Lithium protects against glucocorticoid induced neural progenitor cell apoptosis in the developing cerebellum

Abstract

Respiratory dysfunction is one of the most common causes of death associated with premature birth (Barton et al., 1999). In the United States, 7-10% of pregnant women receive antenatal glucocorticoid (GC) therapy (Matthews et al., 2004), while approximately 19% of very low birth weight infants receive postnatal GC therapy (Jobe, 2009). Clinical research suggests that GC treatment causes permanent neuromotor and cognitive deficits (Yeh et al., 2004) and stunts cerebellar growth (Parikh et al., 2007; Tam et al., 2011). We previously reported that GC-mediated neural progenitor cell (NPC) apoptosis may be responsible for cerebellar neuropathology (Maloney et al., 2011; Noguchi et al., 2008, 2011). The goal of the current study was to determine whether lithium protects NPCs from GC neuroapoptosis in vivo and in vitro. Given that it protects against a range of brain insults, we hypothesized that lithium would significantly attenuate GC induced NPC toxicity. We report that acute lithium pretreatment provides potent, cell-intrinsic neuroprotection against GC induced NPC toxicity in vivo and in vitro.

Keywords: Apoptosis; Cerebellum; Dexamethasone; External granule layer; Glucocorticoid; Lithium; Neural progenitor cell; Neuroprotection.

Copyright © 2013 Elsevier B.V. All rights reserved.

Figures

Figure 1. Lithium protects against NPC apoptosis in the EGL

[A] The external granule layer (EGL) is a proliferative layer responsible for producing a massive number of granule cell neurons during cerebellar development. Once an NPC produces a granule cell neuron, it migrates (white circles with black arrows) past the molecular (ML) and Purkinje cell layers (PCL) before populating the internal granule cell layer (IGL) adjacent to the cerebellar white matter (WM). [B] A 3.0 mg/kg injection of dexamethasone (DEX) dramatically increases neural progenitor cell apoptosis in the EGL as measured by activated caspase-3 immunolabeling (black arrows). [C] Pretreatment with lithium chloride 15 minutes prior to DEX injection significantly lowers EGL apoptosis. [D] Limited amounts of physiological apoptosis can be seen in the saline control group.

Figure 2. Lithium permanently prevents GC induced apoptosis in vivo

[A] Semiquantitative degeneration scores reveal lithium carbonate (Li2CO3) potently blocks dexamethasone (DEX) induced apoptosis compared to saline (SAL) treatment. [B] Lithium chloride (LiCl) and Li2CO3 both potently protect against GC induced apoptosis. [C] Lithium pretreatment significantly reduces DEX induced apoptosis at 4.0 mEq/kg and above in a dose dependent manner. A control (CON) group exposed to no DEX or lithium exhibits low physiological levels of apoptosis which is significantly lower than DEX treatment alone. [D] Lithium protects against DEX induced EGL apoptosis at 6 hours however this toxicity returns at 12 hours. [E] Lithium neuroprotection against corticosterone (which has a shorter half-life than DEX) permanently blocks EGL apoptosis. Numbers in parentheses represent number of animals per group. Bonferroni post-hoc significance levels: *p < 0.001, **p < 0.01.

Figure 3. Dexamethasone induced apoptosis and lithium neuroprotection is cell intrinsic

[A] A concentration response curve shows EGL NPCs cultured in B27 media exhibit a significant increase in the percentage apoptotic cells at 20 μM and above. [B] When EGL NPCs are cultured in N2 media, DEX increases the percentage of apoptotic NPCs at 0.25 μM and above. [C] EGL NPCs cultured in B27 supplement alone show significant increases in the percentage of activated caspase-3 positive cells when compared to N2. [D] DEX concentration has no significant effect on proliferation (as measured by the percentage of KI-67 positive NPCs) in N2 media. [E] Lithium chloride protects against DEX induced apoptosis in a concentration dependent manner in cell culture. [F] The same cells show lithium has no effect on the percentage of KI-67 positive cells. Bonferroni post-hoc significance levels: *p