Differential expression and regulation of myristoylated alanine-rich C kinase substrate (MARCKS) in the hippocampus of C57/BL6J and DBA/2J mice

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in brain that binds the inner surface of the plasma membrane, calmodulin, and cross-links filamentous actin, all in a PKC phosphorylation-reversible manner. MARCKS has been implicated in hippocampal-dependent learning and long-term potentiation (LTP). Previous studies have shown DBA/2 mice to exhibit poor spatial/contextual learning, impaired hippocampal LTP, and hippocampal mossy fiber hypoplasia, as well as reduced hippocampal PKC activity and expression relative to C57BL/6 mice. In the present study, we assessed the expression (mRNA and protein) and subcellular distribution (membrane and cytolsol) of MARCKS in the hippocampus and frontal cortex of C57BL/6 and DBA/2 mice using quantitative western blotting. In the hippocampus, total MARCKS mRNA and protein levels in C57BL/6J mice were significantly lower ( approximately 45%) compared with DBA/2J mice, and MARCKS protein was observed predominantly in the cytosolic fraction. MARCKS expression in frontal cortex did not differ significantly between strains. To examine the dynamic regulation of MARCKS subcellular distribution, mice from each strain were subjected to 60 min restraint stress and MARCKS subcellular distribution was determined 24 h later. Restraint stress resulted in a significant reduction in membrane MARCKS expression in C57BL/6J hippocampus but not in the DBA/2J hippocampus despite similar stress-induced increases in serum corticosterone. Restraint stress did not affect cytosolic or total MARCKS levels in either strain. Similarly, restraint stress (30 min) in rats also induced a significant reduction in membrane MARCKS, but not total or cytosolic MARCKS, in the hippocampus but not in frontal cortex. In rats, chronic lithium treatment prior to stress exposure reduced hippocampal MARCKS expression but did not affect the stress-induced reduction in membrane MARCKS. Collectively these data demonstrate higher resting levels of MARCKS in the hippocampus of DBA/2J mice compared to C57BL/6J mice, and that acute stress leads to a long-term reduction in membrane MARCKS expression in C57BL/6J mice and rats but not in DBA/2J mice. These strain differences in hippocampal MARCKS expression and subcellular translocation following stress may contribute to the differences in behaviors requiring hippocampal plasticity observed between these strains.     

Prenatal alcohol exposure reduces the proportion of newly produced neurons and glia in the dentate gyrus of the hippocampus in female rats

Prenatal alcohol exposure (PAE) alters adult neurogenesis and the neurogenic response to stress in male rats. As the effects of stress on neurogenesis are sexually dimorphic, the present study investigated the effects of PAE on adult hippocampal neurogenesis under both nonstressed and stressed conditions in female rats. Pregnant females were assigned to one of three prenatal treatments: (1) alcohol (PAE)—liquid alcohol (ethanol) diet ad libitum (36% ethanol-derived calories); (2) pair-fed—isocaloric liquid diet, with maltose–dextrin substituted for ethanol, in the amount consumed by a PAE partner (g/kg body wt/day of gestation); and (3) control—lab chow ad libitum. Female offspring were assigned to either nonstressed (undisturbed) or stressed (repeated restraint stress for 9 days) conditions. On day 10, all rats were injected with bromodeoxyuridine (BrdU) and perfused either 24 hours (cell proliferation) or 3 weeks (cell survival) later. We found that PAE did not significantly alter cell proliferation or survival, whereas females from the pair-fed condition exhibited elevated levels of cell survival compared to control females. Importantly, however, the proportion of both new neurons and new glial cells in the hippocampal dentate gyrus was reduced in PAE compared to control females. Exposure to stress did not alter neurogenesis in any of the prenatal treatment groups. In summary, compared to females from the control condition, prenatal dietary restriction enhanced the survival of new neurons, whereas PAE altered the differentiation of newly produced cells in the adult dentate gyrus. Alterations in hippocampal neurogenesis following PAE may contribute to learning and memory deficits seen in individuals with fetal alcohol spectrum disorders.     

Prenatal alcohol exposure reduces the proportion of newly produced neurons and glia in the dentate gyrus of the hippocampus in female rats

Prenatal alcohol exposure (PAE) alters adult neurogenesis and the neurogenic response to stress in male rats. As the effects of stress on neurogenesis are sexually dimorphic, the present study investigated the effects of PAE on adult hippocampal neurogenesis under both nonstressed and stressed conditions in female rats. Pregnant females were assigned to one of three prenatal treatments: (1) alcohol (PAE)—liquid alcohol (ethanol) diet ad libitum (36% ethanol-derived calories); (2) pair-fed—isocaloric liquid diet, with maltose–dextrin substituted for ethanol, in the amount consumed by a PAE partner (g/kg body wt/day of gestation); and (3) control—lab chow ad libitum. Female offspring were assigned to either nonstressed (undisturbed) or stressed (repeated restraint stress for 9 days) conditions. On day 10, all rats were injected with bromodeoxyuridine (BrdU) and perfused either 24 hours (cell proliferation) or 3 weeks (cell survival) later. We found that PAE did not significantly alter cell proliferation or survival, whereas females from the pair-fed condition exhibited elevated levels of cell survival compared to control females. Importantly, however, the proportion of both new neurons and new glial cells in the hippocampal dentate gyrus was reduced in PAE compared to control females. Exposure to stress did not alter neurogenesis in any of the prenatal treatment groups. In summary, compared to females from the control condition, prenatal dietary restriction enhanced the survival of new neurons, whereas PAE altered the differentiation of newly produced cells in the adult dentate gyrus. Alterations in hippocampal neurogenesis following PAE may contribute to learning and memory deficits seen in individuals with fetal alcohol spectrum disorders.     

Prenatal ethanol exposure persistently impairs NMDA receptor-dependent activation of extracellular signal-regulated kinase in the mouse dentate gyrus

The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal-dependent learning deficits and a decreased ability to elicit long-term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade by NMDA receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol-exposed adult animals would have deficits in hippocampal NMDA receptor-dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA-stimulated ERK1/2 activation in acute hippocampal slices prepared from adult fetal alcohol-exposed mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor-dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life-long cognitive deficits, associated with prenatal alcohol exposure.     

Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus

Zika virus (ZIKV) infection during pregnancy is linked to various developmental brain disorders. Infants who are asymptomatic at birth might have postnatal neurocognitive complications. However, animal models recapitulating these neurocognitive phenotypes are lacking, and the circuit mechanism underlying behavioral abnormalities is unknown. Here, we show that ZIKV infection during mouse pregnancy induces maternal immune activation (MIA) and leads to autistic‐like behaviors including repetitive self‐grooming and impaired social memory in offspring. In the medial prefrontal cortex (mPFC), ZIKV‐affected offspring mice exhibit excitation and inhibition imbalance and increased cortical activity. This could be explained by dysregulation of inhibitory neurons and synapses, and elevated neural activity input from mPFC‐projecting ventral hippocampus (vHIP) neurons. We find structure alterations in the synaptic connections and pattern of vHIP innervation of mPFC neurons, leading to hyperconnectivity of the vHIP‐mPFC pathway. Decreasing the activity of mPFC‐projecting vHIP neurons with a chemogenetic strategy rescues social memory deficits in ZIKV offspring mice. Our studies reveal a hyperconnectivity of vHIP to mPFC projection driving social memory deficits in mice exposed to maternal inflammation by ZIKV.     

Redox balance- and radiation-mediated alteration in hippocampal neurogenesis

Changes in the intracellular and extracellular redox balance have been correlated with cell fate decisions in terms of proliferation versus differentiation, entering versus existing cell cycle and survival versus cell death. Adult hippocampal neurogenesis has been correlated with neuronal plasticity of learning and memory; however, the process is exquisitely sensitive to changes in redox balance. Cranial irradiation is an effective modality in treating brain tumours but often leads to deficits in hippocampus-related learning and memory, which is most likely due to sustained elevation of oxygen free radical production and suppression of hippocampal neurogenesis. The subcellular redox environment affecting hippocampal neurogenesis is largely unknown. Using mutant mice deficient in each one of the three superoxide dismutase (SOD, EC 1.15.1.1) isoforms, we have begun to determine the consequences of SOD deficiency in hippocampal neurogenesis and the related functions of learning and memory under normal condition and following cranial irradiation.     

Glucocorticoid Receptors are Localized to Dendritic Spines and Influence Local Actin Signaling

Glucocorticoids affect learning and memory but the cellular mechanisms involved are poorly understood. The present studies tested if the stress-responsive glucocorticoid receptor (GR) is present and regulated within dendritic spines, and influences local signaling to the actin cytoskeleton. In hippocampal field CA1, 13?% of synapses contained GR-immunoreactivity. Three-dimensional reconstructions of CA1 dendrites showed that GR aggregates are present in both spine heads and necks. Consonant with evidence that GR?? mRNA associates with the translation regulator Fragile X Mental Retardation Protein (FMRP), spine GR levels were rapidly increased by group 1 mGluR activation and reduced in mice lacking FMRP. Treatment of cultured hippocampal slices with the GR agonist dexamethasone rapidly (15?C30?min) increased total levels of phosphorylated (p) Cofilin and extracellular signal-regulated kinase (ERK) 1/2, proteins that regulate actin polymerization and stability. Dexamethasone treatment of adult hippocampal slices also increased numbers of PSD95+ spines containing pERK1/2, but reduced numbers of pCofilin-immunoreactive spines. Dexamethasone-induced increases in synaptic pERK1/2 were blocked by the GR antagonist RU-486. These results demonstrate that GRs are present in hippocampal spines where they mediate acute glucocorticoid effects on local spine signaling. Through effects on these actin regulatory pathways, GRs are positioned to exert acute effects on synaptic plasticity.     

Puerarin and Amlodipine Improvement of d-Galactose-Induced Impairments of Behaviour and Neurogenesis in Mouse Dentate Gyrus: Correlation with Glucocorticoid Receptor Expression

Glucocorticoid receptors (GRs) exert actions on the hippocampus that are important for memory formation. There are correlations between vascular dysfunctions and GR-related gene expression. Both vascular dysfunction and GR gene expression decline occur during the ageing process. Therefore, hypotensors, which have effects on improving vascular dysfunction, may be able to ameliorate GR gene expression decline in ageing mice and improve ageing-mediated memory deficits. In this study, we hypothesized that hypotensors could alleviate the decline of GR gene expression and ameliorate age-induced learning and memory deficits in a d-gal-induced ageing mice model. In line with our hypothesis, we found that chronic d-gal treatment decreased GR and DCX expression in the hippocampus, leading to learning and memory deficits. Amlodipine (AM) and puerarin (PU) treatment improved GR gene expression decline in the hippocampus and ameliorated the learning and memory deficits of d-gal-treated mice. These changes correlated with enhanced DCX expression and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Furthermore, PU treatment conveyed better effects than AM treatment, but combination therapy did not enhance the effects on improving GR expression. However, we did not find evidence of these changes in non-d-gal-treated mice that lacked GR gene expression decline. These results suggest that AM and PU could improve d-gal-induced behavioural deficits in correlation with GR gene expression.

     

Redox balance- and radiation-mediated alteration in hippocampal neurogenesis

Abstract

Changes in the intracellular and extracellular redox balance have been correlated with cell fate decisions in terms of proliferation versus differentiation, entering versus existing cell cycle and survival versus cell death. Adult hippocampal neurogenesis has been correlated with neuronal plasticity of learning and memory; however, the process is exquisitely sensitive to changes in redox balance. Cranial irradiation is an effective modality in treating brain tumours but often leads to deficits in hippocampus-related learning and memory, which is most likely due to sustained elevation of oxygen free radical production and suppression of hippocampal neurogenesis. The subcellular redox environment affecting hippocampal neurogenesis is largely unknown. Using mutant mice deficient in each one of the three superoxide dismutase (SOD, EC 1.15.1.1) isoforms, we have begun to determine the consequences of SOD deficiency in hippocampal neurogenesis and the related functions of learning and memory under normal condition and following cranial irradiation.     

Presenilin-1 Dependent Neurogenesis Regulates Hippocampal Learning and Memory

Presenilin-1 (PS1), the catalytic core of the aspartyl protease γ-secretase, regulates adult neurogenesis. However, it is not clear whether the role of neurogenesis in hippocampal learning and memory is PS1-dependent, or whether PS1 loss of function in adult hippocampal neurogenesis can cause learning and memory deficits. Here we show that downregulation of PS1 in hippocampal neural progenitor cells causes progressive deficits in pattern separation and novelty exploration. New granule neurons expressing reduced PS1 levels exhibit decreased dendritic branching and dendritic spines. Further, they exhibit reduced survival. Lastly, we show that PS1 effect on neurogenesis is mediated via β-catenin phosphorylation and notch signaling. Together, these observations suggest that impairments in adult neurogenesis induce learning and memory deficits and may play a role in the cognitive deficits observed in Alzheimer’s disease.     

Cdk5 Contributes to Huntington’s Disease Learning and Memory Deficits via Modulation of Brain Region-Specific Substrates

Cognitive deficits are a major hallmark of Huntington’s disease (HD) with a great impact on the quality of patient’s life. Gaining a better understanding of the molecular mechanisms underlying learning and memory impairments in HD is, therefore, of critical importance. Cdk5 is a proline-directed Ser/Thr kinase involved in the regulation of synaptic plasticity and memory processes that has been associated with several neurodegenerative disorders. In this study, we aim to investigate the role of Cdk5 in learning and memory impairments in HD using a novel animal model that expresses mutant huntingtin (mHtt) and has genetically reduced Cdk5 levels. Genetic reduction of Cdk5 in mHtt knock-in mice attenuated both corticostriatal learning deficits as well as hippocampal-dependent memory decline. Moreover, the molecular mechanisms by which Cdk5 counteracts the mHtt-induced learning and memory impairments appeared to be differentially regulated in a brain region-specific manner. While the corticostriatal learning deficits are attenuated through compensatory regulation of NR2B surface levels, the rescue of hippocampal-dependent memory was likely due to restoration of hippocampal dendritic spine density along with an increase in Rac1 activity. This work identifies Cdk5 as a critical contributor to mHtt-induced learning and memory deficits. Furthermore, we show that the Cdk5 downstream targets involved in memory and learning decline differ depending on the brain region analyzed suggesting that distinct Cdk5 effectors could be involved in cognitive impairments in HD.     

Temporal manipulation of transferrin-receptor-1-dependent iron uptake identifies a sensitive period in mouse hippocampal neurodevelopment

Iron is a necessary substrate for neuronal function throughout the lifespan, but particularly during development. Early life iron deficiency (ID) in humans (late gestation through 2-3 yr) results in persistent cognitive and behavioral abnormalities despite iron repletion. Animal models of early life ID generated using maternal dietary iron restriction also demonstrate persistent learning and memory deficits, suggesting a critical requirement for iron during hippocampal development. Precise definition of the temporal window for this requirement has been elusive due to anemia and total body and brain ID inherent to previous dietary restriction models. To circumvent these confounds, we developed transgenic mice that express tetracycline transactivator regulated, dominant negative transferrin receptor (DNTfR1) in hippocampal neurons, disrupting TfR1 mediated iron uptake specifically in CA1 pyramidal neurons. Normal iron status was restored by doxycycline administration. We manipulated the duration of ID using this inducible model to examine long-term effects of early ID on Morris water maze learning, CA1 apical dendrite structure, and defining factors of critical periods including parvalbmin (PV) expression, perineuronal nets (PNN), and brain-derived neurotrophic factor (BDNF) expression. Ongoing ID impaired spatial memory and resulted in disorganized apical dendrite structure accompanied by altered PV and PNN expression and reduced BDNF levels. Iron repletion at P21, near the end of hippocampal dendritogenesis, restored spatial memory, dendrite structure, and critical period markers in adult mice. However, mice that remained hippocampally iron deficient until P42 continued to have spatial memory deficits, impaired CA1 apical dendrite structure, and persistent alterations in PV and PNN expression and reduced BDNF despite iron repletion. Together, these findings demonstrate that hippocampal iron availability is necessary between P21 and P42 for development of normal spatial learning and memory, and that these effects may reflect disruption of critical period closure by early life ID.     

Region-specific regulation of glucocorticoid receptor/HSP90 expression and interaction in brain

The hippocampal glucocorticoid receptor (GR) is involved in negative feedback regulation of the hypothalamo-pituitary-adrenal axis and is believed to transduce the deleterious effects of glucocorticoids in depression and age-related memory loss. Regulation and intracellular trafficking of the GR are critical determinants of GR action in both health and disease. Here, we show dynamic regulation of GR and its interaction with its principal intracellular chaperone, heat-shock protein (HSP) 90, across the circadian cycle. Our initial experiments indicate that cytosolic hippocampal GR protein is elevated in the evening (PM), whereas nuclear GR and cytosolic HSP90, HSP70 and heat-shock cognate 70 (HSC70), are unchanged. In contrast, there are no changes in examined proteins in the hypothalamus. Immunoprecipitation experiments reveal increased GR-HSP90 associations in the hippocampus in the PM, whereas binding in the hypothalamus is decreased in the PM. Given that GR requires HSP90 for ligand binding, the data suggest that circadian GR signaling capacity is regulated in a region-specific pattern.     

Radiation-induced hyposuppression of the hypothalamic-pituitary-adrenal axis is associated with alterations of hippocampal corticosteroid receptor expression

Therapeutic brain irradiation in children can cause a progressive decline in cognitive functions through a diminished capability to learn and memorize. Because of the known involvement of the hippocampus in memory consolidation, this study was aimed at examining the late effects of gamma radiation on hypothalamic-pituitary-adrenal (HPA) axis activity and hippocampal corticosteroid receptor expression in an animal model of cranial radiotherapy. In the late-response phase, the basal and stress-induced corticosterone levels were not affected by radiation, but the suppression of glucocorticoid negative feedback by dexamethasone was attenuated in irradiated rats. Western blot analyses showed that exposure to radiation led to a decrease of cytosolic glucocorticoid receptor (GR) levels and a concomitant elevation of mineralocorticoid receptor (MR). The results obtained were complemented by those of RT-PCR, since the ratio of GR/MR mRNA was also decreased after radiation exposure. Dexamethasone appeared to be much less effective in shifting GR to the nuclear compartment in irradiated rats than in sham-irradiated animals. However, the expression of chaperones that aid GR intracellular trafficking, Hsp90 and Hsp70, remained unaffected. In conclusion, our data suggest that the hallmark of the late response to gamma radiation is a hyposuppressive state of the HPA axis that is associated with a decrease in both the GR/MR ratio and the nuclear accumulation of dexamethasone-activated GR in the hippocampus.     

Regulation of AMPA receptor function by the human memory-associated gene KIBRA

KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with?α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.     

Overexpression of Human Apolipoprotein A-I Preserves Cognitive Function and Attenuates Neuroinflammation and Cerebral Amyloid Angiopathy in a Mouse Model of Alzheimer Disease

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-β (Aβ) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-β precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aβ in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aβ-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.