Two-photon Imaging as a Platform for the Real-time Evaluation of Human Glial Progenitor Cell Engraftment in the Lysosomal Storage Disorders

Maiken Nedergaard, M.D., D.M.Sc.

University Of Rochester Medical School

Funded in June, 2005: $200000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Determining Whether Embryonic Cells Can Reverse Deadly Inherited Metabolic Disorders

 Researchers will use two-photon cellular imaging in mouse models of two genetically determined “lysosomal storage” diseases, to determine how the diseases lead to accumulation of unprocessed metabolic products from brain cells, resulting in early childhood death.  Additionally, the researchers will graft into the mouse models embryonic “progenitor” cells, which may evolve into cells that clear away the aberrant metabolic products, and will monitor the effectiveness of this treatment through two-photon imaging that extends over several weeks.

Children with genetically determined lysosomal storage diseases, including Tay-Sach’s and Krebbe’s diseases, lack certain enzymes.  Without these enzymes, the children’s brain cells accumulate unprocessed metabolic products, while failing to produce the intended metabolic products needed.  Ordinarily, metabolic products are cleared away from brain cells by “astrocytes,” but in these children this does not occur.  The investigators hypothesize that the accumulation of the unprocessed metabolic products disrupts communication between astrocytes and between astrocytes and brain cells.  They also hypothesize that, by grafting in embryonic progenitor cells, these cells will evolve into functioning astrocytes, which can deliver the missing enzymes and produce clearance of the aberrant metabolic products.  The researchers will use two-photon imaging to test both hypotheses in mouse models of Tay-Sachs and Krabbe's diseases.

Significance:  If grafting embryonic cells into mouse models of these two deadly lipid storage diseases reverses cell damage, the animal model research would provide important evidence for possible human studies of this treatment approach.

ABSTRACT

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Two-photon Imaging as a Platform for the Real-time Evaluation of Human Glial Progenitor Cell Engraftment in the Lysosomal Storage Disorders

The childhood lysosomal storage disorders comprise a group of disorders in which congenital enzymatic deficiencies lead to the accumulation of unprocessed metabolic substrates, which ultimately cause the widespread death of both neurons and glial cells in the brain. These are overwhelmingly diseases of infants and young children, and most result in the unchecked death of the afflicted children. Over the past few years, we have addressed this issue from the standpoint of establishing a cell therapeutic approach to relieving the central nervous system manifestations of the lysosomal storage disorders and of other associated diseases of the cerebral white matter, the pediatric leukodystrophies. This proposal to the Dana Foundation is intended to capitalize upon recent advances in two-photon imaging technology, to test our hypothesis that the misaccumulation of lysosomal storage products in the lysosomal storage diseases may disrupt inter-glial and glial-neuronal communication, and thereby contribute to the poorly understood functional deficits of these disorders.

Using mouse models of both Sandhoff's/Tay-Sachs and Krabbe's diseases, we shall also test the corollary hypothesis that perinatal engraftment by purified preparations of glial progenitor cells might be sufficient to mitigate lysosomal storage product deposition in diseased host cells. To this end, we shall use 2-photon imaging to follow the treatment-associated clearance of mis-accumulated storage products in live host cortices. Together, these studies promise a wealth of new insight into the pathophysiology of neural dysfunction in the lysosomal storage diseases, to be acquired in the context of a potentially exciting new therapeutic strategy based on perinatal brain engraftment by glial progenitor cells.

HYPOTHESIS

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Hypothesis:
We will use 2-photon imaging to test the hypothesis that perinatal engraftment by wild-type glial progenitor cells might be sufficient to ameliorate or reverse lysosomal storage product deposition in diseased host cells in mouse models of both Sandhoff's/Tay-Sachs and Krabbe's diseases.

Goals:
The lysosomal storage disorders comprise a group of disparate disorders in which congenital enzymatic deficiencies lead to the lysosomal accumulation of unprocessed metabolic substrates. Using 2-photon imaging tor therapeutic assessment, we will test the possibility that perinatal engraftment by wild-type glial progenitor cells might be sufficient to ameliorate or reverse lysosomal storage product deposition in diseased host cells, in mouse models of both Sandhoff's/Tay-Sachs and Krabbe's diseases

Methods:
Through implanted cranial windows, we will analyze lysosomal storage products, as well as engrafted human glial progenitor cells by 2-photon imaging. The clearence of lysosomal storage products will be followed as a function of time after engraftment of human glial progenitor cells.

FINDINGS

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Lay Results:
Astrocytes have traditionally been regarded as the housekeeping cell of the nervous system. Novel imaging techniques have, in combination with electrophysiology, shown that astrocytes utilize Ca2+ signaling as a pathway to communicate with themselves and other cell types in the brain. We proposed to study astrocytic signaling in vivo as a platform to evaluate the success of engraftment of glial cell progenitors for treatment of lysosomal storage diseases. We have made progress and established several basic aspects of astrocytic Ca2+ signaling in vivo. Astrocytes respond to physiological stimulation and the responses of astrocytes are essential for activity dependent increases in blood flow. We have also found that astrocytic Ca2+ signaling is severely reduced in two mice models of lysosomal storage disease and partly reverted after implantation of glial cell progenitors.

Scientific Results:
Using funding from the Dana Foundation, we have provided evidence that show that astrocytes are activated during sensory stimulation (Wang et al., 2006). In these experiments, we used 2-photon imaging of astrocytes loaded with Ca2+ indicators in barrel cortex and demonstrated that whisker stimulation evoked increases in astrocytic Ca2+ by activation of mGlu receptors.  A similar approach was employed to show that astrocytes mediate functional hyperemia by release of a COX-1 metabolic product (Takano, 2006). In the last year of funding, we used NADH as an intrinsic marker of the metabolic state of the tissue. We found that the NADH signal was stable in unstimulated animals, but that spreading depression was associated with marked and highly heterogeneous changes in the NADH signal. The changes in NADH closely followed the vasculature and the pattern suggested that cells located most distant to capillaries can experience hypoxia in the absence of artery occlusion. Ongoing experiments test the responses (Ca2+ and NADH) of astrocytes in mice models of lysosomal storage diseases. Our hypothesis is that the misaccumulation of lysosomal storage products in the lysosomal storage diseases disrupts inter-glial and glio-neuronal communication, and thereby contributes to the poorly understood functional deficits of these disorders.  We have established that the responses of astrocytes to whisker stimulation are severely reduced and partly reverted after implantation of glial cell progenitors.

SELECTED PUBLICATIONS

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Cotrina M., Lin J.H., and Nedergaard M.  Adhesive properties of connexin hemichannels. Glia. 2008 Jul 22.

Lin J.H., Lou N., Kang N., Takano T., Hu F., Han X., Xu Q., Lovatt D., Torres A., Willecke K., Yang J., Kang J., and Nedergaard M.  A central role of connexin 43 in hypoxic preconditioning. J Neurosci. 2008 Jan 16;28(3):681-95.

Takano T., Han X., Peng W., Lou N., Lovatt D., Hansen A., Kasischke K., and Nedergaard M.  Cortical spreading depression causes and coincides with tissue hypoxia.  Nat Neurosci. 2007 Jun;10(6):754-62.

Ballabh P., Xu H., Hu F., Braun A., Rivera A., Ungvari Z., Goldman S.A., Csiszar A., and Nedergaard M.  Angiogenic inhibition reduces germinal matrix hemorrhage. Nat Med. 2007 Apr;13(4):477-85.

Lin J.H., Takano T., Arcuino G., Wang X., Hu F., Darzynkiewicz Z., Nunes M., Goldman S.A., and Nedergaard M.  Purinergic signaling regulates neural progenitor cell expansion and neurogenesis. Dev Biol. 2007 Feb 1;302(1):356-66.

Oberheim N.A., Wang X., Goldman S.A., and Nedergaard M.  Astrocytic complexity distinguishes the human brain.  Trends Neurosci. 2006 Oct;29(10):547-53

Wang X., Lou N., Xu Q., Tian G.F., Peng W.G., Han X., Takano T., and Nedergaard M.  Astrocytic Ca2+ signaling evoked by sensory stimulation in vivoNat Neurosci. 2006 Jun;9(6):816-23.

Anderson C. and Nedergaard M.  Emerging challenges of assigning P2X7 receptor function and immunoreactivity in neurons. Trends Neurosci. 2006 May;29(5):257-62.

Takano T., Tian G.F., Peng W., Lou N., Libionka W., Han X., and Nedergaard M.  Astrocyte-mediated control of cerebral blood flow. Nat Neurosci. 2006 Feb;9(2):260-7.