Bi-allelic Variants in RALGAPA1 Cause Profound Neurodevelopmental Disability, Muscular Hypotonia, Infantile Spasms, and Feeding Abnormalities.

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.

RalA controls glucose homeostasis by regulating glucose uptake in brown fat.

Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo.

TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue.

The noncanonical IKK family member TANK-binding kinase 1 (TBK1) is activated by pro-inflammatory cytokines, but its role in controlling metabolism remains unclear. Here, we report that the kinase uniquely controls energy metabolism. Tbk1 expression is increased in adipocytes of HFD-fed mice. Adipocyte-specific TBK1 knockout (ATKO) attenuates HFD-induced obesity by increasing energy expenditure; further studies show that TBK1 directly inhibits AMPK to repress respiration and increase energy storage. Conversely, activation of AMPK under catabolic conditions can increase TBK1 activity through phosphorylation, mediated by AMPK's downstream target ULK1. Surprisingly, ATKO also exaggerates adipose tissue inflammation and insulin resistance. TBK1 suppresses inflammation by phosphorylating and inducing the degradation of the IKK kinase NIK, thus attenuating NF-κB activity. Moreover, TBK1 mediates the negative impact of AMPK activity on NF-κB activation. These data implicate a unique role for TBK1 in mediating bidirectional crosstalk between energy sensing and inflammatory signaling pathways in both over- and undernutrition.

O-linked ß-N-acetylglucosamine (O-GlcNAc) site thr-87 regulates synapsin I localization to synapses and size of the reserve pool of synaptic vesicles.

O-GlcNAc is a carbohydrate modification found on cytosolic and nuclear proteins. Our previous findings implicated O-GlcNAc in hippocampal presynaptic plasticity. An important mechanism in presynaptic plasticity is the establishment of the reserve pool of synaptic vesicles (RPSV). Dynamic association of synapsin I with synaptic vesicles (SVs) regulates the size and release of RPSV. Disruption of synapsin I function results in reduced size of the RPSV, increased synaptic depression, memory deficits, and epilepsy. Here, we investigate whether O-GlcNAc directly regulates synapsin I function in presynaptic plasticity. We found that synapsin I is modified by O-GlcNAc during hippocampal synaptogenesis in the rat. We identified three novel O-GlcNAc sites on synapsin I, two of which are known Ca(2+)/calmodulin-dependent protein kinase II phosphorylation sites. All O-GlcNAc sites mapped within the regulatory regions on synapsin I. Expression of synapsin I where a single O-GlcNAc site Thr-87 was mutated to alanine in primary hippocampal neurons dramatically increased localization of synapsin I to synapses, increased density of SV clusters along axons, and the size of the RPSV, suggesting that O-GlcNAcylation of synapsin I at Thr-87 may be a mechanism to modulate presynaptic plasticity. Thr-87 is located within an amphipathic lipid-packing sensor (ALPS) motif, which participates in targeting of synapsin I to synapses by contributing to the binding of synapsin I to SVs. We discuss the possibility that O-GlcNAcylation of Thr-87 interferes with folding of the ALPS motif, providing a means for regulating the association of synapsin I with SVs as a mechanism contributing to synapsin I localization and RPSV generation.

Tandem lectin weak affinity chromatography for glycoprotein enrichment.

In this chapter we describe the application of lectin weak affinity chromatography (LWAC) for the enrichment of peptides modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a single carbohydrate moiety post-translational modification of intracellular proteins. The stoichiometry of the modification is low and identification of the sites of O-GlcNAc attachment is challenging. To map O-GlcNAc sites we use the approach where a protein sample of interest is digested with trypsin and subjected to LWAC, which employs weak interaction between lectin wheat germ agglutinin and O-GlcNAc. Obtained sample is enriched with O-GlcNAc-modified peptides, which can be identified by means of mass spectrometry.

Swi1 associates with chromatin through the DDT domain and recruits Swi3 to preserve genomic integrity.

Swi1 and Swi3 form the replication fork protection complex and play critical roles in proper activation of the replication checkpoint and stabilization of replication forks in the fission yeast Schizosaccharomyces pombe. However, the mechanisms by which the Swi1-Swi3 complex regulates these processes are not well understood. Here, we report functional analyses of the Swi1-Swi3 complex in fission yeast. Swi1 possesses the DDT domain, a putative DNA binding domain found in a variety of chromatin remodeling factors. Consistently, the DDT domain-containing region of Swi1 interacts with DNA in vitro, and mutations in the DDT domain eliminate the association of Swi1 with chromatin in S. pombe cells. DDT domain mutations also render cells highly sensitive to S-phase stressing agents and induce strong accumulation of Rad22-DNA repair foci, indicating that the DDT domain is involved in the activity of the Swi1-Swi3 complex. Interestingly, DDT domain mutations also abolish Swi1's ability to interact with Swi3 in cells. Furthermore, we show that Swi1 is required for efficient chromatin association of Swi3 and that the Swi1 C-terminal domain directly interacts with Swi3. These results indicate that Swi1 associates with chromatin through its DDT domain and recruits Swi3 to function together as the replication fork protection complex.

Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation.

Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. We therefore speculated that increasing tau O-GlcNAc could be a strategy to hinder pathological tau-induced neurodegeneration. Here we found that treatment of hemizygous JNPL3 tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAc, hindered formation of tau aggregates and decreased neuronal cell loss. Notably, increases in tau O-GlcNAc did not alter tau phosphorylation in vivo. Using in vitro biochemical aggregation studies, we found that O-GlcNAc modification, on its own, hinders tau oligomerization. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease.

Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody.

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-D: -glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.

Human Alzheimer's disease synaptic O-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry.

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer's disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.

Gene expression signatures and response to imatinib mesylate in gastrointestinal stromal tumor.

Despite initial efficacy of imatinib mesylate in most gastrointestinal stromal tumor (GIST) patients, many experience primary/secondary drug resistance. Therefore, clinical management of GIST may benefit from further molecular characterization of tumors before and after imatinib mesylate treatment. As part of a recent phase II trial of neoadjuvant/adjuvant imatinib mesylate treatment for advanced primary and recurrent operable GISTs (Radiation Therapy Oncology Group S0132), gene expression profiling using oligonucleotide microarrays was done on tumor samples obtained before and after imatinib mesylate therapy. Patients were classified according to changes in tumor size after treatment based on computed tomography scan measurements. Gene profiling data were evaluated with Statistical Analysis of Microarrays to identify differentially expressed genes (in pretreatment GIST samples). Based on Statistical Analysis of Microarrays [False Discovery Rate (FDR), 10%], 38 genes were expressed at significantly lower levels in the pretreatment biopsy samples from tumors that significantly responded to 8 to 12 weeks of imatinib mesylate, that is, >25% tumor reduction. Eighteen of these genes encoded Krüppel-associated box (KRAB) domain containing zinc finger (ZNF) transcriptional repressors. Importantly, 10 KRAB-ZNF genes mapped to a single locus on chromosome 19p, and a subset predicted likely response to imatinib mesylate-based therapy in a naïve panel of GIST. Furthermore, we found that modifying expression of genes within this predictive signature can enhance the sensitivity of GIST cells to imatinib mesylate. Using clinical pretreatment biopsy samples from a prospective neoadjuvant phase II trial, we have identified a gene signature that includes KRAB-ZNF 91 subfamily members that may be both predictive of and functionally associated with likely response to short-term imatinib mesylate treatment.

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet-derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10-15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large-scale genomic alterations, but little is known about the mutation-negative tumors. Using genome-wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation-negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation-negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation-negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine-threonine kinase BRAF. The only other mutation-negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT-mutant GISTs identified a minimal overlapping region of deletion of approximately 0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell-cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs.

In vivo modulation of O-GlcNAc levels regulates hippocampal synaptic plasticity through interplay with phosphorylation.

O-Linked N-acetylglucosamine (O-GlcNAc) is a cytosolic and nuclear carbohydrate post-translational modification most abundant in brain. We recently reported uniquely extensive O-GlcNAc modification of proteins that function in synaptic vesicle release and post-synaptic signal transduction. Here we examined potential roles for O-GlcNAc in mouse hippocampal synaptic transmission and plasticity. O-GlcNAc modifications and the enzyme catalyzing their addition (O-GlcNAc transferase) were enriched in hippocampal synaptosomes. Pharmacological elevation or reduction of O-GlcNAc levels had no effect on Schaffer collateral CA1 basal hippocampal synaptic transmission. However, in vivo elevation of O-GlcNAc levels enhanced long term potentiation (LTP), an electrophysiological correlate to some forms of learning/memory. Reciprocally, pharmacological reduction of O-GlcNAc levels blocked LTP. Additionally, elevated O-GlcNAc led to reduced paired-pulse facilitation, a form of short term plasticity attributed to presynaptic mechanisms. Synapsin I and II are presynaptic proteins that increase synaptic vesicle availability for release when phosphorylated, thus contributing to hippocampal synaptic plasticity. Synapsins are among the most extensively O-GlcNAc-modified proteins known. Elevating O-GlcNAc levels increased phosphorylation of Synapsin I/II at serine 9 (cAMP-dependent protein kinase substrate site), serine 62/67 (Erk 1/2 (MAPK 1/2) substrate site), and serine 603 (calmodulin kinase II site). Activation-specific phosphorylation events on Erk 1/2 and calmodulin kinase II, two proteins required for CA1 hippocampal LTP establishment, were increased in response to elevation of O-GlcNAc levels. Thus, O-GlcNAc is a novel regulatory signaling component of excitatory synapses, with specific roles in synaptic plasticity that involve interplay with phosphorylation.

Therapeutic effect of imatinib in gastrointestinal stromal tumors: AKT signaling dependent and independent mechanisms.

Most gastrointestinal stromal tumors (GISTs) possess a gain-of-function mutation in c-KIT. Imatinib mesylate, a small-molecule inhibitor against several receptor tyrosine kinases, including KIT, platelet-derived growth factor receptor-alpha, and BCR-ABL, has therapeutic benefit for GISTs both via KIT and via unknown mechanisms. Clinical evidence suggests that a potential therapeutic benefit of imatinib might result from decreased glucose uptake as measured by positron emission tomography using 18-fluoro-2-deoxy-d-glucose. We sought to determine the mechanism of and correlation to altered metabolism and cell survival in response to imatinib. Glucose uptake, cell viability, and apoptosis in GIST cells were measured following imatinib treatment. Lentivirus constructs were used to stably express constitutively active AKT1 or AKT2 in GIST cells to study the role of AKT signaling in metabolism and cell survival. Immunoblots and immunofluorescent staining were used to determine the levels of plasma membrane-bound glucose transporter Glut4. We show that oncogenic activation of KIT maximizes glucose uptake in an AKT-dependent manner. Imatinib treatment markedly reduces glucose uptake via decreased levels of plasma membrane-bound Glut4 and induces apoptosis or growth arrest by inhibiting KIT activity. Importantly, expression of constitutively active AKT1 or AKT2 does not rescue cells from the imatinib-mediated apoptosis although glucose uptake was not blocked, suggesting that the potential therapeutic effect of imatinib is independent of AKT activity and glucose deprivation. Overall, these findings contribute to a clearer understanding of the molecular mechanisms involved in the therapeutic benefit of imatinib in GIST and suggest that a drug-mediated decrease in tumor metabolism observed clinically may not entirely reflect therapeutic efficacy of treatment.

Analysis of KIT mutations in sporadic and familial gastrointestinal stromal tumors: therapeutic implications through protein modeling.

PURPOSE: Gastrointestinal stromal tumors (GIST) are characterized by expressing a gain-of-function mutation in KIT, and to a lesser extent, PDGFR. Imatinib mesylate, a tyrosine kinase inhibitor, has activity against GISTs that contain oncogenic mutations of KIT. In this study, KIT and PDGFRalpha mutation status was analyzed and protein modeling approaches were used to assess the potential effect of KIT mutations in response to imatinib therapy. EXPERIMENTAL DESIGN: Genomic DNA was isolated from GIST tumors. Exons 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRalpha were evaluated for oncogenic mutations. Protein modeling was used to assess mutations within the juxtamembrane region and the kinase domain of KIT. RESULTS: Mutations in KIT exons 9, 11, and 13 were identified in GISTs with the majority of changes involving the juxtamembrane region of KIT. Molecular modeling indicates that mutations in this region result in disruption of the KIT autoinhibited conformation, and lead to gain-of-function activation of the kinase. Furthermore, a novel germ-line mutation in KIT was identified that is associated with an autosomal dominant predisposition to the development of GIST. CONCLUSIONS: We have used protein modeling and structural analyses to elucidate why patients with GIST tumors containing exon 11 mutations are the most responsive to imatinib mesylate treatment. Importantly, mutations detected in this exon and others displayed constitutive activation of KIT. Furthermore, we have found tumors that are both KIT and PDGFRalpha mutation negative, suggesting that additional, yet unidentified, abnormalities may contribute to GIST tumorigenesis.