Andrographolide promotes hippocampal neurogenesis and spatial memory in the APPswe/PS1ΔE9 mouse model of Alzheimer’s disease

In Alzheimer´s disease (AD) there is a reduction in hippocampal neurogenesis that has been associated to cognitive deficits. Previously we showed that Andrographolide (ANDRO), the main bioactive component of Andrographis paniculate, induces proliferation in the hippocampus of the APPswe/PSEN1ΔE9 (APP/PS1) mouse model of AD as assessed by staining with the mitotic marker Ki67. Here, we further characterized the effect of ANDRO on hippocampal neurogenesis in APP/PS1 mice and evaluated the contribution of this process to the cognitive effect of ANDRO. Treatment of 8-month-old APP/PS1 mice with ANDRO for 4 weeks increased proliferation in the dentate gyrus as evaluated by BrdU incorporation. Although ANDRO had no effect on neuronal differentiation of newborn cells, it strongly increased neural progenitors, neuroblasts and newborn immature neurons, cell populations that were decreased in APP/PS1 mice compared to age-matched wild-type mice. ANDRO had no effect on migration or in total dendritic length, arborization and orientation of immature neurons, suggesting no effects on early morphological development of newborn neurons. Finally, ANDRO treatment improved the performance of APP/PS1 mice in the object location memory task. This effect was not completely prevented by co-treatment with the anti-mitotic drug TMZ, suggesting that other effects of ANDRO in addition to the increase in neurogenesis might underlie the observed cognitive improvement. Altogether, our data indicate that in APP/PS1 mice ANDRO stimulates neurogenesis in the hippocampus by inducing proliferation of neural precursor cells and improves spatial memory performance.

WNK3 Maintains the GABAergic Inhibitory Tone, Synaptic Excitation and Neuronal Excitability via Regulation of KCC2 Cotransporter in Mature Neurons

The activation of chloride (Cl−)permeable gamma (γ)-aminobutyric acid type A(GABAA) receptors induces synaptic inhibition in mature and excitation in immature neurons. This developmental “switch” in GABA function controlled by its polarity depends on the postnatal decrease in intraneuronal Cl− concentration mediated by KCC2, a member of cation-chloride cotransporters (CCCs). The serine-threonine kinase WNK3 (With No Lysine [K]), is a potent regulator of all CCCs and is expressed in neurons. Here, we characterized the functions of WNK3 and its role in GABAergic signaling in cultured embryonic day 18 (E18) hippocampal neurons. We observed a decrease in WNK3 expression as neurons mature. Knocking down of WNK3 significantly hyperpolarized EGABA in mature neurons (DIV13–15) but had no effect on immature neurons (DIV6–8). This hyperpolarized EGABA in WNK3-deficient neurons was not due to the total expression of NKCC1 and KCC2, that remained unchanged. However, there was a reduction in phosphorylated KCC2 at the membrane, suggesting an increase in KCC2 chloride export activity. Furthermore, hyperpolarized EGABA observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence EGABA. Notably, WNK3 knockdown resulted in morphological changes in mature but not immature neurons. Electrophysiological characterization of WNK3-deficient mature neurons revealed reduced capacitances but increased intrinsic excitability and synaptic excitation. Hence, our study demonstrates that WNK3 maintains the “adult” GABAergic inhibitory tone in neurons and plays a role in the morphological development of neurons and excitability.

Shaping the size of a neuronal lineage: the role of Imp and Syp RBPs in the precise elimination of neurons by apoptosis.

Neuronal stem cells produce a finite and stereotyped number of neuronal progenies. This process must be finely regulated during development and adult stages to ensure proper brain function. In Drosophila, stem cells, called Neuroblasts, produce an invariant number of neurons. Two RNA binding proteins, Imp and Syp, play a central role in controlling the speed of division and the end of the proliferative phase of individual NBs, two parameters that influences the final number of neurons produced. Here, we have discovered a novel function for Imp and Syp, where both RBPs shape the number of neurons produced by a stem cell by controlling program cell death (PCD) in immature neurons. By studying a neuroblast lineage, called Lin A/15, which produces motoneurons (MNs) and glia, we have demonstrated that Lin A/15 stem cell spends 40% of its time producing immature MNs which are eliminated by apoptosis. We have revealed that only the first born MNs (Imp +) survive while the last born MNs (Imp- Syp+) are eliminated by apoptosis. Both RBPs play a central role in neuronal survival, Imp promotes neuronal survival while Syp promotes cell death in immature motoneurons. Interestingly their opposite temporal gradient in Lin A/15 stem cell also determines the end of Lin A/15 stem cell neurogenesis by PCD. Both RNA binding proteins are conserved in vertebrates and seem to play a central role in the number of neurons produce during development. The Drosophila model and its genetic tools offer a unique chance to decipher their function in neural stem cell versus immature neurons.

Short high fat diet triggers reversible and region specific effects in DCX+ hippocampal immature neurons of adolescent male mice

Adolescence represents a crucial period for maturation of brain structures involved in cognition. Early in life unhealthy dietary patterns are associated with inferior cognitive outcomes at later ages; conversely, healthy diet is associated with better cognitive results. In this study we analyzed the effects of a short period of hypercaloric diet on newborn hippocampal doublecortin+ (DCX) immature neurons in adolescent mice. Male mice received high fat diet (HFD) or control low fat diet (LFD) from the 5th week of age for 1 or 2 weeks, or 1 week HFD followed by 1 week LFD. After diet supply, mice were either perfused for immunohistochemical (IHC) analysis or their hippocampi were dissected for biochemical assays. Detailed morphometric analysis was performed in DCX+ cells that displayed features of immature neurons. We report that 1 week-HFD was sufficient to dramatically reduce dendritic tree complexity of DCX+ cells. This effect occurred specifically in dorsal and not ventral hippocampus and correlated with reduced BDNF expression levels in dorsal hippocampus. Both structural and biochemical changes were reversed by a return to LFD. Altogether these studies increase our current knowledge on potential consequences of hypercaloric diet on brain and in particular on dorsal hippocampal neuroplasticity.

The PSA-NCAM-Positive “Immature” Neurons: An Old Discovery Providing New Vistas on Brain Structural Plasticity

Studies on brain plasticity have undertaken different roads, tackling a wide range of biological processes: from small synaptic changes affecting the contacts among neurons at the very tip of their processes, to birth, differentiation, and integration of new neurons (adult neurogenesis). Stem cell-driven adult neurogenesis is an exception in the substantially static mammalian brain, yet, it has dominated the research in neurodevelopmental biology during the last thirty years. Studies of comparative neuroplasticity have revealed that neurogenic processes are reduced in large-brained mammals, including humans. On the other hand, large-brained mammals, with respect to rodents, host large populations of special “immature” neurons that are generated prenatally but express immature markers in adulthood. The history of these “immature” neurons started from studies on adhesion molecules carried out at the beginning of the nineties. The identity of these neurons as “stand by” cells “frozen” in a state of immaturity remained un-detected for long time, because of their ill-defined features and because clouded by research ef-forts focused on adult neurogenesis. In this review article, the history of these cells will be reconstructed, and a series of nuances and confounding factors that have hindered the distinction between newly generated and “immature” neurons will be addressed.

Breast Carcinoma-amplified Sequence 2 Regulates Adult Neurogenesis via β-catenin

Background Breast carcinoma-amplified sequence 2 (BCAS2) regulates β-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased β-catenin expression. Because β-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via β-catenin. Methods BCAS2 regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-β-catenin gene therapy. Results To show that BCAS2-regulating adult neurogenesis via β-catenin, first, BCAS2 cKO mice showed low SRY-box 2–positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via β-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased β-catenin expression. Moreover, AAV-β-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2 regulating adult neurogenesis via β-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects. Conclusions BCAS2 can regulate adult neurogenesis in mice via β-catenin.

Active Fraction Combination From Liuwei Dihuang Decoction Improves Adult Hippocampal Neurogenesis and Neurogenic Microenvironment in Cranially Irradiated Mice

Background: Cranial radiotherapy is clinically used in the treatment of brain tumours; however, the consequent cognitive and emotional dysfunctions seriously impair the life quality of patients. LW-AFC, an active fraction combination extracted from classical traditional Chinese medicine prescription Liuwei Dihuang decoction, can improve cognitive and emotional dysfunctions in many animal models; however, the protective effect of LW-AFC on cranial irradiation–induced cognitive and emotional dysfunctions has not been reported. Recent studies indicate that impairment of adult hippocampal neurogenesis (AHN) and alterations of the neurogenic microenvironment in the hippocampus constitute critical factors in cognitive and emotional dysfunctions following cranial irradiation. Here, our research further investigated the potential protective effects and mechanisms of LW-AFC on cranial irradiation–induced cognitive and emotional dysfunctions in mice.Methods: LW-AFC (1.6 g/kg) was intragastrically administered to mice for 14 days before cranial irradiation (7 Gy γ-ray). AHN was examined by quantifying the number of proliferative neural stem cells and immature neurons in the dorsal and ventral hippocampus. The contextual fear conditioning test, open field test, and tail suspension test were used to assess cognitive and emotional functions in mice. To detect the change of the neurogenic microenvironment, colorimetry and multiplex bead analysis were performed to measure the level of oxidative stress, neurotrophic and growth factors, and inflammation in the hippocampus.Results: LW-AFC exerted beneficial effects on the contextual fear memory, anxiety behaviour, and depression behaviour in irradiated mice. Moreover, LW-AFC increased the number of proliferative neural stem cells and immature neurons in the dorsal hippocampus, displaying a regional specificity of neurogenic response. For the neurogenic microenvironment, LW-AFC significantly increased the contents of superoxide dismutase, glutathione peroxidase, glutathione, and catalase and decreased the content of malondialdehyde in the hippocampus of irradiated mice, accompanied by the increase in brain-derived neurotrophic factor, insulin-like growth factor-1, and interleukin-4 content. Together, LW-AFC improved cognitive and emotional dysfunctions, promoted AHN preferentially in the dorsal hippocampus, and ameliorated disturbance in the neurogenic microenvironment in irradiated mice.Conclusion: LW-AFC ameliorates cranial irradiation–induced cognitive and emotional dysfunctions, and the underlying mechanisms are mediated by promoting AHN in the dorsal hippocampus and improving the neurogenic microenvironment. LW-AFC might be a promising therapeutic agent to treat cognitive and emotional dysfunctions in patients receiving cranial radiotherapy.

Galantamine prevents and reverses neuroimmune induction and loss of adult hippocampal neurogenesis following adolescent alcohol exposure

Background Binge ethanol exposure during adolescence reduces hippocampal neurogenesis, a reduction which persists throughout adulthood despite abstinence. This loss of neurogenesis, indicated by reduced doublecortin+ immunoreactivity (DCX+IR), is paralleled by an increase in hippocampal proinflammatory signaling cascades. As galantamine, a cholinesterase inhibitor, has anti-inflammatory actions, we tested the hypothesis that galantamine would prevent (study 1) or restore (study 2) AIE induction of proinflammatory signals within the hippocampus as well as AIE-induced loss of hippocampal neurogenesis. Methods Galantamine (4 mg/kg) or vehicle (saline) was administered to Wistar rats during adolescent intermittent ethanol (AIE; 5.0 g/kg ethanol, 2 days on/2 days off, postnatal day [P] 25-54) (study 1, prevention) or after AIE during abstinent maturation to adulthood (study 2, restoration). Results Results indicate AIE reduced DCX+IR and induced cleaved caspase3 (Casp3) in DCX-expressing immature neurons. Excitingly, AIE induction of activated Casp3 in DCX-expressing neurons is both prevented and reversed by galantamine treatment, which also resulted in prevention and restoration of neurogenesis (DCX+IR). Similarly, galantamine prevented and/or reversed AIE induction of proinflammatory markers, including the chemokine (C-C motif) ligand 2 (CCL2), cyclooxygenase-2 (COX-2), and high mobility group box 1 (HMGB1) protein, suggesting that AIE induction of proinflammatory signaling mediates both cell death cascades and hippocampal neurogenesis. Interestingly, galantamine treatment increased Ki67+IR generally as well as increased pan-Trk expression specifically in AIE-treated rats but failed to reverse AIE induction of NADPH-oxidase (gp91phox). Conclusions Collectively, our studies suggest that (1) loss of neurogenesis after AIE is mediated by persistent induction of proinflammatory cascades which drive activation of cell death machinery in immature neurons, and (2) galantamine can prevent and restore AIE disruptions in the hippocampal environmental milieu to then prevent and restore AIE-mediated loss of neurogenesis.

Doublecortin-Expressing Neurons in Chinese Tree Shrew Forebrain Exhibit Mixed Rodent and Primate-Like Topographic Characteristics

Doublecortin (DCX) is transiently expressed in new-born neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) related to adult neurogenesis in the olfactory bulb (OB) and hippocampal formation. DCX immunoreactive (DCX+) immature neurons also occur in the cerebral cortex primarily over layer II and the amygdala around the paralaminar nucleus (PLN) in various mammals, with interspecies differences pointing to phylogenic variation. The tree shrews (Tupaia belangeri) are phylogenetically closer to primates than to rodents. Little is known about DCX+ neurons in the brain of this species. In the present study, we characterized DCX immunoreactivity (IR) in the forebrain of Chinese tree shrews aged from 2 months- to 6 years-old (n = 18). DCX+ cells were present in the OB, SVZ, SGZ, the piriform cortex over layer II, and the amygdala around the PLN. The numerical densities of DCX+ neurons were reduced in all above neuroanatomical regions with age, particularly dramatic in the DG in the 5–6 years-old animals. Thus, DCX+ neurons are present in the two established neurogenic sites (SVZ and SGZ) in the Chinese tree shrew as seen in other mammals. DCX+ cortical neurons in this animal exhibit a topographic pattern comparable to that in mice and rats, while these immature neurons are also present in the amygdala, concentrating around the PLN as seen in primates and some nonprimate mammals.

Reactive, Adult Neurogenesis From Increased Neural Progenitor Cell Proliferation Following Alcohol Dependence in Female Rats

Hippocampal neurodegeneration is a consequence of excessive alcohol drinking in alcohol use disorders (AUDs), however, recent studies suggest that females may be more susceptible to alcohol-induced brain damage. Adult hippocampal neurogenesis is now well accepted to contribute to hippocampal integrity and is known to be affected by alcohol in humans as well as in animal models of AUDs. In male rats, a reactive increase in adult hippocampal neurogenesis has been observed during abstinence from alcohol dependence, a phenomenon that may underlie recovery of hippocampal structure and function. It is unknown whether reactive neurogenesis occurs in females. Therefore, adult female rats were exposed to a 4-day binge model of alcohol dependence followed by 7 or 14 days of abstinence. Immunohistochemistry (IHC) was used to assess neural progenitor cell (NPC) proliferation (BrdU and Ki67), the percentage of increased NPC activation (Sox2+/Ki67+), the number of immature neurons (NeuroD1), and ectopic dentate gyrus granule cells (Prox1). On day seven of abstinence, ethanol-treated females showed a significant increase in BrdU+ and Ki67+ cells in the subgranular zone of the dentate gyrus (SGZ), as well as greater activation of NPCs (Sox2+/Ki67+) into active cycling. At day 14 of abstinence, there was a significant increase in the number of immature neurons (NeuroD1+) though no evidence of ectopic neurogenesis according to either NeuroD1 or Prox1 immunoreactivity. Altogether, these data suggest that alcohol dependence produces similar reactive increases in NPC proliferation and adult neurogenesis. Thus, reactive, adult neurogenesis may be a means of recovery for the hippocampus after alcohol dependence in females.