Damage Mechanism Of HAmylin On Rat Primary Hippocampal Neurons

Alzheimer's disease?AD?and type II diabetes mellitus?DM2?are the most common aging-related diseases and are characterized by ?-amyloid accumulation in the brain and amylin accumulation in the pancreatic cells,respectively.Several studies have indicated a strong correlation between these two diseases.Amylin oligomerization in the brain appears to be a novel risk factor for the development of AD.Although amylin aggregation has been demonstrated to induce cytotoxicity in pancreatic ?-cell through altering of the homeostasis intracellular free Ca²⁺ concentration([Ca²⁺]_i),its underlying mechanisms have not been fully explored.In this study,we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole-cell patch clamp recordings.Previous studies demonstrated that A? protein in the oligomers formation process on the surface of cell membrane could produce large amounts of ROS,which in turn reduced the stability of cell membrane.We suspected that the damage mechanism of high concentration hAmylin in neurons might be similar.Therefore,we further explored the effect of hAmylin on neuronal membrane stability by immunofluorescence,live-cell imaging system and scanning electron microscopy.Part one The effect of Amylin on [Ca²⁺]_i in rat hippocampal neuronsObjective: To investigate the effects of rat amylin?rAmylin?and human amylin?hAmylin?at different concentrations on [Ca²⁺]_i in rat primary hippocampal neurons and its mechanism.Methods: We cultured the rat fetal hippocampal neurons and used the immunofluorescence technique to identify the neuronal purity and the expression of related receptors.The changes of [Ca²⁺]_i in neurons were recorded by calcium imaging technique.The expression of amylin receptorwas observed by immunofluorescence.The aggregation state of amylin was observed by transmission electron microscopy.Results:1 The purity identification of rat primary hippocampal neuronsNeuronal cell purity > 98%.2 The effects of amylin on [Ca²⁺]_i in rat primary hippocampal neuronsWe investigated the effects of different concentrations of hAmylin on[Ca²⁺]_i in cultured rat hippocampal neurons.Application of 3 to 30 ?M of hAmylin caused a significant [Ca²⁺]_i increase in the hippocampal neurons.The proportion of neurons responded to 3 and 10 ?M of hAmylin were 38.46%and 55.05%,respectively.The R?340/380?increase value was 67.6% ± 17.5%after 10 ?M hAmylin application and the EC50 value was approximately 2.53?M.After administration of 10 ?M hAmylin,the [Ca²⁺]_i levels in the hippocampal neurons peaked within 10 to 15 sec,and sustained at that levels for approximately 50 sec without fading.The [Ca²⁺]_i levels slowly returned to the baseline levels within 30 sec after washout.In addition,the peak value of hAmylin-induced [Ca²⁺]_i increase after repetitive application was similar to the first application,which indicated that no desensitization occurred in the neurons in response to long or repetitive drug application.Elevation in [Ca²⁺]_i levels was observed when 10 ?M of rat amylin?rAmylin?or pramlintide?a hAmylin analogue?was applied.Less than 10% of the total neurons responded to rAmylin and pramlintide.The R?340/380?increase values were 22.1 ± 4.0% in the presence of rAmylin and 24.8 ± 7.0%in the presence of pramlintide,which were significantly lower than the increase induced by application of 10 ?M of hAmylin?P < 0.001?.In addition,the hAmylin receptor antagonists AC187 and AC253?10 ?M?did not affect the [Ca²⁺]_i baseline in hippocampal neurons,but they abolished the Ca²⁺ responses evoked by 1 ?M of hAmylin,10 ?M of pramlintide and 10?M of rAmylin.Surprisingly,AC187 or AC253 failed to abolish the Ca²⁺ response induced by 10 ?M hAmylin.These results suggested that [Ca²⁺]_i increase induced by the low concentration of hAmylin was a amylinreceptor-dependent response whereas the Ca²⁺ response induced by the high concentration of hAmylin was likely through an amylin receptor-independent mechanism.3 The expression of amylin receptor RAMP in hippocampal neurons and adult rat brainUsing immunofluorescence technique,we found that amylin receptor subtype RAMP3 was highly expressed in fetal rat hippocampal neurons,but the expression in adult rat brain hippocampus area was much lower.RAMP1 and RAMP2 subtypes were highly expressed in both hippocampal neurons of rat fetus and adult rat hippocampal slices.4 Concentration-dependent effect on hAmylin aggregationWe speculated that the [Ca²⁺]_i increase induced by 10 ?M of hAmylin might be related to the amyloidogenic property of hAmylin.To test this possibility,we tested the aggregative state of hAmylin using transmission electron microscopy.Both the oligomer and fibrillar forms of amylin were detected in the high?10 ?M?,but not in the low?1 ?M?concentration of hAmylin after 1 or 24 h incubation at 37°C.These results suggested that the receptor-independent mechanism of [Ca²⁺]_i response induced by the high concentration of hAmylin might be related to its misfolding tendency at high concentrations to form its oligomers and fibrous structures.Part two The excitatory regulation of hAmylin on TRPV4Objective: To investigate the receptor-independent regulating mechanism of high concentration of hAmylin on [Ca²⁺]_i increase in neuronsMethods: We recorded the changes in [Ca²⁺]_i using calcium imaging technique and resting potentials using whole-cell patch-clamp technique in cultured rat hippocampal neurons.The role of TRPV4 in hAmylin-induced[Ca²⁺]_i increase was investigated by scRT-PCR and siRNA techniques.Results:1 The calcium channels of plasma membrane involved in hAmylininduced [Ca²⁺]_i increaseIncreases in [Ca²⁺]_i depends on two sources,Ca²⁺ influx from the extrac-ellular space and/or Ca²⁺ release from intracellular Ca²⁺ stores.To investigate whether the hAmylin oligomer-induced increase in Ca²⁺ was due to Ca²⁺ influx from the extracellular space,Ca²⁺ was removed from the extracellular solution and 0.1 mM EGTA was added to chelate the remaining Ca²⁺ .In the extracellular Ca²⁺ -free solution,10 ?M hAmylin did not increase the [Ca²⁺]_i levels in hippocampal neurons,which suggests that Ca²⁺ -entry through Ca²⁺ -permeable ion channels on the cell membrane was responsible for the[Ca²⁺]_i increase induced by the hAmylin oligomer.To further study the contribution of intracellular Ca²⁺ stores to the hAmylin-induced [Ca²⁺]_i increase,hippocampal neurons were pretreated with the sarco/endoplasmic reticulum calcium ATPase?SERCA?blocker CPA?cyclopiazonic acid,5 ?M?.Bath application of CPA for 1 min caused an increase in [Ca²⁺]_i.However,subsequent application of the hAmylin oligomer still induced a rapid [Ca²⁺]_i increase,which was similar to the effect induced by the first application of the hAmylin oligomer before the CPA treatment.Similar results were found in neurons incubated with TG?thapsigargin,1 ?M?,and 10 ?M hAmylin still increased [Ca²⁺]_i in the presence of TG.These results suggested that Ca²⁺ influx from the extracellular space played the prominent role in the high concentration hAmylin-induced [Ca²⁺]_i increase in hippocampal neurons.2 Voltage-gated calcium channels involved in hAmylin-induced [Ca²⁺]_i increaseWe used patch-clamp technique to detect the effect of hAmylin on resting potential of plasma membrane.We found that the resting membrane potential was depolarized from-55.67 ± 2.35 to-35 ± 2.8 mV after bath application of10 ?M of the hAmylin.In addition,there were 3 out of 21 responsive cells accompanied by an action potential discharge.The L-type calcium channels were activated when the membrane resting potential was depolarized to-40 mV.Thus we assumed that voltage-gated calcium channels were involved in hAmylin-induced [Ca²⁺]_i increase in the neurons.The rise in [Ca²⁺]_i triggered by high concentration hAmylin wassignificantly inhibited when neurons were pretreated with 5 ?M amilodipine(a selective blocker of L-type Ca²⁺ channels)or 1 ?M ?-conotoxin GVIA(a selective N-type Ca²⁺ channel blocker)for 1 min.The hAmylin-induced R?340/380?increases were 44.49% ± 13.84% and 52.84% ± 13.23% in the presence of amilodipine and ?-conotoxin GVIA,respectively,which were significantly less than the increases from 10 ?M hAmylin alone?67.98% ±14.85%??P < 0.01?.These results suggested that voltage-gated Ca²⁺ channels,particularly the L-type Ca²⁺ channels,contributed to the high concentration hAmylin-induced [Ca²⁺]_i rise in hippocampal neurons.However,the combination of amilodipine and ?-conotoxin GVIA did not completely abolish the hAmylin-induced [Ca²⁺]_i increase,suggesting that other Ca²⁺ permeable channels might also be involved.3 The hAmylin-induced [Ca²⁺]_i increase depended on extracellular sodiumTo investigate how the high concentration of hAmylin depolarized the membrane potential,Na+ was replaced by NMDG,a large organic cation impermeable to cationic channels.In the Na+-free external solution,the hAmylin oligomer-induced [Ca²⁺]_i increase was significantly decreased.The positive rate dropped to 18.2% and the response changed from 65.39% ±15.91% to 34.34% ± 4.57%?P < 0.001?,which suggested that the [Ca²⁺]_i response induced by the high concentration of hAmylin was dependent on extracellular Na+ entry and followed by membrane depolarization.Interestingly,when 1 ?M of TTX?Na+ channel blocker?was added to the buffer,no expected inhibition was observed in the experiments,which indicated that voltage-gated Na+ channels were not involved in the hAmylin-induced [Ca²⁺]_i increase.In addition,the remaining [Ca²⁺]_i increase was not inhibited by the calcium channel blocker amilodipine.These results implicated that non-selective cation channels that were permeable for Ca²⁺ /Na+,such as transient receptor channels?TRP channels?,were likely involved in the high concentration hAmylin-induced [Ca²⁺]_i increase in hippocampal neurons.4 TRPV4 participated in hAmylin-induced increase in [Ca²⁺]_iAfter the application of the general TRP channel inhibitor,ruthenium red?RR??10 mM?the hAmylin oligomer-induced [Ca²⁺]_i increase was significantly inhibited?P < 0.01?.In the presence of RR,10 ?M hAmylin depolarized the resting membrane potential to 8.8 ± 1.1 mV,which was significantly lower than the depolarization induced by the hAmylin alone?21.4 ± 1.7 mV??P < 0.001?.To further investigate the subtype of the TRP family involved,single cell RT-PCR analysis was used to detect the expression pattern of the TRP channel subtypes in hAmylin-responsive hippocampal neurons.Among all the neurons tested,6 out of 7 cells expressed TRPV4,which was consistent with the findings observed in pancreatic ?-cells.In addition,application of the TRPV4 channel selective inhibitor HC067047?1 ?M?significantly inhibited the hAmylin-induced [Ca²⁺]_i increase?P < 0.01?.In the presence of HC067047,the hAmylin depolarized the resting membrane potential to 4.5 ± 0.5 mV,which was significantly lower than the depolarization induced by the hAmylin oligomer alone?18.1 ± 1.8 mV??P < 0.001?.In the brain slices of adult SD rats,the expression of TRPV4 in the hippocampus was much lower which was consistent with previous reports.On the other hand,the positive expression of TRPV4 was 54% in the primary cultured rat fetal hippocampal neurons,and this value was consistent with the positive response of hAmylin-induced increase in [Ca²⁺]_i.To further confirm that TRPV4 was involved in the hAmylin-induced[Ca²⁺]_i increase,a TRPV4 agonist?GSK1016790A?was applied to the cultured hippocampal neurons.We found that the TRPV4 agonist GSK1016790 A and the high concentration of hAmylin evoked a [Ca²⁺]_i response in the same cells.However,the hAmylin-induced [Ca²⁺]_i response was lower than that induced by TRPV4 agonist GSK1016790A?100 nM?in hippocampal neurons.Therefore,FAM-labeled small interfering RNA?siRNA?for rat TRPV4 was transfected into hippocampal neurons to knock down the expression of TRPV4 channels.The GSK1016790A-induced [Ca²⁺]_i increasewas significant lower in the siRNA-treated hippocampal neurons compared to the scramble siRNA-treated hippocampal neurons?P<0.05?.The hAmylininduced [Ca²⁺]_i increase was also significantly suppressed in the TRPV4siRNA-treated hippocampal neurons,but not in the scrambled siRNA transfected neurons?P < 0.05?.These findings indicated that TRPV4 was likely an early key molecular candidate for the high concentration of hAmylin-induced [Ca²⁺]_i increase in hippocampal neurons,which was similar to the findings in pancreatic ?-cells.To investigate whether hAmylin directly activated TRPV4 channels,we added GSK1016790A?100 nM?and hAmylin in HEK293 cells transfected with TRPV4 channels.GSK1016790 A still induced [Ca²⁺]_i increase but hAmylin did not produce any response of cellular [Ca²⁺]_i.This result indicated that hAmylin was not a direct agonist of TRPV4 channels.Part three The effect of hAmylin on the plasma membrane stability of neuronsObjective: To investigate the mechanism underlying the activation of TRPV4 channels by hAmylin in the neuronsMethods: We used fluorescence microscopy to investigate changes in[Ca²⁺]_i,mitochondrial membrane potential?mt???and reactive oxygen species?ROS?generation in the fetal hippocampal neuronal cultures.We used immunofluorescence,live cell imaging system and scanning electron microscope to observe the influence of hAmylin on the stability of plasma membrane.Results:1 Neuronal cell furnace targeting of hAmylin oligomersAfter an incubation with labeled hAmylin?N-terminal labeled with FAM fluorescence?for 15 min in 37? and the refreshment of culture medium,the fluorescence of aggregated hAmylin was observed.After washing out with fresh medium,the fluorescence disappeared.These results indicated that hAmylin aggregated only on the surface of cell membrane.To investigate whether the aggregation process of hAmylin was reversible,we observed the aggregation of hAmylin for 12 h after incubationwith labeled hAmylin for 15 min.The fluorescence did not decrease,indicating that the process of hAmylin aggregation was irreversible.2 Long-term effect of hAmylin on neuronal morphologyWe used live-cell imaging system to observe neuronal morphology after incubation with high concentrations of hAmylin?10 ?M?for a long period of time.After incubation for about 4 h,karyopyknosis of neurons was observed.After incubation for about 9 h,apoptosis was observed.3 Direct effect of hAmylin on hippocampal tissue in vivo experimentsIn order to observe the direct effect of hAmylin on hippocampus of the rats,we injected 5 ?L of 10 ?M hAmylin into the lateral ventricle of adult rat.The rats were sacrificed 24 h after the injection and the brain was collected for frozen sections.Using immunofluorescence staining,we observed hAmylin-induced hippocampal neuronal loss in rats.In our experiments,microglia migration was not significantly observed,indicating that the inflammatory response was not the main cause of neuronal loss.4 Effect of hAmylin on cell membrane integrityWe hypothesized that hAmylin-induced neuronal [Ca²⁺]_i increase might be associated with the formation of nonselective ion permeation channels on the surface of cell membrane.We did not observe changes in [Ca²⁺]_i after administration with 10 ?M hAmylin for 1 min in HEK293 cells,and hAmylin-induced ionic current change was not observed using the whole-cell voltage clamp.We used immunofluorescence techniques to stain neurons and glial cells.When Triton X-100,the most commonly used permeabilization agent,was replaced by hAmylin,neurons showed fluorescence after incubated with hAmylin for a short period of time?1 min?,but glial cells did not show fluorescence after the same incubation with hAmylin.When we extended the incubation time to 30 min,the fluorescence was observed in both neurons and glial cells.These results indicated that when hAmylin aggregated on the cell surface,it damaged the cell membrane integrity,and the damage effects were cell-type dependent and time-dependent.This might explain why hAmylincould not increase the [Ca²⁺]_i in HEK293 cells or glial cells in a short time.We used scanning electron microscopy to further confirm the effect of hAmylin on cell membrane stability.After hAmylin incubation with cells,the integrity of neuronal and glial cell membrane was destroyed.After transfected with LifeAct plasmid,HEK293 cells was labeled with cytoskeleton?F-actin?.Then,the effect of hAmylin incubation for a long period of time was observed using the living cell imaging system.We observed that 10 ?M hAmylin damaged the integrity of cytoskeleton in HEK293 cells after a long-term incubation.5 Effects of hAmylin on ROS generation and mitochondrial membrane potential in neuronsIt has been reported that the A? protein destroys cell membrane integrity mainly depending its formation of ROS.We hypothesized that the mechanism by which hAmylin destroyed cell membrane integrity might be the same.We used ion imaging technology to observe the generation of ROS in neurons.It was found that high concentration of hAmylin?10 ?M?significantly increased the amount of ROS in neurons?P < 0.001?,while low concentration of hAmylin?1 ?M?did not affect ROS generation.These results indicated that the amount of ROS generation was mainly related to the aggregation of hAmylin.We used JC-1 dye to further observe the effect of hAmylin on neuronal mitochondrial membrane potential.High concentration of hAmylin?10 ?M?significantly reduced mitochondrial membrane potential while low concentration of hAmylin?1 ?M?did not influence the potential.The reduction of mitochondrial membrane potential caused by 10 ?M hAmylin was significantly inhibited?P<0.001?by administration of CsA?mitochondrial PTP opening inhibitor,1 ?M?.But CsA was not able to inhibit [Ca²⁺]_i increase or ROS generation in response to hAmylin.The increase of [Ca²⁺]_i and ROS generation was not related to the decrease of mitochondrial membrane potential.Conclusions:The hAmylin induces [Ca²⁺]_i increase in the rat hippocampal neurons in primary culture through receptor-dependent mechanism mainly responsible for the lower concentration of hAmylin and receptor-independent mechanisms mainly responsible for the high concentration of hAmylin.The receptor-independent mechanism is mainly related to the misfolding and oligomer aggregation of hAmylin at high concentration.hAmylin aggregates precipitated on the neuronal membrane,activated TRPV4 channels and subsequently triggered membrane voltage-gated calcium channel opening followed by membrane depolarization.In the aggregating process on cell surface,the oligomers produce ROS generation,damage of cell membrane integrity and a decrease in mitochondrial membrane potential.