Role of presenilin-1 in cortical lamination and survival of Cajal-Retzius neurons

AbstractPresenilin-1 (PS1), the major causative gene of familial Alzheimer disease, regulates neuronal differentiation and Notch signaling during early neural development. To investigate the role of PS1 in neuronal migration and cortical lamination of the postnatal brain, we circumvented the perinatal lethality of PS1-null mice by generating a conditional knockout (cKO) mouse in which PS1 inactivation is restricted to neural progenitor cells (NPCs) and NPC-derived neurons and glia. BrdU birthdating analysis revealed that many late-born neurons fail to migrate beyond the early-born neurons to arrive at their appropriate positions in the superficial layer, while the migration of the early-born neurons is largely normal. The migration defect of late-born neurons coincides with the progressive reduction of radial glia in PS1 cKO mice. In contrast to the premature loss of Cajal-Retzius (CR) neurons in PS1-null mice, generation and survival of CR neurons are unaffected in PS1 cKO mice. Furthermore, the number of proliferating meningeal cells, which have been shown to be important for the survival of CR neurons, is increased in PS1-null mice but not in PS1 cKO mice. These findings show a cell-autonomous role for PS1 in cortical lamination and radial glial development, and a non-cell-autonomous role for PS1 in CR neuron survival

CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research

CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin--Brief Report

OBJECTIVE: Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. APPROACH AND RESULTS: 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3xFLAG or 3xHA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a protein product of approximately 150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. CONCLUSIONS: This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research

Presenilin Controls CBP Levels in the Adult Drosophila Central Nervous System

Background: Dominant mutations in both human Presenilin (Psn) genes have been correlated with the formation of amyloid plaques and development of familial early-onset Alzheimer’s disease (AD). However, a definitive mechanism whereby plaque formation causes the pathology of familial and sporadic forms of AD has remained elusive. Recent discoveries of several substrates for Psn protease activity have sparked alternative hypotheses for the pathophysiology underlying AD. CBP (CREB-binding protein) is a haplo-insufficient transcriptional co-activator with histone acetly-transferase (HAT) activity that has been proposed to be a downstream target of Psn signaling. Individuals with altered CBP have cognitive deficits that have been linked to several neurological disorders. Methodology/Principal Findings: Using a transgenic RNA-interference strategy to selectively silence CBP, Psn, and Notch in adult Drosophila, we provide evidence for the first time that Psn is required for normal CBP levels and for maintaining specific global acetylations at lysine 8 of histone 4 (H4K8ac) in the central nervous system (CNS). In addition, flies conditionally compromised for the adult-expression of CBP display an altered geotaxis behavior that may reflect a neurological defect. Conclusions/Significance: Our data support a model in which Psn regulates CBP levels in the adult fly brain in a manner that is independent of Notch signaling. Although we do not understand the molecular mechanism underlying th

Presenilin/γ-Secretase Regulates Neurexin Processing at Synapses

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing β-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect β-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS−/− cells rescues the processing of β-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD

Conditional Deletion of PDK1 in the Forebrain Causes Neuron Loss and Increased Apoptosis during Cortical Development

Decreased expression but increased activity of PDK1 has been observed in neurodegenerative disease. To study in vivo function of PDK1 in neuron survival during cortical development, we generate forebrain-specific PDK1 conditional knockout (cKO) mice. We demonstrate that PDK1 cKO mice display striking neuron loss and increased apoptosis. We report that PDK1 cKO mice exhibit deficits on several behavioral tasks. Moreover, PDK1 cKO mice show decreased activities for Akt and mTOR. These results highlight an essential role of endogenous PDK1 in the maintenance of neuronal survival during cortical development

Burden of high fracture probability worldwide: secular increases 2010-2040

SummaryThe number of individuals aged 50 years or more at high risk of osteoporotic fracture worldwide in 2010 was estimated at 158 million and is set to double by 2040.IntroductionThe aim of this study was to quantify the number of individuals worldwide aged 50 years or more at high risk of osteoporotic fracture in 2010 and 2040.MethodsA threshold of high fracture probability was set at the age-specific 10-year probability of a major fracture (clinical vertebral, forearm, humeral or hip fracture) which was equivalent to that of a woman with a BMI of 24 kg/m2 and a prior fragility fracture but no other clinical risk factors. The prevalence of high risk was determined worldwide and by continent using all available country-specific FRAX models and applied the population demography for each country.ResultsTwenty-one million men and 137 million women had a fracture probability at or above the threshold in the world for the year 2010. The greatest number of men and women at high risk were from Asia (55 %). Worldwide, the number of high-risk individuals is expected to double over the next 40 years.ConclusionWe conclude that individuals with high probability of osteoporotic fractures comprise a very significant disease burden to society, particularly in Asia, and that this burden is set to increase markedly in the future. These analyses provide a platform for the evaluation of risk assessment and intervention strategies

More dramatic increases in apoptosis in the lateral cortex of <i>Psen</i> cDKO mice.

<p>Black vertical lines superimposed on an image of a Nissl-stained coronal brain section depict the relative positions of sagittal brain sections spaced 300 µm apart that were analyzed for either Fluoro-Jade B+ or TUNEL+ cells. The average (± s.e.m.) of the three medial-most sections (M) was compared to the average of the three lateral-most sections (L). Scale bar: 1 mm. For each timepoint, 4 mice per genotype (10 sections per mouse) were analyzed. (<i>A, B</i>) Quantification of numbers of Fluoro-Jade B+ cells in sagittal brain sections from mice at 2 months (<i>A</i>) or 4 months (<i>B</i>). At 2 months of age, increases in degenerating neurons are more pronounced in the lateral sections (control: 1.7±0.7; cDKO: 34.1±3.1; p<0.01) than in the medial sections (control: 2.5±0.3; cDKO: 3.4±0.3; p>0.05). By 4 months of age, the number of degenerating neurons increases significantly in medial sections of cDKO mice, compared to controls (control: 2.6±0.3; cDKO: 14.3±1.1; p<0.01). Furthermore, the number of degenerating neurons in lateral sections of cDKO mice is much greater than in medial sections (control: 1.4±0.1 vs. <i>Psen</i> cDKO: 32.1±3.0; p<0.02). *, p<0.05; **, p<0.01; ***, p<0.001. (<i>C, D</i>) Quantification of numbers of TUNEL+ cells in sagittal brain sections from mice age 2 months (<i>C</i>) or 4 months (<i>D</i>). (<i>C</i>) No significant increase was observed in TUNEL+ cells in the <i>Psen</i> cDKO medial cortex at 2 months (control: 3.0±0.3 vs. <i>Psen</i> cDKO: 4.9±0.5; p = 0.05), in contrast to a large increase in lateral <i>Psen</i> cDKO cortex (control: 3.4±0.4 vs. <i>Psen</i> cDKO: 54.8±8.0; p<0.05). (<i>E</i>) At 4 months, lateral <i>Psen</i> cDKO cortex showed a significant increases in TUNEL+ cells (control: 2.3±0.5 vs. <i>Psen</i> cDKO: 32.9±4.1; p<0.05), while medial <i>Psen</i> cDKO cortex showed only a trend toward increased apoptosis (control: 1.3±0.3 vs. <i>Psen</i> cDKO: 11.9±2.3; p = 0.05).</p

Mitochondrial defects in the cerebral cortex of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) (<i>Top</i>) Electron micrographs of mitochondria from the neocortex at 2 months and 6 months of age. Red arrows point to mitochondria typical of those quantified in (<i>A–C</i>). Scale bar, 1 µm. (<i>Bottom</i>) Quantification of mitochondria per 100 µm² in neocortex. The density of <i>Psen</i> cDKO mitochondria was comparable to controls at 2 months (control: 62.5±4.1 vs. <i>Psen</i> cDKO: 68.4±4.1; p>0.05) but reduced by 6 months (control: 61.7±4.8 vs. <i>Psen</i> cDKO: 44.2±4.6; p<0.001), revealing age-dependent loss of mitochondria in <i>Psen</i> cDKO (n = 4 mice per genotype (2 mo) and 3 mice per genotype (6 mo)). (<i>B,C</i>) Analysis of size distribution of cortical mitochondria determined that <i>Psen</i> cDKO has no change in mitochondrial morphology at 2 months (<i>B</i>), but a sizeable reduction in small mitochondria by 6 months of age (<i>C</i>) (0.01–0.1 µm² bin: control, 40.0±4.4 vs. <i>Psen</i> cDKO, 28.6±4.3 (p<0.001); 0.11–0.2 µm² bin: control, 15.5±2.3 vs. <i>Psen</i> cDKO, 7.3±1.6 (p<0.00001)). (<i>D</i>) (<i>Top</i>) High-magnification electron micrographs of cortical mitochondria from mice at 6 months of age. Both control (<i>left</i>) and <i>Psen</i> cDKO samples (<i>middle</i>) have normal mitochondria with clearly visible cristae and outer membrane structures; however, <i>Psen</i> cDKO mice also have a slight increase in the number of swollen mitochondria (<i>right</i>). Scalebar, 0.4 µm (all 3 images). (<i>Bottom</i>) Quantification of large (area >0.3 µm²) mitochondria from mice age 2 months and 6 months. The percentage of large <i>Psen</i> cDKO cortical mitochondria relative to the total number was normal at 2 months of age (control: 0.028±0.017 vs. <i>Psen</i> cDKO: 0.024±0.009; p>0.05), but at 6 months of age, the percentage of large <i>Psen</i> cDKO mitochondria is twice the number observed in controls (control: 0.048±0.018 vs. <i>Psen</i> cDKO: 0.098±0.035; p<0.05), implicating a dysregulation of mitochondrial morphology in the absence of PS function. (n = 4 at 2 months and 3 at 6 months). All data are presented as the mean ± s.e.m.</p