Seizure induces activation of multiple subtypes of neural progenitors and growth factors in hippocampus with neuronal maturation confined to dentate gyrus

Adult hippocampal neurogenesis is altered in response to different physiological and pathological stimuli. GFAP^+ve/nestin^+ve radial glial like Type-1 progenitors are considered to be the resident stem cell population in adult hippocampus. During neurogenesis these Type-1 progenitors matures to GFAP^−ve/nestin^+ve Type-2 progenitors and then to Type-3 neuroblasts and finally differentiates into granule cell neurons. In our study, using pilocarpine-induced seizure model, we showed that seizure initiated activation of multiple progenitors in the entire hippocampal area such as DG, CA1 and CA3. Seizure induction resulted in activation of two subtypes of Type-1 progenitors, Type-1a (GFAP^+ve/nestin^+ve/BrdU^+ve) and Type-1b (GFAP^+ve/nestin^+ve/BrdU^−ve). We showed that majority of Type-1b progenitors were undergoing only a transition from a state of dormancy to activated form immediately after seizures rather than proliferating, whereas Type-1a showed maximum proliferation by 3 days post-seizure induction. Type-2 (GFAP^−ve/nestin^+ve/BrdU^+ve) progenitors were few compared to Type-1. Type-3 (DCX^+ve) progenitors showed increased expression of immature neurons only in DG region by 3 days after seizure induction indicating maturation of progenitors happens only in microenvironment of DG even though progenitors are activated in CA1 and CA3 regions of hippocampus. Also parallel increase in growth factors expression after seizure induction suggests that microenvironmental niche has a profound effect on stimulation of adult neural progenitors.     

Temporal patterns of cortical proliferation of glial cell populations after traumatic brain injury in mice

TBI (traumatic brain injury) triggers an inflammatory cascade, gliosis and cell proliferation following cell death in the pericontusional area and surrounding the site of injury. In order to better understand the proliferative response following CCI (controlled cortical impact) injury, we systematically analyzed the phenotype of dividing cells at several time points post-lesion. C57BL/6 mice were subjected to mild to moderate CCI over the left sensory motor cortex. At different time points following injury, mice were injected with BrdU (bromodeoxyuridine) four times at 3-h intervals and then killed. The greatest number of proliferating cells in the pericontusional region was detected at 3 dpi (days post-injury). At 1 dpi, NG2⁺ cells were the most proliferative population, and at 3 and 7 dpi the Iba-1⁺ microglial cells were proliferating more. A smaller, but significant number of GFAP⁺ (glial fibrillary acidic protein) astrocytes proliferated at all three time points. Interestingly, at 3 dpi we found a small number of proliferating neuroblasts [DCX⁺ (doublecortin)] in the injured cortex. To determine the cell fate of proliferative cells, mice were injected four times with BrdU at 3 dpi and killed at 28 dpi. Approximately 70% of proliferative cells observed at 28 dpi were GFAP⁺ astrocytes. In conclusion, our data suggest that the specific glial cell types respond differentially to injury, suggesting that each cell type responds to a specific pattern of growth factor stimulation at each time point after injury.     

Brain injury induces cholesterol 24-hydroxylase (Cyp46) expression in glial cells in a time-dependent manner

Maintaining the cholesterol homeostasis is essential for normal CNS functioning. The enzyme responsible for elimination of cholesterol excess from the brain is cholesterol 24-hydroxylase (Cyp46). Since cholesterol homeostasis is disrupted following brain injury, in this study we examined the effect of right sensorimotor cortex suction ablation on cellular and temporal pattern of Cyp46 expression in the rat brain. Increased expression of Cyp46 at the lesion site at all post injury time points (2, 7, 14, 28 and 45 days post injury, dpi) was detected. Double immunofluorescence staining revealed colocalization of Cyp46 expression with different types of glial cells in time-dependent manner. In ED1⁺ microglia/macrophages Cyp46 expression was most prominent at 2 and 7 dpi, whereas Cyp46 immunoreactivity persisted in reactive astrocytes throughout all time points post-injury. However, during the first 2 weeks Cyp46 expression was enhanced in both GFAP⁺ and Vim⁺ astrocytes, while at 28 and 45 dpi its expression was mostly associated with GFAP⁺ cells. Pattern of neuronal Cyp46 expression remained unchanged after the lesion, i.e. Cyp46 immunostaining was detected in dendrites and cell body, but not in axons. The results of this study clearly demonstrate that in pathological conditions, like brain injury, Cyp46 displayed atypical expression, being expressed not only in neuronal cells, but also in microglia and astrocytes. Therefore, injury-induced expression of Cyp46 in microglial and astroglial cells may be involved in the post-injury removal of damaged cell membranes contributing to re-establishment of the brain cholesterol homeostasis.     

Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU⁺ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU⁺ cells, very few are mash1⁺ or nestin⁺ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1⁺ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.     

Change of composition of intermediate filaments in rat telencephalon during early postnatal period of ontogenesis

The goal of the present work was to study composition and spatial-temporal distribution of cells containing various proteins of intermediate filaments (nestin, vimentin, GFAP) in various brain areas at the early postnatal period of rat ontogenesis. By using methods of immunohistochemical determination of proteins of intermediate filaments, it has been found that at the early postnatal period of development, in the course of maturation of the nervous tissue, in the cells of cortex, hippocampus, and subventricular area, there occurred changes of immunohistochemical profile of intermediate filaments (ratio of immunopositive (+) and immunonegative (?) cells): nestin⁺/vimentin⁺/GFAP^? cells become nestin^?/vimentin^?/GFAP⁺.     

Yangyin Tongnao granules enhance neurogenesis in the peri-infarct area and upregulate brain-derived neurotrophic factor and vascular endothelial growth factor after focal cerebral ischemic infarction in rats

Yangyin Tongnao granules (YYTNG) have been extensively applied in the treatment of brain injury, mainly due to its antioxidant effects, inhibition of apoptosis, and enhancement of blood circulation. To analyze the effect of YYTNG on the recovery of neurological function and neurogenesis in the peri-infarct area after cerebral ischemic infarction in rats and to elucidate its role in the neuroprotective mechanism of stroke, Sprague–Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. Rats were randomly divided into five groups: sham, MCAO, and YYTNG-treated rats given doses of 0.83, 1.65, or 3.3 g kg^?1 day^?1. The YYTNG-treated groups (1.65 and 3.3 g kg^?1 day^?1) showed higher neurological scores and a lower infarct volume than the MCAO group on day 3 after MCAO. Furthermore, the YYTNG-treated groups (0.83, 1.65, and 3.3 g kg^?1 day^?1) showed higher neurological scores on day 7 after MCAO. The number of BrdU⁺/nestin⁺, BrdU⁺/NeuN⁺, and BrdU⁺/GFAP⁺ cells in the peri-infarct area 7 days after MCAO was significantly increased in the YYTNG-treated groups in a dose-dependent manner. The protein expression levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were significantly higher in all three YYTNG-treated groups than in the MCAO group. Based on these results, administration of YYTNG post ischemia could ameliorate neurological function deficits in rats with MCAO. The therapeutic effect of YYTNG may be due to the promotion of neurogenesis in the peri-infarct area and the upregulation of neuroprotective factors BDNF and VEGF in MCAO rats.

     

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Background

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP^−/−Vim^−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP^−/−Vim^−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

Methodology/Principal Findings

We subjected GFAP^−/−Vim^−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP^−/−Vim^−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN⁺ neurons in the ischemic and contralateral hemisphere was comparable between GFAP^−/−Vim^−/− and wild-type mice. In wild-type mice, the number of S100⁺ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP^−/−Vim^−/− mice, the number of S100⁺ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP^−/−Vim^−/− mice showed an increase in NeuN⁺BrdU⁺ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100⁺BrdU⁺ (surviving newly born) astrocytes.

Conclusions/Significance

Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.     

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo

Background

Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin⁺ cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo.

Methodology/Principal Findings

For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin⁺ microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin⁺ microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU⁺ and nestin⁺, although no further local cell proliferation occurred. In addition, nestin⁺ microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin⁺NG2⁺ microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons.

Conclusions/Significance

The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed.     

Suppressor of Cytokine Signaling-2 (SOCS2) Regulates the Microglial Response and Improves Functional Outcome after Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is frequently characterized by neuronal, axonal and myelin loss, reactive gliosis and neuroinflammation, often associated with functional deficits. Endogenous repair mechanisms include production of new neurons from precursor cells, but usually the new neurons fail to integrate and survive more than a few weeks. This is in part mediated by the toxic and inflammatory environment present in the injured brain which activates precursor cells to proliferate and differentiate but limits survival of the newborn progeny. Therefore, an understanding of mechanisms that regulate production and survival of newborn neurons and the neuroinflammatory response after brain injury may lead to therapeutic options to improve outcomes. Suppressor of Cytokine Signaling 2 (SOCS2) promotes hippocampal neurogenesis and survival of newborn neurons in the adult brain and regulates anti-inflammatory responses in the periphery, suggesting it may be a useful candidate to improve outcomes of TBI. In this study the functional and cellular responses of SOCS2 over-expressing transgenic (SOCS2Tg) mice were compared to wildtype littermates following mild or moderately severe TBI. Unlike wildtype controls, SOCS2Tg mice showed functional improvement on a ladder test, with a smaller lesion volume at 7d post injury and increased numbers of proliferative CD11b⁺ microglia/macrophages at 35d post-injury in the mild injury paradigm. At 7d post-moderately severe injury there was an increase in the area covered by cells expressing an anti-inflammatory M2 phenotype marker (CD206⁺) but no difference in cells with a pro-inflammatory M1 phenotype marker (CD16/32⁺). No effect of SOCS2 overexpression was observed in production or survival of newborn neurons, even in the presence of the neuroprotective agent erythropoietin (EPO). Therefore, SOCS2 may improve outcome of TBI in mice by regulating aspects of the neuroinflammatory response, promoting a more anti-inflammatory environment, although this was not sufficient to enhance survival of newborn cortical neurons.     

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP⁺) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP⁺ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.     

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic?Cduodenal homeobox 1 (Pdx1)⁺/nestin⁺ PPCs on islets. In vitro, co-culturing islets with Pdx1⁺/nestin⁺ PPCs prolonged the former survival from 7 to 14?days. Furthermore, with high glucose (300.8?mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1⁺/nestin⁺ PPCs for 3?days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2?weeks. Without Pdx1⁺/nestin⁺ PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1⁺/nestin⁺ PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo.     

Newborn GnRH neurons in the adult forebrain of the ring dove

The preoptic area of the hypothalamus is a key area that produces gonadotrophin-releasing hormone (GnRH). In birds, the chicken GnRH-I-form neurons are responsible for the hypothalamus-pituitary-gonadal system, which controls reproduction. In the ring dove, electrolytic lesion in the adult hypothalamus induces neurogenesis. In this study, we determined whether adult neurogenesis is involved in repairing GnRH neurons, specifically by generating newborn cells exhibiting GnRH-I immunoreactive properties. We selectively applied electrolytic lesions to three different regions of the diencephalon, including the preoptic area, which contains GnRH-I neurons, and identified new cells (BrdU-positive cells) that co-labeled with GnRH-I-immunoreactive cells. The BrdU⁺/GnRH⁺ double labeled cells were then confirmed with confocal laser analysis. In brains of both male and female ring doves we found new neurons at the lesion site of the preoptic region that were GnRH-I immunoreactive. However, the total number of GnRH neurons in the lesioned brains was less than that of sham-lesioned brains. When two other regions of the diencephalon that contain GnRH-I neurons were damaged, no recruitment of new GnRH-I neurons was detected. The rate of neurogenesis depends on the bird's reproductive phase when the lesion was applied. We found BrdU⁺/GnRH⁺ double-labeled cells almost exclusively during the pre-laying phase when birds are engaged in active courtship that leads to egg laying. Our observations suggest that recruitment of GnRH immunoreactive new neurons is restricted to the hypothalamic region and is sensitive to the reproductive stage of the birds.     

Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1^loxP/yERT2-Cre^GFAP mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI.     

Neonatal neural progenitor cells and their neuronal and glial cell derivatives are fully permissive for human cytomegalovirus infection

Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMV's interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral isolates, Towne (laboratory adapted) and TR (a clinical strain), at a multiplicity of infection of 3. NPCs were fully permissive for both strains, expressing the full range of viral antigens (Ags) and producing relatively large numbers of infectious virions. NPCs infected with TR showed delayed development of cytopathic effects (CPE) and replication centers and shed less virus. This pattern of delay for TR infections held true for all cell types tested. Differentiation of NPCs was carried out for 21 days to obtain either astroglia (>95% GFAP⁺) or a 1:5 mixed neuron/astroglia population (β-tubulin III⁺/GFAP⁺). We found that both of these differentiated populations were fully permissive for HCMV infection and produced substantial numbers of infectious virions. Utilizing a difference in plating efficiencies, we were able to enrich the neuron population to ~80% β-tubulin III⁺ cells. These β-tubulin III⁺-enriched populations remained fully permissive for infection but were very slow to develop CPE. These infected enriched neurons survived longer than either NPCs or astroglia, and a small proportion were alive until at least 14 days postinfection. These surviving cells were all β-tubulin III⁺ and showed viral Ag expression. Surprisingly, some cells still exhibited extended processes, similar to mock-infected neurons. Our findings strongly suggest neurons as reservoirs for HCMV within the developing brain.     

Cell-Specific mRNA Alterations in Na+, K+-ATPase α and β Isoforms and FXYD in Mice Treated Chronically with Carbamazepine, an Anti-Bipolar Drug

Evidence accumulating during almost 50 years suggests Na⁺, K⁺-ATPase dysfunction in bipolar disorder, a disease treatable with chronic administration of lithium salts, carbamazepine or valproic acid. Three Na⁺, K⁺-ATPase α subunits (α1–3) and two β subunits (β1 and β2) are expressed in brain together with the auxiliary protein FXYD7. FXYD7 decreases K⁺ affinity, and thus contributes to stimulation of the enzyme at elevated extracellular K⁺ concentrations. Na⁺, K⁺-ATPase subtype and FXYD7 genes were determined by RT-PCR in mice co-expressing one fluorescent signal with an astrocytic marker or a different fluorescent signal with a neuronal marker and treated for 14 days with carbamazepine. Following fluorescence-activated cell sorting of neurons and astrocytes it was shown that α2 Expression was upregulated in astrocytes and neurons and α1 selectively in neurons, but α3 was unchanged. β1 was upregulated in astrocytes, but not in neurons. β2 was unaffected in astrocytes and absent in neurons. FXYD7 was downregulated specifically in neurons. According to cited literature data these changes should facilitate K⁺ uptake in neurons, without compromising preferential uptake in astrocytes at increased extracellular K⁺ concentrations. This process seems to be important for K⁺ homeostasis of the cellular level of the brain (Xu et al. Neurochem Res E-pub Dec. 12, 2012).     

The embryonic midbrain directs neuronal specification of embryonic stem cells at early stages of differentiation

Specific neuronal differentiation of Embryonic Stem Cells (ESCs) depends on their capacity to interpret environmental cues. At present, it is not clear at which stage of differentiation ESCs become competent to produce multiple neuronal lineages in response to the niche of the embryonic brain. To unfold the developmental potential of ESC-derived precursors, we transplanted these cells into the embryonic midbrain explants, where neurogenesis occurs as in normal midbrain development. Using this experimental design, we show that the transition from ESCs to Embryoid Body (EB) precursors is necessary to differentiate into Lmx1a⁺/Ptx3⁺/TH⁺ dopaminergic neurons around the ventral midline of the midbrain. In addition, EB cells placed at other dorsal-ventral levels of the midbrain give rise to Nkx6.1⁺ red nucleus (RN) neurons, Nkx2.2⁺ ventral interneurons and Pax7⁺ dorsal neurons at the correct positions. Notably, differentiation of ESCs into Neural Precursor Cells (NPCs) prior to transplantation markedly reduces specification at the Lmx1a, Nkx6.1 and Pax7 expression domains, without affecting neuronal differentiation. Finally, exposure to Fgf8 and Shh in vitro promotes commitment of some ESC-derived NPCs to differentiate into putative Lmx1a⁺ dopaminergic neurons in the midbrain. Our data demonstrate intrinsic developmental potential differences among ESC-derived precursor populations.     

Relation Among Neuronal Death,Cell Proliferation and Neuronal Differentiation in the Gerbil Main Olfactory Bulb after Transient Cerebral Ischemia

Neurogenesis occurs during the embryonic stage and throughout life. Brain injuries such as ischemic insults enhance cell proliferation in some areas of the brain. We examined proliferation of newly generated cells in each layer of the gerbil main olfactory bulb (MOB) after 5 min of transient cerebral ischemia using bromodeoxyuridine (BrdU) immunohistochemistry. Ischemia-related neuronal death in the MOB was not detected using Fluoro-Jade B histofluorescence and TUNEL staining. Many BrdU-positive (⁺) cells were found in the rostral migratory stream in control and ischemic MOBs. Significant increase of BrdU⁺ cells was observed in the granule cell layer (GCL) and glomerular layer (GL) from 15 days post-ischemia, and BrdU⁺ cells were very much higher than those of the control group 30 days post-ischemia. At this time point after ischemia/reperfusion, a few BrdU⁺ cells in the GL and GCL were co-localized with calretinin⁺ cells, and many BrdU⁺ cells expressed doublecortin, a marker of immature neurons. These results indicate that cell proliferation is increased in the GCL and GL without apparent neuronal loss from 15 days after transient cerebral ischemia in gerbils.     

Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP⁺¹ antibody. We previously reported that GFAP⁺¹ was expressed in astrocytes and in degenerating neurons in Alzheimer''s disease brains. In this study we aimed at further investigating the neuronal GFAP⁺¹ expression and we started by affinity purifying the GFAP⁺¹ antibody. The purified antibody resulted in a loss of neuronal GFAP⁺¹ signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP⁺¹ expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP⁺¹ antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP⁺¹ antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.     

Sonic Hedgehog Controls the Phenotypic Fate and Therapeutic Efficacy of Grafted Neural Precursor Cells in a Model of Nigrostriatal Neurodegeneration

The expression of soluble growth and survival promoting factors by neural precursor cells (NPCs) is suggested to be a prominent mechanism underlying the protective and regenerative effects of these cells after transplantation. Nevertheless, how and to what extent specific NPC-expressed factors contribute to therapeutic effects is not well understood. Using RNA silencing, the current study investigated the roles of two donor NPC molecules, namely glial cell-line derived neurotrophic factor (GDNF) and sonic hedgehog (SHH), in the protection of substantia nigra dopamine neurons in rats treated with 6-hydroxydopamine (6-OHDA). Analyses indicate that as opposed to the knock-down of GDNF, SHH inhibition caused a profound decline in nigrostriatal neuroprotection. Further, SHH silencing also curbed endogenous neurogenesis and the migration of host brdU⁺/dcx⁺ neural precursors into the striatum, which was present in the animals receiving control or GDNF silenced NPCs. A change in graft phenotype, mainly reflected by a reduced proportion of undifferentiated nestin⁺ cells, as well as a significantly greater host microglial activity, suggested an important role for these processes in the attenuation of neuroprotection and neurogenesis upon SHH silencing. Overall these studies reveal core mechanisms fundamental to grafted NPC-based therapeutic effects, and delineate the particular contributions of two graft-expressed molecules, SHH and GDNF, in mediating midbrain dopamine neuron protection, and host plasticity after NPC transplantation.     

Glial Fatty Acid-Binding Protein 7 (FABP7) Regulates Neuronal Leptin Sensitivity in the Hypothalamic Arcuate Nucleus

The hypothalamus is involved in the regulation of food intake and energy homeostasis. The arcuate nucleus (ARC) and median eminence (ME) are the primary hypothalamic sites that sense leptin and nutrients in the blood, thereby mediating food intake. Recently, studies demonstrating a role for non-neuronal cell types, including astrocytes and tanycytes, in these regulatory processes have begun to emerge. However, the molecular mechanisms involved in these activities remain largely unknown. In this study, we examined in detail the localization of fatty acid-binding protein 7 (FABP7) in the hypothalamic ARC and sought to determine its role in the hypothalamus. We performed a phenotypic analysis of diet-induced FABP7 knockout (KO) obese mice and of FABP7 KO mice treated with a single leptin injection. Immunohistochemistry revealed that FABP7⁺ cells are NG2⁺ or GFAP⁺ in the ARC and ME. In mice fed a high-fat diet, weight gain and food intake were lower in FABP7 KO mice than in wild-type (WT) mice. FABP7 KO mice also had lower food intake and weight gain after a single injection of leptin, and we consistently confirmed that the number of pSTAT3⁺ cells in the ARC indicated that the leptin-induced activation of neurons was significantly more frequent in FABP7 KO mice than in WT mice. In FABP7 KO mice-derived primary astrocyte cultures, the level of ERK phosphorylation was lower after leptin treatment. Collectively, these results indicate that in hypothalamic astrocytes, FABP7 might be involved in sensing neuronal leptin via glia-mediated mechanisms and plays a pivotal role in controlling systemic energy homeostasis.