Kinesin-mediated organelle translocation revealed by specific cellular manipulations.

The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)- positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin- membrane interactions may be of key importance for determining the intracellular distribution of selected organelles

Structure–activity relationship study of 2,4-diaminothiazoles as Cdk5/p25 kinase inhibitors

Cdk5/p25 has emerged as a principle therapeutic target for numerous acute and chronic neurodegenerative diseases, including Alzheimer’s disease. A structure–activity relationship study of 2,4-diaminothiazole inhibitors revealed that increased Cdk5/p25 inhibitory activity could be accomplished by incorporating pyridines on the 2-amino group and addition of substituents to the 2- or 3-position of the phenyl ketone moiety. Interpretation of the SAR results for many of the analogs was aided through in silico docking with Cdk5/p25 and calculating protein hydrations sites using WaterMap. Finally, improved in vitro mouse microsomal stability was also achieved

A path toward understanding neurodegeneration

The specter of neurodegenerative disease, particularly Alzheimer's disease, haunts the developed world and exacts a poorly documented toll on underdeveloped countries. With so little progress made toward finding a cure—or, better, a prevention—it is time to rethink the path to progress. This requires a change in perspective on the type of research that will make a difference. The lesson learned from cancer research is that a new commitment means rethinking the fundamental approach to the disease. Cancer research moved from taking potshots with, usually, cytotoxic drugs to a bottom-up, mechanism-based approach in which newly acquired genetic knowledge played the largest role. Today, that effort has produced a platform of knowledge from which academia and industry are drawing. For neurodegenerative disease, the genetic approach remains valid but the problem must concurrently be approached from a complementary, robust cell biological perspective, focusing on the cellular cascade of events that lead to neuronal cell death

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine

MicroRNA profiling of the murine hematopoietic system

BACKGROUND: MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. RESULTS: We used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions. CONCLUSION: This is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity

Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models

Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3?. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3? in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3? to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting