Impaired Insulin Signaling Alters Mediators of Hippocampal Synaptic Dynamics/Plasticity: A Possible Mechanism of Hyperglycemia-Induced Cognitive Impairment

Abstract

Alzheimer's disease (AD) is a neurological condition that affects the elderly and is characterized by progressive and irreversible neurodegeneration in the cerebral cortex [...].

Keywords: Na+/K+-ATPase; acetylcholine esterase; cognitive behavior; streptozotocin; synapse.

Figure 1

(A) Time spent exploring objects (sec), (B) total time spent exploring objects (sec) as TF + TN objects, and (C) discrimination ratio as DR = TN/(TF + TN) in different groups. Rats with IP-STZ and IVC-STZ injection spent significantly less time exploring novel objects on 4th day of T2 sessions (Veh vs. STZ, ** p < 0.01). ICV-STZ-injected rats demonstrated a significant lack of exploration behavior, indicated by the reduced time spent exploring objects and attenuated discrimination ratio at 3W, compared to the IP-STZ group (p < 0.01). STZ-injected rats showed a significant reduction in interest/curiosity and memory on 4th day of T2 trials (IP-Veh vs. IP-STZ-3W and -6W, ** p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥; p < 0.01). Data are mean ± SD of n = 10 rats/group.

Figure 2

(A) Percentage of errors (incorrect responses), (B) spontaneous alternative correct choices during probe test in different groups. Rats with IP-STZ and IVC-STZ injection demonstrated significantly higher percentages of incorrect responses on the 3rd, 4th, and 5th days of the learning sessions (IP-Veh vs. IP-STZ-6W, * p < 0.05; ICV-Veh-3W vs. ICV-STZ-3W, $ p < 0.05; ICV-Veh-6W vs. ICV-STZ-6W, ¥ p < 0.05). ICV-STZ-injected rats demonstrated significant learning deficits, indicated by a lower percentage of correct choices and higher percentage of incorrect choices at 3W and 6W, compared to the IP-STZ group (p < 0.01). STZ-injected rats showed significant inadequacy in learning and memory on the 5th day, in the probe test (IP-Veh vs. IP-STZ-6W, ** p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01). Data are mean ± SD of n = 10 rats/group.

Figure 3

AChE (A) and Na⁺/K⁺-ATPase (B) activity in STZ- and vehicle-treated (IP or ICV) rats. There was a time-dependent increase in AChE and a significant decrease in Na⁺/K⁺-ATPase in both the IP-STZ and IVC-STZ groups compared to respective vehicle-treated control groups. IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh-1W vs. ICV-STZ-1W, # p < 0.05, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. These changes were more profound in ICV-STZ groups compared to respective IP-STZ groups (p < 0.05). Data are mean ± SD of n = 10 rats/group.

Figure 4

Representative immunoblot of mediators of neuronal survival and synaptic dynamics/plasticity, (A) t-PI3K, (B) p-PI3K, (C) t-Akt, (D) p-Akt, (E) t-GSK-3β, (F) GSK-3β, (G) t-PAK, (H) p-PAK, (I) t-LIMK-1, (J) p-LIMK-1, (K) t-cofilin-1, (L) p-cofilin-1, and (M) β-actin, as a loading control, in hippocampal synaptosomes.

Figure 5

Quantification of t-PI3K (A), p-PI3K (B), and p-PI3K/t-PI3K (C) levels in hippocampal synaptosomal fractions from different groups. The t-PI3K, p-PI3K, and p-PI3K/t-PI3K levels increased in a time-dependent manner in both IP-STZ and IVC-STZ groups compared to respective vehicle control groups. t-PI3K was significantly higher in IP-STZ groups compared to ICV-STZ groups. p-PI3K and p-PI3K/t-PI3K were, on the other hand, significantly lower in the IP-STZ groups compared to the ICV-STZ groups. IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh vs. ICV-STZ-1W, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. Data are mean ± SD of n = 10 rats/group.

Figure 6

Quantification of t-Akt (A), p-Akt (B), and p-Akt/t-Akt (C) levels in hippocampal synaptosomal fractions from different groups. The t-Akt, p-Akt, and p-Akt/t-Akt expression was altered in a time-dependent manner in both IP-STZ and ICV-STZ groups. t-Akt was significantly higher in IP-STZ groups compared to ICV-STZ groups. p-Akt and p-Akt/t-Akt were, on the other hand, significantly lower in the IP-STZ groups compared to the ICV-STZ groups. IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh vs. ICV-STZ-1W, # p < 0.05, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. Data are mean ± SD of n = 10 rats/group.

Figure 7

Quantification of t-GSK-3β (A), p-GSK-3β (B), and p-GSK-3β/t-GSK-3β (C) levels in hippocampal synaptosomal fractions. The t-GSK-3β, p-GSK-3β, and p-GSK-3β/t-GSK-3β levels were altered in a time-dependent manner in both IP-STZ and IVC-STZ groups compared to respective vehicle controls. t-GSK-3β significantly increased in IP-STZ and IVC-STZ groups compared to respective vehicle controls. p-GSK-3β and p-GSK-3β/t-GSK-3β, however, significantly decreased in IP-STZ and IVC-STZ groups. ICV administration of STZ induced more profound changes relative to the IP route. IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh vs. ICV-STZ-1W, # p < 0.05, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $ p < 0.05, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. Data are mean ± SD of n = 10 rats/group.

Figure 8

Changes in t-PAK, p-PAK, and p-PAK/t-PAK levels analyzed in hippocampal synaptosomal fractions. Time-dependent significant changes in t-PAK (A), p-PAK (B), and p-PAK/t-PAK (C) in IP-STZ and IVC-STZ groups. t-PAK was significantly higher in IP-STZ groups compared to ICV-STZ groups (p < 0.01). p-PAK and p-PAK/t-PAK were, on the other hand, significantly lower in the IP-STZ groups compared to the ICV-STZ groups (p < 0.01). IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh vs. ICV-STZ-1W, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. Data are mean ± SD of n = 10 rats/group.

Figure 9

Time course of changes in t-LIMK-1 (A), p-LIMK-1 (B), and p-LIMK-1/t-LIMK-1 (C) in hippocampal synaptosomes. Time-dependent significant changes in t-LIMK-1, p-LIMK-1, and p-LIMK-1/t-LIMK-1 in IP-STZ and IVC-STZ groups as compared to respective vehicle controls. t-LIMK-1 significantly decreased, while p-LIMK-1 and p-LIMK-1/t-LIMK-1 significantly increased in IP-STZ and ICV-STZ groups. The ICV route was more potent than the IP route in inducing these changes. IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh vs. ICV-STZ-1W, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. Data are mean ± SD of n = 10 rats/group.

Figure 10

Changes in the levels of t-cofilin-1, p-cofilin-1, and p-cofilin-1/t-cofilin-1 in response to IP and ICV administration of STZ in hippocampal synaptosomes. Time course analysis showed significant alterations in the levels of t-cofilin-1 (A), p-cofilin-1 (B), and p-cofilin-1/t-cofilin-1 (C) in IP-STZ and IVC-STZ groups compared to corresponding vehicle controls. t-cofilin-1 levels significantly decreased, while p-cofilin-1 and p-cofilin-1/t-cofilin-1 significantly increased in IP-STZ and ICV-STZ groups (p < 0.01). The administration of STZ through the ICV route was more potent than the IP route in inducing these changes. IP-Veh vs. other groups, * p < 0.05, ** p < 0.01; ICV-Veh vs. ICV-STZ-1W, ## p < 0.01; ICV-Veh-3W vs. ICV-STZ-3W, $$ p < 0.01; ICV-Veh-6W vs. ICV-STZ-6W, ¥¥ p < 0.01. Data are mean ± SD of n = 10 rats/group.

Figure 11

Correlation between AChE (A) and Na⁺/K⁺-ATPase (B) activity and cognitive performance (time exploring novel object during T2 test in NOR) in response to STZ administration. Increased acetylcholine metabolizing enzyme (AChE) activity correlated negatively with decreased time spent exploring novel object (r = 0.208), tested in T2 session. Decreased Na⁺/K⁺-ATPase (membrane/synapse potentiation marker) activity correlated positively with the procurement of memory to explore the novel object (r = 0.456). *** p < 0.0001, n = 10 rats/group at 3W and 6W.

Figure 12

Correlation between PI3K and PAK signaling mediators (declined p-PI3K/t-PI3K (A), p-Akt/t-Akt (B), p-GSK-3β/t-GSK-3β (C), and increased p-PAK/t-PAK (D), p-LIMK-1/t-LIMK-1 (E) and p-cofilin-1/t-cofilin-1 (F)) and time exploring novel object in NOR test. Decreased levels of p-PI3K/t-PI3K (r = 0.502), p-Akt/t-Akt (r = 302), and p-GSK-3β/t-GSK-3β (r = 507) correlated positively with decreased time spent exploring novel object (A–C), tested in T2 (with N-object) after the last session in T1 (with F-object) on the 4th day. Increased levels of p-PAK/t-PAK (r = 0.371), p-LIMK-1/t-LIMK-1 (r = 0.538), and p-cofilin-1/t-cofilin-1 (r = 0.504) negatively correlated with a decline in time exploring novel object (D–F). *** p < 0.0001, n = 10 rats/group at 3W and 6W.

Figure 13

Correlation between AChE (A) and Na⁺/K⁺-ATPase (B) activity and cognitive performance (working memory during probe test in T-maze) in response to STZ administration. Increased AChE activity correlated negatively with decreased correct choices and increased percentage of errors (r = 0.332), tested 1 h after the last learning session. Decreased Na⁺/K⁺-ATPase (membrane/synapse potentiation marker) activity correlated positively with working memory. As Na⁺/K⁺-ATPase activity decreased, the percentage of errors in choosing a correct spontaneous alternate turn in the T-maze increased (r = 0.514). *** p < 0.0001, n = 10 rats/group at 3W and 6W.

Figure 14

Correlation of changes in PI3K and PAK signaling mediators (declined p-PI3K/t-PI3K (A), p-Akt/t-Akt (B), p-GSK-3β/t-GSK-3β (C), and increased p-PAK/t-PAK (D), p-LIMK-1/t-LIMK-1 (E) and p-cofilin-1/t-cofilin-1 (F)) with percentage of errors in T-maze. Decreased levels of p-PI3K/t-PI3K (r = 0.377), p-Akt/t-Akt (r = 0.284), and p-GSK-3β/t-GSK-3β (r = 401) correlated negatively with decreased working memory procurement, tested 1 h after the last learning session (A–C). Increased levels of p-PAK/t-PAK (r = 0.492), p-LIMK-1/t-LIMK-1 (r = 0.742), and p-cofilin-1/t-cofilin-1 (r = 0.626) positively correlated with a decline in working memory (D–F), tested in T-maze. *** p < 0.0001, n = 10 rats/group at 3W and 6W.

Figure 15

The mechanism of cognitive dysfunction in IP- and ICV-STZ models of impaired insulin signaling. Under normal conditions, insulin signaling via the insulin receptor (IR) activates PI3K/Akt signaling and inhibits GSK-3β. In addition, insulin signaling attenuates PAK/LIMK-1 signaling, leading to the activation of cofilin-1. Together, this results in improved neuronal survival and synaptic dynamics/plasticity and correlates with normal working memory and interest in new objects. Impaired insulin signaling induced by IP or ICV injections of STZ attenuates PI3K/Akt signaling and enhances GSK-3β activation. Moreover, impaired insulin signaling leads to the activation of the PAK/LIMK-1 arm and inhibition of cofilin-1. These molecular changes have been linked to impaired actin assembly and deteriorated synaptic function. These changes correlate with cognitive deterioration in STZ-treated rats and may explain early synaptic changes in sAD.