Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Reduction in neural performance following recovery from anoxic stress is mimicked by AMPK pathway activation.

Nervous systems are energetically expensive to operate and maintain. Both synaptic and action potential signalling require a significant investment to maintain ion homeostasis. We have investigated the tuning of neural performance following a brief period of anoxia in a well-characterized visual pathway in the locust, the LGMD/DCMD looming motion-sensitive circuit. We hypothesised that the energetic cost of signalling can be dynamically modified by cellular mechanisms in response to metabolic stress. We examined whether recovery from anoxia resulted in a decrease in excitability of the electrophysiological properties in the DCMD neuron. We further examined the effect of these modifications on behavioural output. We show that recovery from anoxia affects metabolic rate, flight steering behaviour, and action potential properties. The effects of anoxia on action potentials can be mimicked by activation of the AMPK metabolic pathway. We suggest this is evidence of a coordinated cellular mechanism to reduce neural energetic demand following an anoxic stress. Together, this represents a dynamically-regulated means to link the energetic demands of neural signaling with the environmental constraints faced by the whole animal

Loss of an activity-dependent hyperpolarization in the DCMD axon following AMPK pathway activation.

<p>(<b>A</b>). The DCMD axon hyperpolarizes during periods of high frequency activity in response to visual stimulation. The shift in membrane potential occurs with no change in the reversal of the afterpotential (side panel), suggesting that this is an electrogenic effect. Black and grey traces in right-side panel are from time points in raw trace indicated by black and grey asterisks, respectively. (<b>B</b>). The membrane potential shows similar hyperpolarizing shifts following electrical stimulation of the axon that generates APs. (<b>C</b>). Electrically stimulated activity fails to evoke a hyperpolarization during ouabain treatment (10⁻⁴ M). (<b>D</b>). There is no change in input resistance before the activity-dependent hyperpolarization (pre) compared to during the event (post). (<b>E</b>). This hyperpolarization is greatly reduced after ouabain application (10⁻⁴ M; P<0.05). (<b>F</b>). Metformin (10 mM) significantly reduces the hyperpolarization in response to 100 Hz electrical stimulation (P<0.05).</p

Performance is temporarily reduced after anoxic coma induced by water immersion.

<p>(<b>A</b>). Raw traces of DCMD activity in response to a looming target stimulus (bottom panel) demonstrate a delay of the response relative to target collision as well as a loss of overall spike activity. (<b>B</b>). The reduction in mean spike count is most prominent at 1 hour of recovery, and gradually returns to control levels over a period of 5 hours. (<b>C</b>). Peristimulus time histogram of grouped data showing instantaneous frequencies of action potentials during target approach with coma recovered animals having a delayed latency and lower peak activity compared to control animals. (<b>D</b>). Illustration of recording arrangement with two extracellular electrodes (ant, anterior; post, posterior) across the mesothoracic ganglion and an intracelullar electrode immediately posterior to the ganglion. Traces are overlayed at the start of the intracellular AP rising phase. (<b>E</b>). An examination of AP properties of the DCMD axon recorded from this region compared between control and coma recovered animals reveal a mild resting membrane depolarization and a significant reduction in AP amplitude in coma-recovered animals. (<b>F</b>). Coma-recovered animals also show a decrease in the conduction velocity between the extracellular electrodes. (<b>G</b>). Frequency-dependent AP amplitude attenuation between events with similar instantaneous frequency from control and coma-recovered animals. APs from coma recovered animals both start with APs of lower amplitude and lose amplitude to a higher degree than controls. (<b>H</b>). Group scatter data of individual APs during a looming response plotted to show the relationship between AP instantaneous frequency, AP amplitude loss, and conduction velocity. Coma-recovered animals show a marked instability of both AP amplitude and conduction velocity with increasing instantaneous frequency. Asterisks denote statistical significance (P<0.05). Number of animals per condition is noted within the bars, valued as mean ± SE.</p

Effect of coma on flight behaviour and basal metabolic rate.

<p>(<b>A</b>). Coma recovered animals have reduced flight duration in the wind tunnel. Box-plot spans 25^th to 75^th percentiles, error bars represent 10^th to 90^th percentile, with solid line as median value. (<b>B</b>). Change in wingbeat frequency as measured through EMG activity from thoracic flight muscles during flight in a wind tunnel. Representative data showing deviations from normal wingbeat frequency for three looms trials in each of a control (black) and a coma-recovered animal (grey). (<b>C</b>). Control animals respond more frequently to the target approach than coma recovered animals. (<b>D</b>). Individual measurement of whole animal metabolic rate using CO2 respirometry prior to anoxic coma and in the recovery period. Basal metabolic rate is initially increased sharply upon return to normoxia, but is then decreased in the following recovery period. Note that prior to coma this animal displayed discontinuous gas exchange characteristic of quiescent animals. Not all locusts showed this pattern and it did not affect the overall CO₂ measured. (<b>E</b>). Metabolic rate is decreased during the post-coma recovery period. Asterisks denote statistical significance (P<0.05).</p

Anoxic preconditioning and AMPK activation is protective against subsequent anoxic events.

<p>Animals were submerged in water and the time until they entered a coma state was measured. Preinjection with 10 µL of 1 mM Compound C shortened the time until coma, and metformin preinjection (10 µL, 500 mM) prolonged the animal's ability to remain active in anoxia. A preconditioning effect was found, in that animals that had recovered for one hour from a previous water submersion showed increased time to coma on subsequent anoxia. The preconditioning effect was similar to preinjection with metformin. Statistical significance is denoted by differing letters (P<0.05).</p

Manipulation of AMPK pathway affects performance in the LGMD/DCMD circuit.

<p>(<b>A</b>). Metformin application (arrow) results in a decreased responsiveness to repeated looming stimuli. (<b>B</b>). Grouped data demonstrating reduced response in metformin (asterisk, P<0.05). (<b>C</b>). Axon recordings of DCMD APs have different waveforms after anoxic coma, or following pharmacological manipulation of the AMPK pathway. Metformin (Met, 10 mM) was used to activate AMPK, Compound C (CC, 0.1 mM) was used to inhibit AMPK. (<b>D</b>). AMPK inhibition (CC, 0.1 mM) attenuates the effects of anoxic coma on AP amplitude, whereas AMPK activation (Met, 10 mM; AICAR, 1 mM) produces APs with a more coma-like waveform. Statistical significance in amplitude differences denoted by differing letters (P<0.05). These treatments do not show a significant difference in the resting membrane potential. (<b>E</b>). Raw trace during repeated 200 Hz stimulation. (<b>F</b>). Grouped data showing AP amplitude drops in metformin treated animals but is sustained in control animals (n = 5 Con, n = 9 Met). (<b>G</b>). Conduction velocity in the DCMD axon during responses to a looming target. Spikes were binned into groups based on instantaneous frequency (<100 Hz, 100–250 Hz, >250 Hz). Measurements were taken before treatment (0 min), after 20 minutes of either a saline control or metformin treatment (as indicated along the x-axis), and again after a 20 minute saline washout (40 min). Conduction velocity was reduced after 20 min of metformin (asterisk, P<0.05), and recovered after washout. (<b>H</b>). Similar effects on conduction velocity are seen with the AMPK activator A-769662, with conduction velocity reduced from pre-treatment levels (0 min) after 15 min of A-769662 and further still after 35 min of A-769662 at all frequency bins (asterisk, P<0.05).</p

Involvement of cAMP in the DCMD axonal response to a metabolic stress.

<p>(<b>A</b>). Pharmacological blockade of cAMP activity by bath application of DDA (1 mM) results in an impairment of conduction velocity down the DCMD axon of otherwise healthy animals (Normoxia; P<0.001). Continual DDA bath application led to conduction failure in the majority of preparations, preventing examination of washout. Quantified responses to DDA across firing frequency are shown in the right panel. (<b>B</b>). Inducing hypoxia by cutting trachea to the thoracic nervous system reduces conduction velocity in the DCMD axon. The loss of conduction velocity can be mitigated by bath application of the cAMP activator forskolin (100 µM). Right panel displays grouped data showing that forskolin protects AP conduction across different firing rates during hypoxia (P<0.05). (<b>C</b>). Reduced conduction velocity in the DCMD axon during responses to a looming target in HCN channel blocker ZD 7288 (100 µM; P<0.001). The effect is reversible and more pronounced during high frequency activity (right panel).</p

Neural performance can be monitored using the visual looming detector circuit, via the descending contralateral movement detector (DCMD).

<p>(A). The timing and electrophysiological properties of APs elicited by a 1 m/s looming stimulus can be recorded from the axon of DCMD. (B). Conduction delay of APs recorded between two extracellular suction electrodes can be used to calculate relative conduction velocity. (C). Quantification of differences in conduction velocity with increasing instantaneous frequency. (D). Activity-dependent hyperpolarization of DCMD (arrow) can be monitored in response to visual stimuli. (E). Electrical stimulation of the axon to evoke 1000 APs (grey arrow) is also sufficient to induce an activity-dependent hyperpolarization (black arrow).</p