The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

Effects of γ-Aminobutyric Acid Type A Receptor Modulation by Flumazenil on Emergence from General Anesthesia.

BACKGROUND: Transitions into conscious states are partially mediated by inactivation of sleep networks and activation of arousal networks. Pharmacologic hastening of emergence from general anesthesia has largely focused on activating subcortical monoaminergic networks, with little attention on antagonizing the γ-aminobutyric acid type A receptor (GABAAR). As the GABAAR mediates the clinical effects of many common general anesthetics, the authors hypothesized that negative GABAAR modulators would hasten emergence, possibly via cortical networks involved in sleep. METHODS: The authors investigated the capacity of the benzodiazepine rescue agent, flumazenil, which had been recently shown to promote wakefulness in hypersomnia patients, to alter emergence. Using an in vivo rodent model and an in vitro GABAAR heterologous expression system, they measured flumazenil's effects on behavioral, neurophysiologic, and electrophysiologic correlates of emergence from isoflurane anesthesia. RESULTS: Animals administered intravenous flumazenil (0.4 mg/kg, n = 8) exhibited hastened emergence compared to saline-treated animals (n = 8) at cessation of isoflurane anesthesia. Wake-like electroencephalographic patterns occurred sooner and exhibited more high-frequency electroencephalography power after flumazenil administration (median latency ± median absolute deviation: 290 ± 34 s) compared to saline administration (473 ± 186 s; P = 0.042). Moreover, in flumazenil-treated animals, there was a decreased impact on postanesthesia sleep. In vitro experiments in human embryonic kidney-293T cells demonstrated that flumazenil inhibited isoflurane-mediated GABA current enhancement (n = 34 cells, 88.7 ± 2.42% potentiation at 3 µM). Moreover, flumazenil exhibited weak agonist activity on the GABAAR (n = 10 cells, 10.3 ± 3.96% peak GABA EC20 current at 1 µM). CONCLUSIONS: Flumazenil can modulate emergence from isoflurane anesthesia. The authors highlight the complex role GABAARs play in mediating consciousness and provide mechanistic links between emergence from anesthesia and arousal.

Metabolic signatures of amyotrophic lateral sclerosis reveal insights into disease pathogenesis.

Metabolic dysfunction is an important modulator of disease course in amyotrophic lateral sclerosis (ALS). We report here that a familial mouse model (transgenic mice over-expressing the G93A mutation of the Cu/Zn superoxide dismutase 1 gene) of ALS enters a progressive state of acidosis that is associated with several metabolic (hormonal) alternations that favor lipolysis. Extensive investigation of the major determinants of H(+) concentration (i.e., the strong ion difference and the strong ion gap) suggests that acidosis is also due in part to the presence of an unknown anion. Consistent with a compensatory response to avert pathological acidosis, ALS mice harbor increased accumulation of glycogen in CNS and visceral tissues. The altered glycogen is associated with fluctuations in lysosomal and neutral α-glucosidase activities. Disease-related changes in glycogen, glucose, and α-glucosidase activity are also found in spinal cord tissue samples of autopsied patients with ALS. Collectively, these data provide insights into the pathogenesis of ALS as well as potential targets for drug development.

Concentration-Dependent Dual Role of Thrombin in Protection of Cultured Rat Cortical Neurons.

Thrombin's role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin's effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin's effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor-1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin's receptor in the setting of direct thrombin inhibitors could be a potential neuroprotective strategy.

Gene transfer to the CNS is efficacious in immune-primed mice harboring physiologically relevant titers of anti-AAV antibodies.

Central nervous system (CNS)-directed gene therapy with recombinant adeno-associated virus (AAV) vectors has been used effectively to slow disease course in mouse models of several neurodegenerative diseases. However, these vectors were typically tested in mice without prior exposure to the virus, an immunological scenario unlikely to be duplicated in human patients. Here, we examined the impact of pre-existing immunity on AAV-mediated gene delivery to the CNS of normal and diseased mice. Antibody levels in brain tissue were determined to be 0.6% of the levels found in systemic circulation. As expected, transgene expression in brains of mice with relatively high serum antibody titers was reduced by 59-95%. However, transduction activity was unaffected in mice that harbored more clinically relevant antibody levels. Moreover, we also showed that markers of neuroinflammation (GFAP, Iba1, and CD3) and histopathology (hematoxylin and eosin (H&E)) were not enhanced in immune-primed mice (regardless of pre-existing antibody levels). Importantly, we also demonstrated in a mouse model of Niemann Pick Type A (NPA) disease that pre-existing immunity did not preclude either gene transfer to the CNS or alleviation of disease-associated neuropathology. These findings support the continued development of AAV-based therapies for the treatment of neurological disorders.

Disease progression in a mouse model of amyotrophic lateral sclerosis: the influence of chronic stress and corticosterone.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron cell loss, muscular atrophy, and a shortened life span. Survival is highly variable, as some patients die within months, while others live for many years. Exposure to stress or the development of a nonoptimal stress response to disease might account for some of this variability. We show in the SOD1(G93A) mouse model of ALS that recurrent exposure to restraint stress led to an earlier onset of astrogliosis and microglial activation within the spinal cord, accelerated muscular weakness, and a significant decrease in median survival (105 vs. 122 d) when compared to nonstressed animals. Moreover, during normal disease course, ALS mice display a cacostatic stress response by developing an aberrant serum corticosterone circadian rhythm. Interestingly, we also found that higher corticosterone levels were significantly correlated with both an earlier onset of paralysis (males: r(2)=0.746; females: r(2)=0.707) and shorter survival times (males: r(2)=0.680; females: r(2)=0.552) in ALS mice. These results suggest that stress is capable of accelerating disease progression and that strategies that modulate glucocorticoid metabolism might be a viable treatment approach for ALS.

AAV4-mediated expression of IGF-1 and VEGF within cellular components of the ventricular system improves survival outcome in familial ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron cell death in the cortex, brainstem, and spinal cord. Extensive efforts have been made to develop trophic factor-based therapies to enhance motor neuron survival; however, achievement of adequate therapeutic delivery to all regions of the corticospinal tract has remained a significant challenge. Here, we show that adeno-associated virus serotype 4 (AAV4)-mediated expression of insulin-like growth factor-1 (IGF-1) or vascular endothelial growth factor (VEGF)-165 in the cellular components of the ventricular system including the ependymal cell layer, choroid plexus [the primary cerebrospinal fluid (CSF)-producing cells of the central nervous system (CNS)] and spinal cord central canal leads to trophic factor delivery throughout the CNS, delayed motor decline and a significant extension of survival in SOD1(G93A) transgenic mice. Interestingly, when IGF-1- and VEGF-165-expressing AAV4 vectors were given in combination, no additional benefit in efficacy was observed suggesting that these trophic factors are acting on similar signaling pathways to modestly slow disease progression. Consistent with these findings, experiments conducted in a recently described in vitro cell culture model of ALS led to a similar result, with both IGF-1 and VEGF-165 providing significant motor neuron protection but in a nonadditive fashion. These findings support the continued investigation of trophic factor-based therapies that target the CNS as a potential treatment of ALS.

Sex effects on behavioral markers of emergence from propofol and isoflurane anesthesia in rats.

Clinical studies have demonstrated sex-related differences in recovery from surgical anesthesia. This study aimed to characterize the emergence pattern following two anesthesia regimens in both sexes of rats. We considered six different markers of emergence from anesthesia: sigh, eye blinking, forelimb movement, mastication, neck extension, and recovery of the righting reflex (RORR). Spontaneous motor activity 24 h after the anesthesia induction was also examined. Our results showed that the rank order of the emergence latency after intraperitoneal propofol, PRO, exposure was forelimb movement < sigh < blink < mastication < neck extension < RORR, while after inhaled isoflurane, ISO, anesthesia the sequence was changed as sigh < blink < mastication < forelimb movement < neck extension < RORR in both male and female rats. Moreover, the latency to emergence after PRO in female rats was significantly higher than male rats, although following ISO there was no difference between the sexes (P < 0.001; P > 0.05, respectively). Open-field testing revealed no difference in PRO and ISO spontaneous locomotor activity due to drug administration (P > 0.05). These two anesthetics presented different emergence sequences. Although clinical data suggests that females arouse faster than males from anesthesia with propofol, our intraperitoneal technique in a rodent model had the opposite effect. Pharmacokinetic analysis demonstrated increased absorption of injected propofol for the female rats in our study, emphasizing the role of sexual dimorphism in drug distribution in rodents. Despite these pharmacokinetic differences, the pharmacodynamic effects of the drugs were remarkably consistent among both sexes through emergence.

Temporal neuropathologic and behavioral phenotype of 6neo/6neo Pompe disease mice.

Pompe disease (glycogen storage disease II) is caused by mutations in the acid alpha-glucosidase gene. The most common form is rapidly progressive with glycogen storage, particularly in muscle, which leads to profound weakness, cardiac failure, and death by the age of 2 years. Although usually considered a muscle disease, glycogen storage also occurs in the CNS. We evaluated the progression of neuropathologic and behavioral abnormalities in a Pompe disease mouse model (6neo/6neo) that displays many features of the human disease. Homozygous mutant mice store excess glycogen within large neurons of hindbrain, spinal cord, and sensory ganglia by the age of 1 month; accumulations then spread progressively within many CNS cell types. "Silver degeneration" and Fluoro-Jade C stains revealed severe degeneration in axon terminals of primary sensory neurons at 3 to 9 months. These abnormalities were accompanied by progressive behavioral impairment on rotorod, wire hanging, and foot fault tests. The extensive neuropathologic alterations in this model suggest that therapy of skeletal and cardiac muscle disorders by systemic enzyme replacement therapy may not be sufficient to reverse functional deficits due to CNS glycogen storage, particularly early-onset, rapidly progressive disease. A better understanding of the basis for clinical manifestations is needed to correlate CNS pathology with Pompe disease manifestations.

Faster emergence behavior from ketamine/xylazine anesthesia with atipamezole versus yohimbine.

Recent interest in reversal of the hypnotic effects of anesthesia has mainly focused on overcoming a surge in GABA-mediated inhibitory signaling through activation of subcortical arousal circuits or antagonizing GABA receptors. Here we examine the reversal of anesthesia produced from non-GABA agents ketamine/xylazine and the effects of antagonists of adrenoreceptors. These antagonists vary in selectivity and produce temporally unique waking behavior post-anesthesia. We compared two antagonists with differential selectivity for α1- vs. α2-receptors, yohimbine (YOH, 1:40 selectivity) and atipamezole (ATI, 1:8500). Adult mice received intraperitoneal injections of either YOH (4.3 mg/kg), ATI (0.4 mg/kg), or saline after achieving sustained loss of righting following injection of ketamine/xylazine (ketamine: 65.0 mg/kg; xylazine: 9.9 mg/kg). Behaviors indicative of the post-anesthesia, re-animation sequence were carefully monitored and the timing of each behavior relative to anesthesia induction was compared. Both YOH and ATI hastened behaviors indicative of emergence, but ATI was faster than YOH to produce certain behaviors, including whisker movement (YOH: 21.9±1.5 min, ATI: 17.5±0.5 min, p = 0.004) and return of righting reflex (RORR) (YOH: 40.6±8.8 min, ATI: 26.0±1.2 min, p<0.001). Interestingly, although YOH administration hastened early behavioral markers of emergence relative to saline (whisking), the completion of the emergence sequence (time from first marker to appearance of RORR) was delayed with YOH. We attribute this effect to antagonism of α1 receptors by yohimbine. Also notable was the failure of either antagonist to hasten the re-establishment of coordinated motor behavior (e.g., attempts to remove adhesive tape on the forepaw placed during anesthesia) relative to the end of emergence (RORR). In total, our work suggests that in addition to pharmacokinetic effects, re-establishment of normal waking behaviors after anesthesia involves neuronal circuits dependent on time and/or activity.

Comparative analysis of acid sphingomyelinase distribution in the CNS of rats and mice following intracerebroventricular delivery.

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we reported that biochemical and functional abnormalities observed in ASM knockout (ASMKO) mice could be partially alleviated by intracerebroventricular (ICV) infusion of hASM. We now show that this route of delivery also results in widespread enzyme distribution throughout the rat brain and spinal cord. However, enzyme diffusion into CNS parenchyma did not occur in a linear dose-dependent fashion. Moreover, although the levels of hASM detected in the rat CNS were determined to be within the range shown to be therapeutic in ASMKO mice, the absolute amounts represented less than 1% of the total dose administered. Finally, our results also showed that similar levels of enzyme distribution are achieved across rodent species when the dose is normalized to CNS weight as opposed to whole body weight. Collectively, these data suggest that the efficacy observed following ICV delivery of hASM in ASMKO mice could be scaled to CNS of the rat.

Relationship between neuropathology and disease progression in the SOD1(G93A) ALS mouse.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1(G93A)). Four phases of the disease were studied in both male and female SOD1(G93A) mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1(G93A) mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons.

Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann-Pick A disease.

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site. As NPA is a global neurometabolic disease, the development of delivery strategies that maximize the distribution of the enzyme throughout the CNS is likely necessary to arrest or delay progression of the disease. To address this challenge, we evaluated the effectiveness of intracerebroventricular (ICV) delivery of recombinant human ASM into ASMKO mice. Our findings showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS. Moreover, a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera. Importantly, we demonstrated that repeated ICV infusions of ASM were effective at improving the disease phenotype in the ASMKO mouse as indicated by a partial alleviation of the motor abnormalities. These findings support the continued exploration of ICV delivery of recombinant lysosomal enzymes as a therapeutic modality for LSDs such as NPA that manifests substrate accumulation within the CNS.

Ability of adeno-associated virus serotype 8-mediated hepatic expression of acid alpha-glucosidase to correct the biochemical and motor function deficits of presymptomatic and symptomatic Pompe mice.

The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe disease.

Combination brain and systemic injections of AAV provide maximal functional and survival benefits in the Niemann-Pick mouse.

Niemann-Pick disease (NPD) is caused by the loss of acid sphingomyelinase (ASM) activity, which results in widespread accumulation of undegraded lipids in cells of the viscera and CNS. In this study, we tested the effect of combination brain and systemic injections of recombinant adeno-associated viral vectors encoding human ASM (hASM) in a mouse model of NPD. Animals treated by combination therapy exhibited high levels of hASM in the viscera and brain, which resulted in near-complete correction of storage throughout the body. This global reversal of pathology translated to normal weight gain and superior recovery of motor and cognitive functions compared to animals treated by either brain or systemic injection alone. Furthermore, animals in the combination group did not generate antibodies to hASM, demonstrating the first application of systemic-mediated tolerization to improve the efficacy of brain injections. All of the animals treated by combination therapy survived in good health to an investigator-selected 54 weeks, whereas the median lifespans of the systemic-alone, brain-alone, or untreated ASM knockout groups were 47, 48, and 34 weeks, respectively. These data demonstrate that combination therapy is a promising therapeutic modality for treating NPD and suggest a potential strategy for treating disease indications that cause both visceral and CNS pathologies.