The F-box protein MEC-15 (FBXW9) promotes synaptic transmission in GABAergic motor neurons in C. elegans.

Ubiquitination controls the activity of many proteins and has been implicated in almost every aspect of neuronal cell biology. Characterizing the precise function of ubiquitin ligases, the enzymes that catalyze ubiquitination of target proteins, is key to understanding distinct functions of ubiquitination. F-box proteins are the variable subunits of the large family of SCF ubiquitin ligases and are responsible for binding and recognizing specific ubiquitination targets. Here, we investigated the function of the F-box protein MEC-15 (FBXW9), one of a small number of F-box proteins evolutionarily conserved from C. elegans to mammals. mec-15 is widely expressed in the nervous system including GABAergic and cholinergic motor neurons. Electrophysiological and behavioral analyses indicate that GABAergic synaptic transmission is reduced in mec-15 mutants while cholinergic transmission appears normal. In the absence of MEC-15, the abundance of the synaptic vesicle protein SNB-1 (synaptobrevin) is reduced at synapses and increased in cell bodies of GABAergic motor neurons, suggesting that MEC-15 affects the trafficking of SNB-1 between cell bodies and synapses and may promote GABA release by regulating the abundance of SNB-1 at synapses.

Voltage-dependent anion channels (VDACs) recruit Parkin to defective mitochondria to promote mitochondrial autophagy.

BACKGROUND: Parkin is recruited to defective mitochondria to promote degradation by an autophagy mechanism (mitophagy). RESULTS: VDACs specifically interact with Parkin on defective mitochondria and are required for efficient targeting of Parkin to mitochondria and subsequent mitophagy. CONCLUSION: VDACs recruit Parkin to defective mitochondria. SIGNIFICANCE: A novel mechanistic aspect of Parkin-dependent mitophagy is proposed that may be relevant to Parkinson disease. Mutations in the ubiquitin ligase Parkin and the serine/threonine kinase PINK1 can cause Parkinson disease. Both proteins function in the elimination of defective mitochondria by autophagy. In this process, activation of PINK1 mediates translocation of Parkin from the cytosol to mitochondria by an unknown mechanism. To better understand how Parkin is targeted to defective mitochondria, we purified affinity-tagged Parkin from mitochondria and identified Parkin-associated proteins by mass spectrometry. The three most abundant interacting proteins were the voltage-dependent anion channels 1, 2, and 3 (VDACs 1, 2, and 3), pore-forming proteins in the outer mitochondrial membrane. We demonstrate that Parkin specifically interacts with VDACs when the function of mitochondria is disrupted by treating cells with the proton uncoupler carbonyl cyanide p-chlorophenylhydrazone. In the absence of all three VDACs, the recruitment of Parkin to defective mitochondria and subsequent mitophagy are impaired. Each VDAC is sufficient to support Parkin recruitment and mitophagy, suggesting that VDACs can function redundantly. We hypothesize that VDACs serve as mitochondrial docking sites to recruit Parkin from the cytosol to defective mitochondria.

An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans.

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking.

Concentration measurements by PULCON using X-filtered or 2D NMR spectra.

Sample concentrations can be measured by nuclear magnetic resonance (NMR) spectroscopy without an internal reference compound using pulse length based concentration determination (PULCON) with 1D NMR spectra. PULCON delivers most accurate results if the spectrum of the sample of interest contains a resolved resonance; but can also be applied to spectral regions with overlapping resonances. If the exact number of lines contributing to the overlapping spectral region is not known, a corresponding error makes the result less precise. The uncertainty about the number of contributing resonances can be reduced with experiments that discriminate different classes of resonances by filtering techniques or by extending PULCON to two-dimensional NMR spectra. We demonstrate the application of PULCON with a 1D 15N-filtered experiment where aromatic resonances of a protein can be observed without interference from 15N-bound protons. Further, we extend PULCON to 2D NMR spectra that permits to determine the exact number of resonances. This extension can readily be applied with sample that contain different solutes.

Measuring protein concentrations by NMR spectroscopy.

In applications of NMR to biological macromolecules in solution, the concentration of the NMR sample is an important parameter describing the sample and providing information for the selection and planning of experiments. Although concentrations can be measured directly by NMR spectroscopy, other methods are usually preferred to measure the concentration of macromolecules in NMR samples. The reasons are the difficulties in the correlation of the sample of interest with the signal intensity representing a known concentration. This correlation is usually obtained by adding to the sample a reference compound with known concentration and comparing the integral over resolved resonance lines of the molecules with known and unknown concentrations. For solutions of biological macromolecules it is very difficult to find a compound that does not interact with the macromolecules and has a resonance outside their spectral range. We introduce PULCON which is a method that correlates the absolute intensities of two spectra measured in different solution conditions. PULCON is easy to implement and apply on all NMR spectrometers; it does not need any special hardware or software. PULCON is very robust and at the same time delivers accurate concentrations of samples in the NMR tube. We demonstrate that PULCON has the potential to replace UV spectroscopy for concentration measurements of NMR samples.

LIN-23-mediated degradation of beta-catenin regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans.

Ubiquitin-mediated protein degradation has been proposed to play an important role in regulating synaptic transmission. Here we show that LIN-23, the substrate binding subunit of a Skp1/Cullin/F Box (SCF) ubiquitin ligase, regulates the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of C. elegans. Mutants lacking lin-23 had an increased abundance of GLR-1 in the ventral cord. The increase of GLR-1 was not caused by changes in the ubiquitination of GLR-1. Instead, SCF(LIN-23) regulates GLR-1 through the beta-catenin homolog BAR-1 and the TCF/Lef transcription factor homolog POP-1. We hypothesize that LIN-23-mediated degradation of BAR-1 beta-catenin regulates the transcription of Wnt target genes, which in turn alter postsynaptic properties.

Ubiquitin and AP180 regulate the abundance of GLR-1 glutamate receptors at postsynaptic elements in C. elegans.

Regulated delivery and removal of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.