The neuromuscular transmission of the SOD1(G93A) mice in the pre-symptomatic phase of the disease.

<p>A) Illustrates raw recordings of 5 EPPs from 4–6 week-old WT and pre-symptomatic SOD1(G93A) mice. B) Shows the frequency histogram of MEPPs amplitudes, which pools the amplitude of all MEPPs recorded at WT (2785 MEPPs) and SOD1(G93A) (2270 MEPPs) fibers. WT – Black bars; SOD1(G93A) – Gray bars. C) Presents the nonlinear regression applied to WT and SOD1(G93A) distributions. As illustrated, both distributions were best fitted with a Gaussian function. In D) are shown examples of a MEPP (<1mV) and a GMEPP (>1mV) and in E) examples of spontaneous events recorded in gap free mode across 10s in WT and SOD1(G93A) neuromuscular junctions.</p

The neuromuscular transmission of the SOD1(G93A) mice in the symptomatic phase of the disease.

<p>A) Illustrates raw recordings of 5 EPPs from adult WT mice and the two groups of symptomatic SOD1(G93A) neuromuscular junctions, SOD1a and SOD1b. B) Shows the frequency histogram of MEPPs amplitudes, that pools the amplitude of all MEPPs recorded at WT (2288 events) and SOD1(G93A) (3674 events) fibers. WT – Black bars; SOD1(G93A) – Gray bars. C) Shows the nonlinear regressions that best shape WT (Gaussian function) and SOD1(G93A) (sum of two Gaussians) data As illustrated, SOD1(G93A) data follows a bimodal distribution with two peak amplitudes, pointing to the existence of 2 groups of neuromuscular junctions. D) After categorizing the two groups, the distributions of MEPPs amplitudes from SOD1a and SOD1b groups were drawn and best-fitted with Gaussian functions. As it is visible, the peak amplitude from both distributions matches the two peak amplitudes from the bimodal curve, validating the grouping. E) Shows examples of spontaneous events recorded in gap free mode across 10s in WT, SOD1a and SOD1b neuromuscular junctions.</p

Comparison of A_2A receptor function upon disease progression in SOD1(G93A) mice and healthy controls; average time course of mean EPP amplitude facilitation by CGS 21680 (5 nM) in (A) pre-(n = 10) and symptomatic (n = 6) SOD1(G93A) rodents and (B) 4–6 weeks (n = 5) and 12–14 weeks (n = 6) old WT mice; effect of CGS 21680 perfusion at 5 nM on: (C) quantal content of EPPs (4–6 weeks old: n = 13, WT, n = 12, SOD1(G93A); 12–14 weeks old: n = 9, WT, n = 7, SOD1(G93A)); (D) MEPP frequency (4–6 weeks old: n = 10, WT, n = 9, SOD1(G93A); 12–14 weeks old: n = 6, WT, n = 5, SOD1(G93A)); and (E) GMEPP frequency (4–6 weeks old: n = 10, WT, n = 11, SOD1(G93A); 12–14 weeks old: n = 4, WT, n = 4, SOD1(G93A)) in both phases of the study from SOD1(G93A) mice and respective healthy controls; *p<0.05 Unpaired <i>t</i>-test; ^∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; ^#p<0.05 Paired <i>t</i>-test (as compared with control value before drug perfusion); control corresponds to

<p>Comparison of A_2A receptor function upon disease progression in SOD1(G93A) mice and healthy controls; average time course of mean EPP amplitude facilitation by CGS 21680 (5 nM) in (A) pre-(n = 10) and symptomatic (n = 6) SOD1(G93A) rodents and (B) 4–6 weeks (n = 5) and 12–14 weeks (n = 6) old WT mice; effect of CGS 21680 perfusion at 5 nM on: (C) quantal content of EPPs (4–6 weeks old: n = 13, WT, n = 12, SOD1(G93A); 12–14 weeks old: n = 9, WT, n = 7, SOD1(G93A)); (D) MEPP frequency (4–6 weeks old: n = 10, WT, n = 9, SOD1(G93A); 12–14 weeks old: n = 6, WT, n = 5, SOD1(G93A)); and (E) GMEPP frequency (4–6 weeks old: n = 10, WT, n = 11, SOD1(G93A); 12–14 weeks old: n = 4, WT, n = 4, SOD1(G93A)) in both phases of the study from SOD1(G93A) mice and respective healthy controls; *p<0.05 Unpaired <i>t</i>-test; ^∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; ^#p<0.05 Paired <i>t</i>-test (as compared with control value before drug perfusion); control corresponds to 100% in all cases.</p

CGS 21680 facilitation of evoked activity is exacerbated in pre-symptomatic mice; (A) representative time-course change of mean EPP amplitude throughout CGS 21680 (5 nM) perfusion and (B) representation of EPP amplitude increase in 4–6 weeks old WT (n = 5) and pre-symptomatic mice (n = 10) upon A_2A receptor activation (CGS 21680 at 5 nM); (C) concentration-response changes in mean EPP amplitude in the presence of CGS 21680 (3 nM: n = 7, WT, n = 7, SOD1G93A; 5 nM: n = 14, WT, n = 13, SOD1G93A; 10 nM: n = 7, WT, n = 5, SOD1G93A) whose effect was blocked by SCH 58261 at 50 nM (n = 5, WT, n = 4, SOD1G93A); (D) raw recording of spontaneous release fluctuations from a 4–6 weeks old WT and pre-symptomatic SOD1G93A neuromuscular junction promoted by CGS 21680 (5 nM); effect of CGS 21680 (5 nM) perfusion regarding (E) MEPP frequency (n = 10, WT, n = 9, SOD1(G93A), (F) quantal Content of EPPs (n = 13, WT, n = 12, SOD1(G93A)) and (G) GMEPP frequency (n = 10, WT, n = 11, SOD1(G93A)) in

<p>CGS 21680 facilitation of evoked activity is exacerbated in pre-symptomatic mice; (A) representative time-course change of mean EPP amplitude throughout CGS 21680 (5 nM) perfusion and (B) representation of EPP amplitude increase in 4–6 weeks old WT (n = 5) and pre-symptomatic mice (n = 10) upon A_2A receptor activation (CGS 21680 at 5 nM); (C) concentration-response changes in mean EPP amplitude in the presence of CGS 21680 (3 nM: n = 7, WT, n = 7, SOD1G93A; 5 nM: n = 14, WT, n = 13, SOD1G93A; 10 nM: n = 7, WT, n = 5, SOD1G93A) whose effect was blocked by SCH 58261 at 50 nM (n = 5, WT, n = 4, SOD1G93A); (D) raw recording of spontaneous release fluctuations from a 4–6 weeks old WT and pre-symptomatic SOD1G93A neuromuscular junction promoted by CGS 21680 (5 nM); effect of CGS 21680 (5 nM) perfusion regarding (E) MEPP frequency (n = 10, WT, n = 9, SOD1(G93A), (F) quantal Content of EPPs (n = 13, WT, n = 12, SOD1(G93A)) and (G) GMEPP frequency (n = 10, WT, n = 11, SOD1(G93A)) in pre-symptomatic SOD1(G93A) mice and respective healthy controls; *p<0.05 Unpaired <i>t</i>-test; ^∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; ^#p<0.05 Paired <i>t</i>-test (as compared with control value before drug perfusion); control corresponds to 100% in all cases.</p

A_2A receptor modulation is lost in symptomatic SOD1(G93A) mice endplates; (A) representative average time-course of mean EPP amplitude change during CGS 21680 (5 nM) bathing and (B) illustrative mean EPP profile facilitation in 12–14 weeks old control (n = 6) and symptomatic mice (n = 6); (C) dose-response alterations in mean EPP amplitude by CGS 21680 (3 nM: n = 8, WT, n = 10, SOD1G93A; 5 nM: n = 10, WT, n = 7, SOD1G93A; 10 nM: n = 11, WT, n = 7, SOD1G93A) were blocked by SCH 58261 at 50 nM in WT mice (n = 4, WT, n = 4, SOD1G93A); (D) SCH 58261 (50 nM) did not affect evoked activity throughout data acquisition (n = 4, WT, n = 7, SOD1G93A); (E) raw recording of spontaneous release variations from a 12–14 weeks old WT and symptomatic SOD1G93A endplate upon CGS 21680 (5 nM) perfusion; effect of A_2A receptor activation by CGS 21680 (5 nM) on (F) MEPP frequency (n = 6, WT, n = 5, SOD1(G93A)) (G) GMEPP frequency (n = 4, WT, n = 4, SOD1(G93A)) and (H) quantal content of

<p>A_2A receptor modulation is lost in symptomatic SOD1(G93A) mice endplates; (A) representative average time-course of mean EPP amplitude change during CGS 21680 (5 nM) bathing and (B) illustrative mean EPP profile facilitation in 12–14 weeks old control (n = 6) and symptomatic mice (n = 6); (C) dose-response alterations in mean EPP amplitude by CGS 21680 (3 nM: n = 8, WT, n = 10, SOD1G93A; 5 nM: n = 10, WT, n = 7, SOD1G93A; 10 nM: n = 11, WT, n = 7, SOD1G93A) were blocked by SCH 58261 at 50 nM in WT mice (n = 4, WT, n = 4, SOD1G93A); (D) SCH 58261 (50 nM) did not affect evoked activity throughout data acquisition (n = 4, WT, n = 7, SOD1G93A); (E) raw recording of spontaneous release variations from a 12–14 weeks old WT and symptomatic SOD1G93A endplate upon CGS 21680 (5 nM) perfusion; effect of A_2A receptor activation by CGS 21680 (5 nM) on (F) MEPP frequency (n = 6, WT, n = 5, SOD1(G93A)) (G) GMEPP frequency (n = 4, WT, n = 4, SOD1(G93A)) and (H) quantal content of EPPs (n = 9, WT, n = 7, SOD1(G93A)); *p<0.05 Unpaired <i>t</i>-test; ^∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; ^#p<0.05 Paired <i>t</i>-test (as compared with control value before drug perfusion); control corresponds to 100% in all cases.</p

Table_3.DOCX

<p>Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca²⁺-dependent proteases, we hypothesized that excessive intracellular Ca²⁺ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ_25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ₂₅₁₃₅. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca²⁺/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption.</p

Table_5.DOCX

<p>Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca²⁺-dependent proteases, we hypothesized that excessive intracellular Ca²⁺ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ_25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ₂₅₁₃₅. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca²⁺/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption.</p

Table_2.DOCX

<p>Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca²⁺-dependent proteases, we hypothesized that excessive intracellular Ca²⁺ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ_25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ₂₅₁₃₅. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca²⁺/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption.</p

Table_4.DOCX

<p>Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca²⁺-dependent proteases, we hypothesized that excessive intracellular Ca²⁺ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ_25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ₂₅₁₃₅. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca²⁺/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption.</p

Table_7.DOCX

<p>Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca²⁺-dependent proteases, we hypothesized that excessive intracellular Ca²⁺ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ_25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ₂₅₁₃₅. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca²⁺/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption.</p