Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction.

Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by protein kinase C (PKC). We have previously shown that the Drosophila adducin, Hu-li tai shao (Hts), plays a role in larval neuromuscular junction (NMJ) growth. Here, we find that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), we show that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.

Neuromuscular dysfunction in the mutant superoxide dismutase mouse model of amyotrophic lateral sclerosis.

To better understand the interaction between motor neuron dysfunction and denervation in amyotrophic lateral sclerosis (ALS), we have evaluated motor neuron number and the retrograde uptake and transport of fluorogold by motor neurons in mice overexpressing mutant superoxide dismutase (mSOD), and wild-type controls. N-CAM immunoreactivity and protein kinase expression were determined in skeletal muscle during denervation. We found that in severely affected mSOD mice, motor neuron loss is moderate (approximate 40% reduction), whereas retrograde uptake/transport as assessed using fluorogold is profoundly impaired (approximately 90% reduction). The impairment in fluorogold uptake/transport corresponds to measures of progressive muscle denervation such as increased N-CAM immunoreactivity of muscle and increased expression of protein kinase B (PKB) in denervated muscle. These data suggest that the debility in the mSOD mouse model of ALS is produced, in part, by impaired retrograde uptake/transport in motor neuron axons in spite of regenerative support from muscle such as elevated expression of PKB.

Heart rate variability of recently concussed athletes at rest and exercise.

PURPOSE: The objective of this study was to assess the neuroautonomic cardiovascular regulation in recently concussed athletes at rest and in response to low-moderate steady-state exercise, using heart rate variability (HRV). METHODS: A 5-min ECG sample was taken at rest from the 14 concussed athletes at 1.8 (+/- 0.2) days postinjury and again at 5 d later. Once asymptomatic at rest, the concussed athletes and their matched controls (N = 14) participated in an exercise protocol. The protocol consisted of a 2-min warm-up with a pedaling frequency between 50 and 60 rpm against a load of 40 W. After the warm-up, the athletes engaged in a low-moderate intensity steady state 10-min exercise bout where the pedaling frequency and load increased to 80-90 rpm and 1.5 W x kg(-1) body weight, respectively. The protocol was repeated 5 d later. A 5-min ECG sample from minutes 4 to 9 of the low-moderate intensity steady state exercise bout was used to assess HRV during exercise. Mixed model ANOVA were used to analyze the data. RESULTS: No difference at rest was detected between the concussed athletes and their matched controls in any of the HRV variables measured. However, across both exercise tests, the concussed group demonstrated a significant decrease in the mean RR interval, and low- and high-frequency power (P < 0.05) in relation to their matched controls. CONCLUSION: Low-moderate steady-state exercise elicits a neuroautonomic cardiovascular dysfunction in concussed athletes that is not present in a rested state. This dysfunction alludes to an exercise induced uncoupling between the autonomic and cardiovascular systems.

Task frequency as a function of age for the powerline technician trade.

It is traditionally believed that worker productivity declines with age. The greatest contributor to this stereotype, particularly in physically demanding jobs, is that older workers can no longer maintain their sustained level of work due to the age-associated decline in physical capacity. The objective of this study was to determine the change in the frequency of task performance as a function of age in the power line technician (PLT) trade. The task requirements of the PLT occupation were determined through the use of a detailed job demands analysis (JDA) and the frequency at which these tasks were performed was acquired through a survey derived from the JDA. The results of the survey were assessed as a function of age. Older lineworkers (> or = 50 years) were found to spend more time working on the ground whereas the younger PLTs (< or = 39 years) more frequently performed the climbing tasks and the corresponding work on the poles and towers. In contrast the older PLTs performed all pushing/pulling tasks at a greater or equal frequency to that of the younger lineworkers. Despite these differences the frequency of task performance is similar across the age groups studied.

Changes in physical capacity as a function of age in heavy manual work.

The objective of this study was to assess the changes in the physical capacity as a function of age in power line technicians (PLTs). The physical test was designed to closely represent the essential physical tasks of the occupation that were identified through a detailed job demand analysis. The results from the physical test showed that six out of nine test variables did not demonstrate a statistical difference between the mean scores of young (< or = 39 years) and old age (50+ years) groups. However the older group scored significantly lower in the aerobic capacity test, one-handed pull down, and both right and left standard handgrip tests. Despite these differences the older PLT appears to meet and exceed the physical requirements necessary to carry out the essential tasks of this trade. However a physical test with a high level of content and construct validity is necessary to accurately evaluate the workers physical capacity in relation to the job demands. Based on the principal of specificity for muscle training and testing, this study has demonstrated that heavy manual work appears to maintain physical capacity specific to the task as age progresses.

Age-related differences in the des IGF-I-mediated activation of Akt-1 and p70 S6K in mouse skeletal muscle.

The ability of des IGF-I to activate Akt-1 and p70 S6K in skeletal muscle with or without acute endurance exercise was examined in young and old mice. Mice were sacrificed 12 h after a moderate intensity treadmill run following an interperitoneal injection of des-IGF-I or saline. Blood and skeletal muscle were collected and IGF-I receptor, Akt-1 and p70 S6K protein contents and their phosphorylation status were determined. Injection of des IGF-I similarly decreased plasma glucose concentration in both young (P<0.01) and old mice (P<0.01) whereas plasma insulin and total IGF-I levels of young and old mice were not significantly changed by des IGF-I. Total IGF-I receptor protein and IGF-1 receptor phosphorylation were lower in aged mice (P<0.05). Basal phosphorylation of Akt-1 was lower in aged skeletal muscle (P<0.01) and this was not caused by changes in Akt-1 protein. In both young (P<0.01) and aged (P<0.05) mice, des IGF-I significantly increased the phosphorylation of Akt-1 at Ser 473. However, a des IGF-I-mediated increase in the p70 S6K phosphorylation (P<0.01) was only seen in young mice. Prior exercise decreased the total plasma IGF-I level in the presence of des IGF-I in aged mice. Des IGF-I-mediated Akt-1 and p70 S6K phosphorylation was not changed by exercise in either young or old mice. It is concluded that there was an aging-related resistance at the p70 S6K level in mouse skeletal muscle that could not be restored by prior exercise and this resistance is associated with lower IGF-I receptor number and Akt-1 phosphorylation in the aged skeletal muscle.

Effects of aging on hepatic IGF-I signaling.

Little is known regarding hepatic insulin-like growth factor-1 IGF-I signaling with aging despite the observation that other tissues demonstrate resistance to IGF-I with aging and declines in liver mass accompany aging. Our aim was to determine if the IGF-I-induced signaling process changes with aging. Young (5 months) and old (24 months) C57BL/6 mice hepatic tissues and blood samples were taken 20 min after an intraperitoneal injection of desIGF-I. Age had no significant effect on plasma glucose, insulin and total IGF-I levels. IRS-1 protein was significantly decreased (33%) with aging. Basal phosphorylation of IRS-1, PKB and ERK were unaffected whereas basal phosphorylation of CREB and FKHR were significantly increased (37 and 33%, respectively) with aging. desIGF-I caused a significant decrease in plasma glucose concentrations in both young (53%) and old (44%) mice. desIGF-I administration significantly increased the phosphorylation of IRS-1 in both young (104%) and old (89%) hepatic tissues. Similarly, the phosphorylation of PKB was dramatically enhanced in both young (527%) and old (350%) hepatic tissues after desIGF-I stimulation. By contrast, desIGF-I administration had no significant effects on the phosphorylation of ERK and phosphorylation of transcription factors CREB and FKHR in both young and old hepatic tissues. These data suggest that aging dose not impair IGF-I signaling in hepatic tissues.