Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 98
Filtrar
1.
Adv Sci (Weinh) ; : e2401392, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874431

RESUMO

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.

2.
Transl Stroke Res ; 2023 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-37462831

RESUMO

Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to Gαq-type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca2+ reporter demonstrated that the majority of the Ca2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels (Trpc3KO compared with wildtype (WT)). This Ca2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP-Trpc3KO reporter brain slice model). Contribution of the Gαq-coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3KO, and Trpc1/3/6/7 quadruple knockout (TrpcQKO), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3KO and TrpcQKO models, with the TrpcQKO showing greatest neuroprotection. These findings demonstrate the contribution of the Gαq-coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.

3.
Front Neurosci ; 17: 1182874, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37274208

RESUMO

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.

4.
Front Neurosci ; 17: 1182845, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37274211

RESUMO

Aminoacyl-tRNA synthetases (ARSs) play an essential role in protein synthesis, being responsible for ligating tRNA molecules to their corresponding amino acids in a reaction known as 'tRNA aminoacylation'. Separate ARSs carry out the aminoacylation reaction in the cytosol and in mitochondria, and mutations in almost all ARS genes cause pathophysiology most evident in the nervous system. Dominant mutations in multiple cytosolic ARSs have been linked to forms of peripheral neuropathy including Charcot-Marie-Tooth disease, distal hereditary motor neuropathy, and spinal muscular atrophy. This review provides an overview of approaches that have been employed to model each of these diseases in vivo, followed by a discussion of the existing animal models of dominant ARS disorders and key mechanistic insights that they have provided. In summary, ARS disease models have demonstrated that loss of canonical ARS function alone cannot fully account for the observed disease phenotypes, and that pathogenic ARS variants cause developmental defects within the peripheral nervous system, despite a typically later onset of disease in humans. In addition, aberrant interactions between mutant ARSs and other proteins have been shown to contribute to the disease phenotypes. These findings provide a strong foundation for future research into this group of diseases, providing methodological guidance for studies on ARS disorders that currently lack in vivo models, as well as identifying candidate therapeutic targets.

5.
J Neurochem ; 2023 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-36977628

RESUMO

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.

6.
Front Mol Neurosci ; 15: 1061257, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36568275

RESUMO

The leukodystrophy Canavan disease is a fatal white matter disorder caused by loss-of-function mutations of the aspartoacylase-encoding ASPA gene. There are no effective treatments available and experimental gene therapy trials have failed to provide sufficient amelioration from Canavan disease symptoms. Preclinical studies suggest that Canavan disease-like pathology can be addressed by either ASPA gene replacement therapy or by lowering the expression of the N-acetyl-L-aspartate synthesizing enzyme NAT8L. Both approaches individually prevent or even reverse pathological aspects in Canavan disease mice. Here, we combined both strategies and assessed whether intracranial adeno-associated virus-mediated gene delivery to a Canavan disease mouse model at 12 weeks allows for reversal of existing pathology. This was enabled by a single vector dual-function approach. In vitro and in vivo biopotency assessment revealed significant knockdown of neuronal Nat8l paired with robust ectopic aspartoacylase expression. Following nomination of the most efficient cassette designs, we performed proof-of-concept studies in post-symptomatic Aspa-null mice. Late-stage gene therapy resulted in a decrease of brain vacuoles and long-term reversal of all pathological hallmarks, including loss of body weight, locomotor impairments, elevated N-acetyl-L-aspartate levels, astrogliosis, and demyelination. These data suggest feasibility of a dual-function vector combination therapy, directed at replacing aspartoacylase with concomitantly suppressing N-acetyl-L-aspartate production, which holds potential to permanently alleviate Canavan disease symptoms and expands the therapeutic window towards a treatment option for adult subjects.

7.
Front Neurol ; 13: 962227, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36226085

RESUMO

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the 'cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, "contralateral suppression" of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24-32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.

8.
Audiol Neurootol ; 27(5): 406-417, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35477110

RESUMO

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.


Assuntos
Miringoplastia , Perfuração da Membrana Timpânica , Adolescente , Adulto , Antropologia Cultural , Baías , Criança , Humanos , Miringoplastia/métodos , Nova Zelândia/epidemiologia , Estudos Retrospectivos , Resultado do Tratamento , Perfuração da Membrana Timpânica/cirurgia
10.
Neurochem Res ; 47(7): 1972-1984, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35357600

RESUMO

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.


Assuntos
Aspartato-tRNA Ligase , Doenças Desmielinizantes , Animais , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Pré-Escolar , Humanos , Camundongos , Mutação , Fenótipo
11.
Front Cell Neurosci ; 15: 661857, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34239416

RESUMO

Central Nervous System (CNS) homeostasis and function rely on intercellular synchronization of metabolic pathways. Developmental and neurochemical imbalances arising from mutations are frequently associated with devastating and often intractable neurological dysfunction. In the absence of pharmacological treatment options, but with knowledge of the genetic cause underlying the pathophysiology, gene therapy holds promise for disease control. Consideration of leukodystrophies provide a case in point; we review cell type - specific expression pattern of the disease - causing genes and reflect on genetic and cellular treatment approaches including ex vivo hematopoietic stem cell gene therapies and in vivo approaches using adeno-associated virus (AAV) vectors. We link recent advances in vectorology to glial targeting directed towards gene therapies for specific leukodystrophies and related developmental or neurometabolic disorders affecting the CNS white matter and frame strategies for therapy development in future.

12.
Neurochem Res ; 45(10): 2527, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32638216

RESUMO

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.

13.
Neurochem Res ; 45(6): 1438-1450, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32424601

RESUMO

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.


Assuntos
Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Dipeptídeos/metabolismo , Metabolismo Energético/fisiologia , Ornitina/metabolismo , Animais , Ácido Aspártico/farmacologia , Encéfalo/efeitos dos fármacos , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Feminino , Cobaias , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ornitina/farmacologia
14.
Brain ; 143(6): 1889-1904, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32375177

RESUMO

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.


Assuntos
Tauopatias/genética , Proteínas tau/genética , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Demência Frontotemporal/genética , Hipocampo/metabolismo , Potenciação de Longa Duração/genética , Masculino , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Fosforilação , Tauopatias/fisiopatologia
15.
Biomolecules ; 10(4)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32316246

RESUMO

Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da-13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.


Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Impedância Elétrica , Venenos de Escorpião/farmacologia , Cafeína/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fluorescência , Células HEK293 , Humanos , Fatores de Tempo
16.
Front Cell Neurosci ; 14: 626610, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33574740

RESUMO

Aminoacyl-tRNA synthetases (ARSs) accurately charge tRNAs with their respective amino acids. As such, they are vital for the initiation of cytosolic and mitochondrial protein translation. These enzymes have become increasingly scrutinized in recent years for their role in neurodegenerative disorders caused by the mutations of ARS-encoding genes. This review focuses on two such genes-DARS1 and DARS2-which encode cytosolic and mitochondrial aspartyl-tRNA synthetases, and the clinical conditions associated with mutations of these genes. We also describe attempts made at modeling these conditions in mice, which have both yielded important mechanistic insights. Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a disease caused by a range of mutations in the DARS2 gene, initially identified in 2003. Ten years later, hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL), caused by mutations of cytosolic DARS1, was discovered. Multiple parallels have been drawn between the two conditions. The Magnetic Resonance Imaging (MRI) patterns are strikingly similar, but still set these two conditions apart from other leukodystrophies. Clinically, both conditions are characterized by lower limb spasticity, often associated with other pyramidal signs. However, perhaps due to earlier detection, a wider range of symptoms, including peripheral neuropathy, as well as visual and hearing changes have been described in LBSL patients. Both HBSL and LBSL are spectrum disorders lacking genotype to phenotype correlation. While the fatal phenotype of Dars1 or Dars2 single gene deletion mouse mutants revealed that the two enzymes lack functional redundancy, further pursuit of disease modeling are required to shed light onto the underlying disease mechanism, and enable examination of experimental treatments, including gene therapies.

17.
Front Cell Neurosci ; 14: 625879, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33551752

RESUMO

Hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL) is a leukodystrophy caused by missense mutations of the aspartyl-tRNA synthetase-encoding gene DARS1. The clinical picture includes the regression of acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Morphologically, HBSL is characterized by a distinct pattern of hypomyelination in the central nervous system including the anterior brainstem, the cerebellar peduncles and the supratentorial white matter as well as the dorsal columns and the lateral corticospinal tracts of the spinal cord. Adequate HBSL animal models are lacking. Dars1 knockout mice are embryonic lethal precluding examination of the etiology. To address this, we introduced the HBSL-causing Dars1 D367Y point mutation into the mouse genome. Surprisingly, mice carrying this mutation homozygously were phenotypically normal. As hypomorphic mutations are more severe in trans to a deletion, we crossed Dars1 D367Y/D367Y mice with Dars1-null carriers. The resulting Dars1 D367Y/- offspring displayed a strong developmental delay compared to control Dars1 D367Y/+ littermates, starting during embryogenesis. Only a small fraction of Dars1 D367Y/- mice were born, and half of these mice died with hydrocephalus during the first 3 weeks of life. Of the few Dars1 D367Y/- mice that were born at term, 25% displayed microphthalmia. Throughout postnatal life, Dars1 D367Y/- mice remained smaller and lighter than their Dars1 D367Y/+ littermates. Despite this early developmental deficit, once they made it through early adolescence Dars1 D367Y/- mice were phenotypically inconspicuous for most of their adult life, until they developed late onset motor deficits as well as vacuolization and demyelination of the spinal cord white matter. Expression levels of the major myelin proteins were reduced in Dars1 D367Y/- mice compared to controls. Taken together, Dars1 D367Y/- mice model aspects of the clinical picture of the corresponding missense mutation in HBSL. This model will enable studies of late onset deficits, which is precluded in Dars1 knockout mice, and can be leveraged to test potential HBSL therapeutics including DARS1 gene replacement therapy.

18.
Front Neurosci ; 13: 691, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447624

RESUMO

The neural interface is a critical factor in governing efficient and safe charge transfer between a stimulating electrode and biological tissue. The interface plays a crucial role in the efficacy of electric stimulation in chronic implants and both electromechanical properties and biological properties shape this. In the case of cochlear implants, it has long been recognized that neurotrophins can stimulate growth of the target auditory nerve fibers into a favorable apposition with the electrode array, and recently such arrays have been re-purposed to enable electrotransfer (electroporation)-based neurotrophin gene augmentation to improve the "bionic ear." For both this acute bionic array-directed electroporation and for chronic conventional cochlear implant arrays, the electric fields generated in target tissue during pulse delivery are central to efficacy, but are challenging to map. We present a computational model for predicting electric fields generated by array-driven DNA electrotransfer in the cochlea. The anatomically realistic model geometry was reconstructed from magnetic resonance images of the guinea pig cochlea and an eight-channel electrode array embedded within this geometry. The model incorporates a description of both Faradaic and non-Faradaic mechanisms occurring at the electrode-electrolyte interface with frequency dependency optimized to match experimental impedance spectrometry measurements. Our simulations predict that a tandem electrode configuration with four ganged cathodes and four ganged anodes produces three to fourfold larger area in target tissue where the electric field is within the range for successful gene transfer compared to an alternate paired anode-cathode electrode configuration. These findings matched in vivo transfection efficacy of a green fluorescent protein (GFP) reporter following array-driven electrotransfer of the reporter-encoding plasmid DNA. This confirms utility of the developed model as a tool to optimize the safety and efficacy of electrotransfer protocols for delivery of neurotrophin growth factors, with the ultimate aim of using gene augmentation approaches to improve the characteristics of the electrode-neural interfaces in chronically implanted neurostimulation devices.

19.
Purinergic Signal ; 15(3): 343-355, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31377959

RESUMO

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.


Assuntos
Adaptação Fisiológica/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Emissões Otoacústicas Espontâneas/fisiologia , Estimulação Acústica , Animais , Camundongos , Camundongos Endogâmicos C57BL , Ruído
20.
Hear Res ; 380: 137-149, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31301514

RESUMO

This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.


Assuntos
Percepção Auditiva , Implante Coclear/instrumentação , Implantes Cocleares , Terapia Genética , Perda Auditiva/reabilitação , Audição , Fatores de Crescimento Neural/genética , Pessoas com Deficiência Auditiva/reabilitação , Animais , Percepção Auditiva/genética , Terapia Combinada , Eletroporação , Técnicas de Transferência de Genes , Audição/genética , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Perda Auditiva/psicologia , Humanos , Pessoas com Deficiência Auditiva/psicologia , Recuperação de Função Fisiológica , Resultado do Tratamento , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...