Progranulin regulates neuronal outgrowth independent of Sortilin

ABSTRACT: BACKGROUND: Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1/) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects. RESULTS: As the first group to evaluate the effect of PGRN loss in Grn knockout primary neuronal cultures, we show neurite outgrowth and branching are significantly decreased in Grn/ neurons compared to wild-type (WT) neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However, the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties of PGRN. In addition, we also present evidence that cleavage of fulllength PGRN into granulin peptides is required for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sort1/ cultures. CONCLUSION: We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth.     

Simulating the effect of evaluation unit size on eligibility to stop mass drug administration for lymphatic filariasis in Haiti

BackgroundThe Transmission Assessment Survey (TAS) is a decision-making tool to determine when transmission of lymphatic filariasis is presumed to have reached a level low enough that it cannot be sustained even in the absence of mass drug administration. The survey is applied over geographic areas, called evaluation units (EUs); existing World Health Organization guidelines limit EU size to a population of no more than 2 million people.Methodology/Principal findingsIn 2015, TASs were conducted in 14 small EUs in Haiti. Simulations, using the observed TAS results, were performed to understand the potential programmatic impact had Haiti chosen to form larger EUs. Nine “combination-EUs” were formed by grouping adjacent EUs, and bootstrapping was used to simulate the expected TAS results.When the combination-EUs were comprised of at least one “passing” and one “failing” EU, the majority of these combination-EU would pass the TAS 79% - 100% of the time. Even in the case when both component EUs had failed, the combination-EU was expected to “pass” 11% of the time.Simulations of mini-TAS, a strategy with smaller power and hence smaller sample size than TAS, resulted in more conservative “passing” and “failing” when implemented in original EUs.Conclusions/SignificanceOur results demonstrate the high potential for misclassification when the average prevalence of lymphatic filariasis in the combined areas differs with regards to the TAS threshold. Of particular concern is the risk of “passing” larger EUs that include focal areas where prevalence is high enough to be potentially self-sustaining. Our results reaffirm the approach that Haiti took in forming smaller EUs. Where baseline or monitoring data show a high or heterogeneous prevalence, programs should leverage alternative strategies like mini-TAS in smaller EUs, or consider gathering additional data through spot check sites to advise EU formation.     

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells

gp96 (GRP94) elicits antigen-presenting cell (APC) activation and can direct peptides into the cross- presentation pathways of APC. These responses arise through interactions of gp96 with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC. Previously, CD91, the alpha2-macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class-A (SR-A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re-presentation are inhibited by the SR-A inhibitory ligand fucoidin, although fucoidin was without effect on alpha2-macroglobulin binding or uptake. Ectopic expression of SR-A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR-A-/- mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR-A in the regulation of cellular responses to heat shock proteins.     

Reduced Functional Reserve in Patients with Age-Related White Matter Changes: A Preliminary fMRI Study of Working Memory

Subcortical age-related white matter changes (ARWMC) are a frequent finding in healthy elderly people suggested to cause secondary tissue changes and possibly affecting cognitive processes. We aimed to determine the influence of the extent of ARWMC load on attention and working memory processes in healthy elderly individuals. Fourteen healthy elderly subjects (MMSE >26; age 55–80 years) performed three fMRI tasks with increasing difficulty assessing alertness, attention (0-back), and working memory (2-back). We compared activation patterns in those with only minimal ARWMC (Fazekas 0–1) to those with moderate to severe ARWMC (Fazekas 2–3). During the fMRI experiments, the study population showed activation in brain areas typically involved in attention and working memory with a recruitment of cortical areas with increasing task difficulty. Subjects with higher lesion load showed a higher activation at all task levels with only sparse increase of signal with increasing complexity. In the lower lesion load group, rising task difficulty lead to a significant and widely distributed increase of activation. Although the number of patients included in the study is small, these findings suggest that even clinically silent ARWMC may affect cognitive processing and lead to compensatory activation during cognitive tasks. This can be interpreted as a reduction of functional reserve and may pose a risk for cognitive decline in these patients.     

Effects of prolonged exercise in highly trained traumatic paraplegic men

This study investigated the cardiovascular and metabolic responses to prolonged wheelchair exercise in a group of highly trained, traumatic paraplegic men. Six endurance-trained subjects with spinal cord lesions from T10 to T12/L3 underwent a maximal incremental exercise test in which they propelled their own track wheelchairs on a motor-driven treadmill to exhaustion to determine maximal O2 uptake (VO2max) and related variables. One week later each subject exercised in the same wheelchair on a motorized treadmill at 60-65% of VO2max for 80 min in a thermoneutral environment (dry bulb 22 degrees C, wet bulb 17 degrees C). Approximately 10 ml of venous blood were withdrawn both 20 min and immediately before exercise (0 min), after 40 and 80 min of exercise, and 20 min postexercise. Venous blood was analyzed for hematocrit (Hct), hemoglobin (Hb), and lactate, and the separated plasma was analyzed for glucose, K+, Na+, Cl-, free fatty acid (FFA), and osmolality. VO2, CO2 production (VCO2), minute ventilation (VE), respiratory exchange ratio (R), net efficiency, and wheelchair strike rate were determined at four intervals throughout the exercise period. Data were analyzed with an analysis of variance repeated-measures design and a Scheffé post hoc test. VO2max was 47.5 +/- 1.8 (SE) ml.min-1.kg-1 with maximal VE BTPS and maximal heart rate (HR) being 100.1 +/- 3.8 l/min and 190 +/- 1 beats/min, respectively. During prolonged exercise there were no significant changes in VO2, VCO2, VE, R, net efficiency, wheelchair strike rate, and lactate, glucose, and Na+ concentrations. Significant increases occurred in HR, FFA, K+, Cl-, osmolality, Hb, and Hct throughout exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

Insulin tolerance test causes hypokalaemia and can provoke cardiac arrhythmias

We report the observation and analysis of a new adverse event during the insulin tolerance test (ITT) and propose additional safety procedures. An 8-year-old girl with growth hormone insufficiency had a cardiac arrest due to ventricular flutter when she was tested for growth hormone deficiency by the ITT. Severe hypokalaemia (K+ 2.6 mmol/l) was observed after resuscitation. Ergometry ECG revealed catecholaminergic polymorphic ventricular tachycardia, a hereditary arrhythmogenic disease. Consecutive measurements of serum potassium during ITT in 29 short children (21 boys) with growth failure revealed a mean decrease of serum potassium by 1.1 +/- 0.4 mmol/l with the nadir at 30 min after the insulin bolus. Hypokalaemia (serum potassium < 3.5 mmol/l) occurred in all but one child; severe hypokalaemia (serum potassium < 2.9 mmol/l) was measured in every third child. This observation indicates that acute hypokalaemia which is induced by insulin and catecholamine excess occurs frequently in ITT. The case shows that the combination of acute hypokalaemia and the adrenergic counterregulation in ITT is a strong trigger of cardiac arrhythmias, which can become life-threatening if the child has an arrhythmogenic disease. Therefore, we recommend ECG monitoring during ITT to enhance the detection of cardiac arrhythmias. In addition, in the case of a comatose child during ITT the determination of the glucose and potassium level as well as adequate treatment are necessary.     

Rectal and esophageal temperatures during upper- and lower-body exercise

This study investigated the question: is core temperature measurement influenced by whether exercise involves predominantly upper- or lower-body musculature? Healthy men were allocated to three groups: treadmill ergometry (T) n=4, cycle ergometry (C) n=6 and arm crank ergometry (AC) n=5. Subjects underwent an incremental exercise test to exhaustion on an exercise-specific ergometer to determine maximum/peak oxygen consumption (V˙O_2max). One week later subjects exercised for 36 min on the same ergometer at approximately 65% V˙O_2max while temperatures at the rectum (T _re) and esophagus (T _es) were simultaneously measured. The V˙O_2max (l · min⁻¹) for groups T [4.76 (0.50)] and C [4.35 (0.30)] was significantly higher than that for the AC group [2.61 (0.24)]. At rest, T _re was significantly higher than T _es in all groups (P<0.05). At the end of submaximal exercise in the C group, T _re [38.32 (0.11)°C] was significantly higher than T _es [38.02 (0.12)°C, P<0.05]. No significant differences between T _re and T _es at the end of exercise were noted for AC and T groups. The temperature difference (T _diff) between T _re and T _es was dissimilar at rest in the three groups; however, by the end of exercise T _diff was approximately 0.2°C for each of the groups, suggesting that at the end of steady-state exercise T _re can validly be used to estimate core temperature. Accepted: 3 November 1997     

Molecular localization of a redox-modulated process regulating plant mitochondrial electron transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.