Limits...
Impairment of adult hippocampal neural progenitor proliferation by methamphetamine: role for nitrotyrosination.

Venkatesan A, Uzasci L, Chen Z, Rajbhandari L, Anderson C, Lee MH, Bianchet MA, Cotter R, Song H, Nath A - Mol Brain (2011)

Bottom Line: Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs).NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH.We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287, USA. avenkat2@jhmi.edu

ABSTRACT
Methamphetamine (METH) abuse has reached epidemic proportions, and it has become increasingly recognized that abusers suffer from a wide range of neurocognitive deficits. Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs). It is now well established that new neurons are continuously generated from NPCs in the adult hippocampus, and accumulating evidence suggests important roles for these neurons in hippocampal-dependent cognitive functions. In a rat hippocampal NPC culture system, we find that METH results in a dose-dependent reduction of NPC proliferation, and higher concentrations of METH impair NPC survival. NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH. We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress. Further, we identify seventeen NPC proteins that are post-translationally modified via 3-nitrotyrosination in response to METH, using mass spectrometric approaches. One such protein was pyruvate kinase isoform M2 (PKM2), an important mediator of cellular energetics and proliferation. We identify sites of PKM2 that undergo nitrotyrosination, and demonstrate that nitration of the protein impairs its activity. Thus, METH abuse may result in impaired adult hippocampal neurogenesis, and effects on NPCs may be mediated by protein nitration. Our study has implications for the development of novel therapeutic approaches for METH-abusing individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired.

Show MeSH

Related in: MedlinePlus

Establishment of NPC culture system in vitro. Adult hippocampal progenitors (NPCs) derived from Fisher rats were maintained under either proliferative or differentiating conditions. A. Under proliferative conditions, over 99% of cells are co-labeled with antibodies to nestin (green, cytoplasmic) and Ki67 (red, nuclear), markers of proliferating cells. B,C. Cells are exposed to proliferative conditions for 24 hrs in the presence of BrdU, followed by 5 days of differentiation conditions stain for BrdU (green), indicating that they were all initially proliferating cells. In addition, some cells in B co-label with Tuj1 (red, neuron), or RIP (blue, oligodendrocyte) and in C co-label with GFAP (blue, astrocyte). D. Immunostaining of cells derived from clonal NPCs grown under proliferative (FGF-2) or differentiating (FBS+RA) conditions yields reproducible percentages of Tuj1 (neuronal), GFAP (astrocytic), and RIP (oligodendroglial) positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3142219&req=5

Figure 1: Establishment of NPC culture system in vitro. Adult hippocampal progenitors (NPCs) derived from Fisher rats were maintained under either proliferative or differentiating conditions. A. Under proliferative conditions, over 99% of cells are co-labeled with antibodies to nestin (green, cytoplasmic) and Ki67 (red, nuclear), markers of proliferating cells. B,C. Cells are exposed to proliferative conditions for 24 hrs in the presence of BrdU, followed by 5 days of differentiation conditions stain for BrdU (green), indicating that they were all initially proliferating cells. In addition, some cells in B co-label with Tuj1 (red, neuron), or RIP (blue, oligodendrocyte) and in C co-label with GFAP (blue, astrocyte). D. Immunostaining of cells derived from clonal NPCs grown under proliferative (FGF-2) or differentiating (FBS+RA) conditions yields reproducible percentages of Tuj1 (neuronal), GFAP (astrocytic), and RIP (oligodendroglial) positive cells.

Mentions: NPCs were isolated from hippocampi of adult rats and maintained in culture media containing fibroblast growth factor (FGF) to ensure proliferation of these cells. Under these conditions, >99% of cells immunostained with antibody to nestin, a marker for NPCs (Figure 1A). These cells also stained for Ki67 a marker of proliferating cells (Figure 1A). To determine if these cells could be differentiated into various neural cell types, we initially treated them with BrdU to label the dividing cells and then changed the media to one containing fetal bovine serum (FBS) and retinoic acid (RA). Under these conditions, we found the presence of neuronal cells as demonstrated by immunostaining for Tuj1 (Figures 1B and 1C), astrocytes that immunostained for GFAP (Figure 1C) and oligodendrocytes that immunostained for RIP (Figure 1B). Nearly equal numbers (15-20%) of each of the cell types were present (Figure 1D). The remainder were undifferentiated NPCs. These self-renewing AHP thus fulfilled the definition of multipotent NPCs and were used for all further experiments.


Impairment of adult hippocampal neural progenitor proliferation by methamphetamine: role for nitrotyrosination.

Venkatesan A, Uzasci L, Chen Z, Rajbhandari L, Anderson C, Lee MH, Bianchet MA, Cotter R, Song H, Nath A - Mol Brain (2011)

Establishment of NPC culture system in vitro. Adult hippocampal progenitors (NPCs) derived from Fisher rats were maintained under either proliferative or differentiating conditions. A. Under proliferative conditions, over 99% of cells are co-labeled with antibodies to nestin (green, cytoplasmic) and Ki67 (red, nuclear), markers of proliferating cells. B,C. Cells are exposed to proliferative conditions for 24 hrs in the presence of BrdU, followed by 5 days of differentiation conditions stain for BrdU (green), indicating that they were all initially proliferating cells. In addition, some cells in B co-label with Tuj1 (red, neuron), or RIP (blue, oligodendrocyte) and in C co-label with GFAP (blue, astrocyte). D. Immunostaining of cells derived from clonal NPCs grown under proliferative (FGF-2) or differentiating (FBS+RA) conditions yields reproducible percentages of Tuj1 (neuronal), GFAP (astrocytic), and RIP (oligodendroglial) positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142219&req=5

Figure 1: Establishment of NPC culture system in vitro. Adult hippocampal progenitors (NPCs) derived from Fisher rats were maintained under either proliferative or differentiating conditions. A. Under proliferative conditions, over 99% of cells are co-labeled with antibodies to nestin (green, cytoplasmic) and Ki67 (red, nuclear), markers of proliferating cells. B,C. Cells are exposed to proliferative conditions for 24 hrs in the presence of BrdU, followed by 5 days of differentiation conditions stain for BrdU (green), indicating that they were all initially proliferating cells. In addition, some cells in B co-label with Tuj1 (red, neuron), or RIP (blue, oligodendrocyte) and in C co-label with GFAP (blue, astrocyte). D. Immunostaining of cells derived from clonal NPCs grown under proliferative (FGF-2) or differentiating (FBS+RA) conditions yields reproducible percentages of Tuj1 (neuronal), GFAP (astrocytic), and RIP (oligodendroglial) positive cells.
Mentions: NPCs were isolated from hippocampi of adult rats and maintained in culture media containing fibroblast growth factor (FGF) to ensure proliferation of these cells. Under these conditions, >99% of cells immunostained with antibody to nestin, a marker for NPCs (Figure 1A). These cells also stained for Ki67 a marker of proliferating cells (Figure 1A). To determine if these cells could be differentiated into various neural cell types, we initially treated them with BrdU to label the dividing cells and then changed the media to one containing fetal bovine serum (FBS) and retinoic acid (RA). Under these conditions, we found the presence of neuronal cells as demonstrated by immunostaining for Tuj1 (Figures 1B and 1C), astrocytes that immunostained for GFAP (Figure 1C) and oligodendrocytes that immunostained for RIP (Figure 1B). Nearly equal numbers (15-20%) of each of the cell types were present (Figure 1D). The remainder were undifferentiated NPCs. These self-renewing AHP thus fulfilled the definition of multipotent NPCs and were used for all further experiments.

Bottom Line: Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs).NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH.We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287, USA. avenkat2@jhmi.edu

ABSTRACT
Methamphetamine (METH) abuse has reached epidemic proportions, and it has become increasingly recognized that abusers suffer from a wide range of neurocognitive deficits. Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs). It is now well established that new neurons are continuously generated from NPCs in the adult hippocampus, and accumulating evidence suggests important roles for these neurons in hippocampal-dependent cognitive functions. In a rat hippocampal NPC culture system, we find that METH results in a dose-dependent reduction of NPC proliferation, and higher concentrations of METH impair NPC survival. NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH. We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress. Further, we identify seventeen NPC proteins that are post-translationally modified via 3-nitrotyrosination in response to METH, using mass spectrometric approaches. One such protein was pyruvate kinase isoform M2 (PKM2), an important mediator of cellular energetics and proliferation. We identify sites of PKM2 that undergo nitrotyrosination, and demonstrate that nitration of the protein impairs its activity. Thus, METH abuse may result in impaired adult hippocampal neurogenesis, and effects on NPCs may be mediated by protein nitration. Our study has implications for the development of novel therapeutic approaches for METH-abusing individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired.

Show MeSH
Related in: MedlinePlus