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Inorganic mercury changes the fate of murine CNS stem cells¹

SABRINA CEDROLA^*,, GIANPAOLO GUZZI, DANIELA FERRARI, ANGELA GRITTI, ANGELO L. VESCOVI, JAMES C. PENDERGRASS and CATERINA A. M. LA PORTA^*,,2

^* Department of General Physiology and Biochemistry, Section of General Pathology, Milan, Italy;
Italian Association for Metal and Biocompatibility Research (AIRMEB), Milan, Italy;
Institute for Stem Cell Research, Department of Biotechnology, San Raffaele Hospital, Milan, Italy; and
Toxic Exposure Study Trust Foundation ALT, Lexington, Kentucky, USA

²Correspondence: Department of General Physiology and Biochemistry, Section of General Pathology, Celoria 26, Milan, Italy. E-mail: caterina.laporta{at}unimi.it

SPECIFIC AIMS

Central nervous system (CNS) stem cells can generate the three major types found in the adult brain—astrocytes, oligodendrocytes, and neurons—independent of the local environment. Environmental stimulation can differentially affect the proliferation, migration, and differentiation of these cells in vivo. We test here whether inorganic mercury influences cellular and molecular characteristics of these cells and their fate. Our data point to new insights about neurotoxicity and cellular injury mediated by mercury on adult neural stem cells (ANSCs). This appears to be of particular interest since there is growing evidence that heavy metals in general and mercury compounds in particular are toxic for the CNS. Therefore, the eventual effect of mercury on ANSCs might participate in the development of a neuropathology.

PRINCIPAL FINDINGS

1. Effect of inorganic mercury on proliferation of adult murine CNS stem cells
ANSCs were plated on 96 multiwells pretreated with Matrigel and incubated for 48 h with solution A or B (20 µg/mL or 50 µg/mL Hg²⁺, respectively, diluted 1:10)._. At the end of the treatments, proliferation rates were evaluated using colorimetric assay (WST-1 or BrdU). Both methods gave the same results. The ANSCs exposed to solution B (50 µg/mL Hg²⁺ diluted 1:10) showed a significant decrease in the proliferation (21%, P<0.001) with respect to untreated control, whereas solution A (20 µg/mL Hg²⁺ diluted 1:10) did not change the proliferative rate.

2. Effect of inorganic mercury on the differentiation pattern of ANSCs
ANSCs grow and expand in culture in an undifferentiated state. Nonetheless, plating stem cell progeny onto an adhesion substrate and removal of growth factors are generally sufficient to promote a spontaneous differentiation process, resulting in production of neuronal and glial cells. Seven days after plating in differentiating conditions, the neuronal/astroglial/oligodendroglial ratio was 15:75:1. We analyzed the possible effects induced by inorganic mercury on the differentiation pattern of ANSCs. Cells were exposed for 48 h to the toxin at different points in their differentiation process: either after the removal of growth factors (early differentiating cells) or 3 days after the removal of growth factor (late differentiating cells). As shown in Fig. 1 , in early differentiating cells treatment with solution A (20 µg/mL Hg²⁺ diluted 1:10) dramatically decreases the neuronal population (34–8.7%, a decrease of 75%). In fact, only scattered neurons are detected in treated vs. untreated cells (Fig. 1A-C , number 2). A similar but more striking effect is induced by solution B (50 µg/mL Hg²⁺, diluted 1:10; % of neuronal population decreased to 5.8%). On the other hand, the number and morphology of glial cells are unchanged after exposure to solution A (20 µg/mL Hg²⁺, diluted 1:10) (Fig. 1C, D compared with Fig. 1A, B , number 3); only after exposure to solution B (50 µg/mL Hg²⁺ diluted 1:10) did we observe a change in the morphology of astrocytes, but not in their total number (Fig. 1E , number 3).

View larger version (64K): [in this window] [in a new window]   Figure 1. The effect of mercury on differentiating ANSC cells.

In late differentiated ANSCs, inorganic mercury exerts a more dramatic effect on neuronal population, which disappears almost completely at both concentrations, whereas the effect on astrocytes is similar to that observed for early cultures.

3. Tubulin/GTP binding and ß-tubulin expression in inorganic mercury treated adult CNS stem cells
Tubulin polymerization, essential in the formation of microtubules, is dependent on GTP binding to theexchangeable E site on the ß-subunit of the tubulin dimer. Photoaffinity labeling of GTP to ß-tubulin was performed by incubating CNS stem cell homogenate with [³²P]GTP-AA for 30 min at 4°C, followed by photolysis for 60 s under UV light (254 nm). Electrophoresis (SDS-PAGE) separated ß-tubulin from other proteins. [³²P]GTP-AA-ß-tubulin interaction (55 kDa band protein) decreases compared with untreated ones, whereas the level of expression of ß-tubulin increases significantly, possibly as a compensatory effect.

4. Induction of heat shock protein (HSP-70/HSC-70) after inorganic mercury exposure
A 70 kDa family of stress proteins is one of the most extensively studied. It leads to protection in several different models of nervous system injury. Untreated CNS stem cells do not express per se HSP/HSC-70 (Fig. 2 ). On the other hand, solution B (50 µg/mL Hg²⁺, diluted 1:10) induces HSP/HSC-70 expression on undifferentiated ANSCs (Fig. 2) as assessed by Western blot. In differentiating and differentiated cells, only astrocytes are positive; neurons are always completely negative (Fig. 2) . Arising from the concentration of inorganic mercury, an increasing number of astrocytes expressing HSP/HSC-70 occurs (Fig. 2) .

View larger version (79K): [in this window] [in a new window]   Figure 2. Levels of expression of HSP-70 on differentiating ANSC cells.

CONCLUSIONS

To the best of our knowledge this is the first study demonstrating that, in ANSCs, inorganic mercury affects either the proliferative capacity or the differentiative pathways leading to neuronal/astrocyte cells. In spite of primate cells that constitutively expressed HSC70, CNS stem cells do not express HSC70 per se. Recently, HSP70 (whose functions are to assist in the maintenance of cellular integrity and viability, preventing, for instance, protein denaturation and incorrect polypeptide aggregation during exposure to physiochemical insults) was demonstrated to lead to protection in several different models of nervous system injury. Our data show that at both concentrations of inorganic mercury, binding of GTP to tubulin is inhibited whereas the level of expression of ß-tubulin increases under inorganic mercury exposure, perhaps as a compensatory effect. Thereby, inorganic mercury inhibiting polymerization of brain tubulin, which is particularly vulnerable to the toxic effect of inorganic mercury because it possesses an extremely reactive sulfhydryl pair, prevents its polymerization into microtubules. Furthermore, exposure to inorganic mercury alters a specific neurochemical process at the nucleotide level, which is involved in maintaining ANSC structure and mitosis.

Another important goal of the present paper was to study the effect of inorganic mercury on the fate of ANSCs. When we expose ANSCs to the toxicant either early or late in the differentiation process, neuronal population always appears significantly decreased at both inorganic mercury concentrations tested. In contrast, only morphological changes in astrocyte cell population occur. Considering that long-term low-level exposure to mercury vapor, the most relevant type of mercury exposure from a toxicological viewpoint, induces both motor and sensory dysfunctions, a plausible mechanism underlying these dysfunctions is that mercury is taken up at the nerve terminals and transported retrogradely in axons to the motor and sensory neurons. Astrocytes are the only cells positive for HSP/HSC70, whereas neurons are absolutely negative. These data confirm therefore that such proteins protect against apoptosis (actually only neuronal cells are decreased), leading to a resistance vs. inorganic mercury.

Figure 3 shows a schematic model suggesting the intracellular mechanism triggered by inorganic mercury that leads to a resistant phenotype of differentiating and differentiated astrocytes. In a high percentage of cells, inorganic mercury actually induces HSP/HSC-70 expression whereas GTP-tubulin activity decreases it. Both factors perhaps help the cells to survive and repair the damages induced by mercuric mercury. In differentiating/differentiated cells, inorganic mercury affects preferentially the neuronal population since they do not express HSP/HSC-70. However, morphological changes occur in astrocytes whose biological significances are unknown.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram.

These data open new perspectives in understanding the biological significance of inorganic mercury and the promotion of neuronal pathology and indicate ANSCs cultures as a model specific for toxicological studies, since it allows us to evaluate the different parameters of toxicity in standardized cultures at predetermined stages of proliferation/differentiation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0491fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0491fje

This Article Full Text (PDF) All Versions of this Article: 17/8/869 02-0491fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by CEDROLA, S. Articles by LA PORTA, C. A. M. Search for Related Content PubMed PubMed Citation Articles by CEDROLA, S. Articles by LA PORTA, C. A. M.