Judith A. Steen

Brain-Specific Phosphorylation of MeCP2 Regulates Activity-Dependent Bdnf Transcription, Dendritic Growth, and Spine Maturation

Mutations or duplications in MECP2 cause Rett and Rett-like syndromes, neurodevelopmental disorders characterized by mental retardation, motor dysfunction, and autistic behaviors. MeCP2 is expressed in many mammalian tissues and functions as a global repressor of transcription; however, the molecular mechanisms by which MeCP2 dysfunction leads to the neural-specific phenotypes of RTT remain poorly understood. Here, we show that neuronal activity and subsequent calcium influx trigger the de novo phosphorylation of MeCP2 at serine 421 (S421) by a CaMKII-dependent mechanism. MeCP2 S421 phosphorylation is induced selectively in the brain in response to physiological stimuli. Significantly, we find that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription. These findings suggest that, by triggering MeCP2 phosphorylation, neuronal activity regulates a program of gene expression that mediates nervous system maturation and that disruption of this process in individuals with mutations in MeCP2 may underlie the neural-specific pathology of RTT.

PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinsons disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.

The Angelman Syndrome Protein Ube3A Regulates Synapse Development by Ubiquitinating Arc

Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.

Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits

Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of β-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.

Pleiotropic effects of miR-183~96~182 converge to regulate cell survival, proliferation and migration in medulloblastoma

Medulloblastomas are the most common malignant brain tumors in children. Several large-scale genomic studies have detailed their heterogeneity, defining multiple subtypes with unique molecular profiles and clinical behavior. Increased expression of the miR-183~96~182 cluster of microRNAs has been noted in several subgroups, including the most clinically aggressive subgroup associated with genetic amplification of MYC. To understand the contribution of miR-183~96~182 to the pathogenesis of this aggressive subtype of medulloblastoma, we analyzed global gene expression and proteomic changes that occur upon modulation of miRNAs in this cluster individually and as a group in MYC-amplified medulloblastoma cells. Knockdown of the full miR-183~96~182 cluster results in enrichment of genes associated with apoptosis and dysregulation of the PI3K/AKT/mTOR signaling axis. Conversely, there is a relative enrichment of pathways associated with migration, metastasis and epithelial to mesenchymal transition, as well as pathways associated with dysfunction of DNA repair in cells with preserved miR-183 cluster expression. Immunocytochemistry and FACS analysis confirm induction of apoptosis upon knockdown of the miR-183 cluster. Importantly, cell-based migration and invasion assays verify the positive regulation of cell motility/migration by the miR-183 cluster, which is largely mediated by miR-182. We show that the effects on cell migration induced by the miR-183 cluster are coupled to the PI3K/AKT/mTOR pathway through differential regulation of AKT1 and AKT2 isoforms. Furthermore, we show that rapamycin inhibits cell motility/migration in medulloblastoma cells and phenocopies miR-183 cluster knockdown. Thus, the miR-183 cluster regulates multiple biological programs that converge to support the maintenance and metastatic potential of medulloblastoma.

FLEXIQuant: A novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides

Measurements of protein abundance and quantitative assessment of multiple post-translational modifications (PTMs) within a single protein are increasingly used to understand the control of protein activity, particularly in metazoan cells. General methods of wide applicability and precision/accuracy for quantitative estimation of protein post-translational regulation are lacking. Protein mass spectrometry has evolved from a high-throughput qualitative technique to a potentially general quantitative tool, but there are still serious limitations in dynamic range and coverage. To address some of these limitations, we introduce a novel MS-based quantitative strategy, FLEXIQuant, (Full-Length Expressed Stable Isotope-labeled Proteins for Quantification), which can track changes in relative peptide abundances as a function of PTM, and determine absolute quantity of a protein from its lysate. We examined two subunits of the anaphase-promoting complex, CDC27 and APC5, as a test of our ability to monitor quantitatively, the PTM status of several peptides over time. We find evidence of differential regulation at different sites, a phenomenon we believe will be very widespread. FLEXIQuant proved itself to be capable of serving as a general quantitative tool.

Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: A quantitative proteomic analysis

The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that during drug arrest the phosphorylation state of the APC changes, indicating that the mitotic arrest is not a static condition. We discuss these findings in terms of the variable efficacy of antimitotic drugs in cancer chemotherapy.

Injury-induced decline of intrinsic regenerative ability revealed by quantitative proteomics

Neurons differ in their responses to injury, but the underlying mechanisms remain poorly understood. Using quantitative proteomics, we characterized the injury-triggered response from purified intact and axotomized retinal ganglion cells (RGCs). Subsequent informatics analyses revealed a network of injury-response signaling hubs. In addition to confirming known players, such as mTOR, this also identified new candidates, such as c-myc, NFκB, and Huntingtin. Similar to mTOR, c-myc has been implicated as a key regulator of anabolic metabolism and is downregulated by axotomy. Forced expression of c-myc in RGCs, either before or after injury, promotes dramatic RGC survival and axon regeneration after optic nerve injury. Finally, in contrast to RGCs, neither c-myc nor mTOR was downregulated in injured peripheral sensory neurons. Our studies suggest that c-myc and other injury-responsive pathways are critical to the intrinsic regenerative mechanisms and might represent a novel target for developing neural repair strategies in adults.

A screen for regulators of survival of motor neuron protein levels

The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK-PI3K-AKT-GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.

NITPICK: peak identification for mass spectrometry data

BackgroundThe reliable extraction of features from mass spectra is a fundamental step in the automated analysis of proteomic mass spectrometry (MS) experiments.ResultsThis contribution proposes a sparse template regression approach to peak picking called NITPICK. NITPICK is a Non-greedy, Iterative Template-based peak PICKer that deconvolves complex overlapping isotope distributions in multicomponent mass spectra. NITPICK is based on fractional averagine, a novel extension to Senkos well-known averagine model, and on a modified version of sparse, non-negative least angle regression, for which a suitable, statistically motivated early stopping criterion has been derived. The strength of NITPICK is the deconvolution of overlapping mixture mass spectra.ConclusionExtensive comparative evaluation has been carried out and results are provided for simulated and real-world data sets. NITPICK outperforms pepex, to date the only alternate, publicly available, non-greedy feature extraction routine. NITPICK is available as software package for the R programming language and can be downloaded from http://hci.iwr.uni-heidelberg.de/mip/proteomics/.