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

Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth.

Antifreeze proteins (AFPs) are extremely efficient at inhibiting ice recrystallization in frozen solutions. Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263] have proposed that this may be an important function of the proteins in freeze-tolerant organisms. We have tested this proposal in vitro by characterizing the influence of AFP on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during warming. Relatively low concentrations (e.g., 5-150 micrograms/ml) of winter flounder (Pseudopleuronectes americanus) AFP enhance survival of red blood cells cryopreserved in hydroxyethyl starch solutions. This effect is most apparent in samples warmed at suboptimal rates, i.e., where ice recrystallization would be exaggerated. Cryomicroscopy demonstrates that AFP inhibits ice recrystallization in the extracellular regions during the latter stages of the warming cycle. AFP concentrations that enhance survival of red cells confer partial inhibition of recrystallization. Relatively high concentrations of AFP (e.g., 1.54 mg/ml) are much more effective at inhibiting extracellular recrystallization. However, extensive growth of ice around the cell, and concomitant cell damage, is noted. The mechanism for this AFP-induced ice growth is unknown. We propose that there is a delicate balance between AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell damage and that this balance hinges on the degrees of inhibition of ice recrystallization and of preferential growth of ice around the cells. We conclude that, under appropriate conditions, one of the proposed functions of AFPs in nature can be emulated, and perhaps have application, in cryopreservation of materials of biomedical interest

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

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

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