Quantitative analysis of the roles of IRM cell adhesion molecules in column formation in the fly brain.

The Drosophila visual center shows columnar structures, basic structural and functional units of the brain, that are shared with the mammalian cerebral cortex. Visual information received in the ommatidia in the compound eye is transmitted to the columns in the brain. However, the developmental mechanisms of column formation are largely unknown. The Irre Cell Recognition Module (IRM) proteins are a family of immunoglobulin cell adhesion molecules. The four Drosophila IRM proteins are localized to the developing columns, the structure of which is affected in IRM mutants, suggesting that IRM proteins are essential for column formation. Since IRM proteins are cell adhesion molecules, they may regulate cell adhesion between columnar neurons. To test this possibility, we specifically knocked down IRM genes in columnar neurons and examined the defects in column formation. We developed a system that automatically extracts the individual column images and quantifies the column shape. Using this system, we demonstrated that IRM genes play critical roles in regulating column shape in a core columnar neuron, Mi1. We also show that their expression in the other columnar neurons, Mi4 and T4/5, is essential, suggesting that the interactions between IRM proteins and multiple neurons shape the columns in the fly brain.

Intracellular trafficking of Notch orchestrates temporal dynamics of Notch activity in the fly brain.

While Delta non-autonomously activates Notch in neighboring cells, it autonomously inactivates Notch through cis-inhibition, the molecular mechanism and biological roles of which remain elusive. The wave of differentiation in the Drosophila brain, the 'proneural wave', is an excellent model for studying Notch signaling in vivo. Here, we show that strong nonlinearity in cis-inhibition reproduces the second peak of Notch activity behind the proneural wave in silico. Based on this, we demonstrate that Delta expression induces a quick degradation of Notch in late endosomes and the formation of the twin peaks of Notch activity in vivo. Indeed, the amount of Notch is upregulated and the twin peaks are fused forming a single peak when the function of Delta or late endosomes is compromised. Additionally, we show that the second Notch peak behind the wavefront controls neurogenesis. Thus, intracellular trafficking of Notch orchestrates the temporal dynamics of Notch activity and the temporal patterning of neurogenesis.

DWnt4 and DWnt10 Regulate Morphogenesis and Arrangement of Columnar Units via Fz2/PCP Signaling in the Drosophila Brain.

Columns are structural and functional units of the brain. However, the mechanism of column formation remains unclear. The medulla of the fly visual center shares features with the mammalian cerebral cortex, such as columnar and layered structures, and provides a good opportunity to study the mechanisms of column formation. Column formation is initiated by three core neurons in the medulla, namely, Mi1, R8, and R7. The proper orientation of neurons is required for the orientation and arrangement of multiple columns. Their orientations may be under the control of planar cell polarity (PCP) signaling, because it is known to regulate the orientation of cells in two-dimensional tissue structures. In this study, we demonstrate that the ligands DWnt4 and DWnt10 expressed specifically in the ventral medulla and dorsal medulla, respectively, globally regulate the columnar arrangement and orientation of Mi1 and R8 terminals through Fz2/PCP signaling in a three-dimensional space.

A continuation method for spatially discretized models with nonlocal interactions conserving size and shape of cells and lattices.

In this paper, we introduce a continuation method for the spatially discretized models, while conserving the size and shape of the cells and lattices. This proposed method is realized using the shift operators and nonlocal operators of convolution types. Through this method and using the shift operator, the nonlinear spatially discretized model on the uniform and nonuniform lattices can be systematically converted into a spatially continuous model; this renders both models point-wisely equivalent. Moreover, by the convolution with suitable kernels, we mollify the shift operator and approximate the spatially discretized models using the nonlocal evolution equations, rendering suitable for the application in both experimental and mathematical analyses. We also demonstrate that this approximation is supported by the singular limit analysis, and that the information of the lattice and cells is expressed in the shift and nonlocal operators. The continuous models designed using our method can successfully replicate the patterns corresponding to those of the original spatially discretized models obtained from the numerical simulations. Furthermore, from the observations of the isotropy of the Delta-Notch signaling system in a developing real fly brain, we propose a radially symmetric kernel for averaging the cell shape using our continuation method. We also apply our method for cell division and proliferation to spatially discretized models of the differentiation wave and describe the discrete models on the sphere surface. Finally, we demonstrate an application of our method in the linear stability analysis of the planar cell polarity model.

Dscam1 establishes the columnar units through lineage-dependent repulsion between sister neurons in the fly brain.

The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. Here, we show that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.

Cullin-4B E3 ubiquitin ligase mediates Apaf-1 ubiquitination to regulate caspase-9 activity.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

Odor preference and olfactory memory are impaired in Olfaxin-deficient mice.

Olfaxin, which is a BNIP2 and Cdc42GAP homology (BCH) domain-containing protein, is predominantly expressed in mitral and tufted (M/T) cells in the olfactory bulb (OB). Olfaxin and Caytaxin, which share 56.3% amino acid identity, are similar in their glutamatergic terminal localization, kidney-type glutaminase (KGA) interaction, and caspase-3 substrate. Although the deletion of Caytaxin protein causes human Cayman ataxia and ataxia in the mutant mouse, the function of Olfaxin is largely unknown. In this study, we generated Prune2 gene mutant mice (Prune2Ex16-/-; knock out [KO] mice) using the CRISPR/Cas9 system, during which the exon 16 containing start codon of Olfaxin mRNA was deleted. Exon 16 has 80 nucleotides and is contained in four of five Prune2 isoforms, including PRUNE2, BMCC1, BNIPXL, and Olfaxin/BMCC1s. The levels of Olfaxin mRNA and Olfaxin protein in the OB and piriform cortex of KO mice significantly decreased. Although Prune2 mRNA also significantly decreased in the spinal cord, the gross anatomy of the spinal cord and dorsal root ganglion (DRG) was intact. Further, disturbance of the sensory and motor system was not observed in KO mice. Therefore, in the current study, we examined the role of Olfaxin in the olfactory system where PRUNE2, BMCC1, and BNIPXL are scarcely expressed. Odor preference was impaired in KO mice using opposite-sex urinary scents as well as a non-social odor stimulus (almond). Results of the odor-aversion test demonstrated that odor-associative learning was disrupted in KO mice. Moreover, the NMDAR2A/NMDAR2B subunits switch in the piriform cortex was not observed in KO mice. These results indicated that Olfaxin may play a critical role in odor preference and olfactory memory.

Improvement of memory recall by quercetin in rodent contextual fear conditioning and human early-stage Alzheimer's disease patients.

Patients with Alzheimer's disease (AD) experience a wide array of cognitive deficits, which typically include the impairment of explicit memory. In previous studies, the authors reported that a flavonoid, quercetin, reduces the expression of ATF4 and delays memory deterioration in an early-stage AD mouse model. In the present study, the effects of long-term quercetin intake on memory recall were assessed using contextual fear conditioning in aged wild-type mice. In addition, the present study examined whether memory recall was affected by the intake of quercetin-rich onion (a new cultivar of hybrid onion 'Quergold') powder in early-stage AD patients. In-vivo analysis indicated that memory recall was enhanced in aged mice fed a quercetin-containing diet. Memory recall in early-stage AD patients, determined using the Revised Hasegawa Dementia Scale, was significantly improved by the intake of quercetin-rich onion (Quergold) powder for 4 weeks compared with the intake of control onion ('Mashiro' white onion) powder. These results indicate that quercetin might influence memory recall.

Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion.

The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax.

CED-4 is an mRNA-binding protein that delivers ced-3 mRNA to ribosomes.

Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.

Quercetin reduces eIF2α phosphorylation by GADD34 induction.

The production of amyloid ß (Aß) in the brain from Aß precursor protein (APP) through γ-secretase is important for the pathogenesis of Alzheimer's disease (AD). Our previous studies have demonstrated that autophagy impairment and endoplasmic reticulum stress increase presenilin 1 expression and enhance γ-secretase activity through the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and the translation of activating transcription factor 4 (ATF4). However, the inhibitory molecules for γ-secretase are largely unknown. Here, we demonstrate that the levels of ATF4 expression are increased in the brain of APP23 AD model mice; furthermore, these levels enhanced in the brain of APP23 mice crossed with obese and diabetic db/db (Lepr(db/db)) mice. A polyhydroxylated flavonoid, quercetin, suppressed presenilin 1 expression and Aß secretion in autophagy-impaired cells by the induction of growth arrest and DNA damaged-inducible gene (GADD) 34, which mediates eIF2α dephosphorylation, leading to decreased ATF4 expression. GADD34 induction was observed in the brain of wild-type mice, and APP23 mice fed quercetin in their diet. After the long-term feeding of quercetin, deterioration in memory assessed by freezing behavior was delayed in APP23 mice. These results indicate that quercetin may reduce eIF2α phosphorylation and ATF4 expression through GADD34 induction in the brain, leading to the improvement of memory in aged mice and the delay of deterioration in memory at the early stage of AD in AD model mice.

Polyglutamine expansion disturbs the endoplasmic reticulum formation, leading to caspase-7 activation through Bax.

The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntington's disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.

Olfaxin as a novel Prune2 isoform predominantly expressed in olfactory system.

Prune homolog 2 (Drosophila) (PRUNE2) encodes a BCH motif-containing protein that shares homology with the Cayman ataxia-related protein Caytaxin. Caytaxin is a substrate of caspase-3 and is specifically expressed at the presynapse of vesicular-type glutamate transporter (VGLUT)-positive neurons, where it plays a role in glutamate neurotransmission primarily in the cerebellum and hippocampus. Here, we showed that a novel Prune2 isoform contains a BCH motif and localizes predominantly to the synaptic cytosol, similar to Caytaxin. However, the isoform is expressed predominantly in the olfactory bulb and layer Ia of the piriform cortex, where Caytaxin is scarcely expressed. The isoform expression is upregulated during development, similar to that in the presynaptic-localizing proteins Synapsin I and Bassoon. Prune2 and its previously identified isoforms have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein, and an antagonist of cellular transformation. In addition, a novel isoform may develop new roles for Prune2 at the synapse in olfactory systems.

Endoplasmic reticulum stress enhances γ-secretase activity.

The endoplasmic reticulum (ER) copes with unfolded proteins in the lumen (ER stress) by activating three distinct intracellular signaling pathways of unfolded protein response (UPR). ER stress contributes to the pathogenesis of obesity and diabetes, which are risk factors for Alzheimer's disease (AD) that accelerate the pathogenesis of AD. However, whether ER stress is involved in the development of AD remains unclear. In this study, we demonstrate that ER stress induces presenilin-1 expression through activating transcription factor 4 (ATF4), resulting in increased amyloid-ß (Aß) secretion by γ-secretase activity, which is suppressed by quercetin by modifying UPR signaling. This result suggests that ER stress may be stimulated in obesity and type 2 diabetes, thereby enhancing γ-secretase activity that is the underlying molecular mechanism affecting the pathogenesis of AD.

The expression and localization of Prune2 mRNA in the central nervous system.

A family of Bcl-2/adenovirus E1B 19kDa-interacting proteins (BNIPs) plays critical roles in several cellular processes such as cellular transformation, apoptosis, neuronal differentiation, and synaptic function, which are mediated by the BNIP2 and Cdc42GAP homology (BCH) domain. Prune homolog 2 (Drosophila) (PRUNE2) and its isoforms -C9orf65, BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1), and BNIP2 Extra Long (BNIPXL) - have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively. However, precise localization of PRUNE2 in the brain remains unclear. Here, we identified the distribution of Prune2 mRNA in the adult mouse brain. Prune2 mRNA is predominantly expressed in the neurons of the cranial nerve motor nuclei and the motor neurons of the spinal cord. The expression in the dorsal root ganglia (DRG) is consistent with the previously described reports. In addition, we observed the expression in another sensory neuron in the mesencephalic trigeminal nucleus. These results suggest that Prune2 may be functional in these restricted brain regions.