BMP9 Protects Septal Neurons from Axotomy-Evoked Loss of Cholinergic Phenotype

Cholinergic projection from the septum to the hippocampus is crucial for normal cognitive function and degeneration of cells and nerve fibers within the septohippocampal pathway contributes to the pathophysiology of Alzheimer's disease. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiating factor during development both in vivo and in vitro.To determine whether BMP9 could protect the adult cholinergic septohippocampal pathway from axotomy-evoked loss of the cholinergic phenotype, we performed unilateral fimbria-fornix transection in mice and treated them with a continuous intracerebroventricular infusion of BMP9 for six days. The number of choline acetyltransferase (CHAT)-positive cells was reduced by 50% in the medial septal nucleus ipsilateral to the lesion as compared to the intact, contralateral side, and BMP9 infusion prevented this loss in a dose-dependent manner. Moreover, BMP9 prevented most of the decline of hippocampal acetylcholine levels ipsilateral to the lesion, and markedly increased CHAT, choline transporter CHT, NGF receptors p75 (NGFR-p75) and TrkA (NTRK1), and NGF protein content in both the lesioned and unlesioned hippocampi. In addition, BMP9 infusion reduced bilaterally hippocampal levels of basic FGF (FGF2) protein.These data indicate that BMP9 administration can prevent lesion-evoked impairment of the cholinergic septohippocampal neurons in adult mice and, by inducing NGF, establishes a trophic environment for these cells

Induction of Cell Signaling Events by the Cholera Toxin B Subunit in Antigen-Presenting Cells▿

Cholera toxin (CT) is one of the most effective and widely studied mucosal adjuvants. Although the ADP-ribosylating A subunit has been implicated in augmenting immune responses, the receptor-binding B subunit (CT-B) has greater immunogenicity and may be a repository of adjuvant activity without potential toxicity. In order to elucidate mechanisms of immune modulation by CT-B alone, primary B cells and macrophages were assessed for responses to CT-B in vitro, as measured by the expression of cell surface markers, cellular signaling events, and cytokine secretion. Increased phosphorylation of multiple signaling molecules, including Erk1/2 and p38, was detected. CT-B also induced transactivation of the transcription elements cyclic AMP-responsive element and NF-κB, the latter of which was inhibited by phosphotyrosine inhibition. While specific inhibition of MEK1/2 did not reduce CT-B induction of cell surface marker expression, it did attenuate CT-B-mediated interleukin-6 secretion. These data show that CT-B induces a set of signaling events related to cellular activation, surface molecule expression, and cytokine production that has potential implications for elucidating CT-B adjuvant activity in the absence of enzymatically active holotoxin

Retinoid-Dependent Restriction of Human Immunodeficiency Virus Type 1 Replication in Monocytes/Macrophages

Vitamin A deficiency has been correlated with increased severity of human immunodeficiency virus type 1 (HIV-1)-associated disease. Moreover, vitamin A supplementation can reduce AIDS-associated morbidity and mortality. Our group and others have shown that retinoids, the bioactive metabolites of vitamin A, repress HIV-1 replication in monocytic cell lines and primary macrophages by blocking long-terminal-repeat (LTR)-directed transcription. Based on these studies, we hypothesize that retinoids are natural repressors of HIV-1 in vivo. We show here that all-trans-retinoic acid (RA)-mediated repression of HIV-1 activation requires pretreatment for at least 12 h and is blocked by the protein synthesis inhibitors cycloheximide and puromycin. Studies of the kinetics of RA-mediated repression in U1 cells and primary monocyte-derived macrophages (MDMs) reveal that the repressive effects of RA on HIV-1 expression are long-lasting but reversible. We demonstrate that HIV-1 expression is activated when U1 cells or MDMs are cultured in retinoid-free synthetic medium and show that physiological concentrations of RA repress this activation. In addition, the synthetic pan-retinoic acid receptor antagonist BMS-204 493 activates HIV-1 replication in U1 cells in a dose-dependent manner, suggesting that RA-induced transactivation of cellular gene expression is required for HIV-1 repression. Together, these data support the hypothesis that retinoids present in tissue culture media in vitro and serum in vivo maintain HIV-1 in a transcriptionally repressed state in monocytes/macrophages

Sections of mouse brain showing a representative image of the unilateral transection of fimbria-fornix (top panel) and the ROI stained with an anti-CHAT antibody (Bottom Panel).

<p>CHAT-positive cells are seen in the medial septum, the diagonal band and in the striatum (bottom panel). The lines show the ROI used for cell count analysis in the medial septum. The ROI was defined by a triangular shape that extended, dorso-ventrally, from the apex of the medial septum (A) to an imaginary line connecting the lower limits of the anterior commissures on each hemisphere (B) and, medio-laterally, from the midline (A) to the outer limits of the medial septal area (C and D).</p

BMP9 protects medial septum cholinergic neurons from axotomy-evoked degeneration.

<p>CHAT-positive cells were counted as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021166#pone-0021166-g001" target="_blank">Fig. 1</a> on both sides of the medial septum and the average cell number on the lesioned side is expressed as % of the average cell number on the intact side. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021166#pone-0021166-g002" target="_blank">Fig. 2</a> for representative sections. Using the best fit to a rectangular hyperbola (R² = 0.89) the EC₅₀ value for BMP9 was 1 ng/h. Note, at 15 ng/h and 38 ng/h BMP9 prevented all loss of CHAT-positive neurons.</p

FGF2 protein levels in hippocampus (HPP).

<p>Mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021166#pone-0021166-g004" target="_blank">Fig. 4</a>. FGF2 was measured by ELISA (* p<0.05 BMP9 vs PBS on the same side).</p

NGFR-p75, TrkA and NGF protein levels in hippocampus (HPP).

<p>Mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021166#pone-0021166-g004" target="_blank">Fig. 4</a>. NGFR-p75 (** p<0.01 BMP vs PBS on the same side) and TrkA (significant treatment effect of BMP9 by two-way ANOVA, p<0.05) were measured by Western blot (top panel) and the data quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021166#pone-0021166-g005" target="_blank">Fig. 5</a>. NGF was measured by ELISA (* p<0.05, ** p<0.01 BMP9 vs PBS on the same side).</p

Representative photomicrographs of anti-CHAT antibody stained basal forebrain areas of mice with a unilateral septohippocampal lesion (right side of the images) following six days of constant intracerebroventricular infusion of vehicle (no BMP9) or BMP9 at doses indicated.

<p>Note a dramatic reduction of the number of CHAT-positive cells in the control brain and sparing of these neurons when BMP9 was administered.</p

Acetylcholine content in hippocampus measured by HPLC in mice with a unilateral septohippocampal lesion following seven days of constant intracerebroventricular infusion of vehicle or 8 ng/h of BMP9.

<p>Note a significant (*p<0.01; 72%) sparing of the loss of hippocampal ACh content in lesioned mice receiving BMP9.</p