TIMP2 protein identified as key player in brain plasticity and memory

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Nov 6 2023

In a recent study published in Neuronal Psychiatry, researchers explored the molecular and cellular processes regulated by tissue inhibitor of metalloproteinases-2 (TIMP2) in the extracellular matrix (ECM) of the adult brain hippocampus.

Background

The hippocampus, a memory-related brain area, depends on intricate biological mechanisms that govern synaptic plasticity. TIMP2, a protein, is involved in these activities, which slow down with age. TIMP2 is a critical component in youthful blood that promotes brain regeneration in old mice, according to researchers. The molecular and cellular details relating TIMP2 to hippocampus function, however, remain unknown. TIMP2 function is critical for understanding its normal functioning and identifying possible therapeutics for age-related brain diseases since its levels diminish with aging.

About the study

In the present study, researchers investigated ways in which TIMP2 regulates hippocampal ECM components involved in processes associated with memory and plasticity.

TIMP2fl/fl, TIMP2 knockout (KO), and wild-type (WT) mice (SynCre/+) mice were utilized for the experiments. Murine hippocampi were dissected to perform ribonucleic acid (RNA) sequencing. Differentially expressed genes (DEGs) were assessed in the Gene Set Enrichment Analysis (GSEA) to gain insights into ways in which transcriptomic alterations may denote altered biological processes.

Weighted gene co-expression network analysis (WGCNA) was performed on the genes to identify modules with correlated expression patterns based on the TIMP2 genotype. Mice were administered 150 milligrams per kg of 5-bromo-2′-deoxyuridine (BrdU) intraperitoneally (i.p.) a day prior to sacrifice to assess cell proliferation or every day for five days, followed by perfusion or sacrifice four weeks later for cellular fate investigation. Microdialysis was performed in vivo to detect TIMP2 levels in the hippocampi of WT mice using high-molecular-weight cut-off probes implanted in the hippocampi.

The tissues were immunohistochemically analyzed and photographed using confocal microscopy to quantify Homer1 and Aggrecan puncta. The dentate gyrus (DG) granular cells were injected with iontophoretic dyes for dendritic spine characterization. Experiments such as novel location recognition, contextual fear conditioning, and the Barnes maze were performed to test hippocampus-based behavior.

The researchers evaluated new location recognition in mice by exposing them to an open-field ground for six minutes before consecutive exposures to two items in fixed places for three trials. For novel site recognition, the discrimination index was manually calculated. Fear conditioning was performed using a two-shock paradigm, in which mice were exposed to paired cue lights and a 1,000 Hz tone for 30 seconds, followed by mild foot shock.

On the following day, the freezing levels were tested. Over four trials, mice navigated a circular labyrinth utilizing visual clues to alternate escape holes in the Barnes maze experiment. Search techniques were classified as serial, localized, random, focused, scanning, direct, targeted, and focal missense. On the third day, the cognitive performances in the trials were scored.

Results

TIMP2 was abundant in the extracellular matrix of the brain and was significantly expressed by CA1 and CA3 hippocampal neurons. TIMP2 deletion resulted in transcriptome alterations in the hippocampal tissues associated with adult neurogenesis and synaptic plasticity processes. The gene set from the most significantly downregulated module, “pale turquoise," was strongly enriched for pathways related to "neurogenesis," "synapse," and "dendritic tree," indicating that TIMP2 may impact plasticity. TIMP2 deletion decreased the number of SRY-Box Transcriptional Factor 2-positive (Sox2+) neural progenitors and doublecortin-positive (DCX+) immature neurons significantly.

TIMP2 deficiency decreased the number of dendritic-type spines in DG cells, resulting in hippocampus-dependent memory deficits. TIMP2 decreased ECM protein accumulation near DG synapses, consistent with the ECM buildup in the hippocampus found with age, as evidenced by substantially increased aggrecan colocalization with Homer1.

Furthermore, TIMP2 deletion impaired neuroblast migration through dense ECM networks in the neurogenic niche, indicating dysregulated extracellular matrix turnover and replicating ECM deposition observed in aged brains, as evidenced by significantly increased matrix metalloproteinase 2 (MMP2) levels and a significant increase in the density of aggrecan puncta in the DG molecular layer.

Most TIMP2-secreting cells stained NeuN-positive, indicating that neuronal cells were the primary source for TIMP2. The murine model for TIMP2 deletion from neurons, which demonstrated the functional abnormalities seen in TIMP2 knockout animals, argued further for the involvement of the TIMP2 neuronal pool in determining the plasticity-related hippocampal function.

TIMP2 knockout mice had a lower preference for training items when moved, showing that TIMP2 plays a vital role in spatial memory. They also displayed less freezing behavior, indicating decreased hippocampus-related contextual discrimination. The Barnes maze experiment demonstrated that TIMP2 KO mice used fewer hippocampus-dependent strategies and had poorer cognitive scores than WT mice, indicating that TIMP2 KO mice have impaired hippocampus-dependent function.

Conclusion

Overall, the study findings showed that neuronal TIMP2 mediated adult neurogenesis, ECM buildup, and hippocampus-dependent memory. TIMP2 remodeling increases synaptic plasticity, crucial for memory. TIMP2's precise timing and involvement in various spine classes could be investigated in future studies. TIMP2 overexpression may help to explain memory issues, and further research into TIMP2 regulation across the lifespan and neuronal subtypes may yield novel insights.