Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.

To define the genetic requirements for pancreatic ductal adenocarcinoma (PDA), we have targeted concomitant endogenous expression of Trp53(R172H) and Kras(G12D) to the mouse pancreas, revealing the cooperative development of invasive and widely metastatic carcinoma that recapitulates the human disease. The primary carcinomas and metastases demonstrate a high degree of genomic instability manifested by nonreciprocal translocations without obvious telomere erosion-hallmarks of human carcinomas not typically observed in mice. No mutations were discovered in other cardinal tumor suppressor gene pathways, which, together with previous results, suggests that there are distinct genetic pathways to PDA with different biological behaviors. These findings have clear implications for understanding mechanisms of disease pathogenesis, and for the development of detection and targeted treatment strategies.

High-Frequency Repetitive Magnetic Stimulation Activates Bactericidal Activity of Macrophages via Modulation of p62/Keap1/Nrf2 and p38 MAPK Pathways.

The effects of repetitive magnetic stimulation (rMS) have predominantly been studied in excitable cells, with limited research in non-excitable cells. This study aimed to investigate the impact of rMS on macrophages, which are crucial cells in the innate immune defense. THP-1-derived macrophages subjected to a 5 min session of 10 Hz rMS exhibited increased Nrf2 activation and decreased Keap1 expression. We found that activation of the Nrf2 signaling pathway relied on rMS-induced phosphorylation of p62. Notably, rMS reduced the intracellular survival of Staphylococcus aureus in macrophages. Silencing Nrf2 using siRNA in THP-1-derived macrophages or utilizing Nrf2 knockout in alveolar macrophages abolished this effect. Additionally, rMS attenuated the expression of IL-1ß and TNF-α inflammatory genes by S. aureus and inhibited p38 MAPK activation. These findings highlight the capacity of rMS to activate the non-canonical Nrf2 pathway, modulate macrophage function, and enhance the host's defense against bacterial infection.

AMPK-Nrf2 Signaling Pathway in Phrenic Motoneurons following Cervical Spinal Cord Injury.

High spinal cord injuries (SCI) induce the deafferentation of phrenic motoneurons, leading to permanent diaphragm paralysis. This involves secondary injury associated with pathologic and inflammatory processes at the site of injury, and at the level of phrenic motoneurons. In the present study, we evaluated the antioxidant response in phrenic motoneurons involving the AMPK-Nrf2 signaling pathway following C2 spinal cord lateral hemi-section in rats. We showed that there is an abrupt reduction in the expression of phosphorylated AMPK and Nrf2 at one hour post-injury in phrenic motoneurons. A rebound is then observed at one day post-injury, reflecting a return to homeostasis condition. In the total spinal cord around phrenic motoneurons, the increase in phosphorylated AMPK and Nrf2 occurred at three days post-injury, showing the differential antioxidant response between phrenic motoneurons and other cell types. Taken together, our results display the implication of the AMPK-Nrf2 signaling pathway in phrenic motoneurons' response to oxidative stress following high SCI. Harnessing this AMPK-Nrf2 signaling pathway could improve the antioxidant response and help in spinal rewiring to these deafferented phrenic motoneurons to improve diaphragm activity in patients suffering high SCI.

Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt-dependent pathway.

CD47 is a membrane receptor that plays pivotal roles in many pathophysiological processes, including infection, inflammation, cell spreading, proliferation, and apoptosis. We show that activation of CD47 increases proliferation of human U87 and U373 astrocytoma cells but not normal astrocytes. CD47 function-blocking antibodies inhibit proliferation of untreated U87 and U373 cells but not normal astrocytes, suggesting that CD47 may be constitutively activated in astrocytoma. CD47 expression levels were similar in our three cell types. CD47 couples to G-proteins in astrocytes and astrocytoma and especially to the Gßγ dimer. Downstream signaling following CD47 activation involves Gßγ dimer-dependent activation of the PI3K/Akt pathway in astrocytoma cells but not in normal astrocytes. This pathway is known to be deregulated in astrocytoma, leading to cell proliferation and enhanced survival signals. Putative PLIC-1 interaction with CD47 in astrocytoma cells but not astrocytes may contribute to the proliferative effect observed upon activation of CD47. Our data indicate that CD47 receptors have a stimulatory role in cell proliferation and demonstrate for the first time that CD47 signals via the PI3K/Akt pathway in cancerous cells but not normal cells.

Effects of aerobic exercise training on muscle plasticity in a mouse model of cervical spinal cord injury.

Cervical spinal cord injury (SCI) results in permanent life-altering motor and respiratory deficits. Other than mechanical ventilation for respiratory insufficiency secondary to cervical SCI, effective treatments are lacking and the development of animal models to explore new therapeutic strategies are needed. The aim of this work was to demonstrate the feasibility of using a mouse model of partial cervical spinal hemisection at the second cervical metameric segment (C2) to investigate the impact of 6 weeks training on forced exercise wheel system on locomotor/respiratory plasticity muscles. To measure run capacity locomotor and respiratory functions, incremental exercise tests and diaphragmatic electromyography were done. In addition, muscle fiber type composition and capillary distribution were assessed at 51 days following chronic C2 injury in diaphragm, extensor digitorum communis (EDC), tibialis anterior (TA) and soleus (SOL) muscles. Six-week exercise training increased the running capacity of trained SCI mice. Fiber type composition in EDC, TA and SOL muscles was not modified by our protocol of exercise. The vascularization was increased in all muscle limbs in SCI trained group. No increase in diaphragmatic electromyography amplitude of the diaphragm muscle on the side of SCI was observed, while the contraction duration was significantly decreased in sedentary group compared to trained group. Cross-sectional area of type IIa myofiber in the contralateral diaphragm side of SCI was smaller in trained group. Fiber type distribution between contralateral and ipsilateral diaphragm in SCI sedentary group was affected, while no difference was observed in trained group. In addition, the vascularization of the diaphragm side contralateral to SCI was increased in trained group. All these results suggest an increase in fatigue resistance and a contribution to the running capacity in SCI trained group. Our exercise protocol could be a promising non-invasive strategy to sustain locomotor and respiratory muscle plasticity following SCI.

Permanent diaphragmatic deficits and spontaneous respiratory plasticity in a mouse model of incomplete cervical spinal cord injury.

High spinal cord injuries (SCI) lead to permanent respiratory insufficiency, and the search for new therapeutics to restore this function is essential. To date, the most documented preclinical model for high SCI is the rat cervical C2 hemisection. However, molecular studies with this SCI model are limited due to the poor availability of genetically modified specimens. The aim of this work was to evaluate the pathophysiology of respiratory activity following a cervical C2 injury at different times post-injury in a C57BL/6 mouse model. No significant spontaneous recovery of diaphragmatic activity was observed up to 30 days post-injury in eupneic condition. However, during a respiratory challenge, i.e. mild asphyxia, a partial restoration of the injured diaphragm was observed at 7 days post-injury, corresponding to the crossed phrenic phenomenon. Interestingly, the diaphragmatic recording between 2 respiratory bursts on the injured side showed an amplitude increase between 1-7 days post-injury, reflecting a change in phrenic motoneuronal excitability. This increase in inter-burst excitability returned to pre-injured values when the crossed phrenic phenomenon started to be effective at 7 days post-injury. Taken together, these results demonstrate the ability of the mouse respiratory system to express long-lasting plasticity following a C2 cervical hemisection and genetically modified animals can be used to study the pathophysiological effects on these plasticity phenomena.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Sulforaphane reduces intracellular survival of Staphylococcus aureus in macrophages through inhibition of JNK and p38 MAPK­induced inflammation.

Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP­1­derived macrophages, primary human peripheral blood mononuclear cell­derived macrophages, and primary mouse bone marrow derived­macrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureus­induced caspases­3/7­dependent cell apoptosis, potentially through downregulation of microRNA (miR)­142­5p and miR­146a­5p. As SFN is a well­known activator of nuclear factor erythroid 2­related factor 2 (Nrf2), Nrf2­/­ BMDMs were used to demonstrate that the SFN­mediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2­deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureus­induced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)­1ß, IL­6, and tumor necrosis factor­α (TNF­α), as well as for the M1 markers C­C motif chemokine receptor 7, IL­23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogen­activated protein kinase (MAPK) (p38) and c­Jun N­terminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureus­induced proinflammatory gene expression levels compared with those observed in the SFN­pretreated macrophages. Furthermore, THP­1­derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNK­inflammatory response and the S. aureus­induced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.

Comparative effectiveness of 4 natural and chemical activators of Nrf2 on inflammation, oxidative stress, macrophage polarization, and bactericidal activity in an in vitro macrophage infection model.

Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their potential or proven anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-1-derived macrophages. We first compared the capacity of sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), and dimethyl fumarate (DMF) to induce the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. Next, we performed a comparative evaluation of the antioxidant and anti-inflammatory efficacies of the 4 selected compounds. THP-1-derived macrophages and LPS-stimulated macrophages were treated with each compound and expression levels of genes coding for inflammatory cytokines IL-1ß, IL-6, and TNF-α were quantified by RT-qPCR. Moreover, expression levels of genes coding for M1 (IL-23, CCR7, IL-1ß, IL-6, and TNF-α) and M2 (PPARγ, MRC1, CCL22, and IL-10) markers were determined in classically-activated M1 macrophages treated with each compound. Finally, the effects of each compound on the intracellular bacterial survival of gram-negative E. coli and gram-positive S. aureus in THP-1-derived macrophages and PBMC-derived macrophages were examined. Our data confirmed the anti-inflammatory and antioxidant effects of SFN, WG, and DMF on LPS-stimulated THP-1-derived macrophages. In addition, SFN or WG treatment of classically-activated THP-1-derived macrophages reduced expression levels of M1 marker genes, while SFN or DMF treatment upregulated the M2 marker gene MRC1. This decrease in expression of M1 marker genes may be correlated with the decrease in intracellular S. aureus load in SFN- or DMF-treated macrophages. Interestingly, an increase in intracellular survival of E. coli in SFN-treated THP-1-derived macrophages that was not observed in PBMC-derived macrophages. Conversely, OTZ exhibited pro-oxidant and proinflammatory properties, and affected intracellular survival of E. coli in THP-1-derived macrophages. Altogether, we provide new potential therapeutic alternatives in treating inflammation and bacterial infection.

N-cadherin and keratinocyte growth factor receptor mediate the functional interplay between Ki-RASG12V and p53V143A in promoting pancreatic cell migration, invasion, and tissue architecture disruption.

The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

Mutant KRAS in the initiation of pancreatic cancer.

Pancreatic ductal adenocarcinoma is the most common pancreatic neoplasm. There are approximately 33,000 new cases of pancreatic ductal adenocarcinoma annually in the United States with approximately the same number of deaths. Surgery represents the only opportunity for cure, but this is restricted to early stage pancreatic cancer. Pancreatic ductal adenocarcinoma evolves from a progressive cascade of cellular, morphological and architectural changes from normal ductal epithelium through preneoplastic lesions termed pancreatic intraepithelial neoplasia (PanIN). These PanIN lesions are in turn associated with somatic alterations in canonical oncogenes and tumor suppressor genes. Most notably, early PanIN lesions and almost all pancreatic ductal adenocarcinomas involve mutations in the K-ras oncogene. Thus, it is believed that activating K-ras mutations are critical for initiation of pancreatic ductal carcinogenesis. This has been proven through elegant genetically engineered mouse models in which a Cre-activated K-Ras(G12D) allele is knocked into the endogenous K-Ras locus and crossed with mice expressing Cre recombinase in pancreatic tissue. As a result, mechanistic insights are now possible into how K-Ras contributes to pancreatic ductal carcinogenesis, what cooperating events are required, and armed with this knowledge, new therapeutic approaches can be pursued and tested.

The mutant K-ras oncogene causes pancreatic periductal lymphocytic infiltration and gastric mucous neck cell hyperplasia in transgenic mice.

A frequent genetic alteration found in premalignant stages of pancreatic adenocarcinoma is K-ras oncogene point mutation. The mechanistic basis for the inability of K-ras mutation to transform pancreatic ductal cells is unclear, although cooperating events with p16 inactivation, p53 mutation, and SMAD 4 mutation are recognized to be necessary. We have generated a novel mouse model in which the cytokeratin 19 promoter, specifically active in pancreatic ductal cells but not other cell types of the pancreas, is fused to mutant K-ras. This is of direct relevance to human pancreatic cancer because premalignant lesions are found specifically in ductal cells. There is dramatic periductal lymphocytic infiltration in the pancreata of transgenic mice, predominantly CD4+ T lymphocytes, which may act as an adaptive immune response to activated ras-mediated signaling. In addition, gene array analysis reveals an induction of N-cadherin in transgenic mice pancreatic ductal cells, the significance of which relates to promotion of cell adhesion and deterrence of cell migration. Apart from these important biological considerations, there is parallel activity of the cytokeratin 19 promoter in the stem cell region of the gastric epithelium, namely in mucous neck cells. Activated K-ras in this context causes mucous neck cell hyperplasia, a precursor to gastric adenocarcinoma. There is concomitant parietal cell decrease, which is a key step toward gastric adenocarcinoma. Taken together, we have defined how mutant K-ras signaling modulates important molecular events in the initiating events of pancreatic and gastric carcinogenesis.

Reorganization of Respiratory Descending Pathways following Cervical Spinal Partial Section Investigated by Transcranial Magnetic Stimulation in the Rat.

High cervical spinal cord injuries lead to permanent respiratory deficits. One preclinical model of respiratory insufficiency in adult rats is the C2 partial injury which causes unilateral diaphragm paralysis. This model allows the investigation of a particular population of respiratory bulbospinal axons which cross the midline at C3-C6 spinal segment, namely the crossed phrenic pathway. Transcranial magnetic stimulation (TMS) is a non-invasive technique that can be used to study supraspinal descending respiratory pathways in the rat. Interestingly, a lateral C2 injury does not affect the amplitude and latency of the largest motor-evoked potential recorded from the diaphragm (MEPdia) ipsilateral to the injury in response to a single TMS pulse, compared to a sham animal. Although the rhythmic respiratory activity on the contralateral diaphragm is preserved at 7 days post-injury, no diaphragm activity can be recorded on the injured side. However, a profound reorganization of the MEPdia evoked by TMS can be observed. The MEPdia is reduced on the non-injured rather than the injured side. This suggests an increase in ipsilateral phrenic motoneurons excitability. Moreover, correlations between MEPdia amplitude and spontaneous contralateral diaphragmatic activity were observed. The larger diaphragm activity correlated with a larger MEPdia on the injured side, and a smaller MEPdia on the non-injured side. This suggests, for the first time, the occurrence of a functional neuroplasticity process involving changes in motoneuron excitability balance between the injured and non-injured sides at a short post-lesional delay.

The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome-cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

Ha-Ras(G12V) induces senescence in primary and immortalized human esophageal keratinocytes with p53 dysfunction.

Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-Ras(G12V)-induced senescence, and p19(ARF-/-) and p53(-/-) cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-Ras(G12V) was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-Ras(G12V) induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated beta-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-Ras(G12V) upregulated p16(INK4a) and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.

Altering FAK-paxillin interactions reduces adhesion, migration and invasion processes.

Focal adhesion kinase (FAK) plays an important role in signal transduction pathways initiated at sites of integrin-mediated cell adhesion to the extracellular matrix. Thus, FAK is involved in many aspects of the metastatic process including adhesion, migration and invasion. Recently, several small molecule inhibitors which target FAK catalytic activity have been developed by pharmaceutical companies. The current study was aimed at addressing whether inhibiting FAK targeting to focal adhesions (FA) represents an efficient alternative strategy to inhibit FAK downstream pathways. Using a mutagenesis approach to alter the targeting domain of FAK, we constructed a FAK mutant that fails to bind paxillin. Inhibiting FAK-paxillin interactions led to a complete loss of FAK localization at FAs together with reduced phosphorylation of FAK and FAK targets such as paxillin and p130Cas. This in turn resulted in altered FA dynamics and inhibition of cell adhesion, migration and invasion. Moreover, the migration properties of cells expressing the FAK mutant were reduced as compared to FAK-/- cells. This was correlated with a decrease in both phospho-Src and phospho-p130Cas levels at FAs. We conclude that targeting FAK-paxillin interactions is an efficient strategy to reduce FAK signalling and thus may represent a target for the development of new FAK inhibitors.

A murine model of cervical spinal cord injury to study post-lesional respiratory neuroplasticity.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Interdisciplinary approaches of transcranial magnetic stimulation applied to a respiratory neuronal circuitry model.

Respiratory related diseases associated with the neuronal control of breathing represent life-threatening issues and to date, no effective therapeutics are available to enhance the impaired function. The aim of this study was to determine whether a preclinical respiratory model could be used for further studies to develop a non-invasive therapeutic tool applied to rat diaphragmatic neuronal circuitry. Transcranial magnetic stimulation (TMS) was performed on adult male Sprague-Dawley rats using a human figure-of-eight coil. The largest diaphragmatic motor evoked potentials (MEPdia) were recorded when the center of the coil was positioned 6 mm caudal from Bregma, involving a stimulation of respiratory supraspinal pathways. Magnetic shielding of the coil with mu metal reduced magnetic field intensities and improved focality with increased motor threshold and lower amplitude recruitment curve. Moreover, transynaptic neuroanatomical tracing with pseudorabies virus (applied to the diaphragm) suggest that connections exist between the motor cortex, the periaqueductal grey cell regions, several brainstem neurons and spinal phrenic motoneurons (distributed in the C3-4 spinal cord). These results reveal the anatomical substrate through which supraspinal stimulation can convey descending action potential volleys to the spinal motoneurons (directly or indirectly). We conclude that MEPdia following a single pulse of TMS can be successfully recorded in the rat and may be used in the assessment of respiratory supraspinal plasticity. Supraspinal non-invasive stimulations aimed to neuromodulate respiratory circuitry will enable new avenues of research into neuroplasticity and the development of therapies for respiratory dysfunction associated with neural injury and disease (e.g. spinal cord injury, amyotrophic lateral sclerosis).

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion.

Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK⁻/⁻ mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130(CAS)/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.