Ago2 and Dicer1 are involved in METH-induced locomotor sensitization in mice via biogenesis of miRNA.

microRNA (miRNA) play important roles in drug addiction and act as a post-transcriptional regulator of gene expression. We previously reported extensive downregulation of miRNAs in the nucleus accumbens (NAc) of methamphetamine (METH)-sensitized mice. However, the regulatory mechanism of this METH-induced downregulation of miRNAs has yet to be elucidated. Thus, we examined METH-induced changes in the expression of miRNAs and their precursors, as well as the expression levels of mRNA and the proteins involved in miRNA biogenesis such as Dicer1 and Ago2, in the nucleus accumbens of METH-induced locomotor sensitized mice. miRNAs and Ago2 were significantly downregulated, while the expression of miRNA precursors remained unchanged or upregulated, which suggests that the downregulation of miRNAs was likely due to a reduction in Ago2-mediated splicing but unlikely to be regulated at the transcription level. Interestingly, the expression level of Dicer1, which is a potential target of METH-induced decreased miRNAs, such as miR-124, miR-212 and miR-29b, was significantly increased. In conclusion, this study indicates that miRNA biogenesis (such as Ago2 and Dicer1) and their miRNA products may have a role in the development of METH addiction.

PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction.

Coiled-coil-helix-coiled-coil-helix domain containing protein 2 (CHCHD2) mutations were linked with autosomal dominant Parkinson's disease (PD) and recently, Alzheimer's disease/frontotemporal dementia. In the current study, we generated isogenic human embryonic stem cell (hESC) lines harboring PD-associated CHCHD2 mutation R145Q or Q126X via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) method, aiming to unravel pathophysiologic mechanism and seek potential intervention strategy against CHCHD2 mutant-caused defects. By engaging super-resolution microscopy, we identified a physical proximity and similar distribution pattern of CHCHD2 along mitochondria with mitochondrial contact site and cristae organizing system (MICOS), a large protein complex maintaining mitochondria cristae. Isogenic hESCs and differentiated neural progenitor cells (NPCs) harboring CHCHD2 R145Q or Q126X mutation showed impaired mitochondria function, reduced CHCHD2 and MICOS components and exhibited nearly hollow mitochondria with reduced cristae. Furthermore, PD-linked CHCHD2 mutations lost their interaction with coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10), while transient knockdown of either CHCHD2 or CHCHD10 reduced MICOS and mitochondria cristae. Importantly, a specific mitochondria-targeted peptide, Elamipretide/MTP-131, now tested in phase 3 clinical trials for mitochondrial diseases, was found to enhance CHCHD2 with MICOS and mitochondria oxidative phosphorylation enzymes in isogenic NPCs harboring heterozygous R145Q, suggesting that Elamipretide is able to attenuate CHCHD2 R145Q-induced mitochondria dysfunction. Taken together, our results suggested CHCHD2-CHCHD10 complex may be a novel therapeutic target for PD and related neurodegenerative disorders, and Elamipretide may benefit CHCHD2 mutation-linked PD.

Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484.

Reorganization of Basolateral Amygdala-Subiculum Circuitry in Mouse Epilepsy Model.

In this study, we investigated the reorganized basolateral amygdala (BLA)-subiculum pathway in a status epilepticus (SE) mouse model with epileptic episodes induced by pilocarpine. We have previously observed a dramatic loss of neurons in the CA1-3 fields of the hippocampus in epileptic mice. Herein, we observed a 43-57% reduction in the number of neurons in the BLA of epileptic mice. However, injection of an anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) into the BLA indicated 25.63% increase in the number of PHA-L-immunopositive terminal-like structures in the ventral subiculum (v-Sub) of epileptic mice as compared to control mice. These data suggest that the projections from the basal nucleus at BLA to the vSub in epileptic mice are resistant to epilepsy-induced damage. Consequently, these epileptic mice exhibit partially impairment but not total loss of context-dependent fear memory. Epileptic mice also show increased c-Fos expression in the BLA and vSub when subjected to contextual memory test, suggesting the participation of these two brain areas in foot shock-dependent fear conditioning. These results indicate the presence of functional neural connections between the BLA-vSub regions that participate in learning and memory in epileptic mice.

Metabotropic glutamate receptor 5 (mGluR5) regulates proliferation and differentiation of neuronal progenitors in the developmental hippocampus.

Metabotropic glutamate receptor 5 (mGluR5) is involved in neural stem cell self-renewal, proliferation, differentiation and survival. In this study, we aimed to further determine the role of mGluR5 in the development of hippocampus using mGluR5 deficit (mGluR5(-/-)) and wild type (mGluR5(+/+)) mice at different developmental ages. We showed that the number of BrdU, NeuroD and DCX immunopositive cells was reduced significantly in mGluR5(-/-) than in mGluR5(+/+) mice from postnatal 7 days (P7) to P28, but not at P60. The length and intensity of DCX immunopositive apical dendrites in the dentate gyrus of mGluR5(-/-) mice were much shorter and lower than in mGluR5(+/+) mice respectively at P14, P21 and P28. NeuN immunostaining indicated an accelerated maturation of hippocampal neurons in mGluR5(-/-) mice. When mGluR5(+/+) mice were treated with 2-methyl-6-(phenylethynyl) pyridine (MPEP), a selective antagonist of mGluR5, decreased proliferation of progenitor cells was observed in the hippocampus at early postnatal developmental stages. At P14, there were more BrdU(+) cells in the stratum granulosum and subgranular layer of the dentate gyrus in mGluR5(+/+) than in mGluR5(-/-) mice, but the percentage of BrdU(+)+NeuroD(+)/BrdU(+) in the dentate gyrus did not change significantly between the two genotypes of mice. Western Blot study suggested that programmed neuronal death was p53-dependent apoptosis in the developmental hippocampus in mGluR5(+/+) mice.

Association of functional polymorphisms of the XRCC4 gene with the risk of breast cancer: a meta-analysis.

OBJECTIVE: X-ray cross-complementing group 4 (XRCC4) is a major repair gene for DNA double-strand breaks (DSB) in the non-homologous end-joining (NHEJ) pathway. Several potentially functional polymorphisms of the XRCC4 gene have been implicated in breast cancer risk, but individually published studies showed inconclusive results. The aim of this meta-analysis was to investigate the association between XRCC4 polymorphisms and the risk of breast cancer. METHODS: The MEDLINE, EMBASE, Web of science and CBM databases were searched for all relevant articles published up to June 20, 2012. Potential associations were assessed with comparisons of the total mutation rate (TMR), complete mutation rate (CMR) and partial mutation rate (PMR) in cases and controls. Statistical analyses were performed using RevMan 5.1.6 and STATA 12.0 software. RESULTS: Five studies were included with a total of 5,165 breast cancer cases and 4,839 healthy controls. Meta-analysis results showed that mutations of rs2075686 (C>T) and rs6869366 (G>T) in the XRCC4 gene were associated with increased risk of breast cancer, while rs2075685 (G>T) and rs10057194 (A>G) might decrease the risk of breast cancer. However, rs1805377 (A>G), rs1056503 (G>T), rs28360317 (ins>del) and rs3734091 (A>G) polymorphisms of XRCC4 gene did not appear to have an influence on breast cancer susceptibility. CONCLUSION: Results from the current meta-analysis suggest that the rs2075685 (G>T) and rs6869366 (G>T) polymorphisms of the XRCC4 gene might increase the risk of breast cancer, whereas rs2075685 (G>T) and rs10057194 (A>G) might be protective factors.

Corticotropin releasing factor (CRF) in the hippocampus of the mouse pilocarpine model of status epilepticus.

We investigated the cellular localization and progressive changes of corticotropin releasing factor (CRF) in the mouse hippocampus, during and after pilocarpine induced status epilepticus (PISE) and subsequent epileptogenesis. We found that CRF gene expression was up-regulated significantly at 2h during and 1d after PISE in comparison to control mice. Immunohistochemical analysis showed that the number of CRF and Fos immunoreactive cells was increased significantly in the strata oriens and pyramidale of CA1 area and in the stratum pyramidale of CA3 area at 2h during and 1d after PISE. CRF was induced in calbindin (CB) or calretinin (CR) immunoreactive interneurons in stratum oriens at 2h during PISE. It suggests that induced CRF may be related to the over excitation of hippocampal neurons and occurrence of status epilepticus. It may also cause excitoneurotoxicity and delayed loss of CA3 and CA1 pyramidal neurons, leading to the onset of epilepsy.

Morpho-physiologic characteristics of dorsal subicular network in mice after pilocarpine-induced status epilepticus.

The goal of this study was to examine the morpho-physiologic changes in the dorsal subiculum network in the mouse model of temporal lobe epilepsy using extracellular recording, juxtacellular and immunofluorescence double labeling, and anterograde tracing methods. A significant loss of total dorsal subicular neurons, particularly calbindin, parvalbumin (PV) and immunopositive interneurons, was found at 2 months after pilocarpine-induced status epilepticus (SE). However, the sprouting of axons from lateral entorhinal cortex (LEnt) was observed to contact with surviving subicular neurons. These neurons had two predominant discharge patterns: bursting and fast irregular discharges. The bursting neurons were mainly pyramidal cells, and their dendritic spine density and bursting discharge rates were increased significantly in SE mice compared with the control group. Fast irregular discharge neurons were PV-immunopositive interneurons and had less dendritic spines in SE mice when compared with the control mice. When LEnt was stimulated, bursting and fast irregular discharge neurons had much shorter latency and stronger excitatory response in SE mice compared with the control group. Our results illustrate that morpho-physiologic changes in the dorsal subiculum could be part of a multilevel pathologic network that occurs simultaneously in many brain areas to contribute to the generation of epileptiform activity.

Cytoarchitectonics and afferent/efferent reorganization of neurons in layers II and III of the lateral entorhinal cortex in the mouse pilocarpine model of temporal lobe epilepsy.

With the mouse pilocarpine model of temporal lobe epilepsy (TLE), we showed a progressive loss of both principal cells and calbindin (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunopositive interneurons in layers II-III of lateral entorhinal cortex (LEnt) from 2 months to 1 year after pilocarpine-induced status epilepticus (PISE). In the efferent pathway of LEnt, more Phaseolus vulgaris leucoagglutinin (PHA-L)-labelled en passant and terminal boutons with larger diameters were shown in the hippocampus and subiculum; in the prefrontal, piriform, and perirhinal cortices; and in the amygdaloid complex in experimental mice at the two time points compared with the control after iontophoretical injection of an anterograde tracer PHA-L into the LEnt. Furthermore, the numbers of CB- or CR-immunopositive neurons contacted by PHA-L-labelled en passant and terminal boutons decreased in most of these areas at 2 months or 1 year after PISE. In the afferent pathway of LEnt, the numbers of retrogradely labelled neurons were reduced significantly in the ipsilateral piriform cortex and endopiriform nucleus at 2 months and 1 year and in the reuniens thalamic nucleus only at 1 year after injection of a retrograde tracer cholera toxin B subunit (CTB) into the LEnt. The percentages of the number of CTB and CB or CR double-labelled neurons of all the retrogradely labelled neurons were also decreased in the reunions thalamic nucleus at 1 year after PISE. It is concluded that both cytoarchitectonic change and reorganization of afferent and efferent pathways in LEnt may be involved in the occurrence of TLE.

[Protective effect of adeno-associated viral vector-mediated expression of human brain-derived neurotrophic factor in rat neurons against beta-amyloid-induced Alzheimer's disease in vitro].

OBJECTIVE: To achieve expression of human brain-derived neurotrophic factor (hBDNF) mediated by recombinant adeno-associated virus (rAAV) and explore the mechanism of its neuroprotective effects in rat neurons against beta-amyloid-induced Alzheimer's disease. METHODS: Using molecular cloning technique, rAAV vector containing hBDNF gene (AAV-hBDNF) was constructed to transfect SD rat hippocampal neurons exposed to beta-amyloid treatment. The changes in cell apoptosis were observed by MTT assay and flow cytometry, and the expression of hBDNF and Bcl-2 protein were determined by immunocytochemical staining. Laser scanning confocal microscopy (LSCM) was used to observe the changes of [Ca(2+)](i). RESULTS: The cultured rat hippocampal neurons were effectively transfected with AAV-hBDNF and expression of BDNF protein was obviously increased. hBNDF expression showed significant protective effects against beta-amyloid-induced neuronal damage, and the expression of Bcl-2 protein was increased significantly and the balance of [Ca(2+)](i) was maintained in BDNF-treated cells with beta-amyloid exposure. CONCLUSION: hBDNF expression can effectively protect cultured rat hippocampal cells from beta-amyloid-induced apoptosis through inhibiting the intracellular calcium overload and increasing the expression of Bcl-2 protein.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy.

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

Reorganization of CA3 area of the mouse hippocampus after pilocarpine induced temporal lobe epilepsy with special reference to the CA3-septum pathway.

We showed that when CA3 pyramidal neurons in the caudal 80% of the dorsal hippocampus had almost disappeared completely, the efferent pathway of CA3 was rarely detectable. We used the mouse pilocarpine model of temporal lobe epilepsy (TLE), and injected iontophoretically the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. In the afferent pathway, the number of neurons projecting to CA3 from medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei increased significantly. In the hippocampus, where CA3 pyramidal neurons were partially lost, calbindin, calretinin, parvalbumin immunopositive back-projection neurons from CA1-CA3 area were observed. Sprouting of Schaffer collaterals with increased number of large boutons in both sides of CA1 area, particularly in the stratum pyramidale, was found. When CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus have almost disappeared completely, surviving CA3 neurons in the rostral 20% of the dorsal hippocampus may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons or to dorsal part of the lateral septum. We concluded that reorganization of CA3 area with its downstream or upstream nuclei may be involved in the occurrence of epilepsy.