iPSC-Derived Dopamine Neurons Reveal Differences between Monozygotic Twins Discordant for Parkinson’s Disease

SummaryParkinson’s disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation (GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins’ fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of “footprint-free” iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin’s neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin

Safety, Pharmacokinetics, and Pharmacodynamics of Oral Venglustat in Patients with Parkinson's Disease and a GBA Mutation: Results from Part 1 of the Randomized, Double-Blinded, Placebo-Controlled MOVES-PD Trial

BACKGROUND: Glucocerebrosidase gene (GBA) mutations influence risk and prognosis of Parkinson's disease (PD), possibly through accumulation of glycosphingolipids, including glucosylceramide (GL-1). Venglustat is a novel, brain penetrant glucosylceramide synthase inhibitor. OBJECTIVE: Evaluate venglustat pharmacology, safety, and tolerability in patients with PD and GBA mutations (GBA-PD). METHODS: Part 1 of the phase 2 MOVES-PD trial (NCT02906020) was a randomized, double-blinded, placebo-controlled, dose-escalation study performed in six countries. Eligible participants included Japanese and non-Japanese patients aged 18-80 years with PD diagnosis and heterozygous GBA mutation. Participants were randomized to three doses of once-daily oral venglustat or placebo and were followed up to 36 weeks (Japanese participants: 52 weeks). Primary endpoint was venglustat safety and tolerability versus placebo. Secondary and exploratory endpoints included venglustat pharmacokinetics and pharmacodynamics. RESULTS: Participants (N = 29) received venglustat (Japanese, n = 9; non-Japanese, n = 13) or placebo (n = 3; n = 4). Eight (89%) Japanese and 12 (92%) non-Japanese venglustat-treated participants experienced at least one adverse event (AE) versus two (67%) and four (100%) participants from the respective placebo groups. Most AEs were mild or moderate; no serious AEs or deaths occurred. Two venglustat-treated non-Japanese participants discontinued due to AEs (confusional state and panic attack). Over 4 weeks, venglustat exposure in plasma and cerebrospinal fluid (CSF) increased, and GL-1 levels in plasma and CSF decreased, both in a dose-dependent manner. At the highest dose, CSF GL-1 decreased by 72.0% in Japanese and 74.3% in non-Japanese participants. CONCLUSION: Venglustat showed favorable safety and tolerability in MOVES-PD Part 1 and target engagement was achieved in CSF

Murine Models of Lysosomal Storage Diseases Exhibit Differences in Brain Protein Aggregation and Neuroinflammation

Genetic, epidemiological and experimental evidence implicate lysosomal dysfunction in Parkinson’s disease (PD) and related synucleinopathies. Investigate several mouse models of lysosomal storage diseases (LSDs) and evaluate pathologies reminiscent of synucleinopathies. We obtained brain tissue from symptomatic mouse models of Gaucher, Fabry, Sandhoff, Niemann–Pick A (NPA), Hurler, Pompe and Niemann–Pick C (NPC) diseases and assessed for the presence of Lewy body-like pathology (proteinase K-resistant α-synuclein and tau aggregates) and neuroinflammation (microglial Iba1 and astrocytic GFAP) by immunofluorescence. All seven LSD models exhibited evidence of proteinopathy and/or inflammation in the central nervous system (CNS). However, these phenotypes were divergent. Gaucher and Fabry mouse models displayed proteinase K-resistant α-synuclein and tau aggregates but no neuroinflammation; whereas Sandhoff, NPA and NPC showed marked neuroinflammation and no overt proteinopathy. Pompe disease animals uniquely displayed widespread distribution of tau aggregates accompanied by moderate microglial activation. Hurler mice also demonstrated proteinopathy and microglial activation. The present study demonstrated additional links between LSDs and pathogenic phenotypes that are hallmarks of synucleinopathies. The data suggest that lysosomal dysregulation can contribute to brain region-specific protein aggregation and induce widespread neuroinflammation in the brain. However, only a few LSD models examined exhibited phenotypes consistent with synucleinopathies. While no model can recapitulate the complexity of PD, they can enable the study of specific pathways and mechanisms contributing to disease pathophysiology. The present study provides evidence that there are existing, previously unutilized mouse models that can be employed to study pathogenic mechanisms and gain insights into potential PD subtypes, helping to determine if they are amenable to pathway-specific therapeutic interventions

Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity

Human Umbilical vein: Involvement of cyclooxygenase-2 pathway in bradykinin B1 receptor sensitized responses

In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-senzitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 μmol/l) or NS-398 (10, 30 μmol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.Fil: Errasti, Andrea Emilse. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rey Ares, Veronica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Daray, Federico Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Rogines Velo, M. P.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Sardi, Sergio Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Paz, Cristina del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Podesta, Ernesto Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Rothlin, Rodolfo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

Characterization of α(1)-adrenoceptor subtypes mediating vasoconstriction in human umbilical vein

1. The present study attempted to characterize pharmacologically the subtypes of α-adrenoceptors mediating contractions in human umbilical vein (HUV). 2. HUV rings were mounted in isolated organ baths and cumulative concentration-response curves were constructed for the α-adrenoceptor agonists phenylephrine and adrenaline. Adrenaline was more potent than phenylephrine (pD(2)=7.29 and 6.04 respectively). 3. Isoproterenol exhibited no agonism on KCl pre-contracted HUV rings. Propranolol (1 μM) and rauwolscine (0.1 μM) did not affect the concentration-response curves to adrenaline. These results demonstrate the lack of involvement of functional β- or α(2)-adrenoceptors in adrenaline-induced vasoconstriction. 4. The non subtype selective α(1)-adrenoceptor antagonist prazosin was evaluated on phenylephrine and adrenaline concentration-response curves. The effects of the competitive α(1A) and α(1D)-adrenoceptor antagonists, 5-methyl urapidil and BMY 7378 and the irreversible α(1B) selective compound chloroethylclonidine (CEC) were also evaluated on adrenaline concentration-response curves. 5. The potencies of prazosin against responses mediated by adrenaline (pA(2)=10.87) and phenylephrine (pA(2)=10.70) indicate the involvement of prazosin-sensitive functional α(1)-adrenoceptor subtype in vasoconstriction of the HUV. 6. The potencies of 5-methyl urapidil (pA(2)=6.70) and BMY 7378 (pA(2)=7.34) were not consistent with the activation of an α(1A)- or α(1D)-adrenoceptor population. 7. Exposure to a relatively low CEC concentration (3 μM) abolished the maximum response to adrenaline suggesting that this response was mediated by an α(1B)-adrenoceptor subtype. 8. We conclude that HUV express a prazosin-sensitive functional α(1)-adrenoceptor resembling the α(1B)-subtype according with the low pA(2) values for both 5-methyl urapidil and BMY 7378 and the high sensitivity to CEC

A <i>Drosophila dGBA1b</i> deletion results in glucocerebrosidase deficiency.

<p>(A) Comparison of protein sequence of human GBA1 and <i>Drosophila</i> dGBA1a and dGBA1b. Gray indicates similar residues, whereas black shading indicates identical residues. (B) Genomic organization of the <i>Drosophila GBA1</i> homologs, <i>dGBA1a</i> and <i>dGBA1b</i>, and the intervening <i>CG31413</i> gene. Orange and blue boxes represent coding and non-coding sequences, respectively. Black arrows indicate direction of transcription. Red arrows designate the breakpoints of the <i>GBA1</i>^<i>ΔTT</i> deletion allele. (C) There was no significant difference in the percentage of <i>GBA1</i>^<i>ΔTT</i> homozygotes and WT controls (<i>GBA1</i>^<i>+/+</i>) that survived the embryo to 1st instar larval transition, the 3^rd instar larval to pupal stage transition, or the pupal to adult stage transition. (D) Relative glucocerebrosidase (GCase) enzyme activity from isolated heads of 14-day-old controls and <i>GBA1</i>^<i>ΔTT</i> homozygotes. (E) Relative GCase enzyme activity from bodies excluding heads of 14-day-old controls and <i>GBA1</i>^<i>ΔTT</i> homozygotes. Error bars represent standard error of the mean (s.e.m.), ns indicates p>0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p

<i>GBA1</i>^<i>ΔTT</i> homozygotes display a memory deficit and neurodegeneration, but do not have dopaminergic neuron loss.

<p>(A) Latency time to initiate courtship in untrained 14-day-old males of indicated genotype. (B) Latency time to initiate courtship in 14-day-old males of indicated genotypes at 1 hour, 6 hours and 24 hours following training using a conditioned mating assay. Note the longer latency times in trained males (B) relative to untrained males (A). (C) Representative paraffin-embedded H&E-stained brain sections from 30-day-old controls and <i>GBA1</i>^<i>ΔTT</i> homozygotes. Yellow arrows indicate vacuoles. (D) Representative image of a projected Z-series of a control adult <i>Drosophila</i> brain stained with anti-Tyrosine Hydroxylase to label dopaminergic (DA) neurons. DA neurons within the PPL1 cluster are indicated by the circled regions. Scale bar, 200 μm. (E) Relative number of DA neurons within the PPL1 cluster of 30-day-old <i>GBA1</i>^<i>ΔTT</i> homozygotes (<i>GBA1</i>^{<i>ΔTT/ΔTT</i>}) <i>N</i> = 19, normalized to age-matched WT controls (<i>GBA1</i>^<i>+/+</i>) <i>N</i> = 21. There was no significant difference between the number of DA neurons within the PPL1 cluster per genotype by Student <i>t</i> test. Error bars represent s.e.m., ns indicates <i>p</i>>0.05, *<i>p</i><0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p

<i>GBA1</i>^<i>ΔTT</i> homozygotes exhibit shortened lifespan and behavioral phenotypes consistent with neuronal dysfunction.

<p>(A) Kaplan-Meier survival curves of WT controls (<i>GBA1</i>^<i>+/+</i>), <i>GBA1</i>^<i>ΔTT</i> heterozygotes (<i>GBA1</i>^{<i>ΔTT/+</i>}), <i>GBA1</i>^<i>ΔTT</i> homozygotes (<i>GBA1</i>^{<i>ΔTT/ΔTT</i>}), WT controls ectopically expressing <i>Drosophila</i> WT <i>dGBA1b</i> using the <i>Actin GAL4</i> driver (<i>Actin-GAL4>UAS-GBA1b;GBA</i>^<i>+/+</i>), <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing <i>Drosophila</i> WT <i>dGBA1b</i> using the <i>Actin GAL4</i> driver <i>(Act-GAL4>UAS-GBA1b;GBA1</i>^{<i>ΔTT/ΔTT</i>}), <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing human WT <i>GBA1</i> using the <i>Actin GAL4</i> driver (<i>Act-GAL4>UAS-hGBA1</i>^<i>WT</i><i>;GBA1</i>^{<i>ΔTT/ΔTT</i>}), and <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing human <i>GBA1</i> harboring the p.<i>N370S</i> mutation using the <i>Actin GAL4</i> driver (<i>Act-GAL4>UAS-hGBA1</i>^<i>N370S</i><i>;GBA1</i>^{<i>ΔTT/ΔTT</i>}). (B) Climbing index of 5-day-old flies of indicated genotypes as described in A. (C) Recovery time from mechanical stress (bang sensitivity) of flies of given genotypes as described in A at given adult ages. (D) Recovery time from heat stress of flies of given phenotypes as described in A at given adult ages. Error bars represent s.e.m., *<i>p</i><0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p