mechanisms, and (c) to modulate glial scaring.

How GRP-derived astrocytes modulate glial scar formation when transplanted into acute spinal cord lesions is part of a study in collaboration with Drs. Mark Noble, Margot Mayer-Proschel, Jeannette and Stephen Davies (University of Colorado). Initial experiments demonstrate a dramatic effect on various parameters of the scar, including the levels of proteoglycan expression, the ultrastructural organization of the scar, and also in the degree of neurite extension and functional recovery obtained as a consequence of cell transplantation. The effects observed depend on which GRP-derived astrocyte cell type is transplanted. As part of our studies on the mechanisms controlling the differentiation of these separate astrocyte cell types, my lab is focusing on the distinct properties of these astrocytes to determine what may be causing this modification of the scar in situ. Our discovery of signaling molecules that enable the specific generation and expansion of astrocyte precursor cell populations now provides a novel new tool for use in CNS repair. The dramatic effects of GRP-derived astrocytes in spinal cord injury repair raise the question of whether astrocyte-specific progenitor cells will be similarly useful in these regards.

Outlook:

The ability to isolate and study glial lineage cells provides a unique opportunity to investigate molecular disease mechanisms in otherwise intractable neurodegenerative diseases. This also provides a tool to develop new therapeutic strategies, whether through direct transplantation of specialized precursors that have a high probability of generating the desired cell type in situ, or as an in vitro screening system for use in drug development. With a focus on astrocyte and glial biology, we are now extending these studies to include human CNS and embryonic stem cells.

1. Li Z, Dong T, Pröschel C, Noble M.
   Chemically diverse toxicants converge on Fyn and c-Cbl to disrupt precursor cell function. PLoS Biol. 2007 Feb;5(2):e35.
   PMID: 17298174 [PubMed - indexed for MEDLINE]

2. Davies JE, Huang C, Proschel C, Noble M, Mayer-Proschel M, Davies SJ.
   Astrocytes derived from glial-restricted precursors promote spinal cord repair. J Biol. 2006;5(3):7. Epub 2006 Apr 27.
   PMID: 16643674 [PubMed - indexed for MEDLINE]

3. Noble M, Mayer-Pröschel M, Pröschel C.
   Redox regulation of precursor cell function: insights and paradoxes. Antioxid Redox Signal. 2005 Nov-Dec;7(11-12):1456-67. Review.
   PMID: 16356108 [PubMed - indexed for MEDLINE]

4. Dietrich J, Lacagnina M, Gass D, Richfield E, Mayer-Pröschel M, Noble M, Torres C, Pröschel C.
   EIF2B5 mutations compromise GFAP+ astrocyte generation in vanishing white matter leukodystrophy. Nat Med. 2005 Mar;11(3):277-83. Epub 2005 Feb 20.
   PMID: 15723074 [PubMed - indexed for MEDLINE]

5. Hill CE, Proschel C, Noble M, Mayer-Proschel M, Gensel JC, Beattie MS, Bresnahan JC.
   Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration. Exp Neurol. 2004 Dec;190(2):289-310.
   PMID: 15530870 [PubMed - indexed for MEDLINE]

6. Noble M, Pröschel C, Mayer-Pröschel M.
   Getting a GR(i)P on oligodendrocyte development.Dev Biol. 2004 Jan 1;265(1):33-52. Review.
   PMID: 14697351 [PubMed - indexed for MEDLINE]

7. Gregori N, Pröschel C, Noble M, Mayer-Pröschel M.
   The tripotential glial-restricted precursor (GRP) cell and glial development in the spinal cord: generation of bipotential oligodendrocyte-type-2 astrocyte progenitor cells and dorsal-ventral differences in GRP cell function. J Neurosci. 2002 Jan 1;22(1):248-56.
   PMID: 11756508 [PubMed - indexed for MEDLINE]

8. Herrera J, Yang H, Zhang SC, Proschel C, Tresco P, Duncan ID, Luskin M, Mayer-Proschel M.
   Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes and oligodendrocytes in vivo. Exp Neurol. 2001 Sep;171(1):11-21.
   PMID: 11520117 [PubMed - indexed for MEDLINE]

9. Frangiskakis JM, Ewart AK, Morris CA, Mervis CB, Bertrand J, Robinson BF, Klein BP, Ensing GJ, Everett LA, Green ED, Pröschel C, Gutowski NJ, Noble M, Atkinson DL, Odelberg SJ, Keating MT.
   LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition. Cell. 1996 Jul 12;86(1):59-69.
   PMID: 8689688 [PubMed - indexed for MEDLINE]

10. Tassabehji M, Metcalfe K, Fergusson WD, Carette MJ, Dore JK, Donnai D, Read AP, Pröschel C, Gutowski NJ, Mao X, Sheer D.
    LIM-kinase deleted in Williams syndrome. Nat Genet. 1996 Jul;13(3):272-3. No abstract available.
    PMID: 8673124 [PubMed - indexed for MEDLINE]

11. Mao X, Jones TA, Williamson J, Gutowski NJ, Pröschel C, Noble M, Sheer D.
    Assignment of the human and mouse LIM-kinase genes (LIMK1; Limk1) to chromosome bands 7q11.23 and 5G1, respectively, by in situ hybridization. Cytogenet Cell Genet. 1996;74(3):190-1. No abstract available.
    PMID: 8941371 [PubMed - indexed for MEDLINE]

12. Pröschel C, Blouin MJ, Gutowski NJ, Ludwig R, Noble M.
    Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro. Oncogene. 1995 Oct 5;11(7):1271-81.
    PMID: 7478547 [PubMed - indexed for MEDLINE]

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