K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

Spatial Mechano-Signaling Regulation of GTPases through Non-Degradative Ubiquitination.

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.

Loss-of-Function Mutations in TRAF7 and KLF4 Cooperatively Activate RAS-Like GTPase Signaling and Promote Meningioma Development.

Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency-driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. SIGNIFICANCE: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.

Syntenin-knock out reduces exosome turnover and viral transduction.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

The Noonan Syndrome Gene Lztr1 Controls Cardiovascular Function by Regulating Vesicular Trafficking.

RATIONALE: Noonan syndrome (NS) is one of the most frequent genetic disorders. Bleeding problems are among the most common, yet poorly defined complications associated with NS. A lack of consensus on the management of bleeding complications in patients with NS indicates an urgent need for new therapeutic approaches. OBJECTIVE: Bleeding disorders have recently been described in patients with NS harboring mutations of LZTR1 (leucine zipper-like transcription regulator 1), an adaptor for CUL3 (CULLIN3) ubiquitin ligase complex. Here, we assessed the pathobiology of LZTR1-mediated bleeding disorders. METHODS AND RESULTS: Whole-body and vascular specific knockout of Lztr1 results in perinatal lethality due to cardiovascular dysfunction. Lztr1 deletion in blood vessels of adult mice leads to abnormal vascular leakage. We found that defective adherent and tight junctions in Lztr1-depleted endothelial cells are caused by dysregulation of vesicular trafficking. LZTR1 affects the dynamics of fusion and fission of recycling endosomes by controlling ubiquitination of the ESCRT-III (endosomal sorting complex required for transport III) component CHMP1B (charged multivesicular protein 1B), whereas NS-associated LZTR1 mutations diminish CHMP1B ubiquitination. LZTR1-mediated dysregulation of CHMP1B ubiquitination triggers endosomal accumulation and subsequent activation of VEGFR2 (vascular endothelial growth factor receptor 2) and decreases blood levels of soluble VEGFR2 in Lztr1 haploinsufficient mice. Inhibition of VEGFR2 activity by cediranib rescues vascular abnormalities observed in Lztr1 knockout mice Conclusions: Lztr1 deletion phenotypically overlaps with bleeding diathesis observed in patients with NS. ELISA screening of soluble VEGFR2 in the blood of LZTR1-mutated patients with NS may predict both the severity of NS phenotypes and potential responders to anti-VEGF therapy. VEGFR inhibitors could be beneficial for the treatment of bleeding disorders in patients with NS.

Stress-induced lncRNA LASTR fosters cancer cell fitness by regulating the activity of the U4/U6 recycling factor SART3.

Dysregulated splicing is a common event in cancer even in the absence of mutations in the core splicing machinery. The aberrant long non-coding transcriptome constitutes an uncharacterized level of regulation of post-transcriptional events in cancer. Here, we found that the stress-induced long non-coding RNA (lncRNA), LINC02657 or LASTR (lncRNA associated with SART3 regulation of splicing), is upregulated in hypoxic breast cancer and is essential for the growth of LASTR-positive triple-negative breast tumors. LASTR is upregulated in several types of epithelial cancers due to the activation of the stress-induced JNK/c-JUN pathway. Using a mass-spectrometry based approach, we identified the RNA-splicing factor SART3 as a LASTR-interacting partner. We found that LASTR promotes splicing efficiency by controlling SART3 association with the U4 and U6 small nuclear ribonucleoproteins (snRNP) during spliceosome recycling. Intron retention induced by LASTR depletion downregulates expression of essential genes, ultimately decreasing the fitness of cancer cells.

LRP1 Has a Predominant Role in Production over Clearance of Aß in a Mouse Model of Alzheimer's Disease.

The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism of the amyloid precursor protein (APP). In cellular models, LRP1 enhances amyloid-ß (Aß) generation via APP internalization and thus its amyloidogenic processing. However, conditional knock-out studies in mice define LRP1 as an important mediator for the clearance of extracellular Aß from brain via cellular degradation or transcytosis across the blood-brain barrier (BBB). In order to analyze the net effect of LRP1 on production and clearance of Aß in vivo, we crossed mice with impaired LRP1 function with a mouse model of Alzheimer's disease (AD). Analysis of Aß metabolism showed that, despite reduced Aß clearance due to LRP1 inactivation in vivo, less Aß was found in cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Further analysis of APP metabolism revealed that impairment of LRP1 in vivo shifted APP processing from the Aß-generating amyloidogenic cleavage by beta-secretase to the non-amyloidogenic processing by alpha-secretase as shown by a decrease in extracellular Aß and an increase of soluble APP-α (sAPP-α). This shift in APP processing resulted in overall lower Aß levels and a reduction in plaque burden. Here, we present for the first time clear in vivo evidence that global impairment of LRP1's endocytosis function favors non-amyloidogenic processing of APP due to its reduced internalization and subsequently, reduced amyloidogenic processing. By inactivation of LRP1, the inhibitory effect on Aß generation overrules the simultaneous impaired Aß clearance, resulting in less extracellular Aß and reduced plaque deposition in a mouse model of AD.

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain.

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.

Single mild traumatic brain injury results in transiently impaired spatial long-term memory and altered search strategies.

Mild traumatic brain injury (mTBI) can lead to diffuse neurophysical damage as well as cognitive and affective alterations. The nature and extent of behavioral changes after mTBI are still poorly understood and how strong an impact force has to be to cause long-term behavioral changes is not yet known. Here, we examined spatial learning acquisition, retention and reversal in a Morris water maze, and assessed search strategies during task performance after a single, mild, closed-skull traumatic impact referred to as "minimal" TBI. Additionally, we investigated changes in conditioned learning in a contextual fear-conditioning paradigm. Results show transient deficits in spatial memory retention, which, although limited, are indicative of deficits in long-term memory reconsolidation. Interestingly, minimal TBI causes animals to relapse to less effective search strategies, affecting performance after a retention pause. Apart from cognitive deficits, results yielded a sub-acute, transient increase in freezing response after fear conditioning, with no increase in baseline behavior, an indication of a stronger affective reaction to aversive stimuli after minimal TBI or greater susceptibility to stress. Furthermore, western blot analysis showed a short-term increase in hippocampal GFAP expression, most likely indicating astrogliosis, which is typically related to injuries of the central nervous system. Our findings provide evidence that even a very mild impact to the skull can have detectable consequences on the molecular, cognitive and affective-like level. However, these effects seemed to be very transient and reversible.

Longitudinal assessment of cerebral perfusion and vascular response to hypoventilation in a bigenic mouse model of Alzheimer's disease with amyloid and tau pathology.

Alzheimer's disease is the most common neurodegenerative disease, and many patients also present with vascular dysfunction. In this study, we aimed to assess cerebral blood flow (CBF) and cerebrovascular response (CVR) as early, pre-symptomatic (3 months of age), imaging markers in a bigenic model of Alzheimer's disease (APP.V717IxTau.P301L, biAT) and in the monogenic parental strains. We further developed our previously published combination of pulsed arterial spin labeling perfusion MRI and hypo-ventilation paradigm, which allows weaning of the mice from the ventilator. Furthermore, the commonly used isoflurane anesthesia induces vasodilation and is thereby inherently a vascular challenge. We therefore assessed perfusion differences in the mouse models under free-breathing isoflurane conditions. We report (i) that we can determine CBF and hypoventilation-based CVR under ketamine/midazolam anesthesia and wean mice from the ventilator, making it a valuable tool for assessment of CBF and CVR in mice, (ii) that biAT mice exhibit lower cortical CBF than wild-type mice at age 3 months, (iii) that CVR was increased in both biAT and APP.V717I mice but not in Tau.P301L mice, identifying the APP genotype as a strong influencer of brain CVR and (iv) that perfusion differences at baseline are masked by the widely used isoflurane anesthesia.

GSK3ß activity alleviates epileptogenesis and limits GluA1 phosphorylation.

BACKGROUND: Glycogen synthase kinase-3ß (GSK3ß) is a key regulator of cellular homeostasis. In neurons, GSK3ß contributes to the control of neuronal transmission and plasticity, but its role in epilepsy remains to be defined. METHODS: Biochemical and electrophysiological methods were used to assess the role of GSK3ß in regulating neuronal transmission and epileptogenesis. GSK3ß activity was increased genetically in GSK3ß[S9A] mice. Its effects on neuronal transmission and epileptogenesis induced by kainic acid were assessed by field potential recordings in mice brain slices and video electroencephalography in vivo. The ion channel expression was measured in brain samples from mice and followed by analysis in samples from patients with temporal lobe epilepsy or focal cortical dysplasia in correlation to GSK3ß phosphorylation. FINDINGS: Higher GSK3ß activity decreased the progression of kainic acid induced epileptogenesis. At the biochemical level, higher GSK3ß activity increased the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 4 under basal conditions and in the epileptic mouse brain and decreased phosphorylation of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 at Serine 831 under basal conditions. Moreover, we found a significant correlation between higher inhibitory GSK3ß phosphorylation at Serine 9 and higher activating GluA1 phosphorylation at Serine 845 in brain samples from epileptic patients. INTERPRETATION: Our data imply GSK3ß activity in the protection of neuronal networks from hyper-activation in response to epileptogenic stimuli and indicate that the anti-epileptogenic function of GSK3ß involves modulation of HCN4 level and the synaptic AMPA receptors pool.

GSK3ß Controls mTOR and Prosurvival Signaling in Neurons.

Glycogen synthase kinases-3ß (GSK3ß) is a key regulator of cell homeostasis. In neurons, GSK3ß contributes to control of neuronal transmission and plasticity. Despite extensive studies in non-neuronal cells, crosstalk between GSK3ß and other signaling pathways remains not well defined in neurons. In the present study, we report that GSK3ß positively affected the activity of effectors of mammalian target of rapamycin complex 1 (mTORC1) and complex 2 (mTORC2), in mature neurons in vitro and in vivo. GSK3ß also promoted prosurvival signaling and attenuated kainic acid-induced apoptosis. Our study identified GSK3ß as a positive regulator of prosurvival signaling, including the mTOR pathway, and indicates the possible neuroprotective role of GSK3ß in models of pharmacologically induced excitotoxicity.

GSK-3ß and MMP-9 Cooperate in the Control of Dendritic Spine Morphology.

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3ß (GSK-3ß) has been implicated. Despite much evidence of the involvement of GSK-3ß in functional synaptic plasticity, it is unclear how GSK-3ß controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3ß in neurons to investigate how GSK-3ß affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3ß activity increased the number of thin spines, whereas lacking GSK-3ß increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3ß activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3ß overexpressing mice. Administration of the nonspecific GSK-3ß inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3ß and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3ß activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3ß-induced synaptic alterations.

Phosphorylation of protein Tau by GSK3ß prolongs survival of bigenic Tau.P301L×GSK3ß mice by delaying brainstem tauopathy.

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3ß with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3ß activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.

Terminal hypothermic Tau.P301L mice have increased Tau phosphorylation independently of glycogen synthase kinase 3α/ß.

The microtubule-associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non-genetic agent that contributes is hypothermia, eventually caused by anesthesia. Remarkably, tauopathy in brains of hibernating mammals is not pathogenic, and, because it is fully reversible, is even considered to be neuroprotective. Here, we assessed the terminal phase of Tau.P301L mice and bigenic crosses with mice lacking glycogen synthase kinase 3 (GSK3)α completely, or GSK3ß specifically in neurons. We also analysed biGT bigenic mice that co-express Tau.P301L with GSK3ß.S9A and develop severe forebrain tauopathy with age. We found that the precocious mortality of Tau.P301L mice was typified by hypothermia that aggravated Tau phosphorylation, but, surprisingly, independently of GSK3α/ß. The important contribution of hypothermia at the time of death of mice with tauopathy suggests that body temperature should be included as a parameter in the analysis of pre-clinical models, and, by extension, in patients suffering from tauopathy.

Early structural and functional defects in synapses and myelinated axons in stratum lacunosum moleculare in two preclinical models for tauopathy.

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer's disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3ß with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer's disease.

Tauopathy differentially affects cell adhesion molecules in mouse brain: early down-regulation of nectin-3 in stratum lacunosum moleculare.

Cell adhesion molecules are important structural substrates, required for synaptic plasticity and synaptogenesis. CAMs differ widely in their expression throughout different brain regions and their specific structural and functional roles in the brain remain to be elucidated. Here, we investigated selected cell adhesion molecules for alterations in expression levels and neuronal localization in validated mouse models for Alzheimer's disease that mimic the age-related progression of amyloid accumulation and tauopathy. Among the cell adhesion molecules analyzed, Nectin-3 expression was affected most and specifically in all mouse models with tauopathy. In particular was Nectin-3 depleted from the specific region of the hippocampus, known as the stratum lacunosum and moleculare, in mice that express wild-type or mutant human protein Tau, either chronically or sub-acutely. Tauopathy progresses from the entorhinal cortex to the hippocampus by unknown mechanisms that could involve transport by the myelinated axons of the temporoammonic and perforant pathways. The decreased expression of Nectin-3 in the stratum lacunosum moleculare is an early marker of impaired transport, and eventual synaptic problems, caused by beginning tauopathy.

Neurological characterization of mice deficient in GSK3α highlight pleiotropic physiological functions in cognition and pathological activity as Tau kinase.

BACKGROUND: GSK3ß is involved in a wide range of physiological functions, and is presumed to act in the pathogenesis of neurological diseases, from bipolar disorder to Alzheimer's disease (AD). In contrast, the GSK3α isozyme remained largely ignored with respect to both aspects. RESULTS: We generated and characterized two mouse strains with neuron-specific or with total GSK3α deficiency. Behavioral and electrophysiological analysis demonstrated the physiological importance of neuronal GSK3α, with GSK3ß not compensating for impaired cognition and reduced LTP. Interestingly, the passive inhibitory avoidance task proved to modulate the phosphorylation status of both GSK3 isozymes in wild-type mice, further implying both to function in cognition. Moreover, GSK3α contributed to the neuronal architecture of the hippocampal CA1 sub-region that is most vulnerable in AD. Consequently, practically all parameters and characteristics indicated that both GSK3 isoforms were regulated independently, but that they acted on the same physiological functions in learning and memory, in mobility and in behavior. CONCLUSIONS: GSK3α proved to be regulated independently from GSK3ß, and to exert non-redundant physiological neurological functions in general behavior and in cognition. Moreover, GSK3α contributes to the pathological phosphorylation of protein Tau.