Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse.

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.

Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice.

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.

A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior.

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Dentate Gyrus Sharp Waves, a Local Field Potential Correlate of Learning in the Dentate Gyrus of Mice.

The hippocampus plays an essential role in learning. Each of the three major hippocampal subfields, dentate gyrus (DG), CA3, and CA1, has a unique function in memory formation and consolidation, and also exhibit distinct local field potential (LFP) signatures during memory consolidation processes in non-rapid eye movement (NREM) sleep. The classic LFP events of the CA1 region, sharp-wave ripples (SWRs), are induced by CA3 activity and considered to be an electrophysiological biomarker for episodic memory. In LFP recordings along the dorsal CA1-DG axis from sleeping male mice, we detected and classified two types of LFP events in the DG: high-amplitude dentate spikes (DSs), and a novel event type whose current source density (CSD) signature resembled that seen during CA1 SWR, but which, most often, occurred independently of them. Because we hypothesize that this event type is similarly induced by CA3 activity, we refer to it as dentate sharp wave (DSW). We show that both DSWs and DSs differentially modulate the electrophysiological properties of SWR and multiunit activity (MUA). Following two hippocampus-dependent memory tasks, DSW occurrence rates, ripple frequencies, and ripple and sharp wave (SW) amplitudes were increased in both, while SWR occurrence rates in dorsal CA1 increased only after the spatial task. Our results suggest that DSWs, like SWRs, are induced by CA3 activity and that DSWs complement SWRs as a hippocampal LFP biomarker of memory consolidation.SIGNIFICANCE STATEMENT Awake experience is consolidated into long-term memories during sleep. Memory consolidation crucially depends on sharp-wave ripples (SWRs), which are local field potential (LFP) patterns in hippocampal CA1 that increase after learning. The dentate gyrus (DG) plays a central role in the process of memory formation, prompting us to cluster sharp waves (SWs) in the DG [dentate SWs (DSWs)] during sleep. We show that both DSW coupling to CA1 SWRs, and their occurrence rates, robustly increase after learning trials. Our results suggest that the DG is directly affected by memory consolidation processes. DSWs may thus complement SWRs as a sensitive electrophysiological biomarker of memory consolidation in mice.

Arc/Arg3.1 mediates a critical period for spatial learning and hippocampal networks.

During early postnatal development, sensory regions of the brain undergo periods of heightened plasticity which sculpt neural networks and lay the foundation for adult sensory perception. Such critical periods were also postulated for learning and memory but remain elusive and poorly understood. Here, we present evidence that the activity-regulated and memory-linked gene Arc/Arg3.1 is transiently up-regulated in the hippocampus during the first postnatal month. Conditional removal of Arc/Arg3.1 during this period permanently alters hippocampal oscillations and diminishes spatial learning capacity throughout adulthood. In contrast, post developmental removal of Arc/Arg3.1 leaves learning and network activity patterns intact. Long-term memory storage continues to rely on Arc/Arg3.1 expression throughout life. These results demonstrate that Arc/Arg3.1 mediates a critical period for spatial learning, during which Arc/Arg3.1 fosters maturation of hippocampal network activity necessary for future learning and memory storage.

Disturbed Prefrontal Cortex Activity in the Absence of Schizophrenia-Like Behavioral Dysfunction in Arc/Arg3.1 Deficient Mice.

Arc/Arg3.1, an activity regulated immediate early gene, is essential for learning and memory, synaptic plasticity, and maturation of neural networks. It has also been implicated in several neurodevelopmental disorders, including schizophrenia. Here, we used male and female constitutive and conditional Arc/Arg3.1 knock-out (KO) mice to investigate the causal relationship between Arc/Arg3.1 deletion and schizophrenia-linked neurophysiological and behavioral phenotypes. Using in vivo local field potential recordings, we observed dampened oscillatory activity in the prefrontal cortex (PFC) of the KO and early conditional KO (early-cKO) mice, in which Arc/Arg3.1 was deleted perinatally. Whole-cell patch-clamp recordings from neurons in PFC slices revealed altered synaptic properties and reduced network gain in the KO mice as possible mechanisms underlying the oscillation deficits. In contrast, we measured normal oscillatory activity in the PFC of late conditional KO (late-cKO) mice, in which Arc/Arg3.1 was deleted during late postnatal development. Our data show that constitutive Arc/Arg3.1 KO mice exhibit no deficit in social engagement, working memory, sensorimotor gating, native locomotor activity, and dopaminergic innervation. Moreover, adolescent social isolation, an environmental stressor, failed to induce deficits in sociability or sensorimotor gating in adult KO mice. Thus, genetic removal of Arc/Arg3.1 per se does not cause schizophrenia-like behavior. Prenatal or perinatal deletion of Arc/Arg3.1 alters cortical network activity, however, without overtly disrupting the balance of excitation and inhibition in the brain and not promoting schizophrenia. Misregulation of Arc/Arg3.1 rather than deletion could potentially tip this balance and thereby promote emergence of schizophrenia and other neuropsychiatric disorders.SIGNIFICANCE STATEMENT The activity-regulated and memory-linked gene Arc/Arg3.1 has been implicated in the pathogenesis of schizophrenia, but direct evidence and a mechanistic link are still missing. The current study asks whether loss of Arc/Arg3.1 can affect brain circuitry and cause schizophrenia-like symptoms in mice. The findings demonstrate that genetic deletion of Arc/Arg3.1 before puberty alters synaptic function and prefrontal cortex activity. Although brain networks are disturbed, genetic deletion of Arc/Arg3.1 does not cause schizophrenia-like behavior, even when combined with an environmental insult. It remains to be seen whether misregulation of Arc/Arg3.1 might critically imbalance brain networks and lead to emergence of schizophrenia.

Creatine, guanidinoacetate and homoarginine in statin-induced myopathy.

Our study evaluated the effect of creatine and homoarginine in AGAT- and GAMT-deficient mice after simvastatin exposure. Balestrino and Adriano suggest that guanidinoacetate might explain the difference between AGAT- and GAMT-deficient mice in simvastatin-induced myopathy. We agree with Balestrino and Adriano that our data shows that (1) creatine possesses a protective potential to ameliorate statin-induced myopathy in humans and mice and (2) homoarginine did not reveal a beneficial effect in statin-induced myopathy. Third, we agree that guanidinoacetate can be phosphorylated and partially compensate for phosphocreatine. In our study, simvastatin-induced damage showed a trend to be less pronounced in GAMT-deficient mice compared with wildtype mice. Therefore, (phospo) guanidinoacetate cannot completely explain the milder phenotype of GAMT-deficient mice, but we agree that it might contribute to ameliorate statin-induced myopathy in GAMT-deficient mice compared with AGAT-deficient mice. Finally, we agree with Balestino and Adriano that AGAT metabolites should further be evaluated as potential treatments in statin-induced myopathy.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, L-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of L-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

The Na+/H+ Exchanger Nhe1 Modulates Network Excitability via GABA Release.

Brain functions are extremely sensitive to pH changes because of the pH-dependence of proteins involved in neuronal excitability and synaptic transmission. Here, we show that the Na+/H+ exchanger Nhe1, which uses the Na+ gradient to extrude H+, is expressed at both inhibitory and excitatory presynapses. We disrupted Nhe1 specifically in mice either in Emx1-positive glutamatergic neurons or in parvalbumin-positive cells, mainly GABAergic interneurons. While Nhe1 disruption in excitatory neurons had no effect on overall network excitability, mice with disruption of Nhe1 in parvalbumin-positive neurons displayed epileptic activity. From our electrophysiological analyses in the CA1 of the hippocampus, we conclude that the disruption in parvalbumin-positive neurons impairs the release of GABA-loaded vesicles, but increases the size of GABA quanta. The latter is most likely an indirect pH-dependent effect, as Nhe1 was not expressed in purified synaptic vesicles itself. Conclusively, our data provide first evidence that Nhe1 affects network excitability via modulation of inhibitory interneurons.

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT(-/-) mice.

Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking L-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT(-/-) (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes.

L-arginine:glycine amidinotransferase deficiency protects from metabolic syndrome.

Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.

Homoarginine supplementation improves blood glucose in diet-induced obese mice.

L-Homoarginine (hArg) is an endogenous amino acid which has emerged as a novel biomarker for stroke and cardiovascular disease. Low circulating hArg levels are associated with increased mortality and vascular events, whereas recent data have revealed positive correlations between circulating hArg and metabolic vascular risk factors like obesity or blood glucose levels. However, it is unclear whether hArg levels are causally linked to metabolic parameters. Therefore, the aim of our study was to investigate whether hArg directly influences body weight, blood glucose, glucose tolerance or insulin sensitivity. Here, we show that hArg supplementation (14 and 28 mg/mL orally per drinking water) ameliorates blood glucose levels in mice on high-fat diet (HFD) by a reduction of 7.3 ± 3.7 or 13.4 ± 3.8 %, respectively. Fasting insulin concentrations were slightly, yet significantly affected (63.8 ± 11.3 or 162.1 ± 39.5 % of control animals, respectively), whereas body weight and glucose tolerance were unaltered. The substantial augmentation of hArg plasma concentrations in supplemented animals (327.5 ± 40.4 or 627.5 ± 60.3 % of control animals, respectively) diminished profoundly after the animals became obese (129.9 ± 16.6 % in control animals after HFD vs. 140.1 ± 8.5 or 206.3 ± 13.6 %, respectively). This hArg-lowering effect may contribute to the discrepancy between the inverse correlation of plasma hArg levels with stroke and cardiovascular outcome, on the one hand, and the direct correlation with cardiovascular risk factors like obesity and blood glucose, on the other hand, that has been observed in human studies. Our results suggest that the glucose-lowering effects of hArg may reflect a compensatory mechanism of blood glucose reduction by hArg upregulation in obese individuals, without directly influencing body weight or glucose tolerance.

Postnatal disruption of the disintegrin/metalloproteinase ADAM10 in brain causes epileptic seizures, learning deficits, altered spine morphology, and defective synaptic functions.

The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid ß (Aß) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPα and increased levels of sAPPß and endogenous Aß peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.

Homoarginine levels are regulated by L-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies.

BACKGROUND: Endogenous arginine homologues, including homoarginine, have been identified as novel biomarkers for cardiovascular disease and outcomes. Our studies of human cohorts and a confirmatory murine model associated the arginine homologue homoarginine and its metabolism with stroke pathology and outcome. METHODS AND RESULTS: Increasing homoarginine levels were independently associated with a reduction in all-cause mortality in patients with ischemic stroke (7.4 years of follow-up; hazard ratio for 1-SD homoarginine, 0.79 [95% confidence interval, 0.64-0.96]; P=0.019; n=389). Homoarginine was also independently associated with the National Institutes of Health Stroke Scale+age score and 30-day mortality after ischemic stroke (P<0.05; n=137). A genome-wide association study revealed that plasma homoarginine was strongly associated with single nucleotide polymorphisms in the L-arginine:glycine amidinotransferase (AGAT) gene (P<2.1 × 10(-8); n=2806), and increased AGAT expression in a cell model was associated with increased homoarginine. Next, we used 2 genetic murine models to investigate the link between plasma homoarginine and outcome after experimental ischemic stroke: (1) an AGAT deletion (AGAT(-/-)) and (2) a guanidinoacetate N-methyltransferase deletion (GAMT(-/-)) causing AGAT upregulation. As suggested by the genome-wide association study, homoarginine was absent in AGAT(-/-) mice and increased in GAMT(-/-) mice. Cerebral damage and neurological deficits in experimental stroke were increased in AGAT(-/-) mice and attenuated by homoarginine supplementation, whereas infarct size in GAMT(-/-) mice was decreased compared with controls. CONCLUSIONS: Low homoarginine appears to be related to poor outcome after ischemic stroke. Further validation in future trials may lead to therapeutic adjustments of homoarginine metabolism that alleviate stroke and other vascular disorders.

Differential regulation of AMPK activation in leptin- and creatine-deficient mice.

AMP-activated protein kinase (AMPK) is a key sensor and regulator of energy homeostasis. Previously, we demonstrated that intracellular energy depletion by L-arginine:glycine amidinotransferase (AGAT) deficiency resulted in AMPK activation and protected from metabolic syndrome. In the present study, we show tissue-specific leptin dependence of AMPK activation by energy depletion. We investigated leptin-dependent AMPK regulation in AGAT- and leptin-deficient (d/d ob/ob) mice. Like ob/ob mice, but unlike d/d mice, d/d ob/ob mice were obese and glucose intolerant. Therefore, leptin is a prerequisite for resistance to metabolic syndrome in AGAT-deficient mice. Quantitative Western blots revealed a 4-fold increase in AMPK activation in skeletal muscle of d/d ob/ob mice (P<0.001). However, AMPK activation was absent in white adipose tissue (WAT) and liver. Compared with blood glucose levels in ob/ob mice, fasting levels were still reduced and therefore did not show leptin dependence (wild-type, 79.4±3.9 mg/dl; d/d, 68.4±3.2 mg/dl; P<0.05). In ob/ob mice and wild-type mice, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), in combination with leptin, augmented glucose tolerance compared with AICAR alone, whereas no improvement was found under conditions of high-fat-diet feeding. These findings reveal a previously unknown synergistic AMPK activation by leptin and intracellular energy depletion, suggesting that AMPK activation can be therapeutically effective in metabolic syndrome only if leptin sensitivity is preserved.

Control of heart rate by cAMP sensitivity of HCN channels.

"Pacemaker" f-channels mediating the hyperpolarization-activated nonselective cation current I(f) are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major I(f)-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4-573X expression causes elimination of the cAMP sensitivity of I(f) and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4-573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of I(f), the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMP-mediated regulation of I(f) determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4-573X-linked SAN dysfunction in humans.

Seizures, behavioral deficits, and adverse drug responses in two new genetic mouse models of HCN1 epileptic encephalopathy.

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the human HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an impairment in inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool toward reaching this objective.

The funny current in genetically modified mice.

Since its first description in 1979, the hyperpolarization-activated funny current (If) has been the object of intensive research aimed at understanding its role in cardiac pacemaker activity and its modulation by the sympathetic and parasympathetic branches of the autonomic nervous system. If was described in isolated tissue strips of the rabbit sinoatrial node using the double-electrode voltage-clamp technique. Since then, the rabbit has been the principal animal model for studying pacemaker activity and If for more than 20 years. In 2001, the first study describing the electrophysiological properties of mouse sinoatrial pacemaker myocytes and those of If was published. It was soon followed by the description of murine myocytes of the atrioventricular node and the Purkinje fibres. The sinoatrial node of genetically modified mice has become a very popular model for studying the mechanisms of cardiac pacemaker activity. This field of research benefits from the impressive advancement of in-vivo exploration techniques of physiological parameters, imaging, genetics, and large-scale genomic approaches. The present review discusses the influence of mouse genetic on the most recent knowledge of the funny current's role in the physiology and pathophysiology of cardiac pacemaker activity. Genetically modified mice have provided important insights into the role of If in determining intrinsic automaticity in vivo and in myocytes of the conduction system. In addition, gene targeting of f-(HCN) channel isoforms have contributed to elucidating the current's role in the regulation of heart rate by the parasympathetic nervous system. This review is dedicated to Dario DiFrancesco on his retirement.