Fig 2: The deficits in basal synaptic transmission and long-term potentiation at Schaffer collateral-CA1 synapses in 5xFAD mice are improved by TIMP-1 administration. A, Schematic diagram showing regions of the hippocampus– CA1, CA2, CA3 and dentate gyrus (DG) and the entorhinal cortex (EC). The dotted lines mark the connecting fibers– Schaffer collateral (SC), mossy fibres (MF), perforant pathway (PP), associational commissural fibers (AC). Stratum radiatum (SR) is the region where the recording electrode is placed in the CA1 region. B, Representative field excitatory post-synaptic potentiation (fEPSP) traces of slices from WT, 5xFAD, WT + TIMP1 and 5xFAD + TIMP-1 treatment groups with increasing current stimulus strength (20–300 µA with 20 µA increments). C, Input-output (I-O) relationship (fEPSP slopes along a step-wise rise in stimulus intensity) representative of the mean fEPSP value of all the slices in each group at each stimulus current strength from 20–300 µA (n = 9–15 slices from 5 or 6 mice/group) D, Area under curve (AUC) from (C); one-way ANOVA, Tukey’s post hoc, mean ± SEM. E, Paired-pulse facilitation (PPF) ratio along increasing inter-stimulus interval (30 to 300 ms with 30 ms increments), n = 9–15 slices from 5 or 6 mice/group. F, AUC from (E); one-way ANOVA, Tukey’s post hoc, mean ± SEM. G, Time course of fEPSP responses with 20 min pre-TBS and 60 min post-TBS. Note the time segments post-TBS demarcated for calculating post-tetanic potentiation (PTP), short-term potentiation (STP) and long-term potentiation (LTP). H, AUC from (G); one-way ANOVA, Tukey’s post hoc, mean ± SEM. I, Representative fEPSP tracings of slices from the four groups. Solid line represents pre-TBS response (baseline) and dotted line represents post-TBS response. J-L, Bar graphs for PTP (3 min post-TBS) (J), STP (21st -30th min) post-TBS (K), LTP (41st -60th min post-TBS) (L); n = 9–12 slices from 5 or 6 mice/group, One-way ANOVA, Tukey’s post hoc, mean ± SEM. *P < 0.05 and **P < 0.01

Fig 3: Exogenous TIMP-1 injection corrects cognitive deficits in 5xFAD mice. A, Representative immunoblots showing endogenous TIMP-1 levels in age-matched wild type (WT) and 5xFAD mice from whole hippocampal tissue lysates at postnatal Day-7 (P07), P21, 2 months (M), 4 M, 6 M and 8 M of age (also refer fig. S1A, B). B, Densitometric analysis for TIMP-1 levels normalized to β-actin (ACTIN). Relative protein levels in 5xFAD mice are compared to WT in each age group (n = 4–6 mice/group; two-tailed unpaired t-test, mean ± SEM). C, Representative immunofluorescence images of coronal sections showing hippocampal regions from WT, 5xFAD, and 5xFAD mice post TIMP-1 injection - one day post injection (5xFAD + TIMP-1(1D)), 7 days post injection (5xFAD + TIMP-1(7D)) and 14 days post injection (5xFAD + TIMP-1(14D)) stained for TIMP-1 (magenta), GFAP (yellow) and MAP2 (white); scale bar-50 μm. D, Quantification for TIMP-1 immunoreactivity in the hippocampus shown as TIMP-1 puncta density normalized to WT (n = 3 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). E, Schematic of stereotactic injection of recombinant TIMP-1 and the behavioral experiment schedule. F-R, Battery of behavioral tests performed with four groups of mice– WT, 5xFAD, 5xFAD + aCSF and 5xFAD + TIMP-1 starting on Day-8 following intracerebroventricular TIMP-1 injection to assess cognitive functions. F, Representative infra-red images of the path travelled by a mouse in each group in the Open-field test (OFT). G, Total distance travelled (cm) in the whole open arena and H, Time spent (sec) in the outer zone of the arena in 10 min of OFT (n = 10–16 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). I, Experimental set-up for elevated-plus maze (EPM) test. J, Latency (sec) of mouse to the closed arm of EPM recorded on Day-1 as initial transfer latency (ITL) and on Day-2 as retention transfer latency (RTL); the difference (ITL-RTL) is shown and compared among groups (n = 10–18 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). K, Experimental set-up for Day-2 of Novel object recognition (NOR) test with one familiar (F) and one novel (N) object for L-N. L, Comparison of the time spent by a mouse with F versus N objects on Day-2 of NOR - exploration time (sec) within each group (n = 11–19 mice/group; two-tailed unpaired t-test, mean ± SEM). M, Discrimination index and N, Preference index (%) calculated to compare among groups from Day-2 data of NOR (n = 11–19 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). For OFT, EPM and NOR also see fig. S2. O, Experimental set-up for Day-1 of contextual fear conditioning (CFC) test. P, Percentage freezing of mice on Day-2 of CFC (n = 8–14 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). Q, Experimental set-up for Day-1 of Cue-dependent fear conditioning (CDFC) test and R, Percentage freezing of mice on Day-2 of CDFC (n = 8–18 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns, not significant

Fig 4: TIMP-1 acts as a multifunctional cytokine with protective signaling and long-term benefits for neurons in 5xFAD mouse. Schematic diagram showing the multiple signaling pathways triggered by intracerebroventricular injection of recombinant TIMP-1 in 5xFAD mouse model of AD and the proposed mechanism that underlies long-term synaptic and cognitive benefits

Fig 5: BDNF signaling pathways is activated by TIMP-1 treatment in 5xFAD mice. A, Schematic diagram for the samples used for BDNF signaling pathway analysis. B-J, Representative immunoblots and corresponding densitometric analyses for Pro-BDNF (B-C), pAkt(Ser473) (D-E), pAkt(Thr308) (F-G) and pERK1(Thr202)/2(Tyr204) (H-J) normalized to corresponding loading controls (n = 4–5 mice/group; one-way ANOVA, Tukey’s post hoc, mean ± SEM). K-S, Representative immunoblots and corresponding densitometric analyses for Pro-BDNF (K-L), pAkt (Ser473) (M-N), pAkt (Thr308) (O-P) and pERK1(Thr202)/2(Tyr204) (Q-S) normalized to corresponding loading controls from hippocampal synaptosomes 14 days post TIMP-1 treatment (n = 4–6 independent synaptosome isolations/group; one-way ANOVA, Tukey’s post hoc). *P < 0.05, **P < 0.01 and ***P < 0.001. Also see S9 for WB results from cortical synaptosomes. T, Schema showing TIMP-1 injection enhanced BDNF and associated signaling pathways in a 5xFAD mouse