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1.
Figure 6

Figure 6. Working hypothesis on how calpain inhibitors (Calp-inh) may be able to improve synaptic dysfunction.. From: Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

(A) Aβ activates a molecular cascade that leads to calpain activation and degradation of enzymes and structural proteins that are involved in learning and memory. Among the consequences of calpain activation, the transcription factor CREB cannot be phosphorylated and initiate transcription. (B) Calpain inhibition blocks the calcium-induced activation of calpain, preventing structural signaling alterations and allowing the phosphorylation of CREB.

Fabrizio Trinchese, et al. J Clin Invest. 2008 August 1;118(8):2796-2807.
2.
Figure 2

Figure 2. The highly selective calpain inhibitor BDA-410 reestablished normal basal frequency of spontaneous neurotransmitter release and rescued impairment of synaptic plasticity in APP/PS1 mouse cultures.. From: Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

(A) Chemical structure of BDA-410. (B) Vehicle-treated APP/PS1 cultures showed approximately 2-fold increase of spontaneous mEPSC frequency (n = 7) compared with vehicle-treated WT cultures (n = 8; P < 0.01 with 1-way ANOVA). BDA-410 did not affect average basal mEPSC frequency in WT cultures (n = 7) and reestablished normal basal frequency of spontaneous neurotransmitter release in APP/PS1 cultures (n = 8; P < 0.01). (C) Application of glutamate (200 μM) did not enhance mEPSC frequency in cultures from APP/PS1 mice compared with cultures from WT mice (n = 7 in APP/PS1 cultures; n = 8 in WT littermate cultures; P < 0.01 with 2-way ANOVA). mEPSC amplitude values were not affected by glutamate (data not shown). Block of calpain activity through BDA-410 was beneficial against the impairment of long-lasting enhancement of synaptic transmission (n = 8; P < 0.01 compared with vehicle-treated APP/PS1 cultures), without affecting it in WT cultures (P > 0.05 compared with vehicle-treated WT cultures).

Fabrizio Trinchese, et al. J Clin Invest. 2008 August 1;118(8):2796-2807.
3.
Figure 3

Figure 3. Calpain inhibition reestablished normal synaptic function following Aβ elevation.. From: Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

(A) E64 improved BST at the CA3-CA1 connection of hippocampal slices from 7-month-old APP/PS1 mice treated for 5 months (10 slices from 9 E64-treated APP/PS1 mice, 7 slices from 7 vehicle-treated APP/PS1 mice, 8 slices from 8 vehicle-treated WT mice; P < 0.01 comparing E64- and vehicle-treated APP/PS1 slices with 2-way ANOVA). BST was not affected in E64-treated WT animals (6 slices from 6 mice; P > 0.05). (B) BDA-410 reestablished normal BST in APP/PS1 mice treated with inhibitor between 8 weeks and 7 months of age (7 slices from 7 BDA-410–treated APP/PS1 mice, 9 slices from 8 vehicle-treated APP/PS1 mice, 8 slices from 7 vehicle-treated WT mice; P < 0.01 comparing BDA-410– and vehicle-treated APP/PS1 slices). BDA-410 did not affect BST in WT littermates (11 slices from 9 mice; P > 0.05). (C) E64 was beneficial against LTP impairment at the same synapses as in A (vehicle-treated APP/PS1 mice versus vehicle-treated WT mice, P < 0.01 with 2-way ANOVA; E64-treated APP/PS1 mice versus vehicle-treated APP/PS1 mice, P < 0.01). The inhibitor did not affect LTP in WT mice (P > 0.05). (D) BDA-410 reestablished normal LTP in APP/PS1 mice treated between 8 weeks and 7 months of age (BDA-410–treated APP/PS1 mice versus vehicle-treated APP/PS1 mice; P < 0.01; same synapses as in B). BDA-410 did not affect LTP in WT littermates (P > 0.05). (E) BDA-410 reestablished normal LTP in APP mice treated between 8 and 11 to 12 months of age (P < 0.05 in 6 slices from 6 BDA-410–treated APP mice versus 7 slices from 7 vehicle-treated APP mice; 7 slices from 7 vehicle-treated WT littermates). BDA-410 did not affect LTP in slices from WT littermates (7 slices from 7 mice; P > 0.05). BST did not vary among the 4 groups of mice (data not shown). (F) BDA-410 reestablished normal LTP in Aβ42-perfused slices (6 slices treated with BDA-410 plus Aβ versus 8 Aβ-treated slices, P < 0.05; 6 vehicle-treated slices versus 7 BDA-410–treated slices). The bar indicates the perfusion with Aβ42.

Fabrizio Trinchese, et al. J Clin Invest. 2008 August 1;118(8):2796-2807.
4.
Figure 4

Figure 4. Calpain inhibition reestablished normal spatial-working memory and associative fear memory in APP/PS1 mice.. From: Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

(A) In RAWM testing APP/PS1 mice treated with E64 for 5 months from 8 weeks of age made the same number of errors on the fourth acquisition (A4) and fifth retention trial (R) as vehicle-treated WT mice (n = 13; P < 0.05 compared with vehicle-treated APP/PS1 mice with 2-way ANOVA; planned comparisons at trial A4, P < 0.05, and trial R, P < 0.01), whereas vehicle-treated double transgenics made more errors than vehicle-treated WT mice (n = 7; P < 0.01 compared with vehicle-treated WT mice). E64 did not affect the performance of WT mice (n = 5; P > 0.05 compared with vehicle-treated WT mice). Visible platform task did not show any sensory-motor or motivational impairment in the same animals (data not shown). (B) BDA-410 reestablished normal spatial-working memory in APP/PS1 mice following 5 months of treatment from 8 weeks of age. BDA-410–treated double transgenics (n = 12) showed similar performance in the RAWM test as vehicle-treated WT littermates (n = 12), whereas vehicle-treated double transgenic mice (n = 11) showed abnormal learning and memory (P < 0.05 in BDA-410–treated APP/PS1 mice compared with vehicle-treated APP/PS1 mice; planned comparisons showed that the 2 groups were significantly different at trial A4, P < 0.05, and trial R, P < 0.01). The inhibitor did not affect the performance of WT mice (n = 14; P > 0.05 compared with vehicle-treated WT mice). The visible platform task did not show any sensory-motor or motivational impairment in all groups (data not shown). (C) BDA-410 reestablished normal contextual learning in APP/PS1 mice following 5 months of treatment from 8 weeks of age (P < 0.01 compared with vehicle-treated APP/PS1 mice). The inhibitor did not affect the performance of WT mice (P > 0.05).

Fabrizio Trinchese, et al. J Clin Invest. 2008 August 1;118(8):2796-2807.
5.
Figure 1

Figure 1. The calpain inhibitor E64 reestablished normal synaptic function in APP/PS1 mouse hippocampal cultures.. From: Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

(A) Calpain 1 immunoreactivity colocalized with immunofluorescence for the presynaptic protein, synapsin I. Calpain 1–immunoreactive puncta (left panel). Synapsin I–immunoreactive puncta (middle panel). Colocalization of calpain-immunoreactive puncta with synapsin I–immunoreactive puncta (so that the puncta appear yellow) (right panel). Scale bar: 15 μm. (B) Western blot demonstrated that E64 annulled calpain cleavage of spectrin to its 150-kDa fragment (n = 5 per group). (C) Quantitative western blot analysis of the 150-kDa fragment showed a 32% increase in APP/PS1 cultures compared with WT cultures (n = 5 for both; P < 0.05; data normalized against α-tubulin). (D) Vehicle-treated (veh-treated) APP/PS1 cultures showed approximately 2-fold increase of spontaneous mEPSC frequency (n = 10) compared with vehicle-treated WT cultures (n = 10; P < 0.01, with 1-way ANOVA). E64 did not affect average basal mEPSC frequency in WT cultures (n = 10; P > 0.05 with t test) but reestablished normal basal frequency of spontaneous neurotransmitter release in APP/PS1 cultures (n = 6). (E) Application of glutamate no longer enhanced mEPSC frequency in cultures from vehicle-treated APP/PS1 mice compared with cultures from vehicle-treated WT mice (n = 10 in APP/PS1 cultures; n = 10 in WT littermate cultures; P < 0.01 with 2-way ANOVA) without affecting mEPSC amplitude in either genotype (data not shown). Block of calpain activity through E64 ameliorated the deficit in long-lasting enhancement of synaptic transmission (n = 10; P < 0.01 compared with vehicle-treated APP/PS1 cultures), without affecting it in WT cultures (n = 10; P > 0.05 compared with vehicle-treated WT cultures).

Fabrizio Trinchese, et al. J Clin Invest. 2008 August 1;118(8):2796-2807.
6.
Figure 5

Figure 5. Calpain inhibition reestablished the increase in CREB phosphorylation during synaptic plasticity in APP/PS1 mice and produced a normal distribution of the synaptic protein synapsin I.. From: Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

(A) Western blot for pCREB at Ser-133 from cultured hippocampal neurons after 5-minute glutamate treatment. Five-day-old cultures were either treated with vehicle, E64, or BDA-410 for 3 days prior to applying glutamate. (B) Quantitative western blot analysis of data shown in A. pCREB levels in vehicle-treated WT cultures were increased after glutamate (n = 11; P < 0.01). APP/PS1 cultures failed to present the pCREB increase (n = 7; P > 0.01 with 1-way ANOVA). However, both E64 and BDA-410 reestablished normal pCREB values (n = 5 and 8, respectively). E64 and BDA-410 did not affect phosphorylation in WT cultures (n = 5 and 7, respectively). All samples were normalized against α-tubulin. (C) Examples of hippocampal slices stained with a pCREB antibody and fixed 60 minutes after tetanus in WT and APP/PS1 animals treated for 5 months from 8 weeks of age with E64, BDA-410, or vehicle. Lower-power (original magnification, ×4) view of the entire slice (left and middle panels). Higher-power (original magnification, ×16) view of CA1 cell pyramidal area (right panels). (D) Plot showing blockade of CA1-pCREB increase after tetanus in APP/PS1 slices (n = 6 both for WT and APP/PS1 slices; P < 0.01), whereas treatment with both E64 and BDA-410 for 5 months from 8 weeks of age reestablished the tetanus-induced pCREB increase (n = 6 for both; P < 0.01 compared with tetanized slices from vehicle-treated APP/PS1 mice). Both E64 and BDA-410 had no effect on WT mice after tetanus (n = 6 for both). In the absence of theta-burst, neither E64 nor BDA-410 induced changes in WT or APP/PS1 mice (n = 4 for each group; data not shown). IF, immunofluorescence. (E) The number of synapsin I–immunoreactive puncta was increased in vehicle-treated APP/PS1 cultures (n = 6) compared with vehicle-treated WT cultures (n = 6; P < 0.01). However, the basal synapsin I–immunoreactive puncta number was normal after exposure to E64 (n = 7) or BDA-410 (n = 5). The inhibitors did not affect synapsin I immunoreactivity in WT cultures (E64, n = 6; BDA-410, n = 7; P < 0.01 for both compared with vehicle-treated APP/PS1 cultures). (F) Glutamate failed to increase synapsin I–immunoreactive puncta numbers in vehicle-treated APP/PS1 cultures (n = 8) compared with vehicle-treated WT cultures (n = 7, P < 0.01), whereas both E64 and BDA-410 reestablished the glutamate-induced immunoreactivity increase in transgenic cultures (E64, n = 9; BDA-410, n = 9; P < 0.01 for both compared with vehicle-treated APP/PS1 cultures). Both E64 and BDA-410 had no effect on glutamate-induced immunoreactivity increase in WT cultures (E64, n = 9; BDA-410, n = 8).

Fabrizio Trinchese, et al. J Clin Invest. 2008 August 1;118(8):2796-2807.

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