Progressive Degenerative Myopathy and Myosteatosis in ASNSD1-Deficient Mice.

Mice with an inactivating mutation in the gene encoding asparagine synthetase domain containing 1 (ASNSD1) develop a progressive degenerative myopathy that results in severe sarcopenia and myosteatosis. ASNSD1 is conserved across many species, and whole body gene expression surveys show maximal expression levels of ASNSD1 in skeletal muscle. However, potential functions of this protein have not been previously reported. Asnsd1-/- mice demonstrated severe muscle weakness, and their normalized body fat percentage on both normal chow and high fat diets was greater than 2 SD above the mean for 3651 chow-fed and 2463 high-fat-diet-fed knockout (KO) lines tested. Histologic lesions were essentially limited to the muscle and were characterized by a progressive degenerative myopathy with extensive transdifferentiation and replacement of muscle by well-differentiated adipose tissue. There was minimal inflammation, fibrosis, and muscle regeneration associated with this myopathy. In addition, the absence of any signs of lipotoxicity in Asnsd1-/- mice despite their extremely elevated body fat percentage and low muscle mass suggests a role for metabolic dysfunctions in the development of this phenotype. Asnsd1-/- mice provide the first insight into the function of this protein, and this mouse model could prove useful in elucidating fundamental metabolic interactions between skeletal muscle and adipose tissue.

Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Ligation of mouse L4 and L5 spinal nerves produces robust allodynia without major motor function deficit.

Spinal nerve L5/L6 ligation (SNL) in rats has become the standard for mechanistic studies of peripheral neuropathy and screening for novel analgesics. Conventional SNL in our hybrid mice resulted in a wide range of allodynia. Anatomical evaluation indicated that a variable number of lumbar vertebrae existed, resulting in L4/L5 or L5/L6 being ligated. Surprisingly, L4/L5 ligation did not result in ipsilateral hind limb paralysis and produced robust allodynia. Following a recent report that the mouse L4 neural segment is homologous with rat L5 we generated L4, L5 or both L4 and L5 (L4/L5) ligations in C57 mice after establishing a modified set of surgical landmarks. In contrast to rats, L4 ligation in these mice did not result in hind limb paralysis. Robust allodynia was observed in all three ligation groups. Nerve degeneration confirmed that L4 and L5, respectively, are primary contributors to the tibial and sural branches of the sciatic nerve in mice. A larger von Frey sensitive area reflected the wider distribution of Wallerian degeneration in the hindlimb of L4- compared to L5-ligated mice. Ligation of mouse L4 and L5 spinal nerves produces consistent, robust neuropathic pain behaviors and is suitable as a model for investigating mechanisms of neuropathic pain and for testing of novel analgesics. Gabapentin, used as a validation drug in neuropathic pain models and as a reference compound for novel analgesics, significantly reduced allodynia in the mice tested (L4/L5 ligations). Given the ease of surgery, robust allodynia, and larger von Frey sensitive area, we conclude that combined ligation of spinal nerves L4 and L5 optimizes the SNL model in mice.

Transmembrane and ubiquitin-like domain containing 1 (Tmub1) regulates locomotor activity and wakefulness in mice and interacts with CAMLG.

Tmub1 (C7orf21/HOPS) encodes a protein containing a ubiquitin-like domain. Tmub1 is highly expressed in the nervous system. To study its physiological function, we generated mice with Tmub1 deleted by homologous recombination. The knockout mice were grossly normal and viable. In a comprehensive behavioral testing battery, the only knockout phenotype displayed was a strong increase in home cage locomotor activity during the dark phase (subjective day) of the light:dark (L:D) cycle. There were no changes in activity during the light period. There were no changes in locomotor activity observed in other assays, e.g. novel open-field. The increase in dark phase locomotor activity persisted during a seven day D:D (complete darkness) challenge, and remained largely confined to the normally dark period. Telemetric recording in freely moving subjects for one 24 hr L:D cycle, revealed the same increase in locomotor activity in the dark phase. In addition, EEG analysis showed that the knockout mice exhibited increased waking and decreased NREM & REM times during the dark phase, but the EEG was otherwise normal. Using lacZ as a reporter we found Tmub1 expression prominent in a few brain structures including the thalamus, a region known to drive wakefulness and arousal via its projections to the cortex. We identified calcium modulating cyclophilin ligand CAMLG/CAML as a binding partner by a yeast two-hybrid screen of a brain library. The interaction of Tmub1 and CAMLG was confirmed by co-immunoprecipitation assays in HEK cells. The two proteins were also found to be co-localized to the cytoplasm when expressed in HEK cells. Both Tmub1 and CAMLG have been recently described in the regulation of membrane trafficking of specific receptors. Taken together our results implicate Tmub1 in the regulation of locomotor activity and wakefulness and suggest that Tmub1 binds to and functions together with CAMLG.

Loss of the putative catalytic domain of HDAC4 leads to reduced thermal nociception and seizures while allowing normal bone development.

Histone deacetylase 4 (HDAC4) has been associated with muscle & bone development [1]-[6]. N-terminal MEF2 and RUNX2 binding domains of HDAC4 have been shown to mediate these effects in vitro. A complete gene knockout has been reported to result in premature ossification and associated defects resulting in postnatal lethality [6]. We report a viral insertion mutation that deletes the putative deacetylase domain, while preserving the N-terminal portion of the protein. Western blot and immuno-precipitation analysis confirm expression of truncated HDAC4 containing N-terminal amino acids 1-747. These mutant mice are viable, living to at least one year of age with no gross defects in muscle or bone. At 2-4 months of age no behavioral or physiological abnormalities were detected except for an increased latency to respond to a thermal nociceptive stimulus. As the mutant mice aged past 5 months, convulsions appeared, often elicited by handling. Our findings confirm the sufficiency of the N-terminal domain for muscle and bone development, while revealing other roles of HDAC4.

AMPA and NMDA receptor-mediated currents in developing dentate gyrus granule cells.

Granule cells (GCs) of the hippocampal dentate gyrus (DG) undergo postnatal neurogenesis such that cells at different maturational stages can be studied within an anatomically restricted region and a narrow animal age epoch. Using whole cell patch clamp recordings in hippocampal slices, we have previously found that input resistance (IR) correlates inversely with morphometric indicators of GC maturity. Using IR as an index of maturity we measured developmental changes in synaptic currents mediated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in GCs from 5- to 12-day rats. Peak NMDA and AMPA EPSC amplitudes increased, and the NMDA/AMPA ratio reversed with advancing cell age. NMDA EPSCs showed a maturational decrease in rise time but no change in decay time, whereas AMPA EPSCs showed neither rise nor decay time changes with development. Ifenprodil, a high affinity selective inhibitor of NR1/NR2B diheteromeric NMDA receptors, blocked approximately 50% of the peak amplitude of evoked NMDA EPSCs in all tested GCs regardless of their maturity and did not affect the measured kinetic properties. These data suggest that development of glutamatergic synapses follows distinct schedules. AMPA receptors possessed mature kinetics and became the dominant glutamatergic current within the age epoch studied, whereas NMDA receptors showed maturational changes in rise times but not decay kinetics. The reported modifications of EPSC properties are consistent with changes in receptor and synapse number, and relative quantities of AMPA and NMDA receptors. Changes in the subunit composition that determines NMDA decay kinetics may occur beyond the early neonatal period.

Severe retinal degeneration associated with disruption of semaphorin 4A.

PURPOSE: Semaphorin 4A (Sema4A) is a member of the transmembrane class 4 family of semaphorins. It has recently been shown to participate in cell-cell communication in the immune system. High levels of sema4A are also present in brain and eye, but its function in the central nervous system has not been studied. To investigate the function of Sema4A, we generated mice deficient in this transmembrane signaling molecule. METHODS: An embryonic stem (ES) cell clone with a retroviral gene-trap insertion in the sema4A gene was used to generate mice lacking this transmembrane semaphorin. Fundus photography, fluorescein angiography, and electroretinography were used to evaluate retinal anatomy and physiology in mice lacking Sema4A. Electron microscopy and immunohistochemistry with cell-type-specific markers were used to characterize retinal development. In situ hybridization with sema4A-specific riboprobes was used to localize expression of this gene in the developing and adult eye. RESULTS: Fundus photography performed at 14 weeks of age revealed severe retinal degeneration, attenuated retinal vessels, and depigmentation in mice lacking Sema4A. At this age, the outer nuclear layer was reduced to a single row of photoreceptor cells, and the outer plexiform layer was thin and disorganized. Disruption of Sema4A also compromised the physiological function of both rod and cone photoreceptors. Developmental studies in Sema4A-deficient mice revealed abnormal morphology of photoreceptor outer segments during the time at which they establish contacts with apical microvilli of the retinal pigment epithelium (RPE). Sema4A is expressed in the inner retina and RPE during the time at which photoreceptor outer segments elongate. CONCLUSIONS: These findings identify a previously unknown function of Sema4A in the developing visual system and provide a useful model for understanding cell-cell interactions that occur between photoreceptors and the RPE.