Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5 weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

Agrin isoforms with distinct amino termini: differential expression, localization, and function.

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

The synaptic protein syntaxin1 is required for cellularization of Drosophila embryos.

Syntaxins are membrane proteins involved in vesicle trafficking and are required for the release of neurotransmitter at nerve terminals. The presence of syntaxins on target membranes has been hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins, the synaptobrevins or VAMPS. We have mutagenized syntaxin1 in Drosophila and have found that it links the mechanism of synaptic transmission to a distinct cell biological process: the cellularization of early embryos. This specialized form of cell division separates the 6,000 nuclei of the syncytial blastoderm into separate cells through the invagination of the surface membrane of the embryo. During this process, syntaxin1 protein is present on the newly forming lateral cell surfaces and invaginating cleavage furrows. This protein is derived both from maternal deposition of mRNA and protein and from early zygotic transcription. To analyze syntaxin1's role in early development, female germ line mosaics mutant for syntaxin1 expression were generated by mitotic recombination to reduce the maternal contribution. Visualizing the actin cytoskeleton and glycosylated surface proteins reveals that embryos with insufficient syntaxin1 have large acellular patches. The patches do not appear until cellularization begins, and the process fails entirely within these regions. These results provide genetic evidence that membrane trafficking is required for the cellularization of the syncytial blastoderm. We propose that the invagination of the surface membrane proceeds by the fusion of intracellular membrane vesicles with the surface. This reaction uses the same syntaxin1 protein as is required for neurotransmitter secretion at synapses. Thus, a single syntaxin can participate in trafficking steps that are functionally as distinct as synaptic transmission and cell division.

Alternatively spliced isoforms of nerve- and muscle-derived agrin: their roles at the neuromuscular junction.

Agrin induces synaptic differentiation at the skeletal neuromuscular junction (NMJ); both pre- and postsynaptic differentiation are drastically impaired in its absence. Multiple alternatively spliced forms of agrin that differ in binding characteristics and bioactivity are synthesized by nerve and muscle cells. We used surgical chimeras, isoform-specific mutant mice, and nerve-muscle cocultures to determine the origins and nature of the agrin required for synaptogenesis. We show that agrin containing Z exons (Z+) is a critical nerve-derived inducer of postsynaptic differentiation, whereas neural isoforms containing a heparin binding site (Y+) and all muscle-derived isoforms are dispensable for major steps in synaptogenesis. Our results also suggest that the requirement of agrin for presynaptic differentiation is mediated indirectly by its ability to promote postsynaptic production or localization of appropriate retrograde signals.

Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos.

Approximately half of the motoneurons produced during development die before birth or shortly after birth. Although it is believed that survival depends on a restricted supply of a trophic sustenance produced by the synaptic target tissue (i.e., muscle), it is unclear whether synapse formation per se is involved in motoneuron survival. To address this issue, we counted cranial motoneurons in a set of mutant mice in which formation of neuromuscular junctions is dramatically impaired (i.e., null mutants for agrin, nerve-derived agrin, rapsyn, and MuSK). We demonstrate that in the absence of synaptogenesis, there is an 18-34% increase in motoneuron survival in the facial, trochlear, trigeminal motor, and hypoglossal nuclei; the highest survival occurred in the MuSK-deficient animals in which synapse formation is most severely compromised. There was no change in the size of the mutant motoneurons as compared with control animals, and the morphology of the mutant motoneurons appeared normal. We postulate that the increased axonal branching observed in these mutants leads to a facilitated "access" of the motoneurons to muscle-derived trophic factors at sites other than synapses or that inactivity increases the production of such factors. Finally, we examined motoneurons in double mutants of CNTFRalpha(-/-) (in which there is a partial loss of motoneurons) and MuSK(-/-) (in which there is an increased survival of motoneurons). The motoneuron numbers in the double mutants parallel those of the single MuSK-deficient mice, indicating that synapse disruption can even overcome the deleterious effect of CNTFRalpha ablation.

Differential expression of transcripts from syb, a Drosophila melanogaster gene encoding VAMP (synaptobrevin) that is abundant in non-neuronal cells.

VAMP (synaptobrevin) is a highly conserved membrane protein originally described as a component of brain synaptic vesicles. The Drosophila melanogaster VAMP-encoding gene (syb) comprises five exons. Splicing exons 1,2,3,4,5 (syb-b) results in a protein with a C-terminal hydrophobic domain and a negligible intraluminal domain. Splicing exons 1,2,3,5 (syb-a) predicts a protein with a 20-amino-acid luminal domain at the C terminus. The ratio of syb-a to syb-b transcripts is highly regulated during development. The syb transcripts show no enrichment in the nervous system and are present in very early embryos, well before neurogenesis. The greatest concentration of syb transcripts was found in cells of the gut and malpighian tubules. Thus, syb may have a general role in membrane trafficking and, perhaps, a role in the secretion of digestive enzymes.

Development of the neuromuscular junction: genetic analysis in mice.

Formation of the skeletal neuromuscular junction is a multi-step process that requires communication between the nerve and muscle. Studies in many laboratories have led to identification of factors that seem likely to mediate these interactions. 'Knock-out' mice have now been generated with mutations in several genes that encode candidate transsynaptic messengers and components of their effector mechanisms. Using these mice, it is possible to test hypotheses about the control of synaptogenesis. Here, we review our studies on neuromuscular development in mutant mice lacking agrin alpha CGRP, rapsyn, MuSK, dystrophin, dystrobrevin, utrophin, laminin alpha 5, laminin beta 2, collagen alpha 3 (IV), the acetylcholine receptor epsilon subunit, the collagenous tail of acetylcholinesterase, fibroblast growth factor-5, the neural cell adhesion molecule, and tenascin-C.

Hexokinase levels in discrete regions of rat brain: evaluation by quantitative immunofluorescence using interactive laser cytometry and comparison with basal rates of glucose utilization.

Relative levels of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been determined in 16 discrete regions of adult rat brain by a quantitative immunofluorescence method. The distribution of immunofluorescence in brain sections was determined by interactive laser cytometry and related to hexokinase content by comparison with standard sections containing known amounts of the enzyme. In many of these regions, referred to here as group I regions, hexokinase content was correlated with previously reported basal rates of glucose utilization. However, several regions (group II regions) in which hexokinase content exceeded that expected from basal rates of glucose utilization were also detected. Compared with the corresponding regions from albino rat brain, higher hexokinase levels were found in the dorsal and ventral lateral geniculate (group I regions) of pigmented Norway rats, a result reflecting previously reported increased glucose utilization by these regions in pigmented rats. There was no difference in hexokinase levels in the superior colliculus, a group II structure, from albino and pigmented rats, a finding implying that a reported increase in rate of glucose utilization in the superior colliculus of pigmented rats is effected without an increase in hexokinase content. It is suggested that group II regions may be adapted to sustain increases in rates of glucose utilization that are, relative to basal rates, considerably greater than those experienced by group I regions.

External abdominal aortic compression: a study of a resuscitation manoeuvre for postpartum haemorrhage.

External aortic compression is an emergency manoeuvre proposed to reduce postpartum haemorrhage and permit time for resuscitation and control of bleeding. To assess this technique, a prospective study was performed on twenty normal non-bleeding parturients. The abdominal aorta was compressed by firm pressure with a closed fist just above the umbilicus. Leg and arm arterial blood pressures were measured and femoral artery pulsation felt before, during and after compression. Leg blood pressure was completely obliterated in 55% and significantly reduced (P < 0.01) in a further 10% of subjects. All of these subjects with reduced or absent leg blood pressure also had obliteration of the femoral pulse with compression. Systemic arterial blood pressure was not significantly elevated by successful aortic occlusion. Discomfort with the manoeuvre was significantly increased (P < 0.05) in the group of subjects that had successful aortic occlusion. It is recommended that external aortic compression be considered in severe life-threatening postpartum haemorrhage, particularly during stabilisation or transport of the patient. This simple manoeuvre may be used as an adjunct to other measures and could prove of benefit, especially in locations or situations where advanced medical assistance is geographically or temporally removed.

Choice of incision and pain following gallbladder surgery.

A prospective randomized trial compared pain in the first 24 h after gallbladder surgery via an upper midline or a transverse incision. Pain was measured by the patients' self-administered consumption of pethidine, degree of postoperative respiratory impairment and a visual analogue pain scale. The upper midline incision group self-administered significantly more pethidine than the transverse incision group (P less than 0.001), but there was no difference between the groups in respiratory function or visual analogue pain scale results 24 h after operation. Length of hospital stay was not different. An upper midline incision is more painful than a transverse incision in the first 24 h following gallbladder surgery.

Acid aspiration prophylaxis in Australian obstetric hospitals--a survey.

During 1987 a confidential survey of all hospitals in Australia providing obstetric services was undertaken to determine the antacid medications used routinely as prophylaxis against acid aspiration pneumonitis. Of the 567 hospitals surveyed, 379 (67%) responded. Of these, 243 hospitals provide an obstetric service which includes caesarean section, and 67% of these perform less than 500 deliveries per annum. Aspiration prophylaxis during labour was used in 22.4% of responding hospitals. Prior to elective caesarean section, 11.5% used no prophylaxis, and 39.4% used particulate antacids such as magnesium trisilicate mixture (33.3%) or Mylanta (6.1%). Sodium citrate mixture was the most popular therapy (37%). Results were similar in the emergency caesarean section group. The use of cimetidine or ranitidine was uncommon in all groups. Results of this survey suggest marked differences in attitudes towards acid aspiration prophylaxis between Australian and British obstetric anaesthetic practices.

Monitoring epidural analgesia in the parturient.

Appropriate monitoring during obstetric epidural analgesia consists of: 1. Indirect BP and pulse monitoring before epidural insertion, frequently after every dose, and intermittently thereafter. 2. The aspiration test before all injections. 3. Frequent clinical monitoring for signs of intravascular injection during administration of small intermittent doses (not more than 3-5 ml at a time). 4. Frequent clinical monitoring for sympathetic, sensory and motor signs indicating upward extension of the block. 5. Frequent monitoring of the fetal heart rate (FHR) and other signs of fetal welfare. In many instances continuous tocogram with fetal heart rate (CTG) monitoring is useful. We do not believe CTG use is mandatory for epidural analgesia in the uncomplicated pregnancy, but we do advocate that it (and other appropriate fetal monitoring techniques) be used when risk factors or complications, either fetal or maternal, are present or suspected. The anaesthetist should be familiar with fetal monitoring techniques, their use and interpretation. He or she should be prepared to recommend their use when it is considered appropriate to do so.

Post-operative respiratory failure due to bilateral phrenic nerve palsy.

A 65 year old female patient developed a large left pleural effusion and a sternal split dehiscence following aorto-coronary artery bypass grafting. A second operation was performed to investigate and drain the pleural effusion and to repair the sternum. Subsequent to this operation the patient was in acute respiratory failure due to bilateral phrenic palsy. It is probable that the left phrenic nerve was damaged in the initial operation and the right nerve in the second. The patient's subsequent progress is described.

Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin.

Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+ influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To investigate the mechanism of transmitter release, we have examined the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion. We generated a series of mutations, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous "minis" that are seen at the synapse can be generated by a protein complex that is distinct from that required for an evoked synaptic response.

Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse.

The development of chemical synapses is regulated by interactions between pre- and postsynaptic cells. At the vertebrate skeletal neuromuscular junction, the organization of an acetylcholine receptor (AChR)-rich postsynaptic apparatus has been well studied. Much evidence suggests that the nerve-derived protein agrin activates muscle-specific kinase (MuSK) to cluster AChRs through the synapse-specific cytoplasmic protein rapsyn. But how postsynaptic differentiation is initiated, or why most synapses are restricted to an 'end-plate band' in the middle of the muscle remains unknown. Here we have used genetic methods to address these issues. We report that the initial steps in postsynaptic differentiation and formation of an end-plate band require MuSK and rapsyn, but are not dependent on agrin or the presence of motor axons. In contrast, the subsequent stages of synaptic growth and maintenance require nerve-derived agrin, and a second nerve-derived signal that disperses ectopic postsynaptic apparatus.

Identification and characterization of Drosophila genes for synaptic vesicle proteins.

Proteins associated with synaptic vesicles are likely to control the release of neurotransmitter. Because synaptic transmission is fundamentally similar between vertebrates and invertebrates, vesicle proteins from vertebrates that are important for synaptic transmission should be present in Drosophila as well. This investigation describes Drosophila homologs of vamp, synaptotagmin, and rab3 that are expressed in a pattern consistent with a function in Drosophila neurotransmission. One previously reported candidate (syb), a Drosophila homolog of the vamp or synaptobrevin proteins, has been shown to be expressed at very low levels in neurons and is most abundant in the gut. A neuronal Drosophila vamp (n-syb) is described here and is localized to chromosome band 62A. Northern analysis and in situ hybridizations to mRNA indicate that the novel vamp, as well as the genes for synaptotagmin (syt) and rab3 (drab3), is expressed in the Drosophila nervous system. These genes are widely (perhaps ubiquitously) expressed in the nervous system and we have no evidence of additional neuronal isoforms of synaptotagmin, vamp, or rab3. Immunoreactivity for synaptotagmin and vamp is located in synaptic regions of the nervous system. This distribution suggests that these molecules are components of synaptic vesicles in Drosophila. The conserved structure and neuronal expression pattern of these genes indicate that they may function in processes that are required for both vertebrate and invertebrate synaptic transmission. Because of their distribution in the nervous system and because n-syb, synaptotagmin, and drab3 do not appear to be in a family of functionally redundant homologs, we predict that mutation of these genes will have a profound neurological phenotype and that they are therefore good candidates for a genetic dissection in Drosophila.

Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled.

We identified a seven-pass transmembrane receptor of the cadherin superfamily, designated Flamingo (Fmi), localized at cell-cell boundaries in the Drosophila wing. In the absence of Fmi, planar polarity was distorted. Before morphological polarization of wing cells along the proximal-distal (P-D) axis, Fmi was redistributed predominantly to proximal and distal cell edges. This biased localization of Fmi appears to be driven by an imbalance of the activity of Frizzled (Fz) across the proximal/distal cell boundary. These results, together with phenotypes caused by ectopic expression of fz and fmi, suggest that cells acquire the P-D polarity by way of the Fz-dependent boundary localization of Fmi.