Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Translational challenges in advancing regenerative therapy for treating neurological disorders using nanotechnology.

The focus of regenerative therapies is to replace or enrich diseased or injured cells and tissue in an attempt to replenish the local environment and function, while slowing or halting further degeneration. Targeting neurological diseases specifically is difficult, due to the complex nature of the central nervous system, including the difficulty of bypassing the brain's natural defense systems. While cell-based regenerative therapies show promise in select tissues, preclinical and clinical studies have been largely unable to transfer these successes to the brain. Advancements in nanotechnologies have provided new methods of central nervous system access, drug and cell delivery, as well as new systems of cell maintenance and support that may bridge the gap between regenerative therapies and the brain. In this review, we discuss current regenerative therapies for neurological diseases, nanotechnology as nanocarriers, and the technical, manufacturing, and regulatory challenges that arise from inception to formulation of nanoparticle-regenerative therapies.

The effect of phenytoin on parathyroid hormone stimulated cAMP activity in cultured murine osteoblasts.

Cells were isolated by sequential collagenase digestion from the parietal segments of one day old mice (Swiss albino BNL strain) and characterized for osteoblast parameters by alkaline phosphatase histochemistry and bovine parathyroid hormone (bPTH-(1-34] induced cAMP activity (protein binding assay). Phenytoin (DPH) reduced PTH stimulated cAMP activity nearly 3-fold in the presence and nearly 1.5-fold in the absence of added calcium. In the absence of PTH, DPH exerted no significant effect. Bay-K-8644, a calcium channel activator, appeared to approximate the PTH stimulation of cAMP activity, even in the presence of DPH. This study demonstrates that DPH has a direct effect on PTH stimulated cAMP activity in cultured murine osteoblasts.

Calcitonin stimulates cAMP accumulation in chicken osteoclasts.

Calcitonin causes an increase in the accumulation of cAMP in mammalian osteoclasts leading to an inhibition of bone resorption. Increases in cAMP subsequent to calcitonin stimulation have not been detected in previous studies of chicken osteoclasts as the only source of large numbers of highly purified cells. In this report, we studied the effects of salmon calcitonin (sCT), bovine parathyroid hormone -(1-34) [(bPTH -(1-34)] and forskolin (FSK) on cAMP accumulation in freshly isolated osteoclasts obtained non-enzymatically from the metaphysis of 5-7 week old rachitogenic chickens. Parathyroid hormone did not stimulate the accumulation of intracellular cAMP. Calcitonin and forskolin treatment caused a nearly 2.5 and 3.5-fold increase in cAMP respectively. This study demonstrates that chicken osteoclasts respond to calcitonin with an increase in cAMP accumulation and that the rachitogenic chicken may be a valuable source of hormonally sensitive cells for the study of osteoclast biology.

Cardiac muscle ultrastructure and cyclic AMP reactions to altered gravity conditions.

Morphological and biochemical analyses of heart muscle of rats subjected to microgravity on Spacelab 3 (SL-3) flight and rats born and reared under increased gravity (1.7 G) conditions were compared with 1-G controls. Electronmicroscopic studies showed an increase in the number of lipid droplets and in areas of glycogen storage. Distribution changes of microtubules and cytoskeletal elements from both SL-3 and 1.7-G groups were observed. The high Km cyclic AMP phosphodiesterase activity was lower (P less than 0.05) in SL-3 heart muscle, and low Km activity was lower in 1.7-G males but was unaltered in females. Cyclic AMP-dependent protein kinase (cA-PK) activity was decreased in subcellular fractions of heart muscle of SL-3 animals. Recompartmentalization of cA-PK activity occurred in particulate tissue fraction of 1.7-G animals (70.3% of total for 1.7 G vs. 35.9% for controls). Phosphorylation of endogenous low-mobility proteins increased in SL-3 heart-soluble fractions. Photoaffinity labeling (18 h, 4 degrees C) decreased in type II cA-PK regulatory (R) subunits in both SL-3 and in 1.7-G male heart tissue particulate fractions. The 1.7-G female heart R subunit distribution did not differ from controls. These findings indicate that in heart muscle altered gravity conditions influenced physiological reactions similar to catecholamine-induced receptor-mediated hormonal responses.

Changes in cAMP phosphodiesterase activity during oral mucosal regeneration.

Preliminary studies (Lineweaver-Burk, Ca2+ calmodulin sensitivity) suggest that oral mucosa contains at least two cyclic AMP phosphodiesterases, one a high affinity (low Km) enzyme and the other a low affinity (high Km) enzyme. Analysis of the distribution of both enzymatic forms during oral mucosal regeneration revealed that the low Km, and high Km cAMP phosphodiesterase activities were significantly elevated prior to the first wave of cAMP accumulation and during the second cAMP wave. Although the cAMP peaks declined between 20-24 h, both cAMP phosphodiesterases remained significantly elevated at the wound site. The apparent Km of the low Km form increased from 5.3 to 7.5 microM, while that of the high Km form remained essentially unaltered 20 h after wounding. The low Km, and high Km cAMP phosphodiesterase activities in normal rat oral mucosa were not affected by epinephrine or insulin; were slightly inhibited by glucagon, and significantly inhibited by methylprednisolone. Imidazole and histamine activated both forms and theophylline was inhibitory to the enzyme. The high Km cAMP phosphodiesterase was sensitive, and the low Km form insensitive to Ca2+ calmodulin stimulation.

Effect of inflammation upon human gingival cyclic AMP metabolism.

The effect of the increasing degree of human gingival inflammation on adenylate cyclase (basal, fluoride stimulated) and low Km and high Km cAMP phosphodiesterase activities were evaluated in separate studies. Human gingival biopsies were classified by the Löe Bleeding Index as mildly, moderately, and markedly inflamed. Basal and F- stimulated adenylate cyclase (cAMP synthesis) activities were found to be unaltered by the increasing degree of inflammation when the data were expressed on either a mg wet wt, or mg protein basis. A significant loss of F- stimulated adenylate cyclase (mg protein) activity was observed in the moderately inflamed group when the data were compared with either the mildly or markedly inflamed groups of tissue. The low Km, and high Km cAMP phosphodiesterase activities (cAMP degradation) were found to be unaffected by gingival inflammation. This suggests that neither cAMP synthesis, nor degradation are stimulated in human gingiva by inflammation.

Adenylate cyclase localization in unfixed specimens of rat oral mucosa and isolated mitochondria.

The cytochemical localization of adenylate cyclase (E.C.4.6.1.1) was demonstrated in unfixed rat oral mucosal specimens and isolated mitochondrial fractions by use of a modified cytochemical reaction mixture. The adenylate cyclase reaction product was observed in the mitochondrial cristae of intact epithelial and fibroblast cells, and in isolated mitochondria after a 10-min incubation in the reaction mixture. The validity of the cytochemical adenylate cyclase method was supported by biochemical studies.

Elastase +/- soybean trypsin inhibitor dissociation of rat oral mucosa: ultrastructural and oxidative metabolic destructive changes in isolated, epithelial and dermal mitochondria after dissociation.

Epithelial and connective tissue compartments of rat oral mucosa were dissociated after incubation with elastase +/- soybean trypsin inhibitor (SBTI). Elastase + SBTI induced greater ultrastructural damage within the dissociated compartments than elastase alone. The basal lamina remained with the epithelial layer after elastase separation and was destroyed after exposure to elastase + SBTI. Isolated epithelial mitochondria were more severely damaged ultrastructurally after elastase + SBTI separation of the compartment than those prepared after exposure to elastase alone. Isolated fibroblast mitochondria were damage to the same extent after dissociation of the compartment with either medium. Oxidative metabolism and mitochondrial recoveries declined significantly after exposure to either dissociating medium. Cytochrome oxidase activity was significantly greater than succinic cytochrome c reductase in the control and experimental groups. Oxidative metabolism was found to be significantly greater in the connective tissue compartment than the epithelial compartment after dissociation of immature rat oral mucosa. Our data suggests that caution be utilized in assessing cellular viability and oxidative metabolism in tissue compartments immediately after their dissociation by proteolytic enzymes.

Trauma affects cyclic AMP and DNA levels at injured and non-injured distal oral mucosal sites.

Experiments in rats were conducted to test the hypothesis that gingival trauma affects cyclic AMP and DNA levels at the gingival wound, and non-injured distal (gingival, palatal) sites. Cyclic AMP and DNA levels rose and fell in a cyclic fashion during the time (0.5-24 h) periods analyzed. Significant increases in cAMP levels occurred at 8 and 20 h and at 8 and 16 h, respectively, at the wound and non-injured palatal site, peripheral to the wound. Similar increases (not significant) in cAMP levels were also noted at the non-injured gingival contralateral site at the same time intervals. DNA distributions were found to be significantly greater 10 and 16 h after injury at the gingival wound, and distal non-injured gingival and palatal sites.

Oxidative phosphorylation in rat oral mucosal mitochondria.

Oral mucosal mitochondria were isolated and characterized morphologically by electron microscopy. Polarographic measurements were made of respiration and oxidative phosphorylation in the mitochondrial preparations. ADP:O ratios approaching or slightly exceeding the theoretical maxima and stabilized respiratory control ratios were achieved with malate + glutamate, succinate and ascorbate-N,N,N1N1 tetramethyl-p-phenylenediamine (TMPD) as substrates. Inhibition by rotenone, antimycin A, azide, and cyanide established the classical electron transport chain as the major pathway of mitochondrial respiration. Respiration of the oral mucosal mitochondria was stimulated by DNP in the presence of succinate. DNP-stimulated respiration exceeded that observed in the presence of ADP plus Pi and increasing the concentration of DNP progressively inhibited respiration.