Michael Fauler

GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak

Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.

K-dependent paradoxical membrane depolarization and Na overload, major and reversible contributors to weakness by ion channel leaks

Normal resting potential (P1) of myofibers follows the Nernst equation, exhibiting about −85 mV at a normal extracellular K+ concentration ([K+]o) of 4 mM. Hyperpolarization occurs with decreased [K+]o, although at [K+]o < 1.0 mM, myofibers paradoxically depolarize to a second stable potential of −60 mV (P2). In rat myofiber bundles, P2 also was found at more physiological [K+]o and was associated with inexcitability. To increase the relative frequency of P2 to 50%, [K+]o needed to be lowered to 1.5 mM. In the presence of the ionophore gramicidin, [K+]o reduction to only 2.5 mM yielded the same effect. Acetazolamide normalized this increased frequency of P2 fibers. The findings mimic hypokalemic periodic paralysis (HypoPP), a channelopathy characterized by hypokalemia-induced weakness. Of myofibers from 7 HypoPP patients, up to 25% were in P2 at a [K+]o of 4 mM, in accordance with their permanent weakness, and up to 99% were in P2 at a [K+]o of 1.5 mM, in accordance with their paralytic attacks. Of 36 HypoPP patients, 25 had permanent weakness and myoplasmic intracellular Na+ ([Na+]i) overload (up to 24 mM) as shown by in vivo 23Na-MRI. Acetazolamide normalized [Na+]i and increased muscle strength. HypoPP myofibers showed a nonselective cation leak of 12–19.5 μS/cm2, which may explain the Na+ overload. The leak sensitizes myofibers to reduced serum K+, and the resulting membrane depolarization causes the weakness. We postulate that the principle of paradoxical depolarization and loss of function upon [K+]o reduction may apply to other tissues, such as heart or brain, when they become leaky (e.g., because of ischemia).

Sodium channelopathies of skeletal muscle result from gain or loss of function

Five hereditary sodium channelopathies of skeletal muscle have been identified. Prominent symptoms are either myotonia or weakness caused by an increase or decrease of muscle fiber excitability. The voltage-gated sodium channel NaV1.4, initiator of the muscle action potential, is mutated in all five disorders. Pathogenetically, both loss and gain of function mutations have been described, the latter being the more frequent mechanism and involving not just the ion-conducting pore, but aberrant pores as well. The type of channel malfunction is decisive for therapy which consists either of exerting a direct effect on the sodium channel, i.e., by blocking the pore, or of restoring skeletal muscle membrane potential to reduce the fraction of inactivated channels.

Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons

See Borgkvist et al. (doi:10.1093/brain/awu150) for a scientific commentary on this article. D2 autoreceptors and L-type calcium channels are both implicated in Parkinson’s disease, but how they interact is unclear. Dragicevic et al. reveal that L-type calcium channels can modulate D2-autoreceptor responses via the neuronal calcium sensor NCS-1. This dopamine-dependent signalling network is altered in Parkinson’s disease and could represent a therapeutic target.

Mutual exacerbation of peroxisome proliferator-activated receptor γ coactivator 1α deregulation and α-synuclein oligomerization

Aggregation of α‐synuclein (α‐syn) and α‐syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC‐1α in mitochondria, no studies have addressed whether PGC‐1α directly influences oligomerization of α‐syn or whether α‐syn oligomers impact PGC‐1α expression.

Ion channels and ion transporters of the transverse tubular system of skeletal muscle

This review focuses on the electrical properties of the transverse (T) tubular membrane of skeletal muscle, with reference to the contribution of the T-tubular system (TTS) to the surface action potential, the radial spread of excitation and its role in excitation-contraction coupling. Particularly, the most important ion channels and ion transporters that enable proper depolarization and repolarization of the T-tubular membrane are described. Since propagation of excitation along the TTS into the depth of the fibers is a delicate balance between excitatory and inhibitory currents, the composition of channels and transporters is specific to the TTS and different from the surface membrane. The TTS normally enables the radial spread of excitation and the signal transfer to the sarcoplasmic reticulum to release calcium that activates the contractile apparatus. However, due to its structure, even slight shifts of ions may alter its volume, Nernstian potentials, ion permeabilities, and consequently T-tubular membrane potential and excitability.

Neutralization of a negative charge in the S1–S2 region of the KV7.2 (KCNQ2) channel affects voltage-dependent activation in neonatal epilepsy

The voltage‐gated potassium channels KV7.2 and KV7.3 (genes KCNQ2 and KCNQ3) constitute a major component of the M‐current controlling the firing rate in many neurons. Mutations within these two channel subunits cause benign familial neonatal convulsions (BFNC). Here we identified a novel BFNC‐causing mutation (E119G) in the S1–S2 region of KV7.2. Electrophysiological investigations in Xenopus oocytes using two‐microelectrode voltage clamping revealed that the steady‐state activation curves for E119G alone and its coexpressions with KV7.2 and/or KV7.3 wild‐type (WT) channels were significantly shifted in the depolarizing direction compared to KV7.2 or KV7.2/KV7.3. These shifts reduced the relative current amplitudes for mutant channels particularly in the subthreshold range of an action potential (about 45% reduction at −50 mV for E119G compared to KV7.2, and 33% for E119G/KV7.3 compared to KV7.2/KV7.3 channels). Activation kinetics were significantly slowed for mutant channels. Our results indicate that small changes in channel gating at subthreshold voltages are sufficient to cause neonatal seizures and demonstrate the importance of the M‐current for this voltage range. This was confirmed by a computer model predicting an increased burst duration for the mutation. On a molecular level, these results reveal a critical role in voltage sensing of the negatively charged E119 in S1–S2 of KV7.2, a region that – according to molecular modelling – might interact with a positive charge in the S4 segment.

Orchestrated increase of dopamine and PARK mRNAs but not miR-133b in dopamine neurons in Parkinson's disease.

Progressive loss of substantia nigra dopamine neurons (SN DA) is a hallmark of aging and of Parkinsons disease (PD). Mutations in PARK genes cause familial PD forms. Increased expression of alpha-synuclein (PARK4) is a disease-triggering event in familial PD and also observed in SN DA neurons in sporadic PD but related transcriptional changes are unknown. With optimized single-cell quantitative real-time polymerase chain reaction analysis, we compared messenger RNA and microRNA levels in SN DA neurons from sporadic PD patients and controls. Non-optimally matched donor ages and RNA integrities are common problems when analyzing human samples. We dissected the influence of distinct ages and RNA integrities of our samples by applying a specifically-optimized, linear-mixed-effects model to quantitative real-time polymerase chain reaction-data. We identified that elevated alpha-synuclein messenger RNA levels in SN DA neurons of human PD brains were positively correlated with corresponding elevated levels of mRNAs for functional compensation of progressive SN DA loss and for enhanced proteasomal (PARK5/UCHL1) and lysosomal (PARK9/ATPase13A2) function, possibly counteracting alpha-synuclein toxicity. In contrast, microRNA miR-133b levels, previously implicated in transcriptional dysregulation in PD, were not altered in SN DA neurons in PD.

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen‐Tawil syndrome (ATS), a familial disorder leading to stress‐triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time‐resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patients individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.—Seebohm, G., Strutz‐Seebohm, N., Ursu, O. N., Preisig‐Müller, R., Zuzarte, M., Hill, E. V., Kienitz, M.‐C., Bendahhou, S., Fauler, M., Tapken, D., Decher, N., Collins, A., Jurkat‐Rott, K., Steinmeyer, K., Lehmann‐Horn, F., Daut, J., Tavaré, J. M., Pott, L., Bloch,W., Lang, F. Altered stress stimulation of inward rectifier potassium channels in Andersen‐Tawil syndrome. FASEB J. 26, 513–522 (2012). www.fasebj.org

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.