Jana Vukovic

Microglia Modulate Hippocampal Neural Precursor Activity in Response to Exercise and Aging

Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX3CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX3CL1 receptor, CX3CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX3CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX3CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX3CL1–CX3CR1 axis critically contributes toward this process.

Striking Denervation of Neuromuscular Junctions without Lumbar Motoneuron Loss in Geriatric Mouse Muscle

Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1–L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ∼2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia.

Immature Doublecortin-Positive Hippocampal Neurons Are Important for Learning But Not for Remembering

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.

Prolactin Stimulates Precursor Cells in the Adult Mouse Hippocampus

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.

Activation of neural precursors in the adult neurogenic niches

The generation of new neurons within the dentate gyrus of the mature hippocampus is critical for spatial learning, object recognition and memory, whereas new neurons born in the subventricular zone (SVZ) contribute to olfactory function. Adult neurogenesis is a multistep process that begins with the activation and proliferation of a pool of stem/precursor cells. Although the presence of self-renewing and multipotent neural precursors is well established in the SVZ, it is only recently that the existence of such a precursor population has been demonstrated in the hippocampus, the region of the brain involved in learning and memory. Determining how this normally latent pool can be activated therefore offers considerable potential for the development of targeted neurogenic-based therapeutics to ameliorate the cognitive decline associated with hippocampal dysfunction in several neurodegenerative diseases. In this review, we summarize the effects of neural activity, various molecular factors and pharmaceutical agents, as well as voluntary exercise, in activating endogenous neural precursors in the two neurogenic niches of the adult brain, and highlight the role of activation-driven enhancement of neurogenesis for the treatment of psychiatric illness and aging dementia.

CX3CL1/fractalkine regulates branching and migration of monocyte-derived cells in the mouse olfactory epithelium

The olfactory epithelium (OE) is a site of massive adult neurogenesis where olfactory sensory neurons (OSNs) are continuously turned over. Tissue macrophages have been implicated in phagocytosis of degenerating cells but the molecular mechanisms that allow for their recruitment while maintaining a neurogenic microenvironment are poorly understood. This study reports that the neuroprotective chemokine CX3CL1 is expressed by OSNs and olfactory ensheathing cells. Monocyte-derived cells in the OE depend on CX3CL1-signalling for intraepithelial migration and apical dendrite expression. These observations are first to demonstrate phenotypic differences in appearance and distribution of monocyte-derived cells in nervous tissue due to CX3CR1 deficiency.

CX3CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX₃CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX₃CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX₃CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX₃CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX₃CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX₃CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX₃CL1. Collectively, our data indicate that signaling through the CX₃CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

GH Mediates Exercise-Dependent Activation of SVZ Neural Precursor Cells in Aged Mice

Here we demonstrate, both in vivo and in vitro, that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation.

Influence of adult Schwann cells and olfactory ensheathing glia on axon--target cell interactions in the CNS: a comparative analysis using a retinotectal co-graft model.

We used an in vivo transplant approach to examine how adult Schwann cells and olfactory ensheathing glia OEG influence the specificity of axon-target cell interactions when they are introduced into the CNS. Populations of either Schwann cells or OEG were mixed with dissociated fetal tectal cells presumptive superior colliculus and, after reaggregation, pieces were grafted onto newborn rat superior colliculus. Both glial types were prelabeled with lentiviral vectors encoding green fluorescent protein. Grafts rapidly established fiber connections with the host and retinal projections into co-grafts were assessed 656 days posttransplantation by injecting cholera toxin B into host eyes. In control rats that received pure dissociated-reaggregated tectal grafts, retinal ganglion cell RGC axons selectively innervated defined target areas, corresponding to the retinorecipient layer in normal superior colliculus. The pattern of RGC axon ingrowth into OEG containing co-grafts was similar to that in control grafts. However, in Schwann cell co-grafts there was reduced host retinal input into presumptive target areas and many RGC axons were scattered throughout the neuropil. Given that OEG in co-grafts had minimal impact on axon-target cell recognition, OEG might be an appropriate cell type for direct transplantation into injured neuropil when attempting to stimulate specific pathway reconstruction.

Lack of fibulin-3 alters regenerative tissue responses in the primary olfactory pathway

The adult olfactory epithelium has maintained the ability to reconstitute its olfactory sensory neurons (OSNs) from a basal progenitor cell compartment. This allows for life-long turnover and replacement of receptor components as well as repair of the primary olfactory pathway in response to injury and environmental insults. The present study investigated whether fibulin-3, a glycoprotein in the extracellular matrix and binding partner of tissue inhibitor of metalloproteinases-3 (TIMP-3), plays a role in ongoing plasticity and regenerative events in the adult primary olfactory pathway. In wild-type control mice, fibulin-3 protein was detected on IB4(+)CD31(+) blood vessels, nerve fascicles and the basement membrane underneath the olfactory epithelium. After target ablation (olfactory bulbectomy), fibulin-3 was also abundantly present in the central nervous system (CNS) scar tissue that occupied the bulbar cavity. Using two different lesion models, i.e. intranasal Triton X-100 lesion and olfactory bulbectomy, we show that fibulin-3 deficient (Efemp1(-/-)) mice have impaired recovery of the olfactory epithelium after injury. Ten days post-injury, Efemp1(-/-) mice showed altered basal stem/progenitor cell proliferation and increased overall numbers of mature (olfactory marker protein (OMP) -positive) versus immature OSNs. However, compromised regenerative capacity of the primary olfactory pathway in Efemp1(-/-) mice was evidenced by reduced numbers of mature OSNs at the later time point of 42 days post-injury. In addition to these neural differences there were consistent changes in blood vessel structure in the olfactory lamina propria of Efemp1(-/-) mice. Overall, these data suggest a role for fibulin-3 in tissue maintenance and regeneration in the adult olfactory pathway.