Age‐related changes in cerebellar and hypothalamic function accompany non‐microglial immune gene expression, altered synapse organization, and excitatory amino acid neurotransmission deficits

We describe age-related molecular and neuronal changes that disrupt mobility or energy balance based on brain region and genetic background. Compared to young mice, aged C57BL/6 mice exhibit marked locomotor (but not energy balance) impairments. In contrast, aged BALB mice exhibit marked energy balance (but not locomotor) impairments. Age-related changes in cerebellar or hypothalamic gene expression accompany these phenotypes. Aging evokes upregulation of immune pattern recognition receptors and cell adhesion molecules. However, these changes do not localize to microglia, the major CNS immunocyte. Consistent with a neuronal role, there is a marked age-related increase in excitatory synapses over the cerebellum and hypothalamus. Functional imaging of these regions is consistent with age-related synaptic impairments. These studies suggest that aging reactivates a developmental program employed during embryogenesis where immune molecules guide synapse formation and pruning. Renewed activity in this program may disrupt excitatory neurotransmission, causing significant behavioral deficits

Pattern Recognition Receptors, Immune Proteins, and NF-κB Signaling Regulate Behaviors Associated With Aging Phenotypes

The aging process is accompanied by functional impairments, including reduced locomotor function, fragmentation of active states, and alterations in energy balance. Our lab has demonstrated that immune proteins are increased in specific regions of the mouse brain that correlate with strain specific deficits. These immune proteins include toll-like receptors (Tlr), class I major histocompatibility complex proteins (MHC I), and complement proteins. There is an increasing appreciation for the role of immune proteins in neurodevelopment; however, their involvement in age-associated deficits is poorly understood. Here, we present data demonstrating that 1) activation of a specific immune receptor (Tlr2) leads to changes in cell signaling that may underlie age related functional deficits, and 2) loss of specific immune proteins (Tlr2 and C3) and mutation of MHC I H2-Kb lead to impairments in behaviors commonly effected by aging. First, we present data demonstrating that activation of toll-like receptor 2 by a synthetic agonists leads to activation of NF-κB signaling in cerebellar granule cells. Our data, among others, suggests that NF-κB signaling is dysregulated in the aging brain. Additionally, we suggest that accumulation of amyloid β in the aging brain may act as an endogenous agonist for Tlr2 activation. This data reveals that a less appreciated pathway, neuronal immune receptor signaling, may play a role in age related NF-κB dysregulation. Second, analysis of mice lacking specific immune proteins revealed that loss of complement protein 3 leads to deficits in locomotor function characterized by reduced gait speed and markers of gait ataxia. Additionally, loss of complement 3 leads to changes in cerebellar granule cell synapse density and excitability in vitro. Loss of toll-like receptor 2 results in consolidation of active and inactive states in young mice, suggesting that toll-like receptor 2 may play a role in hypothalamic functions. Mutation of major histocompatibility complex isoform H2-Kb leads to a progressive obesity phenotype characterized by reduced activity and deficits in orexin neuron function. Collectively, this data demonstrates the involvement of immune proteins in regulating behaviors often disrupted in aging, including locomotor function, active state regulation, and energy balance

Toll-Like Receptor 2 Is a Regulator of Circadian Active and Inactive State Consolidation in C57BL/6 Mice

Regulatory systems required to maintain behavioral arousal remain incompletely understood. We describe a previously unappreciated role that toll-like receptor 2 (Tlr2, a membrane bound pattern recognition receptor that recognizes specific bacterial, viral, and fungal peptides), contributes toward regulation of behavioral arousal. In 4–4.5 month old mice with constitutive loss of Tlr2 function (Tlr2−/− mice), we note a marked consolidation in the circadian pattern of both active and inactive states. Specifically, Tlr2−/− mice demonstrated significantly fewer but longer duration active states during the circadian dark cycle, and significantly fewer but longer duration inactive states during the circadian light cycle. Tlr2−/− mice also consumed less food and water, and moved less during the circadian light cycle. Analysis of circadian rhythms further suggested that Tlr2−/− mice demonstrated less day-to-day variability in feeding, drinking, and movement behaviors. Reevaluation of this same mouse cohort at age 8–8.5 months revealed a clear blunting of these differences. However, Tlr2−/− mice were still noted to have fewer short-duration active states during the circadian dark cycle, and continued to demonstrate significantly less day-to-day variability in feeding, drinking, and movement behaviors. These results suggest that Tlr2 function may have a role in promoting transitions between active and inactive states. Prior studies have demonstrated that Tlr2 regulates sickness behaviors including hypophagia, hyperthermia, and decreased activity. Our work suggests that Tlr2 function also evokes behavioral fragmentation, another aspect of sickness behavior and a clinically significant problem of older adults

Complement Component C3 Loss leads to Locomotor Deficits and Altered Cerebellar Internal Granule Cell In Vitro Synaptic Protein Expression in C57BL/6 mice

Complement component 3 (C3) expression is increased in the cerebellum of aging mice that demonstrate locomotor impairments and increased excitatory synapse density. However, C3 regulation of locomotion, as well as C3 roles in excitatory synapse function, remain poorly understood. Here, we demonstrate that constitutive loss of C3 function in mice evokes a locomotor phenotype characterized by decreased speed, increased active state locomotor probability, and gait ataxia. C3 loss does not alter metabolism or body mass composition. No evidence of significant muscle weakness or degenerative arthritis was found in C3 knockout mice to explain decreased gait speeds. In an enriched primary cerebellar granule cell culture model, loss of C3 protein results in increased excitatory synaptic density and increased response to KCl depolarization. Our analysis of excitatory synaptic density in the cerebellar internal granule cell and molecular layers did not demonstrate increased synaptic density in vivo, suggesting the presence of compensatory mechanisms regulating synaptic development. Functional deficits in C3 knockout mice are therefore more likely to result from altered synaptic function and/or connectivity than gross synaptic deficits. Our data demonstrate a novel role for complement proteins in cerebellar regulation of locomotor output and control

Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome

The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in vivo model of ocular hypertensive glaucoma. Co-culture of rat and human bone marrow-derived mesenchymal stem cells with retinal explants increased retinal ganglion cell survival, after 7 days ex vivo, by ~2-fold and was associated with reduced apoptosis and increased nerve fibre layer and inner plexiform layer thicknesses. These effects were not demonstrated by co-culture with human or mouse fibroblasts. Conditioned media from mesenchymal stem cells conferred neuroprotection, suggesting that the neuroprotection is mediated, at least partly, by secreted factors. We compared the concentrations of 29 factors in human mesenchymal stem cell and fibroblast conditioned media, and identified 11 enriched in the mesenchymal stem cell secretome. Includes Supplementary material (Figure S1)