The characterization of the Atxn2-CAG42-knock-in mouse as a model for Spinocerebellar Ataxia Type 2

Die Ursache der neurodegenerativen Erkrankung Spinozerebelläre Ataxie Typ 2 (SCA2) ist eine expandierte Polyglutamin-Domäne im humanen ATXN2-Gen von normalerweise 22 auf über 31 CAGs. Von der Degeneration sind vorwiegend die zerebellären Purkinje Neuronen betroffen, in denen zunehmend zytoplasmatische Aggregate sichtbar werden. Auch wenn die genaue Funktion von ATXN2 und die zugrunde liegenden molekularen Mechanismen noch immer ungeklärt sind, werden ein toxischer Funktionsgewinn sowie der Verlust der normalen Proteinfunktion als mögliche Ursachen diskutiert.rnUm ein wirklichkeitsgetreues Tiermodell für die SCA2 zu haben, wurde eine knock-in Maus generiert, deren einzelnes CAG im Atxn2-Gen durch 42 CAGs ersetzt wurde. Dieses Mausmodell ist durch eine stabile Vererbung der Expansion charakterisiert. Weiterhin zeigt sie ein verringertes Körpergewicht sowie eine spät beginnende motorische Inkoordination, was dem Krankheitsbild von SCA2 entspricht. rnIm Weiteren konnte gezeigt werden, dass, obwohl die Atxn2 mRNA-Spiegel in Großhirn und Kleinhirn erhöht waren, die Menge an löslichem ATXN2 im Laufe der Zeit abnahm und dies mit einem Auftreten an unlöslichem ATXN2 korrelierte. Dieser im Kleinhirn progressive Prozess resultierte schließlich in zytoplasmatischen Aggregaten innerhalb der Purkinje Neuronen alter Mäuse. Der Verlust an löslichem ATXN2 könnte Effekte erklären, die auf einen partiellen Funktionsverlust von ATXN2 zurückzuführen sind, wobei die Aggregatbildung einen toxischen Funktionsgewinn wiederspiegeln könnte. Neben ATXN2 wurde auch sein Interaktor PABPC1 zunehmend unlöslich. Während dies im Großhirn eine Erhöhung der PABPC1 mRNA- und löslichen Proteinspiegel zur Folge hatte, konnte keine kompensatorische Veränderung seiner mRNA und zudem eine Verminderung an löslichem PABPC1 im Kleinhirn beobachtet werden. Auch PABPC1 wurde in Aggregate sequestriert. Diese Unterschiede zwischen Großhirn und Kleinhirn könnten zu der spezifischen Vulnerabilität des Kleinhirns beitragen.rnUm die Folgen auf mRNA-Prozessierung zu untersuchen, wurde ein Transkriptomprofil im mittleren sowie fortgeschrittenen Alter der Mäuse erstellt. Hierbei war eine erhöhte Expression von Fbxw8 im Kleinhirn alter Mäuse auffällig. Als Komponente eines Ubiquitin-E3-Ligase-Komplexes, hilft FBXW8 in der Degradierung von Zielproteinen und könnte somit die Toxizität des expandieren ATXN2 verringern. rnZur näheren Beschreibung der physiologischen Funktion von ATXN2, konnte in ATXN2-knock-out Mäusen gezeigt werden, dass das Fehlen von ATXN2 zu einer reduzierten globalen Proteinsyntheserate führte und somit eine Rolle als Translationsaktivator möglich erscheint. Kompensatorisch wurde eine erhöhte S6-Phosphorylierung gemessen.

Nerve excitability studies characterize Kv1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1

Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K(+) channel subunit K(v)1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, K(v)1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of K(v)1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify K(v)1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.

Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A.

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.

Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A.

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel CaV2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant CaV2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.

Neuronal calcium channel mutation in a patient with congenital ataxia and attacks of hemiplegic migraine with cerebral edema

Background: Familial Hemiplegic Migraine (FHM), characterized by a prolonged unilateral hemiparesis, mainly results from mutations in the alpha-1a subunit of the calcium channel gene CACNA1A that can also cause two other dominantly inherited neurological disorders, Episodic Ataxia type 2 (EA2, with sometimes migrainous headaches) and Spinocerebellar Ataxia type 6 (SCA6, late-onset and progressive). A same mutation can have different clinical expression in a family (hemiplegic migraine, migraine-coma, cerebellar ataxia). CACNA1A mutations in FHM are usually missense, leading to gain-of-function, while truncating mutations leading to loss-of-function are usually associated with EA2. Case report: This 9-year-old girl was seen as a baby for hypotonia and transient vertical nystagmus. Her first brain MRI was normal. She evolved as a congenital ataxia, but since the age of two, she had attacks of coma, hemiparesis (either side), partial seizures, dystonic movements and fever. Attacks were initially triggered by minor head bumps, subsequently spontaneous. Brain MRIs in the acute stage always showed transient unilateral hemisphere swelling. Follow-up images revealed atrophic lesions in the temporo-occipital regions and cerebellar atrophy. A prophylactic trial with flunarizine was ineffective. Acetazolamide was recently introduced. Methods: Since our patient shared features of both FHM and EA2, we studied the CACNA1A gene by direct sequencing in the patient's and parents' DNA. Results: We identified an unreported de novo heterozygous deletion of three base pairs (c.4503_4505delCTT) predicting the deletion of one amino acid (p.Phe1502del). The CACNA1A protein contains 4 domains, each formed by six transmembrane segments. The deletion is located in a highly conserved region in segment 6 (S6) of the third domain. Mutations in S6 segments of calcium channels change single-channel conductance and channel selectivity, most resulting in loss-of-function. Outlook: In vitro expression studies of the identified mutation are underway, aiming at understanding its functional consequences and finding an efficient treatment.

Autofluorescence Findings Of A Novel Maculopathy In Patient With Spinocerebellar Ataxia Type 1 And Of A Cone - Rod Type Retinal Dysfunction In Three Generation Family With Spinocerebellar Ataxia Type 7

Purpose: To report a novel maculopathy in a patient with SCA1. To describe autofluorescence findings in family with SCA7 and associated cone-rod retinal dysfunction.Methods: 4 affected patients from two families were assessed to investigate a progressive loss of visual acuity (VA). Examinations included fundus photography, autofluorescence (AF) fundus fluorescein angiogragraphy (FFA) and optical coherence tomography. Electroretinogram (full-field) was performed in 2 affected patients. All patients had color vision testing using Ishihara pseudoisochromatic plates. Molecular analysis was performed in family 2.Results: The patient with known diagnosis of SCA1 had a visual acuity of 20/200 bilaterally and dyschromatopsia. He had saccadic pursuit. Fundus examination showed mild retinal pigment epithelium (RPE) changes at the macula. OCT showed bilateral macular serous detachment, which was not obvious at the FFA and explained his VA. AF imaging showed a central hyperfluorescence. The 45 year old proband from family 2 had a visual acuity of 200/20 and dyschromatopsia. ERG testing showed cone type dysfunction of photoreceptors. Her daughter affected at a younger age had the same ERGs findings. Fundus examination showed mild RPE changes in proband, normal findings in her daughter. AF imaging of both patients showed a ring of high density AF around the fovea. The ring was also obvious on near infrared AF. Later onset of gait imbalance led to the diagnosis of SCA7Conclusions: Within the group of spinocerebellar ataxias, only the type 7 is associated with retinal dysfunction. We present the first report of maculopathy associated with SCA1 causing severe vision loss. The ring of high density AF in SCA7 confirmed an early retinal photoreceptor dysfunction in patient with normal fundus.

Intravenous dipyrone for the acute treatment of episodic tension-type headache: A randomized, placebo-controlled, double-blind study

Acute headaches are responsible for a significant percentage of the case load at primary care units and emergency rooms in Brazil. Dipyrone (metamizol) is easily available in these settings, being the most frequently used drug. We conducted a randomized, placebo-controlled, double-blind study to assess the effect of dipyrone in the acute treatment of episodic tension-type headache. Sixty patients were randomized to receive placebo (intravenous injection of 10 ml saline) or 1 g dipyrone in 10 ml saline. We used seven parameters of analgesic evaluation. The patients receiving dipyrone showed a statistically significant improvement (P<0.05) of pain compared to placebo up to 30 min after drug administration. The therapeutic gain was 30% in 30 min and 40% in 60 min. The number of patients needed to be treated for at least one to have benefit was 3.3 in 30 min and 2.2 in 60 min. There were statistically significant reductions in the recurrence (dipyrone = 25%, placebo = 50%) and use of rescue medication (dipyrone = 20%, placebo = 47.6%) for the dipyrone group. Intravenous dipyrone is an effective drug for the relief of pain in tension-type headache and its use is justified in the emergency room setting.

Type 2 diabetes and impaired glucose tolerance are associated with word memory source monitoring recollection deficits but not simple recognition familiarity deficits following water, low glycaemic load, and high glycaemic load breakfasts.

Isolated source monitoring recollection deficits indicate that abnormalities in glucose metabolism are not detrimental for global episodic memory processes. This enhances our understanding of how metabolic disorders are associated with memory impairments.

Non B-DNA structures and genomic instability associated with myotonic dystrophy type 2 (CCTG).(CAGG) repeats

There are many diseases associated with the expansion of DNA repeats in humans. Myotonic dystrophy type 2 is one of such diseases, characterized by expansions of a (CCTG)•(CAGG) repeat tract in intron 1 of zinc finger protein 9 (ZNF9) in chromosome 3q21.3. The DM2 repeat tract contains a flanking region 5' to the tract that consists of a polymorphic repetitive sequence (TG)14-25(TCTG)4-11(CCTG) n. The (CCTG)•(CAGG) repeat is typically 11-26 repeats in persons without the disease, but can expand up to 11,000 repeats in affected individuals, which is the largest expansion seen in DNA repeat diseases to date. This DNA tract remains one of the least characterized disease-associated DNA repeats, and mechanisms causing the repeat expansion in humans have yet to be elucidated. Alternative, non B-DNA structures formed by the expanded repeats are typical in DNA repeat expansion diseases. These sequences may promote instability of the repeat tracts. I determined that slipped strand structure formation occurs for (CCTG)•(CAGG) repeats at a length of 42 or more. In addition, Z-DNA structure forms in the flanking human sequence adjacent to the (CCTG)•(CAGG) repeat tract. I have also performed genetic assays in E. coli cells and results indicate that the (CCTG)•(CAGG) repeats are more similar to the highly unstable (CTG)•(CAG) repeat tracts seen in Huntington's disease and myotonic dystrophy type 1, than to those of the more stable (ATTCT)•(AGAAT) repeat tracts of spinocerebellar ataxia type 10. This instability, however, is RecA-independent in the (CCTG)•(CAGG) and (ATTCT)•(AGAAT) repeats, whereas the instability is RecA-dependent in the (CTG)•(CAG) repeats. Structural studies of the (CCTG)•(CAGG) repeat tract and the flanking sequence, as well as genetic selection assays may reveal the mechanisms responsible for the repeat instability in E. coli, and this may lead to a better understanding of the mechanisms contributing to the human disease state. ^

Elevated Micronucleus Frequency In Patients With Type 2 Diabetes, Dyslipidemia And Periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Undulatory Physical Resistance Training Program Increases Maximal Strength In Elderly Type 2 Diabetics.

To investigate the effects of a specific protocol of undulatory physical resistance training on maximal strength gains in elderly type 2 diabetics. The study included 48 subjects, aged between 60 and 85 years, of both genders. They were divided into two groups: Untrained Diabetic Elderly (n=19) with those who were not subjected to physical training and Trained Diabetic Elderly (n=29), with those who were subjected to undulatory physical resistance training. The participants were evaluated with several types of resistance training's equipment before and after training protocol, by test of one maximal repetition. The subjects were trained on undulatory resistance three times per week for a period of 16 weeks. The overload used in undulatory resistance training was equivalent to 50% of one maximal repetition and 70% of one maximal repetition, alternating weekly. Statistical analysis revealed significant differences (p<0.05) between pre-test and post-test over a period of 16 weeks. The average gains in strength were 43.20% (knee extension), 65.00% (knee flexion), 27.80% (supine sitting machine), 31.00% (rowing sitting), 43.90% (biceps pulley), and 21.10% (triceps pulley). Undulatory resistance training used with weekly different overloads was effective to provide significant gains in maximum strength in elderly type 2 diabetic individuals.

Surgical Treatment Of Type 2 Diabetes In Subjects With Mild Obesity: Mechanisms Underlying Metabolic Improvements.

This study aims to assess the clinical and physiological effects of Roux-en-Y gastric bypass (RYGBP) on type 2 diabetes associated with mild obesity (body mass index [BMI] 30-34.9 kg/m(2)) over 24 months postsurgery. In this prospective trial, 36 mildly obese subjects (19 males) with type 2 diabetes using oral antidiabetic drugs with (n = 24) or without insulin (n = 12) underwent RYGBP. Follow-up was conducted at baseline and 3, 6, 12, and 24 months postsurgery. The following endpoints were considered: changes in HbA1c, fasting glucose and insulin, antidiabetic therapy, BMI, oral glucose insulin sensitivity [OGIS, from meal tolerance test (MTT)], beta-cell secretory function [ΔCP(0-30)/ΔGlu(0-30) (ΔC-peptide/Δglucose ratio, MTT 0-30 min), disposition index (DI = OGIS [Symbol: see text] ΔCP(0-30)/ΔGlu(0-30)], glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) [incremental area under the curve (AUCi)], adiponectin, C-reactive protein, and lipids. All subjects achieved normal-to-overweight BMI after 3 months. Over 24 months, 31/36 (86 %) subjects presented HbA1c <7 % [complete and partial remission of diabetes in 9/36 (22 %) and 1/36 (3 %), respectively]. Since 3 months postsurgery, improvements were observed in OGIS [290 (174) to 373 (77) ml/min/m(2), P = 0.009], ΔCP(0-30)/ΔGlu(0-30) [0.24 (0.19) to 0.52 (0.34) ng/mg, P = 0.001], DI [7.16 (8.53) to 19.8 (15.4) (ng/mg) (ml/min/m(2)), P = 0.001], GLP-1 AUCi [0.56 (0.64) to 3.97 (3.86) ng/dl [Symbol: see text] 10 min [Symbol: see text] 103, P = 0.000], and GIP AUCi [30.2 (12.6) to 27.0 (20.2) ng/dl [Symbol: see text] 10 min [Symbol: see text] 103, P = 0.004]. At baseline and after 12 months, subjects with diabetes nonremission had longer diabetes duration, higher HbA1c, lower beta-cell secretory function, and higher first 30-min GIP AUCi, compared with those with remission. RYGBP improves the glucose metabolism in subjects with type 2 diabetes and mild obesity. This effect is associated with improvement of insulin sensitivity, beta-cell secretory function, and incretin secretion.