BACE1 inhibition more effectively suppresses initiationthan progression of β-amyloid pathology

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Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

A tool for examining the role of the zinc finger myelin transcription factor 1 (Myt1) in neural development: Myt1 knock-in mice

The Myt1 family of transcription factors is unique among the many classes of zinc finger proteins in how the zinc-stabilized fingers contact the DNA helix. To examine the function of Myt1 in the developing nervous system, we generated mice in which Myt1 expression was replaced by an enhanced Green Fluorescent Protein fused to a Codon-improved Cre recombinase as a protein reporter. Myt1 knock-in mice die at birth, apparently due to improper innervation of their lungs. Elimination of Myt1 did not significantly affect the number or distribution of neural precursor cells that normally express Myt1 in the embryonic spinal cord. Nor was the general pattern of differentiated neurons altered in the embryonic spinal cord. The Myt1 knock-in mice should provide an important tool for identifying the in vivo targets of Myt1 action and unraveling the role of this structurally distinct zinc finger protein in neural development

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting

High LRRK2 Levels Fail to Induce or Exacerbate Neuronal Alpha-Synucleinopathy in Mouse Brain

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson’s disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy

Ionotropic Glutamate Receptor AMPA 1 Is Associated with Ovulation Rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy

Excess α-synuclein worsens disease in mice lacking ubiquitin carboxy-terminal hydrolase L1

Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species

Surfactant secretion in LRRK2 knock-out rats : changes in lamellar body morphology and rate of exocytosis

Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca2+ release upon ATP treatment and significant triggering of LB exocytosis. These findings are in line with the strong Ca2+-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca2+ signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP