Martino’s lab has contributed to the field of stem cell biology by demonstrating that adult neural stem/precursor cells (NPCs) posses a constitutive and inducible ‘immunotrophic’ signature which makes these cells – once in vivo transplanted – capable to protect the central nervous system (CNS) from inflammatory and degenerative insults. The concept of therapeutic plasticity emerged as the capacity of in vivo transplanted NPCs to act as therapeutic weapons not only by replacing damaged cells but also by promoting neuroprotection via the release of immunomodulatory and neurotrophic molecules. 

 

Our team investigates the cellular and molecular mechanisms of myelin repair in the central nervous system (CNS), with a strong emphasis on the role of stem cells from both the CNS and PNS in the remyelination of demyelinated lesions.

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In X-ALD, mutations of the ABCD1 gene lead to impaired β-oxidation of VLCFA in virtually all cell types. Inactivation of the homologous gene in mice shows a very late onset neuropathy starting at 16 months that is reminiscent of Adrenomyeloneuropathy (AMN), the adult onset form of X-ALD [1]. Interestingly, when peroxisomal functions are completely inactivated in oligodendrocytes (Cnp-Cre*Pex5), the mouse model exhibits axonal degeneration and progressive subcortical demyelination, very similar to the early onset cerebral childhood form of X-ALD [2]. Moreover, the disease phenotype of the conditional knockout mice is reminiscent of X-ALD patients regarding inflammation with infiltration of B and T lymphocytes into the brain lesions. Thus, human cerebral X-ALD may also be caused by a complete oligodendroglial dysfunction of peroxisomes. A similar but less severe phenotype is observed in astroglia-specific PEX5 knockout mice, while neuron-specific PEX5 mice lack an overt neuropathology [3]. These mouse models suggest that oligodendroglial and to a minor extend astroglial peroxisomes contribute to the disease phenotype of X-ALD.

We hypothesize that a critical step of X-ALD (i.e. secondary loss of peroxisomal function) occurs selectively in oligodendrocytes, (astroglia, miccroglia and adrenal cells), in which ABCD-1 deficient peroxisomes accumulate additional functional defects, most likely as a result of the cell type-specific metabolism and high degree of lipid and cholesterol turnover. These secondary defects are not linked to known ABCD1 functions, but they are likely to trigger demyelination, axonal loss, and neuroinflammation ("disease outbreak").

Such a secondary peroxisomal dysfunction caused by ABCD1-deficiency might be prevented by enhancing peroxisomal turnover, thereby shortening the lifetime of single peroxisomes and reducing the number of “old” peroxisomes that have acquired a secondary defect.

 

Experimental increase of peroxisomal turnover in the brain should be possible by the activation of peroxisome proliferator-activated receptors (PPARs). Thus, we are analyzing the effects of PPAR agonists that can cross the blood-brain-barrier on oligodendroglia and astroglia in cell-culture.We treated oli-neu cells, an oligodendrocyte progenitor cell line, primary oligodendrocytes, and primary astrocytes separately with specific ligands for all three PPAR isoforms. We used PPAR-α agonist fenofibrate, the synthetic PPAR-β/δ agonistGW0742, and pioglitazone as PPAR-γ agonist.

1)    PPAR agonist treatment in cell culture: Western blot analysis of primary astrocytes revealed a higher amount of peroxisomal membrane protein 70 (PMP70) after treatment with PPARaor PPARgagonist for 72 hours. When the cell culture conditions for oli-neu cells were adapted, we detected equivalent changes also in this oligodendroglial precursor cell line, indicating an increase of peroxisome steady-state number. Simultaneously, another peroxisomal membrane protein PEX11b, which is an essential mediator of peroxisomal division, was down regulated on protein and RNA level. Together these findings suggest that peroxisomal proliferation is achieved by enhanced peroxisomalde novo synthesis, in contrast to peroxisomal division. However, we measured only a moderate increase in the essential peroxisomal (de novo) biogenesis factor PEX16. At present, we are addressing the question of peroxisomal lifespan. To this end we use live-cell imaging of untreated versus agonist treated cells with fluorescently labeled peroxisomes. Additionally, the experiments that were performed on oli-neu cells and astrocytes are being repeated on microglia and primary oligodendrocytes, of which we have in the meantime optimized the quality (with respect to amount and purity) to analyze peroxisome-related gene expression by western blot and quantitative real-time PCR.

2)    PPAR agonist treatment in vivo: 9 treatment groups consisting of Abcd1 knockout and wildtype mice were treated, for one month, with different concentrations of the 3 PPAR agonists. Currently we are analyzing these 120 mice. The animals have tolerated the treatment well: Neither daily weight recordings, nor weekly motor performance tests have revealed any toxic effect of the applied substances. Preliminary data show great promise to efficiently increase peroxisomal number in vivo in the CNS.  

 

In summary, our in vitro andin vivo findings support the hypothesis that the turnover of myelin peroxisomes [4] can be enhanced by PPAR agonist treatment. We anticipate that an increased production (and thus turnover) of new peroxisomes will decrease the number of those organelles that are about to develop secondary dysfunction. Hence, any future therapeutic trial with PPAR agonists is an attempt to circumvent the underlying disease mechanism due to ABCD1 deficiency. Translated to humans, such a therapy could prevent disease in asymptomatic boys, and possibly stop progression from AMN to cerebral AMN.

 

References:

[1]           Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002). Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum Mol Genet 11, 499-505.

[2]           Kassmann, C.M. et al. (2007). Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes. Nat Genet 39, 969-76.

[3]           Bottelbergs, A., Verheijden, S., Hulshagen, L., Gutmann, D.H., Goebbels, S., Nave, K.-A., Kassmann, C. and Baes, M. (2010). Axonal integrity in the absence of functional peroxisomes from projection neurons and astrocytes. Glia 58, 1532-43.

[4]           Kassmann, C.M. et al. (2011). A role for myelin-associated peroxisomes in maintainingparanodal loops and axonal integrity. FEBS Lett. 585(14):2205-11.

 

Over the last 12 months we have continued our work of gaining a better understanding of how brain stem cells become oligodendrocytes - the cells that make myelin and that are lost in demyelinating diseases such as the leukodystrophies and multiple sclerosis. The highlights are three two major studies we have published (or that will be published soon). The first is in a collaboration with Charles ffrench-Constant (University of Edinburgh) and others describing a molecule, RXR, that encourages brain stem cells to become oligodendrocytes (Huang et al., 2011). The second, in collaboration with Steve Fancy and David Rowitch (UCSF), describes a pathway, the wnt pathway, which prevents stem cells becoming oligodendrocytes (Fancy et al., 2011). In both studies we have shown how these effects can be manipulated in such a way that the possibility of developing drugs that promote myelin regeneration (remyelination) now seems feasible. So, for example, if we either encourage RXR function or inhibit wnt function using small molecules that might form the basis of future drug development we can make remyelination work more efficiently in animal models of demyelination. These approaches may turn out to be important adjuncts to cell based therapies for leukodystrophies, helping transplanted cells to become new myelin-forming oligodendrocytes. In a third paper, a collaboration with Amy Wagers at Harvard, we have shown that the profound negative effects of age on remyelination are reversible (Ruckh et al., 2012). Over the next 12 months we hope to secure funds that will allow us to start to convert our exciting laboratory findings into new drug-based therapies that will directly help those affected with myelin diseases.

 

Fancy, S.P.J., Harrington, E.P., Yuen, T.J., Silbereis, J.C., Zhao, C., Baranzini, S.E., Bruce, C.C., Otero, J.J., Huang, E.J., Nusse, R., et al. (2011). Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination. Nat Neurosci14, 1009-1016.

Huang, J.K., Jarjour, A.A., Nait Oumesmar, B., Kerninon, C., Williams, A., Krezel, W., Kagechika, H., Bauer, J., Zhao, C., Baron van Evercooren, A., et al. (2011). Retinoid X receptor gamma signaling accelerates CNS remyelination. Nature Neuroscience14, 45-53.

Ruckh, J.M., Zhao, J.W., Shadrach, J.L., van Wijngaarden, P., Rao, T.N., Wagers, A.J., and Franklin, R.J.M. (2012). Rejuvenation of regeneration in the aging central nervous system. Cell Stem Cellin press.

 

We are focusing major efforts into the investigation of endogenous remyelination in the model of irradiated food ingestion with subsequent global demyelination and remyelination.  We are trying to determine whether adult oligodendrocytes are able to take part in remyelination.  The model will also be used to study the effects of relapsing disease on remyelination by studying multiple episodes.  We also plan to study this in mature/old animals to explore the affect of aging on repair.  In addition, we are using state-of-the-art MRI to evaluate demyelination and remyelination in the model.

 

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