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

Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C- terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT- immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.</jats:p

In Vitro and In Vivo Characterization of Two C-11-Labeled PET Tracers for Vesicular Acetylcholine Transporter

PURPOSE: The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective (11)C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo. PROCEDURES: For both (−)-[(11)C]2 and (−)-[(11)C]6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (−)-[(11)C]2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI. RESULTS: The resolved enantiomers (−)-2 and (−)-6 were very potent and selective for VAChT in vitro (K(i)<5 nM for VAChT with >35-fold selectivity for VAChT vs. σ receptors); both radioligands, (−)-[(11)C]2 and (−)-[(11)C]6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (−)-[(11)C]2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550±0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)-2, striatal uptake of (−)-[(11)C]2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (−)-[(11)C]2 displayed favorable in vivo stability in rat blood and brain. PET studies of (−)-[(11)C]2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold. CONCLUSIONS: The radioligand (−)-[(11)C]2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects

Syntheses and Radiosyntheses of Two Carbon-11 Labeled Potent and Selective Radioligands for Imaging Vesicular Acetylcholine Transporter

PURPOSE: The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. The syntheses and C-11 labeling of two potent enantiopure VAChT inhibitors are reported here. PROCEDURES: Two VAChT inhibitors, (±)-2 and (±)-6, were successfully synthesized. A chiral HPLC column was used to resolve the enantiomers from each corresponding racemic mixture for in vitro characterization. The radiosyntheses of (−)-[(11)C]2 and (−)-[(11)C]6 from the corresponding desmethyl phenol precursor was accomplished using [(11)C]methyl iodide or [(11)C]methyl triflate, respectively. RESULTS: The synthesis of (−)-[(11)C]2 was accomplished with 40–50 % radiochemical yield (decay-corrected), SA>480 GBq/μmol (EOB), and radiochemical purity >99 %. Synthesis of (−)-[(11)C]6 was accomplished with 5–10 % yield, SA>140 GBq/μmol (EOB), and radiochemical purity >97 %. The radiosynthesis and dose formulation of each tracer was completed in 55–60 min. CONCLUSIONS: Two potent enantiopure VAChT ligands were synthesized and (11)C-labeled with good radiochemical yield and specific activity