Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF.

Neurodegenerative diseases can have long preclinical phases and insidious progression patterns, but the mechanisms of disease progression are poorly understood. Because quantitative accounts of neuronal circuitry affected by disease have been lacking, it has remained unclear whether disease progression reflects processes of stochastic loss or temporally defined selective vulnerabilities of distinct synapses or axons. Here we derive a quantitative topographic map of muscle innervation in the hindlimb. We show that in two mouse models of motoneuron disease (G93A SOD1 and G85R SOD1), axons of fast-fatiguable motoneurons are affected synchronously, long before symptoms appear. Fast-fatigue-resistant motoneuron axons are affected at symptom-onset, whereas axons of slow motoneurons are resistant. Axonal vulnerability leads to synaptic vesicle stalling and accumulation of BC12a1-a, an anti-apoptotic protein. It is alleviated by ciliary neurotrophic factor and triggers proteasome-dependent pruning of peripheral axon branches. Thus, motoneuron disease involves predictable, selective vulnerability patterns by physiological subtypes of axons, episodes of abrupt pruning in the target region and compensation by resistant axons.

An intrinsic distinction in neuromuscular junction assembly and maintenance in different skeletal muscles.

We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed "Fast Synapsing" (FaSyn) and "Delayed Synapsing" (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.

Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cows.

The objective of our study was to evaluate 2 pregnancy-associated glycoprotein (PAG)-based enzyme-linked immunosorbent assays (ELISAs) for use with either blood or milk. From 12 dairy farms, 116 Montbéliarde or Holstein cows were selected that had either undergone artificial insemination (AI; n = 102) or had calved (n = 14) 2-3 months earlier and had not undergone any further AI. Serum, plasma, and milk were obtained from all cows; serum and plasma were analyzed using the blood pregnancy test and milk using the milk pregnancy test. No false-positive results were observed when samples of the 14 noninseminated cows were tested. Cows undergoing AI were sampled at ~16, 30, and 41 days post-AI. An additional milk sample was taken at ~53 days post-AI. To establish whether the inseminated cows were pregnant, the cows were subjected to transrectal ultrasonography (TU) on or around day 41. Of the 102 inseminated cows, 63 were confirmed pregnant by TU. By day 30, the serum, plasma, and milk ELISAs demonstrated 100%, 100%, and 98.1% sensitivity and 88.6%, 88.9%, and 90.3% specificity, respectively, with potential pregnancy losses 30-41 days post-AI. Accuracy obtained on serum, plasma, and milk at ~41 days post-AI and on milk at ~53 days post-AI ranged from 97.4% to 100%. There were no differences of practical significance in performance between the blood and milk ELISAs for the sampling dates chosen. This new diagnostic capability with milk samples offers a major improvement in bovine reproductive management.

Real-time investigation of cytochrome c release profiles in living neuronal cells undergoing amyloid beta oligomer-induced apoptosis.

Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid ß oligomer (AßO)-induced apoptosis, as a model Alzheimer's disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy.

Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction.

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.