Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.     

Activity-dependent release of phosphorylated human tau from Drosophila neurons in primary culture

Neuronal activity can enhance tau release and thus accelerate tauopathies. This activity-dependent tau release can be used to study the progression of tau pathology in Alzheimer''s disease (AD), as hyperphosphorylated tau is implicated in AD pathogenesis and related tauopathies. However, our understanding of the mechanisms that regulate activity-dependent tau release from neurons and the role that tau phosphorylation plays in modulating activity-dependent tau release is still rudimentary. In this study, Drosophila neurons in primary culture expressing human tau (hTau) were used to study activity-dependent tau release. We found that hTau release was markedly increased by 50 mM KCl treatment for 1 h. A similar level of release was observed using optogenetic techniques, where genetically targeted neurons were stimulated for 30 min using blue light (470 nm). Our results showed that activity-dependent release of phosphoresistant hTau^S11A was reduced when compared with wildtype hTau. In contrast, release of phosphomimetic hTau^E14 was increased upon activation. We found that released hTau was phosphorylated in its proline-rich and C-terminal domains using phosphorylation site-specific tau antibodies (e.g., AT8). Fold changes in detectable levels of total or phosphorylated hTau in cell lysates or following immunopurification from conditioned media were consistent with preferential release of phosphorylated hTau after light stimulation. This study establishes an excellent model to investigate the mechanism of activity-dependent hTau release and to better understand the role of phosphorylated tau release in the pathogenesis of AD since it relates to alterations in the early stage of neurodegeneration associated with increased neuronal activity.     

Endogenously expressed estrogen receptors mediate neuroprotection in hippocampal cells (HT22)

Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism.     

Changes in Ca, Cu, Fe, Mg, and Zn contents in mouse brain tissues after prolonged oral ingestion of brick tea liquor containing a high level of Al

A study was conducted to analyze the regional distribution of Ca, Cu, Fe, Mg, and Zn contents in brain tissues after animals were given liquor of brick tea that contained a high Al content. In 25 normal adult male mice given either water or 0.9% NaCl for 1 mo or 2 mo, the metal concentrations in the serum, liver, frontal cortex, hippocampus, cerebellum, and brain stem were comparable (p>0.05). When the drinking water was replaced by a 1% brick tea liquor, which contained a high Al content, serum Al concentration was increased significantly 1 mo after the onset of the experiment and remained high at the end of the second month. The level of Al was also elevated in both the cortex and hippocampus at 1 mo after replacing tea for drinking water. In addition to Al, there were a significant increase in hippocampal Zn and a decrease in Cu contents. There was no change in tissue Mg or Fe contents, but there was a significant increase in Ca content in every brain region studied. It was suggested that the increase in Ca might be the result of the effect of other components in tea. Unlike the brain, there was no change in the concentration of any of the metals, including Al, in the liver, which further demonstrated that the changes observed in the brain was specific. The results of the present study confirmed that Al, when given orally in the form of tea, could be absorbed into the bloodstream. The absorbed Al could accumulate in selected brain regions. The presence of Al might also change the tissue content of endogenous trace metals.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

Developmentally regulated expression of a cell surface protein,neuropilin, in the mouse nervous system

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons. © 1996 John Wiley & Sons, Inc.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.     

Functional morphology and neuronal innervation of the prothoracic defence gland in Timema

Timema is the most basal genus of Phasmatodea and the sister group to the remaining stick and leaf-insects (Euphasmatodea). An autapomorphy of all phasmids is the paired prothoracic exocrine defence glands. In this study, the anatomy and innervation of the defence glands in Timema petita and Timema chumash are described and compared with the data on Euphasmatodea. In all phasmids, the glands consist of a cuticular epithelium, a secretory epithelium and muscular fibres that compress the lumen. In Timematodea, the muscular part of the gland is less developed than in Euphasmatodea and the ejection of the defence secretion depends on the dorsal longitudinal neck muscles. On the neuroanatomical level, Timema petita and Timema chumash lack neurons that are involved in the independent contraction of the gland in euphasmids. In both studied species of Timema, neck muscles play an active role in the gland function which is not observed in any other phasmid. Considering the basal position of this genus, this supports the hypothesis that in euphasmids, the muscular part of the gland evolved from the dorsal longitudinal neck muscles. Additionally, the same nerves that innervate the dorsal longitudinal neck muscles in all Polyneoptera also innervate the defence glands in phasmids.     

Elevated Prolactin during Pregnancy Drives a Phenotypic Switch in Mouse Hypothalamic Dopaminergic Neurons

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