The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for >30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler ɑ-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS's ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic KRAS. Methods: Mice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D ). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion. Results: Mice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients. Conclusions: Our results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS's ability to promote pancreatic intraepithelial lesion formation.

Sox9 knockout mice have improved recovery following stroke.

The partial recovery that can occur after a stroke has been attributed to structural and functional plasticity that compensate for damage and lost functions. This plasticity is thought to be limited in part by the presence of growth inhibitors in the central nervous system. Blocking or reducing signals from inhibitors of axonal sprouting such as Nogo and chondroitin sulfate proteoglycans (CSPGs) increases post-stroke axonal sprouting and improves recovery. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and demonstrated that conditional Sox9 ablation leads to increased axonal sprouting and improved recovery after spinal cord injury. In the present study we evaluate the effect of conditional Sox9 ablation in a transient middle cerebral artery occlusion (MCAO) model of stroke. We demonstrate that conditional Sox9 ablation leads to reduced CSPG levels, increased tissue sparing and improved post-stroke neurological recovery. Anterograde tract tracing studies demonstrate that in the Sox9 conditional knockout mice corticorubral and corticospinal projections from the contralateral, uninjured cortex increase projections to targets in the midbrain and spinal cord denervated by the injury. These results suggest that targeting SOX9 is a viable strategy to promote reparative axonal sprouting, neuroprotection and recovery after stroke.

Conditional Sox9 ablation improves locomotor recovery after spinal cord injury by increasing reactive sprouting.

The absence of axonal regeneration after spinal cord injury (SCI) has been attributed to the up-regulation of axon-repelling molecules, such as chondroitin sulfate proteoglycans (CSPGs) present in the glial scar that forms post-SCI. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and also demonstrated that conditional Sox9 ablation leads to decreased CSPG levels and improved recovery of hind limb function after SCI. We herein demonstrate increased neural input onto spinal neurons caudal to the lesion in spinal cord injured Sox9 conditional knock out mice as indicated by increased levels of the presynaptic markers synaptophysin and vesicular glutamate transporter 1 (VGLUT1) compared to controls. Axonal sparing, long-range axonal regeneration and reactive sprouting were investigated as possible explanations for the increase in neural inputs caudal to the lesion and for the improved locomotor outcomes in spinal cord-injured Sox9 conditional knock out mice. Whereas retrograde tract-tracing studies failed to reveal any evidence for increased axonal sparing or for long-range regeneration in the Sox9 conditional knock out mice, anterograde tract-tracing experiments demonstrated increased reactive sprouting caudal to the lesion after SCI. Finally we demonstrate that application of a broad spectrum MMP inhibitor to reduce CSPG degradation in Sox9 conditional knock out mice prevents the improvements in locomotor recovery observed in untreated Sox9 conditional knock out mice. These results suggest that improved recovery of locomotor function in Sox9 conditional knock out mice after SCI is due to increased reactive sprouting secondary to reduced CSPG levels distal to the lesion.

CD11d integrin blockade reduces the systemic inflammatory response syndrome after traumatic brain injury in rats.

Traumatic CNS injury triggers a systemic inflammatory response syndrome (SIRS), in which circulating inflammatory cells invade body organs causing local inflammation and tissue damage. We have shown that the SIRS caused by spinal cord injury is greatly reduced by acute intravenous treatment with an antibody against the CD11d subunit of the CD11d/CD18 integrin expressed by neutrophils and monocyte/macrophages, a treatment that reduces their efflux from the circulation. Traumatic brain injury (TBI) is a frequently occurring injury after motor vehicle accidents, sporting and military injuries, and falls. Our studies have shown that the anti-CD11d treatment diminishes brain inflammation and oxidative injury after moderate or mild TBI, improving neurological outcomes. Accordingly, we examined the impact of this treatment on the SIRS triggered by TBI. The anti-CD11d treatment was given at 2h after a single moderate (2.5-3.0 atm) or 2 and 24h after each of three consecutive mild (1.0-1.5 atm) fluid percussion TBIs. Sham-injured, saline-treated rats served as controls. At 24h, 72 h, and 4 or 8 weeks after the single TBI and after the third of three TBIs, lungs of rats were examined histochemically, immunocytochemically and biochemically for downstream effects of SIRS including inflammation, tissue damage and expression of oxidative enzymes. Lung sections revealed that both the single moderate and repeated mild TBI caused alveolar disruption, thickening of inter-alveolar tissue, hemorrhage into the parenchyma and increased density of intra-and peri-alveolar macrophages. The anti-CD11d treatment decreased the intrapulmonary influx of neutrophils and the density of activated macrophages and the activity of myeloperoxidase after these TBIs. Moreover, Western blotting studies showed that the treatment decreased lung protein levels of oxidative enzymes gp91(phox), inducible nitric oxide synthase and cyclooxygenase-2, as well as the apoptotic pathway enzyme caspase-3 and levels of 4-hydroxynonenal-bound proteins (an indicator of lipid peroxidation). Decreased expression of the cytoprotective transcription factor Nrf2 reflected decreased lung oxidative stress. Anti-CD11d treatment also diminished the lung concentration of free radicals and tissue aldehydes. In conclusion, the substantial lung component of the SIRS after single or repeated TBIs is significantly decreased by a simple, minimally invasive and short-lasting anti-inflammatory treatment.

Evaluation of the calmodulin-SOX9 interaction by "magnetic fishing" coupled to mass spectrometry.

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site.

Nesca, a novel neuronal adapter protein, links the molecular motor kinesin with the pre-synaptic membrane protein, syntaxin-1, in hippocampal neurons.

Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, ßIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.

CD11d Antibody Treatment Improves Recovery in Spinal Cord-Injured Mice.

Acute administration of a monoclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. This has been chiefly attributed to the reduced infiltration of neutrophils into the injured spinal cord in treated rats. More recently, treating spinal cord-injured mice with a Ly-6G neutrophil-depleting antibody was demonstrated to impair neurological recovery. These disparate results could be due to different mechanisms of action utilized by the two antibodies, or due to differences in the inflammatory responses between mouse and rat that are triggered by SCI. To address whether the anti-CD11d treatment would be effective in mice, a CD11d mAb (205C) or a control mAb (1B7) was administered intravenously at 2, 24, and 48 h after an 8-g clip compression injury at the fourth thoracic spinal segment. The anti-CD11d treatment reduced neutrophil infiltration into the injured mouse spinal cord and was associated with increased white matter sparing and reductions in myeloperoxidase (MPO) activity, reactive oxygen species, lipid peroxidation, and scar formation. These improvements in the injured spinal cord microenvironment were accompanied by increased serotonin (5-HT) immunoreactivity below the level of the lesion and improved locomotor recovery. Our results with the 205C CD11d mAb treatment complement previous work using this anti-integrin treatment in a rat model of SCI.

Nerve growth factor activation of the TrkA receptor induces cell death, by macropinocytosis, in medulloblastoma Daoy cells.

Ectopic expression of the TrkA receptor tyrosine kinase in tumors of the nervous system can mediate nerve growth factor (NGF)-dependent cell death by apoptosis and /or autophagy. Herein, we demonstrate that TrkA can also induce cell death in medulloblastoma Daoy cells by a caspase-independent mechanism that involves the hyperstimulation of macropinocytosis. Specifically, NGF-stimulates the uptake of AlexaFluor546-dextran into lysosome-associated membrane protein-1 positive vacuoles which fuse with microtubule associated protein light chain 3 (LC3) positive autophagosomes, to form large intracellular vacuoles (> 1 mum), which then fuse with lysotracker positive lysosomes. While LC3 cleavage and the appearance of LC3 positive vacuoles suggest the induction of autophagy, siRNA reduced expression of four proteins essential to autophagy (beclin-1, Atg5, LC3 and Atg9) neither blocks NGF-induced vacuole formation nor cell death. TrkA activated cell death does not require p38, JNK or Erk1/2 kinases but does require activation of class III PI-3 kinase and is blocked by the casein kinase 1 (CK1) inhibitor, D4476. This inhibitor does not interfere with TrkA activation but does block NGF-dependent AlexaFluor546-dextran uptake and CK1 dependent phosphorylation of beta-catenin. Collectively, these data demonstrate that TrkA stimulates cell death by a novel mechanism involving CK1-dependent hyperstimulation of macropinocytosis.

The fibroblast growth factor receptor substrate 3 adapter is a developmentally regulated microtubule-associated protein expressed in migrating and differentiated neurons.

Fibroblast growth factor (FGF) mediated signaling is essential to many aspects of neural development. Activated FGF receptors signal primarily through the FGF receptor substrate (Frs) adapters, which include Frs2/Frs2alpha and Frs3/Frs2beta. While some studies suggest that Frs3 can compensate for the loss of Frs2 in transfected cells, the lack of an effective Frs3 specific antibody has prevented efforts to determine the role(s) of the endogenous protein. To this end, we have generated a Frs3 specific antibody and have characterized the pattern of Frs3 expression in the developing nervous system, its subcellular localization as well as its biochemical properties. We demonstrate that Frs3 is expressed at low levels in the ventricular zone of developing cortex, between E12 and E15, and it co-localizes with nestin and acetylated alpha-tubulin in radial processes in the ventricular/subventricular zones as well as with betaIII tubulin in differentiated cortical neurons. Subcellular fractionation studies demonstrate that endogenous Frs3 is both soluble and plasma membrane associated while Frs3 expressed in 293T cells associates exclusively with lipid rafts. Lastly, we demonstrate that neuronal Frs3 binds microtubules comparable to the microtubule-associated protein, MAP2, while Frs2 does not. Collectively, these data suggest that neuronal Frs3 functions as a novel microtubule binding protein and they provide the first biochemical evidence that neuronal Frs3 is functionally distinct from Frs2/Frs2alpha.

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons.

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Talpha1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.

Human tumorous imaginal disc 1 (TID1) associates with Trk receptor tyrosine kinases and regulates neurite outgrowth in nnr5-TrkA cells.

The human tumorous imaginal disc 1 (TID1) proteins including TID1(L) and TID1(S), members of the DnaJ domain protein family, are involved in multiple intracellular signaling pathways such as apoptosis induction, cell proliferation, and survival. Here we report that TID1 associates with the Trk receptor tyrosine kinases and regulates nerve growth factor (NGF)-induced neurite outgrowth in PC12-derived nnr5 cells. Binding assays and transfection studies showed that the carboxyl-terminal end of TID1 (residues 224-429) bound to Trk at the activation loop (Tyr(P)(683)-Tyr(684)(P)(684) in rat TrkA) and that TID1 was tyrosine phosphorylated by Trk both in yeast and in transfected cells. Moreover endogenous TID1 was also tyrosine phosphorylated by and co-immunoprecipitated with Trk in neurotrophin-stimulated primary rat hippocampal neurons. Overexpression studies showed that both TID1(L) and TID1(S) significantly facilitated NGF-induced neurite outgrowth in TrkA-expressing nnr5 cells possibly through a mechanism involving increased activation of mitogen-activated protein kinase. Consistently knockdown of endogenous TID1, mediated with specific short hairpin RNA, significantly reduced NGF-induced neurite growth in nnr5-TrkA cells. These data provide the first evidence that TID1 is a novel intracellular adaptor that interacts with the Trk receptor tyrosine kinases in an activity-dependent manner to facilitate Trk-dependent intracellular signaling.