PTEN inhibition promotes robust growth of bulbospinal respiratory axons and partial recovery of diaphragm function in a chronic model of cervical contusion spinal cord injury.

High spinal cord injury (SCI) leads to persistent and debilitating compromise in respiratory function. Cervical SCI not only causes the death of phrenic motor neurons (PhMNs) that innervate the diaphragm, but also damages descending respiratory pathways originating in the rostral ventral respiratory group (rVRG) located in the brainstem, resulting in denervation and consequent silencing of spared PhMNs located caudal to injury. It is imperative to determine whether interventions targeting rVRG axon growth and respiratory neural circuit reconnection are efficacious in chronic cervical contusion SCI, given that the vast majority of individuals are chronically-injured and most cases of SCI involve contusion-type damage to the cervical region. We therefore employed a rat model of chronic cervical hemicontusion to test therapeutic manipulations aimed at reconstructing damaged rVRG-PhMN-diaphragm circuitry to achieve recovery of respiratory function. At a chronic time point post-injury, we systemically administered: an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential; an antagonist peptide directed against receptor-type protein tyrosine phosphatase sigma (PTPσ), another important negative regulator of axon growth capacity; or a combination of these two peptides. PTEN antagonist peptide (PAP4) promoted partial recovery of diaphragm motor activity out to nine months post-injury (though this effect depended on the anesthetic regimen used during recording), while PTPσ peptide did not impact diaphragm function after cervical SCI. Furthermore, PAP4 promoted robust growth of descending bulbospinal rVRG axons caudal to the injury within the denervated portion of the PhMN pool, while PTPσ peptide did not affect rVRG axon growth at this location that is critical to control of diaphragmatic respiratory function. In conclusion, we find that, when PTEN inhibition is targeted at a chronic time point following cervical contusion, our non-invasive PAP4 strategy can successfully promote significant regrowth of damaged respiratory neural circuitry and also partial recovery of diaphragm motor function.

PTEN inhibition promotes robust growth of bulbospinal respiratory axons and partial recovery of diaphragm function in a chronic model of cervical contusion spinal cord injury.

High spinal cord injury (SCI) leads to persistent and debilitating compromise in respiratory function. Cervical SCI not only causes the death of phrenic motor neurons (PhMNs) that innervate the diaphragm, but also damages descending respiratory pathways originating in the rostral ventral respiratory group (rVRG) located in the brainstem, resulting in denervation and consequent silencing of spared PhMNs located caudal to injury. It is imperative to determine whether interventions targeting rVRG axon growth and respiratory neural circuit reconnection are efficacious in chronic cervical contusion SCI, given that the vast majority of individuals are chronically-injured and most cases of SCI involve contusion-type damage to the cervical region. We therefore employed a clinically-relevant rat model of chronic cervical hemicontusion to test therapeutic manipulations aimed at reconstructing damaged rVRG-PhMN-diaphragm circuitry to achieve recovery of respiratory function. At a chronic time point post-injury, we systemically administered: an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential; an antagonist peptide directed against receptor-type protein tyrosine phosphatase sigma (PTPσ), another important negative regulator of axon growth capacity; or a combination of these two peptides. PTEN antagonist peptide (PAP4) promoted partial recovery of diaphragm motor activity out to nine months post-injury, while PTPσ peptide did not impact diaphragm function after cervical SCI. Furthermore, PAP4 promoted robust growth of descending bulbospinal rVRG axons caudal to the injury within the denervated portion of the PhMN pool, while PTPσ peptide did not affect rVRG axon growth at this location that is critical to control of diaphragmatic respiratory function. In conclusion, we find that, when PTEN inhibition is targeted at a chronic time point following cervical contusion that is most relevant to the SCI clinical population, our non-invasive PAP4 strategy can successfully promote significant regrowth of damaged respiratory neural circuitry and also partial recovery of diaphragm motor function. HIGHLIGHTS: PTEN antagonist peptide promotes partial diaphragm function recovery in chronic cervical contusion SCI.PTPσ inhibitory peptide does not impact diaphragm function recovery in chronic cervical contusion SCI.PTEN antagonist peptide promotes growth of bulbospinal rVRG axons in chronic cervical contusion SCI.PTPσ peptide does not affect rVRG axon growth in chronic cervical contusion SCI.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1-G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

Short-term high-fat diet feeding of mice suppresses catecholamine-stimulated Ca2+ signalling in hepatocytes and intact liver.

Excess consumption of carbohydrates, fat and calories leads to non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance; these are major factors in the pathogenesis of type II diabetes. Hormones and catecholamines acting through G-protein coupled receptors (GPCRs) linked to phospholipase C (PLC) and increases in cytosolic Ca2+ ([Ca2+ ]c ) regulate many metabolic functions of the liver. In the intact liver, catabolic hormones such as glucagon, catecholamines and vasopressin integrate and synergize to regulate the frequency and extent to which [Ca2+ ]c waves propagate across hepatic lobules to control metabolism. Dysregulation of hepatic Ca2+ homeostasis has been implicated in the development of metabolic disease, but changes in hepatic GPCR-dependent Ca2+ signalling have been largely unexplored in this context. We show that short-term, 1-week, high-fat diet (HFD) feeding of mice attenuates noradrenaline-stimulated Ca2+ signalling, reducing the number of cells responding and suppressing the frequency of [Ca2+ ]c oscillations in both isolated hepatocytes and intact liver. The 1-week HFD feeding paradigm did not change basal Ca2+ homeostasis; endoplasmic reticulum Ca2+ load, store-operated Ca2+ entry and plasma membrane Ca2+ pump activity were unchanged compared to low-fat diet (LFD)-fed controls. However, noradrenaline-induced inositol 1,4,5-trisphosphate production was significantly reduced after HFD feeding, demonstrating an effect of HFD on receptor-stimulated PLC activity. Thus, we have identified a lesion in the PLC signalling pathway induced by short-term HFD feeding, which interferes with hormonal Ca2+ signalling in isolated hepatocytes and the intact liver. These early events may drive adaptive changes in signalling, which lead to pathological consequences in fatty liver disease. KEY POINTS: Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic. In healthy liver, the counteracting effects of catabolic and anabolic hormones regulate metabolism and energy storage as fat. Hormones and catecholamines promote catabolic metabolism via increases in cytosolic Ca2+ ([Ca2+ ]c ). We show that 1 week high-fat diet (HFD) feeding of mice attenuated the Ca2+ signals induced by physiological concentrations of noradrenaline. Specifically, HFD suppressed the normal pattern of periodic [Ca2+ ]c oscillations in isolated hepatocytes and disrupted the propagation of intralobular [Ca2+ ]c waves in the intact perfused liver. Short-term HFD inhibited noradrenaline-induced inositol 1,4,5-trisphosphate generation, but did not change basal endoplasmic reticulum Ca2+ load or plasma membrane Ca2+ fluxes. We propose that impaired Ca2+ signalling plays a key role in the earliest phases of the etiology of NAFLD, and is responsible for many of the ensuing metabolic and related dysfunctional outcomes at the cellular and whole tissue level.

Response of Astrocyte Subpopulations Following Spinal Cord Injury.

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species.

High content, quantitative AFM analysis of the scalable biomechanical properties of extracellular vesicles.

Extracellular vesicles (EVs) are studied extensively as natural biomolecular shuttles and for their diagnostic and therapeutic potential. This exponential rise in interest has highlighted the need for highly robust and reproducible approaches for EV characterisation. Here we optimise quantitative nanomechanical tools and demonstrate the advantages of EV population screening by atomic force microscopy (AFM). Our high-content informatics analytical tools are made available for use by the EV community for widespread, standardised determination of structural stability. Ultracentrifugation (UC) and sonication, the common mechanical techniques used for EV isolation and loading respectively, are used to demonstrate the utility of optimised PeakForce-Quantitative Nano Mechanics (PF-QNM) analysis. EVs produced at an industrial scale exhibited biochemical and biomechanical alterations after exposure to these common techniques. UC resulted in slight increases in physical dimensions, and decreased EV adhesion concurrent with a decrease in CD63 content. Sonicated EVs exhibited significantly reduced levels of CD81, a decrease in size, increased Young's modulus and decreased adhesive force. These biomechanical and biochemical changes highlight the effect of EV sample preparation techniques on critical properties linked to EV cellular uptake and biological function. PF-QNM offers significant additional information about the structural information of EVs following their purification and downstream processing, and the analytical tools will ensure consistency of analysis of AFM data by the EV community, as this technique continues to become more widely implemented.

Facial grimace testing as an assay of neuropathic pain-related behavior in a mouse model of cervical spinal cord injury.

A major portion of individuals affected by traumatic spinal cord injury (SCI) experience one or more types of chronic neuropathic pain (NP), which is often intractable to currently available treatments. The availability of reliable behavioral assays in pre-clinical models of SCI-induced NP is therefore critical to assess the efficacy of new potential therapies. Commonly used assays to evaluate NP-related behavior in rodents, such as Hargreaves thermal and von Frey mechanical testing, rely on the withdrawal response to an evoked stimulus. However, other assays that test spontaneous/non-evoked NP-related behavior or supraspinal aspects of NP would be highly useful for a more comprehensive assessment of NP following SCI. The Mouse Grimace Scale (MGS) is a tool to assess spontaneous, supraspinal pain-like behaviors in mice; however, the assay has not been characterized in a mouse model of SCI-induced chronic NP, despite the critical importance of mouse genetics as an experimental tool. We found that beginning 2 weeks after cervical contusion, SCI mice exhibited increased facial grimace features compared to laminectomy-only control mice, and this grimace phenotype persisted to the chronic time point of 5 weeks post-injury. We also found a significant relationship between facial grimace score and the evoked forepaw withdrawal response in both the Hargreaves and von Frey tests at 5 weeks post-injury when both laminectomy-only and SCI mice were included in the analysis. However, within only the SCI group, there was no correlation between grimace score and Hargreaves or von Frey responses. These results indicate both that facial grimace analysis can be used as an assay of spontaneous NP-related behavior in the mouse model of SCI and that the information provided by the MGS may be different than that provided by evoked tests of sensory function.

The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum.

Cytosolic Ca2+ regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage Plasmodium falciparum, yet our understanding of Ca2+ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically encoded Ca2+ indicator GCaMP3, we have investigated the dynamics of Ca2+ release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca2+ from intracellular stores in P. falciparum parasites; (ii) Thg is able to induce Ca2+ release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca2+ influx from extracellular media, through a mechanism resembling store-operated Ca2+ entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca2+ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone or the release of acidic Ca2+ stores with nigericin. These data suggest the presence of two Ca2+ pools in P. falciparum with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pump inhibitors, and only the release of the Thg-sensitive Ca2+ store induces Ca2+ influx. Activation of the store-operated Ca2+ entry-like Ca2+ influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca2+ levels for their activation.

Combination therapy with monoamine oxidase inhibitors and other antidepressants or stimulants: strategies for the management of treatment-resistant depression.

Treatment-resistant depression (TRD) is a major health concern. More than 40% of patients treated for major depressive disorder with an appropriate antidepressant dose for an adequate duration fail to respond. Further, approximately half of adults with major depressive disorder fail to achieve sustained remission despite various medication trials. The utilization of monoamine oxidase inhibitors (MAOIs) for the treatment of depression in clinical practice today is low due to their widely known adverse effects, some of which may be life threatening, and the risk for dietary and drug interactions. For these reasons, MAOIs are not recommended to be prescribed along with other antidepressants or certain prescription or nonprescription drugs. Pharmacologic options are limited for individuals with TRD, however, and there is a paucity of data on the efficacy of MAOIs in combination with other antidepressants for the management of TRD. We performed a search of the PubMed database (inception through January 25, 2015) to identify cases that illustrate the potential utility, as well as risks, of combination treatment with MAOIs and other antidepressants for the management of TRD; 18 articles met the criteria for our search. In addition, we performed a retrospective case series by reviewing the medical records of 29 adults treated for depression with an MAOI plus another psychotropic agent (an antidepressant or stimulant medication) between 2003 and 2012 at a large Midwestern teaching hospital. We compared the findings of the published experience with our local experience to allow for more informed decisions regarding pharmacotherapy in patients with TRD. We separated the local experience into two groups: 15 cases with the selective MAO type B inhibitor selegiline combined with medications presumed to increase the risk of serotonin syndrome and 14 cases with nonselective MAOIs (phenelzine and tranylcypromine) combined with other contraindicated medications. Although risks of combination treatment certainly exist with selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, or clomipramine, the current literature supports cautious use of combining MAOIs with other antidepressants in patients with TRD who have failed multiple treatment modalities. In addition, the data from the 29 patients receiving combination therapy with an MAOI and another antidepressant or stimulant medication revealed that 21% improved significantly, with no complications. This case series and literature review suggest that when used under close supervision and under the care of an experienced clinician in psychiatry, combination therapy may be a consideration for the management of TRD in patients not responding to monotherapy or other combinations of antidepressants.