Manganese Health Research Program: Phase 2, Core 15
|Research Core Project Number:|
|Research Core Project:||Effect of manganese on glial-neuronal interactions|
|Core Principal Investigator (CPI):||Lucio G. Costa
University of Washington
Dept. of Environmental and Occupational Health Sciences
4225 Roosevelt #100, Seattle, WA 98105
Specific aims were formulated to address the following questions: 1) Does Mn exposure of astrocytes impair their ability to induce axonal and neurite outgrowth in neurons? 2) Is modulation of extracellular matrix proteins by Mn involved in its effect? 3) Does Mn exposure of astrocytes impair their ability to promote formation of synapses?
Manganese (Mn) neurotoxicity is characterized by a variety of psychiatric, cognitive and motor disturbances, the latter resembling parkinsonian symptoms. Mn is transported in brain by different mechanisms, and accumulates in the striatum, globus pallidus and substantia nigra, where concentrations of 200-300 uM can be found. Among brain cells, astrocytes accumulate this metal and concentrations of Mn 50-60-fold higher than in neurons can be indeed found. The exact mechanism(s) of Mn neurotoxicity are not known, but there is evidence that Mn can elicit oxidative stress, cause mitochondrial dysfunction, alter the homeostasis of glutamate, cause astrocytic swelling and alter the expression of a number of genes. There is emerging and convincing evidence that astrocytes play an essential role in fostering the development and survival of neurons. Indeed, astrocytes express and release a variety of factors, including neurotrophins, cytokines, growth factors, extracellular matrix proteins, proteoglycans and cholesterol, that have profound effects on neuronal proliferation, differentiation and survival of neurons, on neurite outgrowth and on synaptogenesis. By targeting astrocytes, neurotoxic compounds may thus indirectly affect neurons, by inhibiting several aspects of astrocyte-neuron interactions that are vital for the “well-being” of neuronal cells.The general hypothesis of this proposal is that Mn would impair the ability of these cells to promote differentiation of neurons. The proposed studies will thus focus of the rather novel paradigm of glial-neuronal interactions, which may provide important insights in the mechanisms of Mn neurotoxicity. Specific aims will address the following questions: 1) Does Mn exposure of astrocytes impair their ability to induce axonal and neurite outgrowth in neurons? 2) Is modulation of extracellular matrix proteins (fibronectin and laminin) and of PAI-1 by Mn involved in its effect? 3) Does Mn exposure of astrocytes impair their ability to promote formation of synapses, and is altered thrombospondin-1 involved in this effect? 4) Is oxidative stress involved in the effects of Mn on astrocytes?
We have developed both an in vivo and an in vitro model to understand the contribution of manganese to Parkinsonian symptoms. The principle goal of the in vivo studies was to determine if the behavioral phenotype of manganese exposure is due to selective loss of dopaminergic cells. To this end, Dr. Michael Aschner's laboratory prepared C57BL/6 mice animals that were given intraperitoneal injections of MnCl2 (5 mg/kg/day) or saline daily for 30 days. These animals were used to determine if manganese induced alterations in protein expression in the basal ganglia circuit, which would be consistent with a specific loss of dopaminergic neurons. The McLaughlin and Aschner labs collaboratively developed immunohistochemical analyses and cell counting techniques in which we determined there was a 17% reduction in neurons expressing tyrosine hydroxylase, an enzyme required for the production of dopamine, in the substantia nigra (SN) of Mn-treated animals. Quantification of Nissl bodies through cresyl-violet staining revealed a widespread reduction total neuronal number throughout the SN. One of the consequences of loss of dopaminergic cells would be a loss of GAD67, the rate-limiting enzyme in GABA production in the striatum, which receives projections from the substantia nigra. We observed decreases in numbers of GAD67 expressing interneurons within the STR and the GP of Mn-treated mice. This decrease was 39.4% in the STR and 14% in the GP. While we did not observe changes in the activated form of the cell death protein caspase 3 in the substantia nigra, striatum or other regions, we hypothesize that this was likely due to the fact that cleavage of this protease is an earlier event in the chronic exposure paradigm. The results of these studies suggest that Mn exposure can produce neurochemical dysfunction in key areas of the basal ganglia, including the striatum and globus pallidus. Ongoing experiments using primary cultures of ventral midbrain neurons are elucidating the cell signaling mechanism responsible for the selective vulnerability observed with Mn exposure.
|Project started: 4/15/2008|
|Scheduled completion date: 1/31/2010|
Stankowski J., Leitch D., Aschner M., McLaughlin B. and Stanwood G. D. (2008) Selective vulnerability of dopaminergic systems to Manganese: Relevance to occupational exposure. Neurotoxicology And Teratology 30, 259.
Key research accomplishments:
- Exposure of rat cortical astrocytes to Mn, followed by wash-out, decreased their ability to promote neurite outgrowth in hippocampal neurons.
- This effect of Mn was observed at concentrations that did not alter the viability of astrocytes and neurons.
- Anti-oxidants reversed the effect of Mn, while GSH depletion potentiated its effect, suggesting an involvement of Mn-induced oxidative stress in astrocytes.
- Mn caused a decrease in the levels of fibronectin protein and mRNA, which was also antagonized by antioxidants.
- Other oxidative stress-inducing compounds caused effects similar to Mn
- Results indicate that by targeting astrocytes, Mn impairs their ability to promote neuronal differentiation.
Publications/Presentations arising from project:
- Giordano G, Pizzurro D, VanDeMark K, Guizzetti M, Costa LG. Manganese inhibits the ability of astrocytes to promote neuronal differentiation. Toxicol. Appl. Pharmacol. 240: 226-235, 2009.
- Costa LG, Pizzurro D, Dao K, Guizzetti M, Giordano G. Manganese impairs the ability of astrocytes to promote neurite outgrowth in rat hippocampal primary neurons. Society of Toxicology Annual Meeting, Baltimore, MD, March 2009 (Toxicologist 108: 40, 2009).
- Pizzurro D, Giordano G, Guizzetti M, Costa LG. Manganese impairs fibronectin and plasminogen activator inhibitor-1 (PAI-1) expression in astrocytes. Pacific Northwest Association of Toxicologists (PANWAT) Annual Meeting, Seattle, WA, September 2009.
Glial-neuronal interactions are increasingly being recognized as playing a primary role in normal brain function and development. Our results show that exposure of astrocytes to Mn impairs their ability to promote differentiation of hippocampal neurons. Astrocytes are known to act as a “sink” for Mn. At concentrations that do not alter astrocyte viability, Mn affects their ability to promote neurite outgrowth in hippocampal neurons. This effect of Mn in astrocytes is most likely mediated by its ability to induce oxidative stress in these cells, and involves an effect of Mn on fibronectin, an extracellular matrix protein which has a neurite-promoting action. Other compunds causing oxidative stress also caused the same astrocyte-mediated impairment of neuritogenesis and of fibronectin expression. The effect of Mn was due solely to its action on astrocytes as direct exposure of hippocampal neurons to Mn did not affect neuritogenesis. These results show that by targeting astrocytes, Mn can alter an important aspect of glial-neuronal interactions, contributing to its overall neurotoxicity and developmental neurotoxicity.
Last updated: July 2011