Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging.

Peripheral administration of tissue inhibitor of metalloproteinases 2 (TIMP2), a protein inhibitor of matrix metalloproteinases (MMPs), has previously been shown to have beneficial effects on cognition and neurons in aged mice. Here, to better understand the potential of recombinant TIMP2 proteins, an IgG4Fc fusion protein (TIMP2-hIgG4) was developed to extend the plasma half-life of TIMP2. Following one month of administration of TIMP2 or TIMP2-hIgG4 via intraperitoneal injections, 23-month-old male C57BL/6J mice showed improved hippocampal-dependent memory in a Y-maze, increased hippocampal cfos gene expression, and increased excitatory synapse density in the CA1 and dentate gyrus (DG) of the hippocampus. Thus, fusion to hIgG4 extended the half-life of TIMP2 while retaining the beneficial cognitive and neuronal effects. Moreover, it retained its ability to cross the blood-brain barrier. To deepen the mechanistic understanding of the beneficial function of TIMP2 on neuronal activity and cognition, a TIMP2 construct lacking MMP inhibitory activity, Ala-TIMP2, was generated, which provides steric hindrance that prevents inhibition of MMPs by the TIMP2 protein while still allowing MMP binding. A comprehensive assessment of the MMP inhibitory and binding capacity of these engineered proteins is outlined. Surprisingly, MMP inhibition by TIMP2 was not essential for its beneficial effects on cognition and neuronal function. These findings both confirm previously published research, expand on the potential mechanism for the beneficial effects of TIMP2, and provide important details for a therapeutic path forward for TIMP2 recombinant proteins in aging-related cognitive decline.

Molecular and histological correlates of cognitive decline across age in male C57BL/6J mice.

INTRODUCTION: Increasing age is the number one risk factor for developing cognitive decline and neurodegenerative disease. Aged humans and mice exhibit numerous molecular changes that contribute to a decline in cognitive function and increased risk of developing age-associated diseases. Here, we characterize multiple age-associated changes in male C57BL/6J mice to understand the translational utility of mouse aging. METHODS: Male C57BL/6J mice from various ages between 2 and 24 months of age were used to assess behavioral, as well as, histological and molecular changes across three modalities: neuronal, microgliosis/neuroinflammation, and the neurovascular unit (NVU). Additionally, a cohort of 4- and 22-month-old mice was used to assess blood-brain barrier (BBB) breakdown. Mice in this cohort were treated with a high, acute dose of lipopolysaccharide (LPS, 10 mg/kg) or saline control 6 h prior to sacrifice followed by tail vein injection of 0.4 kDa sodium fluorescein (100 mg/kg) 2 h later. RESULTS: Aged mice showed a decline in cognitive and motor abilities alongside decreased neurogenesis, proliferation, and synapse density. Further, neuroinflammation and circulating proinflammatory cytokines were increased in aged mice. Additionally, we found changes at the BBB, including increased T cell infiltration in multiple brain regions and an exacerbation in BBB leakiness following chemical insult with age. There were also a number of readouts that were unchanged with age and have limited utility as markers of aging in male C57BL/6J mice. CONCLUSIONS: Here we propose that these changes may be used as molecular and histological readouts that correspond to aging-related behavioral decline. These comprehensive findings, in the context of the published literature, are an important resource toward deepening our understanding of normal aging and provide an important tool for studying aging in mice.

Abnormalities in cortical interneuron subtypes in ephrin-B mutant mice.

To explore roles for ephrin-B/EphB signaling in cortical interneurons, we previously generated ephrin-B (Efnb1/b2/b3) conditional triple mutant (TMlz ) mice using a Dlx1/2.Cre inhibitory neuron driver and green fluorescent protein (GFP) reporters for the two main inhibitory interneuron groups distinguished by expression of either glutamic acid decarboxylase 1 (GAD1; GAD67-GFP) or 2 (GAD2; GAD65-GFP). This work showed a general involvement of ephrin-B in migration and population of interneurons into the embryonic neocortex. We now determined whether specific interneurons are selectively affected in the adult brains of TMlz .Cre mice by immunostaining with antibodies that identify the different subtypes. The results indicate that GAD67-GFP-expressing interneurons that also express parvalbumin (PV), calretinin (CR) and, to a lesser extent, somatostatin (SST) and Reelin (Rln) were significantly reduced in the cortex and hippocampal CA1 region in TMlz .Cre mutant mice. Neuropeptide Y (NPY) interneurons that also express GAD67-GFP were reduced in the hippocampal CA1 region, but much less so in the cortex, although these cells exhibited abnormal cortical layering. In GAD65-GFP-expressing interneurons, CR subtypes were reduced in both cortex and hippocampal CA1 region, whereas Rln interneurons were reduced exclusively in hippocampus, and the numbers of NPY and vasoactive intestinal polypeptide (VIP) subtypes appeared normal. PV and CR subtype interneurons in TMlz .Cre mice also exhibited reductions in their perisomatic area, suggesting abnormalities in dendritic/axonal complexity. Altogether, our data indicate that ephrin-B expression within forebrain interneurons is required in specific subtypes for their normal population, cortical layering and elaboration of cell processes.

Autonomous and non-autonomous roles for ephrin-B in interneuron migration.

While several studies indicate the importance of ephrin-B/EphB bidirectional signaling in excitatory neurons, potential roles for these molecules in inhibitory neurons are largely unknown. We identify here an autonomous receptor-like role for ephrin-B reverse signaling in the tangential migration of interneurons into the neocortex using ephrin-B (EfnB1/B2/B3) conditional triple mutant (TMlz) mice and a forebrain inhibitory neuron specific Cre driver. Inhibitory neuron deletion of the three EfnB genes leads to reduced interneuron migration, abnormal cortical excitability, and lethal audiogenic seizures. Truncated and intracellular point mutations confirm the importance of ephrin-B reverse signaling in interneuron migration and cortical excitability. A non-autonomous ligand-like role was also identified for ephrin-B2 that is expressed in neocortical radial glial cells and required for proper tangential migration of GAD65-positive interneurons. Our studies thus define both receptor-like and ligand-like roles for the ephrin-B molecules in controlling the migration of interneurons as they populate the neocortex and help establish excitatory/inhibitory (E/I) homeostasis.