Dysregulated pathways in spinocerebellar ataxia type 2 and ataxia telangectasia

Cerebellar ataxias are a group of neurodegenerative disorders primarily affecting the cerebellum. Although causative mutations in several genes have been identified there is currently no cure for ataxias. The first part of this dissertation is focused on Spinocerebellar ataxia type 2 (SCA2). SCA2 is a dominant ataxia caused by repeat expansion mutations in the ATXN2 gene, which encodes the protein Ataxin2 (ATXN2). A polyglutamine (polyQ) tract consisting of CAG repeats interrupted by CAA was identified at exon 1 of ATXN2. Healthy individuals have between 22 and 23 glutamines, while expansions longer than 33 CAG repeats cause SCA2. The most noticeable symptom that SCA2 patients show is ataxic gait; however, they also show cerebellar dysarthria, dysdiadochokinesia, and ocular dysmetria caused by the progressive cerebellar degeneration. To model the SCA2 disease, we generated a new mouse model where 100 CAG repeats were introduced in the mouse Atxn2 gene via homologous recombination. The characterization of this mouse model, Atxn2-CAG100-KIN, demonstrated that it reproduces the symptomatology observed in SCA2 patients. These animals showed significant loss of weight over time, brain atrophy, and motor deficits. In addition, ATXN2 intermediate expansions have been linked to the pathology of Amyotrophic lateral sclerosis (ALS) as a risk factor. ALS is a fatal neurodegenerative disease where the motor neurons in the brain and spinal cord degenerate. A hallmark of ALS is the presence of TDP43-positive inclusions in neurons and glia. Further studies of post mortem spinal cord samples from SCA2 patients showed severe and widespread neurodegeneration of the central somatosensory system. Therefore, it was of interest to further investigate the pathology affection of this tissue in the Atxn2-CAG100-KIN line and the relationship between ATXN2 and TDP43. The characterization of the spinal cord pathology via protein quantification, transcript quantification, and immunohistochemistry showed a preferential affection of RNA binding proteins (RBP) in the spinal cord rather than the cerebellum. The ALS-linked factors TDP43 and TIA1 showed time-dependent co-aggregation with ATXN2 in spinal cord sections together with an increase of CASP3 levels. Therefore, this mouse model can help develop new therapies and evaluate their effect in differently affected areas. A transcriptome data set from Atxn2-CAG100-KIN spinal cord samples at the final disease stage of this mouse model showed a strong up-regulation of RNA toxicity-, immune- and lysosome-implicated factors. These data pointed to a pathological reactivation of the synaptic pruning and phagocytosis in microglia. ATXN2-positive aggregates were found in microglia from spinal cord sections of 14-month-old Atxn2-CAG100-KIN via immunohistochemistry. The characterization of microglial response and the potentially deleterious effects of the expanded ATXN2 in this cell type could lead to therapies to improve patients’ living standards or delay the symptoms’ onset. The second part of this thesis was focused on an autosomal recessive form of cerebellar ataxia, Ataxia Telangiectasia (A-T), with childhood onset. A-T patients show severe cerebellar atrophy manifesting as ataxia when the child starts to walk. The genetic cause of A-T is loss-of-function-mutations in the Ataxia Telangiectasia Mutated gene (ATM). ATM is a kinase involved in DNA damage response, oxidative stress, insulin resistance, autophagy via mTOR signaling, and synaptic function. Working with proteome data from cerebrospinal fluid of 12 A-T patients and 12 healthy controls, we aimed to define novel biomarkers that would allow following the neurodegeneration in extracellular fluid. Additional validation efforts with ~2-month-old Atm-knock-out (Atm-/-) cerebellar samples helped us to define a scenario were the deficit of vesicle-associated ATM alters the secretion of ApoB, reelin, and glutamate. As extracellular factors, apolipoproteins and their cargo such as vitamin E may be useful for neuroprotective interventions.Der erste Teil dieser Dissertation befasste sich mit der spinozerebellären Ataxie Typ 2 (SCA2). SCA2 ist eine dominante Ataxie, die durch wiederholte Expansionsmutationen im ATXN2-Gen verursacht wird, welches Protein Ataxin2 (ATXN2) kodiert. Ein Polyglutamin (polyQ) -Trakt, der aus durch CAA unterbrochenen CAG-Wiederholungen bestand, wurde im Exon 1 von ATXN2 identifiziert. Gesunde Personen haben zwischen 22 und 23 Glutamine, während Expansionen, die länger als 33 CAG-Wiederholungen sind, SCA2 verursachen. Das auffälligste Symptom bei SCA2-Patienten ist der ataxische Gang. Sie zeigen jedoch auch Kleinhirn-Dysarthrie, Dysdiadochokinese und Augendysmetrie, die durch die fortschreitende Kleinhirn-Degeneration verursacht werden. Um die SCA2-Krankheit zu modellieren, haben wir ein neues Mausmodell erstellt, bei dem 100 CAG-Wiederholungen über homologe Rekombination in das Maus-Atxn2-Gen eingeführt wurden. Die Charakterisierung dieses Mausmodells, Atxn2-CAG100-KIN, zeigte, dass es die bei SCA2-Patienten beobachtete Symptomatik reproduziert. Diese Tiere zeigten im Laufe der Zeit einen signifikanten Gewichtsverlust, eine Hirnatrophie und motorische Defizite. Darüber hinaus wurden ATXN2-Zwischenexpansionen mit der Pathologie der Amyotrophen Lateralsklerose (ALS) als Risikofaktor in Verbindung gebracht. ALS ist eine tödliche neurodegenerative Erkrankung, bei der die Motoneuronen im Gehirn und Rückenmark entartet sind. Ein Kennzeichen von ALS ist das Vorhandensein von TDP43-positiven Einschlüssen in Neuronen und Glia. Weitere Studien an post mortem-Rückenmarkproben von SCA2-Patienten zeigten eine schwere und weit verbreitete Neurodegeneration des zentralen somatosensorischen Systems. Daher war es von Interesse, die Pathologie dieses Gewebes in der Atxn2-CAG100-KIN-Linie und die Beziehung zwischen ATXN2 und TDP43 weiter zu untersuchen. Die Charakterisierung der Pathologie des Rückenmarks mittels Proteinquantifizierung, Transkriptquantifizierung und Immunhistochemie zeigte eine bevorzugte Beeinflussung von RNA-bindenden Proteinen im Rückenmark anstelle des Kleinhirns. Die ALS-verknüpften Faktoren TDP43 und TIA1 zeigten eine zeitabhängige Co-Aggregation mit ATXN2 in Rückenmarksschnitten zusammen mit einem Anstieg der CASP3-Spiegel. Daher kann dieses Mausmodell dazu beitragen, neue Therapien zu entwickeln und deren Wirkung in unterschiedlich betroffenen Bereichen zu bewerten. Die Erzeugung eines Transkriptomdatensatzes aus Atxn2-CAG100-KIN-Rückenmarksproben im Endstadium der Erkrankung dieses Mausmodells zeigte eine starke Hochregulation der RNA-Toxizitäts-, Immun- und Lysosomen-implizierten Faktoren. Diese Daten wiesen auf eine pathologische Reaktivierung des synaptischen Schnitts und der Phagozytose in Mikroglia hin. ATXN2-positive Aggregate wurden in Mikroglia aus Rückenmarksschnitten des 14 Monate alten Atxn2-CAG100-KIN mittels Immunhistochemie gefunden. Die Charakterisierung der Mikroglia-Reaktion und die potenziell schädlichen Wirkungen des expandierten ATXN2 in diesem Zelltyp könnten zu Therapien führen, mit denen der Lebensstandard der Patienten verbessert oder der Beginn der Symptome verzögert werden kann. Der zweite Teil dieser Arbeit befasste sich mit einer autosomal rezessiven Form der Kleinhirnataxie, Ataxia Telangiectasia (A-T), mit Beginn während der Kindheit. A-T-Patienten zeigen eine schwere Atrophie des Kleinhirns, die sich als Ataxie manifestiert, wenn das Kind zu laufen beginnt. Die genetische Ursache von A-T sind Funktionsverlustmutationen im Ataxia Telangiectasia Mutated Gen (ATM). ATM ist eine Kinase, die an DNA-Schadensantwort, oxidativem Stress, Insulinresistenz, Autophagie über mTOR-Signalübertragung und synaptischer Funktion beteiligt ist. In Zusammenarbeit mit Proteomdaten aus der Liquor cerebrospinalis von 12 A-T-Patienten und 12 gesunden Kontrollpersonen sollten neue Biomarker definiert werden, mit denen die Neurodegeneration in der extrazellulären Flüssigkeit verfolgt werden kann. Zusätzliche Validierungsbemühungen mit ~2 Monate alten Atm-Knock-out Kleinhirnproben halfen uns, ein Szenario zu definieren, in dem das Defizit von vesikelassoziierten ATM die Sekretion von ApoB, Reelin und Glutamat verändert. Als extrazelluläre Faktoren können Apolipoproteine und ihre Ladung wie Vitamin E für neuroprotektive Interventionen nützlich sein

Expression of GAD2 in excitatory neurons projecting from the ventrolateral periaqueductal gray to the locus coeruleus

Summary: The ventrolateral periaqueductal gray (vlPAG) functionally projects to diverse brain regions, including the locus coeruleus (LC). Excitatory projections from the vlPAG to the LC are well described, while few studies have indicated the possibility of inhibitory projections. Here, we quantified the relative proportion of excitatory and inhibitory vlPAG-LC projections in male and female mice, and found an unexpected overlapping population of neurons expressing both GAD2 and VGLUT2. Combined in vitro optogenetic stimulation and electrophysiology of LC neurons revealed that vlPAG neurons expressing channelrhodopsin-2 under the GAD2 promoter release both GABA and glutamate. Subsequent experiments identified a population of GAD2+/VGLUT2+ vlPAG neurons exclusively releasing glutamate onto LC neurons. Altogether, we demonstrate that ∼25% of vlPAG-LC projections are inhibitory, and that there is a significant GAD2 expressing population of glutamatergic projections. Our findings have broad implications for the utility of GAD2-Cre lines within midbrain and brainstem regions, and especially within the PAG

Generation of an Atxn2-CAG100 knock-in mouse reveals N-acetylaspartate production deficit due to early Nat8l dysregulation

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion mutations in the ATXN2 gene, mainly affecting motor neurons in the spinal cord and Purkinje neurons in the cerebellum. While the large expansions were shown to cause SCA2, the intermediate length expansions lead to increased risk for several atrophic processes including amyotrophic lateral sclerosis and Parkinson variants, e.g. progressive supranuclear palsy. Intense efforts to pioneer a neuroprotective therapy for SCA2 require longitudinal monitoring of patients and identification of crucial molecular pathways. The ataxin-2 (ATXN2) protein is mainly involved in RNA translation control and regulation of nutrient metabolism during stress periods. The preferential mRNA targets of ATXN2 are yet to be determined. In order to understand the molecular disease mechanism throughout different prognostic stages, we generated an Atxn2-CAG100-knock-in (KIN) mouse model of SCA2 with intact murine ATXN2 expression regulation. Its characterization revealed somatic mosaicism of the expansion, with shortened lifespan, a progressive spatio-temporal pattern of pathology with subsequent phenotypes, and anomalies of brain metabolites such as N-acetylaspartate (NAA), all of which mirror faithfully the findings in SCA2 patients. Novel molecular analyses from stages before the onset of motor deficits revealed a strong selective effect of ATXN2 on Nat8l mRNA which encodes the enzyme responsible for NAA synthesis. This metabolite is a prominent energy store of the brain and a well-established marker for neuronal health. Overall, we present a novel authentic rodent model of SCA2, where in vivo magnetic resonance imaging was feasible to monitor progression and where the definition of earliest transcriptional abnormalities was possible. We believe that this model will not only reveal crucial insights regarding the pathomechanism of SCA2 and other ATXN2-associated disorders, but will also aid in developing gene-targeted therapies and disease prevention

Pannexin-1 channel inhibition alleviates opioid withdrawal in rodents by modulating locus coeruleus to spinal cord circuitry

Abstract Opioid withdrawal is a liability of chronic opioid use and misuse, impacting people who use prescription or illicit opioids. Hyperactive autonomic output underlies many of the aversive withdrawal symptoms that make it difficult to discontinue chronic opioid use. The locus coeruleus (LC) is an important autonomic centre within the brain with a poorly defined role in opioid withdrawal. We show here that pannexin-1 (Panx1) channels expressed on microglia critically modulate LC activity during opioid withdrawal. Within the LC, we found that spinally projecting tyrosine hydroxylase (TH)-positive neurons (LCspinal) are hyperexcitable during morphine withdrawal, elevating cerebrospinal fluid (CSF) levels of norepinephrine. Pharmacological and chemogenetic silencing of LCspinal neurons or genetic ablation of Panx1 in microglia blunted CSF NE release, reduced LC neuron hyperexcitability, and concomitantly decreased opioid withdrawal behaviours in mice. Using probenecid as an initial lead compound, we designed a compound (EG-2184) with greater potency in blocking Panx1. Treatment with EG-2184 significantly reduced both the physical signs and conditioned place aversion caused by opioid withdrawal in mice, as well as suppressed cue-induced reinstatement of opioid seeking in rats. Together, these findings demonstrate that microglial Panx1 channels modulate LC noradrenergic circuitry during opioid withdrawal and reinstatement. Blocking Panx1 to dampen LC hyperexcitability may therefore provide a therapeutic strategy for alleviating the physical and aversive components of opioid withdrawal

A Bayesian reanalysis of the Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial

Background Timing of initiation of kidney-replacement therapy (KRT) in critically ill patients remains controversial. The Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial compared two strategies of KRT initiation (accelerated versus standard) in critically ill patients with acute kidney injury and found neutral results for 90-day all-cause mortality. Probabilistic exploration of the trial endpoints may enable greater understanding of the trial findings. We aimed to perform a reanalysis using a Bayesian framework. Methods We performed a secondary analysis of all 2927 patients randomized in multi-national STARRT-AKI trial, performed at 168 centers in 15 countries. The primary endpoint, 90-day all-cause mortality, was evaluated using hierarchical Bayesian logistic regression. A spectrum of priors includes optimistic, neutral, and pessimistic priors, along with priors informed from earlier clinical trials. Secondary endpoints (KRT-free days and hospital-free days) were assessed using zero–one inflated beta regression. Results The posterior probability of benefit comparing an accelerated versus a standard KRT initiation strategy for the primary endpoint suggested no important difference, regardless of the prior used (absolute difference of 0.13% [95% credible interval [CrI] − 3.30%; 3.40%], − 0.39% [95% CrI − 3.46%; 3.00%], and 0.64% [95% CrI − 2.53%; 3.88%] for neutral, optimistic, and pessimistic priors, respectively). There was a very low probability that the effect size was equal or larger than a consensus-defined minimal clinically important difference. Patients allocated to the accelerated strategy had a lower number of KRT-free days (median absolute difference of − 3.55 days [95% CrI − 6.38; − 0.48]), with a probability that the accelerated strategy was associated with more KRT-free days of 0.008. Hospital-free days were similar between strategies, with the accelerated strategy having a median absolute difference of 0.48 more hospital-free days (95% CrI − 1.87; 2.72) compared with the standard strategy and the probability that the accelerated strategy had more hospital-free days was 0.66. Conclusions In a Bayesian reanalysis of the STARRT-AKI trial, we found very low probability that an accelerated strategy has clinically important benefits compared with the standard strategy. Patients receiving the accelerated strategy probably have fewer days alive and KRT-free. These findings do not support the adoption of an accelerated strategy of KRT initiation

Initiation of continuous renal replacement therapy versus intermittent hemodialysis in critically ill patients with severe acute kidney injury: a secondary analysis of STARRT-AKI trial

Background: There is controversy regarding the optimal renal-replacement therapy (RRT) modality for critically ill patients with acute kidney injury (AKI). Methods: We conducted a secondary analysis of the STandard versus Accelerated Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial to compare outcomes among patients who initiated RRT with either continuous renal replacement therapy (CRRT) or intermittent hemodialysis (IHD). We generated a propensity score for the likelihood of receiving CRRT and used inverse probability of treatment with overlap-weighting to address baseline inter-group differences. The primary outcome was a composite of death or RRT dependence at 90-days after randomization. Results: We identified 1590 trial participants who initially received CRRT and 606 who initially received IHD. The composite outcome of death or RRT dependence at 90-days occurred in 823 (51.8%) patients who commenced CRRT and 329 (54.3%) patients who commenced IHD (unadjusted odds ratio (OR) 0.90; 95% confidence interval (CI) 0.75-1.09). After balancing baseline characteristics with overlap weighting, initial receipt of CRRT was associated with a lower risk of death or RRT dependence at 90-days compared with initial receipt of IHD (OR 0.81; 95% CI 0.66-0.99). This association was predominantly driven by a lower risk of RRT dependence at 90-days (OR 0.61; 95% CI 0.39-0.94). Conclusions: In critically ill patients with severe AKI, initiation of CRRT, as compared to IHD, was associated with a significant reduction in the composite outcome of death or RRT dependence at 90-days

Regional Practice Variation and Outcomes in the Standard Versus Accelerated Initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) Trial: A Post Hoc Secondary Analysis.

ObjectivesAmong patients with severe acute kidney injury (AKI) admitted to the ICU in high-income countries, regional practice variations for fluid balance (FB) management, timing, and choice of renal replacement therapy (RRT) modality may be significant.DesignSecondary post hoc analysis of the STandard vs. Accelerated initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial (ClinicalTrials.gov number NCT02568722).SettingOne hundred-fifty-three ICUs in 13 countries.PatientsAltogether 2693 critically ill patients with AKI, of whom 994 were North American, 1143 European, and 556 from Australia and New Zealand (ANZ).InterventionsNone.Measurements and main resultsTotal mean FB to a maximum of 14 days was +7199 mL in North America, +5641 mL in Europe, and +2211 mL in ANZ (p p p p p p p p = 0.007).ConclusionsAmong STARRT-AKI trial centers, significant regional practice variation exists regarding FB, timing of initiation of RRT, and initial use of continuous RRT. After adjustment, such practice variation was associated with lower ICU and hospital stay and 90-day mortality among ANZ patients compared with other regions