AMP-activated protein kinase activators have compound and concentration-specific effects on brain metabolism.

AMP-activated protein kinase (AMPK) is a key sensor of energy balance playing important roles in the balancing of anabolic and catabolic activities. The high energy demands of the brain and its limited capacity to store energy indicate that AMPK may play a significant role in brain metabolism. Here, we activated AMPK in guinea pig cortical tissue slices, both directly with A769662 and PF 06409577 and indirectly with AICAR and metformin. We studied the resultant metabolism of [1-13 C]glucose and [1,2-13 C]acetate using NMR spectroscopy. We found distinct activator concentration-dependent effects on metabolism, which ranged from decreased metabolic pool sizes at EC50 activator concentrations with no expected stimulation in glycolytic flux to increased aerobic glycolysis and decreased pyruvate metabolism with certain activators. Further, activation with direct versus indirect activators produced distinct metabolic outcomes at both low (EC50 ) and higher (EC50 × 10) concentrations. Specific direct activation of ß1-containing AMPK isoforms with PF 06409577 resulted in increased Krebs cycle activity, restoring pyruvate metabolism while A769662 increased lactate and alanine production, as well as labelling of citrate and glutamine. These results reveal a complex metabolic response to AMPK activators in brain beyond increased aerobic glycolysis and indicate that further research is warranted into their concentration- and mechanism-dependent impact.

Developmental delay and late onset HBSL pathology in hypomorphic Dars1M256L mice.

The leukodystrophy Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is caused by recessive mutations of the DARS1 gene, which encodes the cytoplasmic aspartyl-tRNA synthetase. HBSL is a spectrum disorder with disease onset usually during early childhood and no available treatment options. Patients display regression of previously acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Gene-function studies in mice revealed that homozygous Dars1 deletion is embryonically lethal, suggesting that successful modelling of HBSL requires the generation of disease-causing genocopies in mice. In this study, we introduced the pathogenic DARS1 M256L mutation located on exon nine of the murine Dars1 locus. Despite causing severe illness in humans, homozygous Dars1 M256L mice were only mildly affected. To exacerbate HBSL symptoms, we bred Dars1 M256L mice with Dars1-null 'enhancer' mice. The Dars1 M256L/- offspring displayed increased embryonic lethality, severe developmental delay, reduced body weight and size, hydrocephalus, anophthalmia, and vacuolization of the white matter. Remarkably, the Dars1 M256L/- genotype affected energy metabolism and peripheral organs more profoundly than the nervous system and resulted in reduced body fat, increased respiratory exchange ratio, reduced liver steatosis, and reduced hypocellularity of the bone marrow. In summary, homozygous Dars1 M256L and compound heterozygous Dars1 M256L/- mutation genotypes recapitulate some aspects of HBSL and primarily manifest in developmental delay as well as metabolic and peripheral changes. These aspects of the disease might have been overlooked in HBSL patients with severe neurological deficits but could be included in the differential diagnosis of HBSL in the future.

Audiological and Surgical Correlates of Myringoplasty Associated with Ethnography in the Bay of Plenty, New Zealand.

INTRODUCTION: This retrospective cohort study of myringoplasty performed at Tauranga Hospital, Bay of Plenty, New Zealand from 2010 to 2020 sought to identify predictive factors for successful myringoplasty with particular consideration given to the known high prevalence of middle ear conditions in New Zealand Maori. METHODS: Outcomes were surgical success (perforation closure at 1 month) and hearing improvement, which were correlated against demographic, pathological, and surgical variables. RESULTS: 174 patients underwent 221 procedures (139 in children under 18 years old), with 66.1% of patients being New Zealand Maori and 24.7% New Zealand European ethnicity. Normalized by population demographics, New Zealand Maori were 2.3 times overrepresented, whereas New Zealand Europeans were underrepresented by 0.34 times (a 6.8 times relative treatment differential). The rate of surgical success was 84.6%, independent of patient age, gender, and ethnicity. A postauricular approach and the use of temporalis fascia grafts were both correlated with optimal success rates, whereas early postoperative infection (<1 month) was correlated with ∼3 times increased failure. Myringoplasty improved hearing in 83.1% of patients (average air-bone gap reduction of 10.7 dB). New Zealand Maori patients had ∼4 times greater preoperative conductive hearing loss compared to New Zealand Europeans, but benefited the most from myringoplasty. DISCUSSION/CONCLUSION: New Zealand Maori and pediatric populations required greater access to myringoplasty, achieving good surgical and audiological outcomes. Myringoplasty is highly effective and significantly improves hearing, particularly for New Zealand Maori. Pediatric success rates were equivalent to adults, supporting timely myringoplasty to minimize morbidity from untreated perforations.

Onset of hippocampal network aberration and memory deficits in P301S tau mice are associated with an early gene signature.

Hyperphosphorylation and deposition of tau in the brain characterizes frontotemporal dementia and Alzheimer's disease. Disease-associated mutations in the tau-encoding MAPT gene have enabled the generation of transgenic mouse models that recapitulate aspects of human neurodegenerative diseases, including tau hyperphosphorylation and neurofibrillary tangle formation. Here, we characterized the effects of transgenic P301S mutant human tau expression on neuronal network function in the murine hippocampus. Onset of progressive spatial learning deficits in P301S tau transgenic TAU58/2 mice were paralleled by long-term potentiation deficits and neuronal network aberrations during electrophysiological and EEG recordings. Gene-expression profiling just prior to onset of apparent deficits in TAU58/2 mice revealed a signature of immediate early genes that is consistent with neuronal network hypersynchronicity. We found that the increased immediate early gene activity was confined to neurons harbouring tau pathology, providing a cellular link between aberrant tau and network dysfunction. Taken together, our data suggest that tau pathology drives neuronal network dysfunction through hyperexcitation of individual, pathology-harbouring neurons, thereby contributing to memory deficits.

Correction to: L-Aspartate, L-Ornithine and L-Ornithine-L-Aspartate (LOLA) and Their Impact on Brain Energy Metabolism.

The original version of this published article, the bottom right hand panels of Figs. 3-6 were labelled as "Isotopomers formed from [1-13C]D-glucose". This is incorrect and should read "Isotopomers formed from [1,2-13C]acetate". This has been corrected by publishing this correction article.

L-Aspartate, L-Ornithine and L-Ornithine-L-Aspartate (LOLA) and Their Impact on Brain Energy Metabolism.

L-Ornithine-L-aspartate (LOLA), a crystalline salt, is used primarily in the management of hepatic encephalopathy. The degree to which it might penetrate the brain, and the effects it might have on metabolism in brain are poorly understood. Here, to investigate the effects of LOLA on brain energy metabolism we incubated brain cortical tissue slices from guinea pig (Cavea porcellus) with the constituent amino acids of LOLA, L-ornithine or L-aspartate, as well as LOLA, in the presence of [1-13C]D-glucose and [1,2-13C]acetate; these labelled substrates are useful indicators of brain metabolic activity. L-Ornithine produced significant "sedative" effects on brain slice metabolism, most likely via conversion of ornithine to GABA via the ornithine aminotransferase pathway, while L-aspartate showed concentration-dependent excitatory effects. The metabolic effects of LOLA reflected a mix of these two different processes and were concentration-dependent. We also investigated the effect of an intraperitoneal bolus injection of L-ornithine, L-aspartate or LOLA on levels of metabolites in kidney, liver and brain cortex and brain stem in mice (C57Bl6J) 1 h later. No significant changes in metabolite levels were seen following the bolus injection of L-aspartate, most likely due to rapid metabolism of aspartate before reaching the target tissue. Brain cortex glutamate was decreased by L-ornithine but no other brain effects were observed with any other compound. Kidney levels of aspartate were increased after injection of L-ornithine and LOLA which may be due to interference by ornithine with the kidney urea cycle. It is likely that without optimising chronic intravenous infusion, LOLA has minimal impact on healthy brain energy metabolism due to systemic clearance and the blood - brain barrier.

Human Brain Region-Specific Alternative Splicing of TRPC3, the Type 3 Canonical Transient Receptor Potential Non-Selective Cation Channel.

Canonical transient receptor potential (TRPC) non-selective cation channels are broadly expressed by neurons, glia and the microvasculature of the brain. In neurons and astrocytes, these ion channels are coupled to group I metabotropic glutamate receptors via Gαq-phospholipase C signal transduction. In the mouse cerebellar Purkinje neurons, TRPC channels assembled as tetramers of TRPC3 subunits exclusively mediate this glutamatergic signalling mechanism and regulation of alternative splicing results in dominance of a high Ca2+ conducting TRPC3c isoform. This regional control of TRPC3 transcript type likely has physiological and pathophysiological sequelae. The current study provides a quantitative comparison of the TRPC3c splice variant and the TRPC3b full-length isoform expression across seven regions of the human brain. This shows that the cerebellum has the highest expression level of both isoforms and that regulation of alternative splicing results in a higher propensity of the TRPC3c isoform in the cerebellum relative to the TRPC3b isoform (in a 1:3 ratio). This compares with the other regions (motor cortex, hippocampus, midbrain subregions, pons and medulla) where the prevalence of TRPC3c relative to TRPC3b is typically less than half as abundant. The finding here of a bias in the high-conductance TRPC3c isoform in the cerebellum is consistent with the enhanced vulnerability of the cerebellum to ischaemic injury.

Onset kinetics of noise-induced purinergic adaptation of the 'cochlear amplifier'.

A major component of slowly reversible hearing loss which develops with sustained exposure to noise has been attributed to release of ATP in the cochlea activating P2X2 receptor (P2X2R) type ATP-gated ion channels. This purinergic humoral adaptation is thought to enable the highly sensitive hearing organ to maintain function with loud sound, protecting the ear from acoustic overstimulation. In the study that established this hearing adaptation mechanism as reported by Housley et al. (Proc Natl Acad Sci U S A 110:7494-7499, 2013), the activation kinetics were determined in mice from auditory brainstem response (ABR) threshold shifts with sustained noise presentation at time points beyond 10 min. The present study was designed to achieve finer resolution of the onset kinetics of purinergic hearing adaptation, and included the use of cubic (2f1-f2) distortion product otoacoustic emissions (DPOAEs) to probe whether the active mechanical outer hair cell 'cochlear amplifier' contributed to this process. We show that the ABR and DPOAE threshold shifts were largely complete within the first 7.5 min of moderate broadband noise (85 dB SPL) in wildtype C57Bl/6J mice. The ABR and DPOAE adaptation rates were both best fitted by a single exponential function with ~ 3 min time constants. ABR and DPOAE threshold shifts with this noise were minimal in mice null for the P2rx2 gene encoding the P2X2R. The findings demonstrate a considerably faster purinergic hearing adaptation to noise than previously appreciated. Moreover, they strongly implicate the outer hair cell as the site of action, as the DPOAEs stem from active cochlear electromotility.

Resistance to neomycin ototoxicity in the extreme basal (hook) region of the mouse cochlea.

Aminoglycoside ototoxicity results in permanent loss of the sensory hair cells in the mammalian cochlea. It usually begins at the basal turn causing high-frequency hearing loss. Here we describe previously unreported resistance of hair cells to neomycin ototoxicity in the extreme basal (hook) region of the developing cochlea of the C57BL/6 mouse. Organ of Corti explants from mice at postnatal day 3 were incubated (37 °C, 5% CO2) in normal culture medium for 19.5 h prior to and after exposure to neomycin (1 mM, 3 h). To study neomycin uptake in the hair cells, cochlear explants were incubated with Neomycin Texas-red (NTR) conjugate. As expected, exposure to neomycin significantly reduced the survival of inner (IHC) and outer hair cells (OHC). IHC survival rate was high in the apical segment and low in the basal segment. OHC were well preserved in the apical and hook regions, with substantial OHC loss in the basal segment. The NTR uptake study demonstrated that the high survival rate in the extreme basal turn OHC was associated with low NTR uptake. Treatment with a calcium chelator (BAPTA), which disrupts the opening of mechanoelectrical (MET) transduction channels, abolished or reduced NTR uptake in the hair cells throughout the cochlea. This confirmed the essential role of MET channels in neomycin uptake and implied that the transduction channels could be impaired in the hook region of the developing mouse cochlea, possibly as a result of the cadherin 23 mutation responsible for the progressive deafness in C57BL/6 mice.

Uncoupling N-acetylaspartate from brain pathology: implications for Canavan disease gene therapy.

N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease-a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.

In vivocharacterization of the aspartyl-tRNA synthetase DARS: Homing in on the leukodystrophy HBSL.

BACKGROUND: The recently diagnosed leukodystrophy Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL) is caused by mutations of the cytoplasmic aspartyl-tRNA synthetase geneDARS. The physiological role of DARS in translation is to accurately pair aspartate with its cognate tRNA. Clinically, HBSL subjects show a distinct pattern of hypomyelination and develop progressive leg spasticity, variable cognitive impairment and epilepsy. To elucidate the underlying pathomechanism, we comprehensively assessed endogenous DARS expression in mice. Additionally, aiming at creating the first mammalian HBSL model, we genetically engineered and phenotyped mutant mice with a targetedDarslocus. RESULTS: DARS, although expressed in all organs, shows a distinct expression pattern in the adult brain with little immunoreactivity in macroglia but enrichment in neuronal subpopulations of the hippocampus, cerebellum, and cortex. Within neurons, DARS is mainly located in the cell soma where it co-localizes with other components of the translation machinery. Intriguingly, DARS is also present along neurites and at synapses, where it potentially contributes to local protein synthesis.Dars-null mice are not viable and die before embryonic day 11. Heterozygous mice with only one functionalDarsallele display substantially reduced DARS levels in the brain; yet these mutants show no gross abnormalities, including unchanged motor performance. However, we detected reduced pre-pulse inhibition of the acoustic startle response indicating dysfunction of attentional processing inDars+/-mice. CONCLUSIONS: Our results, for the first time, show an in-depth characterization of the DARS tissue distribution in mice, revealing surprisingly little uniformity across brain regions or between the major neural cell types. The complete loss of DARS function is not tolerated in mice suggesting that the identified HBSL mutations in humans retain some residual enzyme activity. The mild phenotype of heterozygousDars-null carriers indicates that even partial restoration of DARS levels would be therapeutically relevant. Despite the fact that they do not resemble the full spectrum of clinical symptoms, the robust pre-pulse inhibition phenotype ofDars+/-mice will be instrumental for future preclinical therapeutic efficacy studies. In summary, our data is an important contribution to a better understanding of DARS function and HBSL pathology.

ß-Hydroxybutyrate Boosts Mitochondrial and Neuronal Metabolism but is not Preferred Over Glucose Under Activated Conditions.

The ketone body, ß-hydroxybutyrate (ßOHB), is metabolised by the brain alongside the mandatory brain fuel glucose. To examine the extent and circumstances by which ßOHB can supplement glucose metabolism, we studied guinea pig cortical brain slices using increasing concentrations of [U-13C]D-ßOHB in conjunction with [1-13C]D-glucose under conditions of normo- and hypoglycaemia, as well as under high potassium (40 mmol/L K+) depolarization in normo- and hypoglycaemic conditions. The contribution of ßOHB to synthesis of GABA was also probed by inhibiting the synthesis of glutamine, a GABA precursor, with methionine sulfoximine (MSO). [U-13C]D-ßOHB at lower concentrations (0.25 and 1.25 mmol/L) stimulated mitochondrial metabolism, producing greater total incorporation of label into glutamate and GABA but did not have a similar effect in the cytosolic compartment where labelling of glutamine was reduced at 1.25 mmol/L [U-13C]D-ßOHB. At higher concentrations (2.5 mmol/L) [U-13C]D-ßOHB inhibited metabolism of [1-13C]D-glucose, and reduced total label incorporation and total metabolite pools. When glucose levels were reduced, ßOHB was able to partially restore the loss of glutamate and GABA caused by hypoglycaemia, but was not able to supplement levels of lactate, glutamine or alanine or to prevent the increase in aspartate. Under depolarizing conditions glucose was the preferred substrate over ßOHB, even in hypoglycaemic conditions where comparatively less ßOHB was incorporated except into aspartate isotopomers. Inhibition of glutamine synthesis with MSO had no significant effect on incorporation of label from [U-13C]D-ßOHB into GABA C2,1 indicating that the majority of this GABA was synthesized in GABAergic neurons from [U-13C]D-ßOHB rather than from Gln C4,5 imported from astrocytes.

ATP-gated ion channels mediate adaptation to elevated sound levels.

The sense of hearing is remarkable for its auditory dynamic range, which spans more than 10(12) in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2-null mice. At higher sound levels (≥95 dB), additional processes dominated TTS, and P2RX2-null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2-null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.

Mutation of the ATP-gated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise.

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X(2) receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X(2) receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X(2) function is required for life-long normal hearing and for protection from exposure to noise.

Properties of ATP-gated ion channels assembled from P2X2 subunits in mouse cochlear Reissner's membrane epithelial cells.

In the cochlea, Reissner's membrane separates the scala media endolymphatic compartment that sustains the positive endocochlear potential and ion composition necessary for sound transduction, from the scala vestibuli perilymphatic compartment. It is known that with sustained elevated sound levels, adenosine 5'-triphosphate (ATP) is released into the endolymph and ATP-gated ion channels on the epithelial cells lining the endolymphatic compartment shunt the electrochemical driving force, contributing to protective purinergic hearing adaptation. This study characterises the properties of epithelial cell P2X(2)-type ATP-activated membrane conductance in the mouse Reissner's membrane, which forms a substantial fraction of the scale media surface. The cells were found to express two isoforms (a and b) of the P2X(2) subunit arising from alternative splicing of the messenger RNA (mRNA) transcript that could contribute to the trimeric subunit assembly. The ATP-activated conductance demonstrated both immediate and delayed desensitisation consistent with incorporation of the combination of P2X(2) subunit isoforms. Activation by the ATP analogue 2meSATP had equipotency to ATP, whereas α,ß-meATP and adenosine 5'-diphosphate (ADP) were ineffective. Positive allosteric modulation of the P2X(2) channels by protons was profound. This native conductance was blocked by the P2X(2)-selective blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and the conductance was absent in these cells isolated from mice null for the P2rX2 gene encoding the P2X(2) receptor subunit. The activation and desensitisation properties of the Reissner's membrane epithelial cell ATP-gated P2X(2) channels likely contribute to the sensitivity and kinetics of purinergic control of the electrochemical driving force for sound transduction invoked by noise exposure.

Gene Electrotransfer via Conductivity-Clamped Electric Field Focusing Pivots Sensori-Motor DNA Therapeutics: "A Spoonful of Sugar Helps the Medicine Go Down".

Viral vectors and lipofection-based gene therapies have dispersion-dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close-field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of "naked" plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near-field conductivity ("conductivity-clamping"-CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic-ready bionics-derived CC-GET device achieved neurotrophin-encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics-based hearing. The performance of CC-GET is evaluated in the brain, an organ problematic for pulsed electric field-based plasmid DNA delivery, due to high required currents causing Joule-heating and damaging electroporation. Here CC-GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red-shifted channelrhodopsin and a genetically-encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders.

Alternative splicing of the TRPC3 ion channel calmodulin/IP3 receptor-binding domain in the hindbrain enhances cation flux.

Canonical transient receptor potential (TRPC3) nonselective cation channels are effectors of G-protein-coupled receptors (GPCRs), activated via phospholipase C-diacylglycerol signaling. In cerebellar Purkinje cells, TRPC3 channels cause the metabotropic glutamate receptor (mGluR)-mediated slow EPSC (sEPSC). TRPC3 channels also provide negative feedback regulation of cytosolic Ca(2+), mediated by a C terminus "calmodulin and inositol trisphosphate receptor binding" (CIRB) domain. Here we report the alternative splicing of the TRPC3 mRNA transcript (designated TRPC3c), resulting in omission of exon 9 (approximately half of the CIRB domain) in mice, rats, and guinea pigs. TRPC3c expression is brain region specific, with prevalence in the cerebellum and brainstem. The TRPC3c channels expressed in HEK293 cells exhibit increased basal and GPCR-activated channel currents, and increased Ca(2+) fluorescence responses, compared with the previously characterized (TRPC3b) isoform when activated via either the endogenous M3 muscarinic acetylcholine receptor, or via coexpressed mGluR1. GPCR-induced TRPC3c channel opening rate (cell-attached patch) matched the maximum activation achieved with inside-out patches with zero cytosolic Ca(2+), whereas the GPCR-induced TRPC3b activation frequency was significantly less. Both TRPC3 channel isoforms were blocked with 2 mm Ca(2+), attributable to CIRB domain regulation. In addition, genistein blocked Purkinje cell (S)-2-amino-2-(3,5-dihydroxyphenyl) acetic acid (mGluR1)-activated TPRC3 current as for recombinant TRPC3c current. This novel TRPC3c ion channel therefore has enhanced efficacy as a neuronal GPCR-Ca(2+) signaling effector, and is associated with sensorimotor coordination, neuronal development, and brain injury.

Canonical transient receptor potential channel subtype 3-mediated hair cell Ca(2+) entry regulates sound transduction and auditory neurotransmission.

The physiological significance of canonical transient receptor potential (TRPC) ion channels in sensory systems is rapidly emerging. Heterologous expression studies show that TRPC3 is a significant Ca(2+) entry pathway, with dual activation via G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol second messenger signaling, and through negative feedback, whereby a fall in cytosolic Ca(2+) releases Ca(2+) -calmodulin channel block. We hypothesised that the latter process contributes to cochlear hair cell cytosolic Ca(2+) homeostasis. Confocal microfluorimetry with the Ca(2+) indicator Fluo-4 acetoxymethylester showed that, when cytosolic Ca(2+) was depleted, Ca(2+) re-entry was significantly impaired in mature TRPC3(-/-) inner and outer hair cells. The impact of this disrupted Ca(2+) homeostasis on sound transduction was assessed with the use of distortion product otoacoustic emissions (DPOAEs), which constitute a direct measure of the outer hair cell transduction that underlies hearing sensitivity and frequency selectivity. TRPC3(-/-) mice showed significantly stronger DPOAE (2f1  - f2 ) growth functions than wild-type (WT) littermates within the frequency range of best hearing acuity. This translated to hyperacusis (decreased threshold) measured by the auditory brainstem response (ABR). TRPC3(-/-) and WT mice did not differ in the levels of temporary and permanent threshold shift arising from noise exposure, indicating that potential GPCR signaling via TRPC3 is not pronounced. Overall, these data suggest that the Ca(2+) set-point in the hair cell, and hence membrane conductance, is modulated by TRPC3s through their function as a negative feedback-regulated Ca(2+) entry pathway. This TPRC3-regulated Ca(2+) homeostasis shapes the sound transduction input-output function and auditory neurotransmission.

Recessive aminoacyl-tRNA synthetase disorders: lessons learned from in vivo disease models.

Protein synthesis is a fundamental process that underpins almost every aspect of cellular functioning. Intriguingly, despite their common function, recessive mutations in aminoacyl-tRNA synthetases (ARSs), the family of enzymes that pair tRNA molecules with amino acids prior to translation on the ribosome, cause a diverse range of multi-system disorders that affect specific groups of tissues. Neurological development is impaired in most ARS-associated disorders. In addition to central nervous system defects, diseases caused by recessive mutations in cytosolic ARSs commonly affect the liver and lungs. Patients with biallelic mutations in mitochondrial ARSs often present with encephalopathies, with variable involvement of peripheral systems. Many of these disorders cause severe disability, and as understanding of their pathogenesis is currently limited, there are no effective treatments available. To address this, accurate in vivo models for most of the recessive ARS diseases are urgently needed. Here, we discuss approaches that have been taken to model recessive ARS diseases in vivo, highlighting some of the challenges that have arisen in this process, as well as key results obtained from these models. Further development and refinement of animal models is essential to facilitate a better understanding of the pathophysiology underlying recessive ARS diseases, and ultimately to enable development and testing of effective therapies.