CXCR4 prevents dispersion of granule neuron precursors in the adult dentate gyrus.

Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one-third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4-GFP expression in NSCs, neuronal-committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12-promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2-positive neural stem/early progenitor cells, NeuroD-positive neuronal-committed progenitors, and DCX-positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one-third of the GCL as differentiation area for immature granule neurons.

A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.

Human neuromelanin induces neuroinflammation and neurodegeneration in the rat substantia nigra: implications for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN). It has been suggested that microglial inflammation augments the progression of PD. Neuromelanin (NM), a complex polymer pigment found in catecholaminergic neurons, has sparked interest because of the suggestion that NM is involved in cell death in Parkinson's disease, possibly via microglia activation. To further investigate the possible role of NM in the pathogenesis of PD, we conducted in vivo experiments to find out whether microglial cells become activated after injection of human neuromelanin (NM) into (1) the cerebral cortex or (2) the substantia nigra to monitor in this PD-relevant model both microglial activation and possible neurodegeneration. In this study, adult male Wistar rats received an intracerebral injection of either NM, bacterial lipopolysaccharide (LPS, positive control), phosphate-buffered saline (PBS, negative control) or colloidal gold suspension (negative particular control). After different survival times (1, 8 or 12 weeks), brain slices from the cerebral cortex or substantia nigra (SN, 1 week) were stained with Iba-1 and/or GFAP antibody to monitor microglial and astrocytic reaction, and with tyrosine hydroxylase (TH) to monitor dopaminergic cell survival (SN group only). The injection of LPS induced a strong inflammatory response in the cortex as well in the substantia nigra. Similar results could be obtained after NM injection, while the injection of PBS or gold suspension showed only moderate or no glial activation. However, the inflammatory response declined during the time course. In the SN group, there was, apart from strong microglia activation, a significant dopaminergic cell loss after 1 week of survival time. Our findings clearly indicate that extracellular NM could be one of the key molecules leading to microglial activation and neuronal cell death in the substantia nigra. This may be highly relevant to the elucidation of therapeutic strategies in PD.

TNF-alpha antibody infusion impairs survival of stroke-generated neuroblasts in adult rat brain.

Stroke induced by 2 h middle cerebral artery occlusion triggers increased striatal and hippocampal neurogenesis in adult rats. We investigated the effect of tumor necrosis factor-alpha (TNF-alpha) inhibition on the survival of the new neurons. The mitotic marker BrdU was given on days 5 to 7, and TNF-alpha antibody or control protein was infused into the lateral ventricle of the ischemic hemisphere from day 8 to 14 after stroke. At the end of infusions, the TNF-alpha antibody-treated rats showed markedly fewer new striatal and hippocampal neurons, as compared to animals given control protein. The present findings suggest that TNF-alpha, probably acting via its receptor TNFR2, can promote the survival of stroke-generated hippocampal and striatal neurons.

Microglia-derived tumor necrosis factor-alpha exaggerates death of newborn hippocampal progenitor cells in vitro.

Production of new hippocampal neurons continues in adult mammals and different brain insults can significantly increase this process. However, many hippocampal progenitor cells (HPC) die shortly after birth. Here we investigated the possibility that increased release of cytokines by activated microglia contributes to the death of HPC. We showed that addition of tumor necrosis factor-alpha (TNFalpha) to the medium of a cultured HPC line (HiB5) shortly after the cells stopped division causes significant apoptotic cell death. Conditioned medium from an activated microglial cell line (BV-2) had a similar effect, though conditioned medium from nonactivated microglia increased the survival of HPC. Reverse transcription-PCR indicated that HPC and microglial cells express both TNF receptors, TNF-R1 and TNF-R2. Coculturing of HPC with activated microglial cells aggravated death of hippocampal progenitors and also caused death of microglial cells themselves. Our data indicate that activated microglia-released TNFalpha might be an important contributor in inflammation-induced exaggeration of death of newly formed HPC in the adult brain after an insult.

Inflammation is detrimental for neurogenesis in adult brain.

New hippocampal neurons are continuously generated in the adult brain. Here, we demonstrate that lipopolysaccharide-induced inflammation, which gives rise to microglia activation in the area where the new neurons are born, strongly impairs basal hippocampal neurogenesis in rats. The increased neurogenesis triggered by a brain insult is also attenuated if it is associated with microglia activation caused by tissue damage or lipopolysaccharide infusion. The impaired neurogenesis in inflammation is restored by systemic administration of minocycline, which inhibits microglia activation. Our data raise the possibility that suppression of hippocampal neurogenesis by activated microglia contributes to cognitive dysfunction in aging, dementia, epilepsy, and other conditions leading to brain inflammation.