Trigeminal neuropathic pain development and maintenance in rats are suppressed by a positive modulator of alpha 6 GABA(A) receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors containing the alpha 6 subunit are located in trigeminal ganglia, and their reduction by small interfering RNA increases inflammatory temporomandibular and myofascial pain in rats. We thus hypothesized that enhancing their activity may help in neuropathic syndromes originating from the trigeminal system. Here, we performed a detailed electrophysiological and pharmacokinetic analysis of two recently developed deuterated structurally similar pyrazoloquinolinone compounds. DK-I-56-1 at concentrations below 1 mu M enhanced gamma-aminobutyric acid (GABA) currents at recombinant rat alpha 6 beta 3 gamma 2, alpha 6 beta 3 delta and alpha 6 beta 3 receptors, whereas it was inactive at most GABA(A) receptor subtypes containing other alpha subunits. DK-I-87-1 at concentrations below 1 mu M was inactive at alpha 6-containing receptors and only weakly modulated other GABA(A) receptors investigated. Both plasma and brain tissue kinetics of DK-I-56-1 were relatively slow, with half-lives of 6 and 13 hr, respectively, enabling the persistence of estimated free brain concentrations in the range 10-300 nM throughout a 24-hr period. Results obtained in two protocols of chronic constriction injury of the infraorbital nerve in rats dosed intraperitoneally with DK-I-56-1 during 14 days after surgery or with DK-I-56-1 or DK-I-87-1 during 14 days after trigeminal neuropathy were already established, demonstrated that DK-I-56-1 but not DK-I-87-1 significantly reduced the hypersensitivity response to von Frey filaments. Significance Neuropathic pain induced by trigeminal nerve damage is poorly controlled by current treatments. DK-I-56-1 that positively modulates alpha 6 GABA(A) receptors is appropriate for repeated administration and thus may represent a novel treatment option against the development and maintenance of trigeminal neuropathic pain

Biomarkers of probable Alzheimer’s disease

S staranjem prebivalstva postaja demenca vedno večji problem. Kar 70 %, od več kot 47 milijonov, primerov demence spada pod diagnozo Alzheimerjeve bolezni. Pod zgodnjo obliko Alzheimerjeve bolezni spada le od 1 % do 5 % vseh primerov. Za to obliko bolezni je značilna mendelska oblika dedovanja. Pri pozni obliki Alzheimerjeve bolezni je genetska komponenta šibkejša. Blaga kognitivna motnja je diagnoza, postavljena ljudem, ki imajo težave s kognicijo, vendar le-ti ne vplivajo na njihovo vsakdanje življenje. Ti ljudje imajo povišano tveganje za razvoj Alzheimerjeve bolezni. Če oseba opaža zmanjšanje kognitivne zmogljivosti, vendar klinični testi ne kažejo upada, se osebo uvrsti pod diagnozo subjektivne kognitivne pritožbe. Tveganje, ki ga ta skupina ljudi nosi za razvoj demence, še vedno ni znano. Najdenih je bilo že več kot dvajset genov, ki verjetno vplivajo na tveganje za pojav pozne oblike Alzheimerjeve bolezni, od katerih je najmočnejšo povezavo imel gen z zapisom za apolipoprotein E (ApoE). Od treh alelov je alel ApoE E4 povezan s povečanjem tveganja za razvoj bolezni, pri čemer je tveganje pri heterozigotih ApoE E4/- kar 3-krat višje kot pri homozigotih ApoE E3/E3, pri homozigotih ApoE E4/E4 pa kar od 8 do 12-krat. V naši raziskavi smo si zadali dva cilja: I) določiti frekvence alelov ApoE pri bolnikih z Alzheimerjevo boleznijo v Sloveniji in II) najti nove potencialne biološke označevalce v že obstoječih podatkovnih zbirkah, ki bi jih bilo možno uporabiti pri diagnosticiranju verjetne Alzheimerjeve bolezni. V raziskavi je sodelovalo 113 bolnikov, obravnavanih v ambulantah Centra za kognitivne motnje na Kliničnem oddelku za bolezni živčevja, Nevrološke klinike, Univerzitetnega kliničnega centra Ljubljana. Preiskovancem smo odvzeli kri, iz katere je bila izolirana DNA. Obenem smo zbrali tudi njihove klinične podatke. Zaradi prenizkega števila udeležencev s postavljeno diagnozo Alzheimerjeve bolezni, smo analizo opravili na bolnikih z diagnozami subjektivne kognitivne pritožbe, blage kognitivne motnje in kognitivnega upada. Tri skupine so se med seboj razlikovale v starosti in rezultatih testov kognicije, vendar ne v frekvencah genotipov in alelov ApoE. Genotipizirana populacija se po frekvencah genotipov ApoE statistično pomembno razlikuje od splošne evropske populacije Da bi našli nove potencialne biološke označevalce, smo zbrali že obstoječe asociacijske študije celotnega genoma, opravljene na populacijah bolnikov z Alzheimerjevo boleznijo. Rezultate teh študij smo analizirali s pomočjo spletnega orodja Integratomics (http://genepark.mf.uni-lj.si/integratomics/home). Najvišji rezultat si je delilo trinajst odsekov genoma, v katerih se nahaja devetnajst genov. Ti so bili preverjeni na neodvisnih podatkih iz literature. Le sedem genov je bilo predhodno povezanih s povišanim tveganjem za pojav Alzheimerjeve bolezni. Zaradi nizkega števila udeležencev, frekvenc alelov ApoE pri bolnikih z verjetno Alzheimerjevo boleznijo ni bilo možno določiti. V prihodnosti bi morali analizo ponoviti z večjim vzorcem. V integratomski analizi smo dobili kandidatne gene, ki bi jih morali potrditi s pomočjo genotipizacije pri bolnikih z Alzheimerjevo boleznijo in zdravih kontrolah. Zanimivo bi bilo tudi, da se v analizo vključi dodatne raziskave, ki niso asociacijske študije celotnega genoma.Dementia is becoming ever-bigger problem as the population ages. 70 % of 47 million cases of dementia is attributed to Alzheimer’s disease. Early onset Alzheimer’s disease accounts for about 1 % to 5 % of all cases. This type of the disease is typicaly inherited in a mendelian fashion. The genetic component is much weaker in cases of late onset Alzheimer’s disease. People with mild cognitive impairment usually have troubles with cognition but these troubles do not affect their everyday life. People with this diagnosis carry a higher risk for the development of Alzheimer’s disease. If a person notices a decline in cognitive abilities, although the clinical tests do not show it, a person is diagnosed with subjective cognitive complaint. The risk for developing dementia that people with subjective cognitive complaint carry is still not known. More than 20 genes were already found to probably affect the risk of developing late onset Alzheimer’s disease. The apolipoprotein E (ApoE) gene had the strongest association. From the three alleles, ApoE E4 is associated with higher risk for Alzheimer’s disease, with 3 times higher risk in heterozygotes ApoE E4/- and 8 to 12 times higher risk in homozygotes ApoE E4/E4, compared to homozygotes ApoE E3/E3. In our project, we formed two goals: I) assigning the frequencies of ApoE alleles in Slovene population of patients with Alzheimer’s disease and II) finding new potential biomarkers in preexistent databases, which could be used for the diagnosis of possible Alzheimer’s disease. In the project, we included 113 patients, treated in the clinic of Center for cognitive impairments at the Department of neurology, University medical centre, Ljubljana. We took the blood from the patients and extracted from it the DNA. We also acquired their clinical data. Because of the low number of patients, diagnosed with Alzheimer’s disease, we performed the analysis on patients, diagnosed with subjective cognitive complaint, mild cognitive impairment and cognitive decline. The three groups differed in age and results of cognitive tests, but not in the frequencies of ApoE genotypes and alleles. The genotyped population differed with statistical significance from general European population in the frequencies of ApoE genotypes. To find new potential biomarkers, we collected preexisting genome wide association studies, performed on populations of Alzheimer’s disease patients. We analyzed the results of these studies using an online tool Integratomics (http://genepark.mf.uni-lj.si/integratomics/home). The 13 regions, which shared the highest score, contained 19 genes. These were further verified in independent literature data. Of the 19 genes, only seven were described beforehand to have an association with the risk of Alzheimer’s disease. Because of a low number of participants, it was not possible to assess the frequencies of ApoE alleles in patients with Alzheimer’s disease. In the future, we would need to repeat the analysis with a higher number of participants. The integratomic analysis resulted in a list of candidate genes, which should be verified in a follow up, consisting of a genotyping of patients with Alzheimer’s disease and healthy controls. It would also be interesting to include additional non-genome wide association studies in the analysis

Zero-fluoroscopy catheter ablation of focal atrial tachycardia in a pregnant woman with tachycardia induced cardiomyopathy

Introduction: Occurrence of tachycardias increase during pregnancy in line with the increased propensity to ectopic activity. Case presentation: We present a case of a 30-year-old woman in the 18th week of pregnancy with atrial tachycardia and tachycardia-induced cardiomyopathy that was successfully treated with zero fluoroscopy catheter ablation. Discussion: The described method is safe and efficient and could be used in the future when drug refractory tachycardias occur during pregnancy

a6-Containing GABAA Receptors: Functional Roles and Therapeutic Potentials

GABAA receptors containing the α6 subunit are highly expressed in cerebellar granule cells and less abundantly in many other neuronal and peripheral tissues. Here, we for the first time summarize their importance for the functions of the cerebellum and the nervous system. The cerebellum is not only involved in motor control but also in cognitive, emotional, and social behaviors. α6βγ2 GABAA receptors located at cerebellar Golgi cell/granule cell synapses enhance the precision of inputs required for cerebellar timing of motor activity and are thus involved in cognitive processing and adequate responses to our environment. Extrasynaptic α6βδ GABAA receptors regulate the amount of information entering the cerebellum by their tonic inhibition of granule cells, and their optimal functioning enhances input filtering or contrast. The complex roles of the cerebellum in multiple brain functions can be compromised by genetic or neurodevelopmental causes that lead to a hypofunction of cerebellar α6-containing GABAA receptors. Animal models mimicking neuropsychiatric phenotypes suggest that compounds selectively activating or positively modulating cerebellar α6-containing GABAA receptors can alleviate essential tremor and motor disturbances in Angelman and Down syndrome as well as impaired prepulse inhibition in neuropsychiatric disorders and reduce migraine and trigeminal-related pain via α6-containing GABAA receptors in trigeminal ganglia. Genetic studies in humans suggest an association of the human GABAA receptor α6 subunit gene with stress-associated disorders. Animal studies support this conclusion. Neuroimaging and post-mortem studies in humans further support an involvement of α6-containing GABAA receptors in various neuropsychiatric disorders, pointing to a broad therapeutic potential of drugs modulating α6-containing GABAA receptors

Comparison of ADVIA Centaur ultra-sensitive and high-sensitive assays for troponin I in serum

Cardiac troponin I (cTnI) is a standard biomarker for the diagnosis of acute myocardial infarction (AMI). While older, ultra-sensitive cTnI (us-cTnI) assays use the 99th percentile as the reference threshold, newer high-sensitive cTnI (hs-cTnI) assays use the limit of detection or functional sensitivity instead. However, little has been done to systematically compare these two methods. The present study also served as a validation of hs-cTnI in our laboratory. Here, we compared the results obtained from the blood serum obtained from 8810 patients using the us-cTnI and the hs-cTnI assays run in tandem on the ADVIA Centaur XP analyser. We found that in 2279 samples the concentration of cTnI measured with the ultra-sensitive method was below the detection limit, while with the high-sensitive method, only 540 were below the detection limit. We also compared results from these assays with the ultimate diagnosis of a subset of individuals. The analysis of the results below cut-off with the ultra-sensitive method showed that this method would not detect 96 cases related to heart disorder. Overall, the main finding of our research is that hs-cTnI is the preferable option and is able to be deployed effectively in the laboratory setting

Towards functional selectivity for 632 GABAA receptors: a series of novel pyrazoloquinolinones

Background and Purpose The GABAA receptors are ligandgated ion channels, which play an important role in neurotransmission. Their variety of binding sites serves as an appealing target for many clinically relevant drugs. Here, we explored the functional selectivity of modulatory effects at specific extracellular +/ interfaces, using a systematically varied series of pyrazoloquinolinones. Experimental Approach Recombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABAelicited currents by the newly synthesized and reference compounds were investigated by the twoelectrode voltage clamp method. Key Results We identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at 632 GABAA receptors with nearly no residual activity at the other x32 (x = 15) subtypes. This modulation was independent of affinity for +/ interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular +/ interfaces. Conclusion and Implications These results constitute a major step towards a potential selective positive modulation of certain 6containing GABAA receptors, which might be useful to elicit their physiological role. Furthermore, these studies pave the way towards insights into molecular principles that drive positive versus negative allosteric modulation of specific GABAA receptor isoforms.(VLID)480595

Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity