Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.     

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. The host immune response toward aerosol infection is to quarantine tubercle bacilli in a granulomatous structure (1, 2). However, granuloma-associated mycobacteria can switch to a non-replicative, “dormant” state and successfully evade immune response for decades after infection (3, 4). The metabolic events that permit tubercle bacilli to enter host cells and revive from states of persistence suggest that lipids are utilized as a carbon source (5–7). During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG)¹-enriched lipid droplets (8–10). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.Comparative sequence analysis of the Mtb genome has revealed that it contains 250 genes encoding enzymes involved in lipid metabolism compared with only 50 enzymes in Escherichia coli, which has a genome of comparable size. Among these genes, 150 are predicted to encode proteins involved in lipid catabolism (12, 13). A family of 24 carboxyl ester hydrolases called “lip” genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15–18).Tetrahydrolipstatin, a serine esterase inhibitor, covalently binds to and inhibits mammalian lipases and fatty acid synthase (FAS) and is marketed as “Orlistat” for the treatment of severe forms of obesity (19). THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20–22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23–25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria. Importantly, we systematically compared readouts of fluorescently tagged THL-proteins (7 bands on one-dimensional SDS-PAGE) with those of mass spectrometry-based peptide identification of enriched protein fractions (247 in growing cells). This comparison led to the identification of 14 THL targets, two of which were further validated experimentally. We furthermore provide a conceptual framework for the evaluation of this target list using both experimental as well as bioinformatics approaches in two examples, lipH and tesA. Overall, our data indicate that THL is an anti-mycobacterial drug because of its potential to (1) bind to a relatively wide range of lipolytic enzymes and (2) prevent bacilli from resuscitating from a nonreplicating persistent (NRP) state when lipid metabolism is particularly important.     

Anatomical substrate for neurotensin-acetylcholine interactions in the rat basal forebrain

We have previously shown by combined radioautography and acetylcholinesterase histochemistry that the distribution of ¹²⁵I-neurotensin (NT) binding sites was in register with that of cholinergic neurons in the rat nucleus basalis magnocellularis (NBM). The present study utilized three experimental approaches to elaborate on the type and cellular localization of NT binding sites in the NBM. Competition studies using levocabastine, a selective blocker of the low affinity NT binding component, revealed that most of the ¹²⁵I-NT binding sites labeled in the NBM are of the levocabastine-insensitive high affinity type, known to correspond to the physiologically active receptor. Ibotenic acid-induced lesions of the NBM produced a marked reduction in both cholinesterase reactivity and cellular ¹²⁵I-NT binding suggesting that most of the labeled sites are associated with the cholinergic neurons themselves rather than with an afferent input to those cells. Finally, examination of the high resolution radioautographic distribution of ¹²⁵I-NT binding sites in semithin sections revealed that a proportion of ¹²⁵I-NT-labeled receptors is associated with the plasma membrane of magnocellular perikarya and proximal processes, thereby providing an anatomical substrate for a local action of NT in the NBM.