Gene Triplication Research Articles

Mitochondria-targeted oligomeric α-synuclein induces TOM40 degradation and mitochondrial dysfunction in Parkinson’s disease and parkinsonism-dementia of Guam

Mitochondrial dysfunction is a central aspect of Parkinson’s disease (PD) pathology, yet the underlying mechanisms are not fully understood. This study investigates the link between α-Synuclein (α-Syn) pathology and the loss of translocase of the outer mitochondrial membrane 40 (TOM40), unraveling its implications for mitochondrial dysfunctions in neurons. We discovered that TOM40 protein depletion occurs in the brains of patients with Guam Parkinsonism-Dementia (Guam PD) and cultured neurons expressing α-Syn proteinopathy, notably, without corresponding changes in TOM40 mRNA levels. Cultured neurons expressing α-Syn mutants, with or without a mitochondria-targeting signal (MTS) underscores the role of α-Syn’s mitochondrial localization in inducing TOM40 degradation. PDe-related etiological factors, such as 6-hydroxydopamine or ROS/metal ions stress, which promotes α-Syn oligomerization, exacerbate TOM40 depletion in PD patient-derived cells with SNCA gene triplication. Although α-Syn interacts with both TOM40 and TOM20 in the outer mitochondrial membrane, degradation is selective for TOM40, which occurs via the ubiquitin-proteasome system (UPS) pathway. Our comprehensive analyses using Seahorse technology, mitochondrial DNA sequencing, and damage assessments, demonstrate that mutant α-Syn-induced TOM40 loss results in mitochondrial dysfunction, characterized by reduced membrane potential, accumulation of mtDNA damage, deletion/insertion mutations, and altered oxygen consumption rates. Notably, ectopic supplementation of TOM40 or reducing pathological forms of α-Syn using ADP-ribosylation inhibitors ameliorate these mitochondrial defects, suggesting potential therapeutic avenues. In conclusion, our findings provide crucial mechanistic insights into how α-Syn accumulation leads to TOM40 degradation and mitochondrial dysfunction, offering insights for targeted interventions to alleviate mitochondrial defects in PD.

Optical genome mapping of structural variants in Parkinson’s disease-related induced pluripotent stem cells

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants, as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast, larger SVs, which may significantly influence human phenotypes, have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study, we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines, which were not identified by long-read sequencing. Additionally, various exon deletions were confirmed by OGM in the PRKN gene of iPSCs, of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs, no gene fusions, no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary, OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques, which is supportive for OGM’s future use in gene discovery and iPSC line screening.

Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

Down syndrome and DYRK1A overexpression: relationships and future therapeutic directions.

Down syndrome is a genetic-based disorder that results from the triplication of chromosome 21, leading to an overexpression of many triplicated genes, including the gene encoding Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A). This protein has been observed to regulate numerous cellular processes, including cell proliferation, cell functioning, differentiation, and apoptosis. Consequently, an overexpression of DYRK1A has been reported to result in cognitive impairment, a key phenotype of individuals with Down syndrome. Therefore, downregulating DYRK1A has been explored as a potential therapeutic strategy for Down syndrome, with promising results observed from in vivo mouse models and human clinical trials that administered epigallocatechin gallate. Current DYRK1A inhibitors target the protein function directly, which tends to exhibit low specificity and selectivity, making them unfeasible for clinical or research purposes. On the other hand, antisense oligonucleotides (ASOs) offer a more selective therapeutic strategy to downregulate DYRK1A expression at the gene transcript level. Advances in ASO research have led to the discovery of numerous chemical modifications that increase ASO potency, specificity, and stability. Recently, several ASOs have been approved by the U.S. Food and Drug Administration to address neuromuscular and neurological conditions, laying the foundation for future ASO therapeutics. The limitations of ASOs, including their high production cost and difficulty delivering to target tissues can be overcome by further advances in ASO design. DYRK1A targeted ASOs could be a viable therapeutic approach to improve the quality of life for individuals with Down syndrome and their families.

Dissecting the contribution of human chromosome 21 syntenic regions to recognition memory processes in adult and aged mouse models of Down syndrome.

Trisomy of human chromosome 21 (Hsa21) results in a constellation of features known as Down syndrome (DS), the most common genetic form of intellectual disability. Hsa21 is orthologous to three regions in the mouse genome on mouse chromosome 16 (Mmu16), Mmu17 and Mmu10. We investigated genotype-phenotype relationships by assessing the contribution of these three regions to memory function and age-dependent cognitive decline, using three mouse models of DS, Dp1Tyb, Dp(17)3Yey, Dp(10)2Yey, that carry an extra copy of the Hsa21-orthologues on Mmu16, Mmu17 and Mmu10, respectively. Prior research on cognitive function in DS mouse models has largely focused on models with an extra copy of the Mmu16 region and relatively little is known about the effects of increased copy number on Mmu17 and Mmu10 on cognition and how this interacts with the effects of aging. As aging is is a critical contributor to cognitive and psychiatric changes in DS, we hypothesised that ageing would differentially impact memory function in Dp1Tyb, Dp(17)3Yey, and Dp(10)2Yey, models of DS. Young (12-13 months and old (18-20 months mice Dp1Tyb, Dp(17)3Yey and Dp(10)2Yey mice were tested on a battery of object recognition memory test that assessed object novelty detection, novel location detection and associative object-in place memory. Following behavioral testing, hippocampal and frontal cortical tissue was analysed for expression of glutamatergic receptor proteins using standard immunoblot techniques. Young (12-13 months and old (18-20 months mice Dp1Tyb, Dp(17)3Yey and Dp(10)2Yey mice were tested on a battery of object recognition memory test that assessed object novelty detection, novel location detection and associative object-in place memory. Following behavioral testing, hippocampal and frontal cortical tissue was analysed for expression of glutamatergic receptor proteins using standard immunoblot techniques. Our results show that distinct Hsa21-orthologous regions contribute differentially to cognitive dysfunction in DS mouse models and that aging interacts with triplication of Hsa21-orthologous genes on Mmu10.

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson's Disease Animal Model.

Parkinson's disease (PD) caused by SNCA gene triplication (3XSNCA) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3XSNCA and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3XSNCA, a gene-edited isogenic line with a frame-shift mutation on all SNCA alleles (SNCA 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the SNCA 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3XSNCA and SNCA 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, SNCA gene alterations-either amplification or knock-out-negatively impact Th+ dopaminergic neuron maturation. These findings highlight SNCA's critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.

®-Thalassemia intermedia: implication of presenting 〈-globin gene triplication

®-Thalassemia intermedia: implication of presenting 〈-globin gene triplication

Complete F9 Gene Deletion, Duplication, and Triplication Rearrangements: Implications for Factor IX Expression and Clinical Phenotypes.

Factor IX (FIX) plays a critical role in blood coagulation. Complete deletion of F9 results in severe hemophilia B, whereas the clinical implications of complete F9 duplication and triplication remain understudied. To investigate the rearrangement mechanisms underlying complete F9 deletion (cases 1 and 2), duplication (cases 3 and 4), and triplication (case 5), and to explore their association with FIX expression levels and clinical impacts. Plasma FIX levels were detected using antigen and activity assays. CNVplex technology, optical genome mapping, and long-distance polymerase chain reaction were employed to characterize the breakpoints of the chromosomal rearrangements. Cases 1 and 2 exhibited FIX activities below 1%. Case 3 displayed FIX activities within the reference range. However, cases 4 and 5 showed a significant increase in FIX activities. Alu-mediated nonallelic homologous recombination was identified as the cause of F9 deletion in case 1; FoSTeS/MMBIR (Fork Stalling and Template Switching/microhomology-mediated break-induced replication) contributed to both F9 deletion and tandem duplication observed in cases 2 and 3; BIR/MMBIR (break-induced replication/microhomology-mediated break-induced replication) mediated by the same pair of low-copy repeats results in similar duplication-triplication/inversion-duplication (DUP-TRP/INV-DUP) rearrangements in cases 4 and 5, leading to complete F9 duplication and triplication, respectively. Large deletions involving the F9 gene exhibit no apparent pattern, and the extra-hematologic clinical phenotypes require careful analysis of other genes within the deletion. The impact of complete F9 duplication and triplication on FIX expression might depend on the integrity of the F9 upstream sequence and the specific rearrangement mechanisms. Notably, DUP-TRP/INV-DUP rearrangements significantly elevate FIX activity and are closely associated with thrombotic phenotypes.

Luspatercept in the Treatment of Beta Thalassemia in Italy: Lights and Shadows in Clinical Practice

Luspatercept has been demonstrated to reduce the need for blood transfusion with manageable side effects in clinical trials, thus receiving the approval for the treatment of anemia associated with transfusion dependent beta thalassemia in adults. However, very little data on its safety and efficacy in real life have been hitherto published. Special warnings and precautions for use refer to the possibility of occurrence of thromboembolic events, extramedullary hemopoiesis masses and increased blood pressure. Ten Italian Specialized Centres for the treatment of Hemoglobinopathies, with the patronage of the “Società Italiana Talassemie ed Emoglobinopatie” (SITE), evaluated the effects of luspatercept in terms of safety and blood transfusion reduction in 116 patients (Table 1) who received the first dose of the drug after its marketing. The median duration of treatment was 158 days (min-max: 15-442). β globin genotype was β 0/β 0 in 42 subjects (36.2%), β 0/ β + in 33 (28.4%), β +/ β + in 23 (19.8%), β 0/β wt plus α gene triplication in 4 (3.4%) and unknown in 14 (12%). Sixty-four patients (55.2%) were splenectomized. At the time of data collection, 32 patients (27.6%) had already discontinued the drug. Discontinuation had occurred after a median time of 132 days of treatment (min-max: 15-338) due to insufficient efficacy in the judgement of the physician (10) or patient (3), side effects attributed to luspatercept (16) that were detrimental to the continuation of therapy in the judgement of the physician (6) or patient (10), pregnancy (1) and other causes (2). There were 13 patients with a transfusion-free interval of at least 8 weeks during the treatment period (median 12 weeks, min-max range 9-48). Their median age at baseline was 37.6 years (min-max range 18.4 - 51.6) and age at first transfusion 24 years (min-max range 1 - 33). β globin genotype was β 0/ β 0 in 1 (7.7 %), β 0/ β + in 2 (15.4 %), β +/ β + in 7 (53.8 %), β 0/ β wt plus α gene triplication in 3 (23.1 %). Patients who decreased transfusion requirements by ≥ 33% and ≥ 50% in at least one of the follow-up periods of 12-weeks were 52 (44.8%) and 36 (31%), respectively. When considering the 13-24 week interval (considered as the primary endpoint of the Believe study), of the 66 patients who completed the second follow-up, 31 of 66 patients (47.0%) reduced their transfusion load by ≥33% and 20 of 66 patients (30%) by ≥50%. Focusing on the first two quarters of treatment, the pairwise t test showed a progressive and significant (p<0.05) reduction in the number of transfused units and pure red blood cell volumes (figure). There were no significant differences between the pre-treatment hemoglobin value and that during treatment. Serum ferritin also decreased progressively (p<0.05) during treatment (basal: median 576 ng/mL (Q1-Q3: 289-992); weeks 1-12: 479 ng/mL (232- 848); weeks 13-24: 385 ng/mL (212-644). The pregnancy that was initiated during the luspatercept treatment is still ongoing and no definitive information can be given. Sixty patients (51.7%) reported at least one side effect potentially related to luspatercept. The most frequent side effects were bone pain (40), asthenia-fatigue (11), hypertension requiring therapy or modification of therapy (11), joint pain (11), folic acid deficiency (6), heart palpitations (4) and edema (4). In two patients, an asymptomatic increased volume of masses of extramedullary erythropoiesis was reported. Another two patients presented with thromboembolic events: ischemic stroke in one case and superficial venous thrombosis in the other. Both were splenectomized with no other known risk factors. Despite the limitations of the short duration of follow-up, the analysis reports several significant results. The high percentage of patients who decided not to continue treatment underlines the need for a proper management of patient expectations and selection. The drug proves to be effective also in real life and those who respond best in the short and medium term seem to be who already have residual erythropoiesis, as seen from the age at the start of transfusions and the genotype of those who have had a long period of transfusion independence. Although the drug is generally well-tolerated, some adverse events appear more frequent than reported in Believe. Finally, the report of the two patients who developed a thromboembolic event including stroke, suggests the importance of continuing to be cautious in this regard.

The Cell Wall-Related Gene Families of Wheat (Triticum aestivum)

Wheat crops provide 20% of calories worldwide. Cell walls function in plant growth, are part of biotic and abiotic stress resistance, and provide plant mechanical strength and adaptability. These functions factor into the productivity of wheat. The genes that produce and maintain the plant cell wall are up to 10% of the genome in many varied families. Previously, curated cell wall gene families have been published for maize and rice, two other important crop grasses. Here, 81 cell wall-related wheat gene families curated via sequence similarity to maize and rice and unique family protein motif searches are presented. A total of 4086 wheat, 1118 maize, 1036 rice, and 955 Arabidopsis genes were aligned and placed into gene family trees to present homologs for all four species. Due to hexaploidy, many wheat cell wall gene families show expected triplication of genes per family over maize, rice, and Arabidopsis. However, several families contained more wheat genes than expected. The utility of this research is demonstrated with an example from a pre-harvest sprouting study to identify specific gene families rather than the less descriptive identification available with standard bioinformatic searches.

Prevalence and genetic analysis of triplicated α-globin gene in Ganzhou region using high-throughput sequencing.

α-globin gene triplication carriers were not anemic in general, while some studies found that α-globin gene triplication coinherited with heterozygous β-thalassemia may cause adverse clinical symptoms, which yet lacks sufficient evidence in large populations. In this study, we investigated the prevalence and distribution of α-globin gene triplication as well as the phenotypic characteristics of α-globin gene triplication coinherited with heterozygous β-thalassemia in Ganzhou city, southern China. During 2021-2022, a total of 73,967 random individuals who received routine health examinations before marriage were genotyped for globin gene mutations by high-throughput sequencing. Among them, 1,443 were α-globin gene triplication carriers, with a carrier rate of 1.95%. The most prevalent mutation was αααanti3.7/αα (43.10%), followed by αααanti4.2/αα (38.12%). 42 individuals had coinherited α-globin gene triplication and heterozygous β-thalassemia. However, they did not differ from the individuals with heterozygous β-thalassemia and normal α-globin (αα/αα) in terms of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels. In addition, heterogenous clinical phenotypes were found in two individuals with the same genotype. Our study established a database of Ganzhou α-globin gene triplication and provided practical advice for the clinical diagnosis of α-globin gene triplication.

Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

Fragile X messenger ribonucleoprotein 1 protein (FMRP) binds many mRNA targets in the brain. The contribution of these targets to fragile X syndrome (FXS) and related autism spectrum disorder (ASD) remains unclear. Here, we show that FMRP deficiency leads to elevated microtubule-associated protein 1B (MAP1B) in developing human and non-human primate cortical neurons. Targeted MAP1B gene activation in healthy human neurons or MAP1B gene triplication in ASD patient-derived neurons inhibit morphological and physiological maturation. Activation of Map1b in adult male mouse prefrontal cortex excitatory neurons impairs social behaviors. We show that elevated MAP1B sequesters components of autophagy and reduces autophagosome formation. Both MAP1B knockdown and autophagy activation rescue deficits of both ASD and FXS patients’ neurons and FMRP-deficient neurons in ex vivo human brain tissue. Our study demonstrates conserved FMRP regulation of MAP1B in primate neurons and establishes a causal link between MAP1B elevation and deficits of FXS and ASD.

Molecular basis of a high Hb A2/Hb Fβ-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication.

β 0-thalassemia deletion removing 5´β-globin promoter usually presents phenotype with high hemoglobin (Hb) A2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the β 0-thalassemia with 3.4 kb deletion. A total of 148 subjects, including 127 heterozygotes, 20 Hb E-β-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at A γ-globin promoter, rs5006884 on OR51B6 gene, -158 G γ-XmnI, BCL11A binding motifs (TGGTCA) between 3´A γ-globin gene and 5´δ-globin gene. It was found that heterozygous β 0-thalassemia and Hb E-β 0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous β 0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-β 0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous β 0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain β-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´β-globin promoter may likely be responsible for this unusual phenotype. The results indicate that β 0-thalassemia with 3.4 kb deletion is a mild β-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis.

TBX1 copy number variant of unknown significance presenting as severe esophageal stricture and recurrent candidiasis

IntroductionTBX1 Is involved in pharyngeal pouch development affecting thymus, parathyroid, craniofacial, and cardiac outflow tract development. TBX1 deletions present with phenotype of Di George syndrome. We present the case of a patient with esophageal stricture and candidiasis showing a novel TBX1 gene triplication VUS. Case descriptionA 27-year-old male was hospitalized for hematemesis and progressive dysphagia. The physical exam was remarkable for short stature, underweight, dental hypoplasia, alopecia and occipital vitiligo. Calcium and lymphocyte numbers were normal. Severe esophageal strictures precluded upper endoscopy. He received IV fluconazole for two weeks to continue oral antifungal indefinitely. Since infancy he has had multiple candida infections in nails and oral mucosa requiring fluconazole, and medical history of asthma and atopic dermatitis. No history of cardiac abnormalities. At 14 years old he started with progressive dysphagia. Barium swallow and endoscopies showed severe acute and chronic esophagitis and thoracic esophageal strictures with severe stenosis. Patient's sister has history of candida infections, hypocalcemia due to hypoparathyroidism, and hypothyroidism. There is consanguinity among paternal grandparents. Invitae PID panel showed VUS with TBX1 gene triplication, not previously reported. Familial genetic testing was requested for her sister to support the diagnosis of DiGeorge Syndrome. DiscussionTBX1 gene deletions present a spectrum clinical phenotype including thymic hypoplasia, hypocalcemia, and congenital heart defects. TBX1 duplication and gain of function TBX1 heterozygous mutation led to protein dysfunction and a similar phenotype caused by loss-of-function mutations or deletions. TBX1 triplication could plausibly lead to dysfunctional protein expression, which requires confirmation. TBX1 is a negative regulator of thymic epithelial cell differentiation and proliferation, which could affect immune regulation and explain patient's chronic candida infections. Feeding difficulties and esophageal dysmotility have been reported in Di George syndrome. Sibling's hypocalcemia due to hypoparathyroidism could be explained by TBX1 dysfunction. APECED was considered in differential diagnosis due to chronic mucocutaneous candidiasis and sister's endocrinopathies, nevertheless, no AIRE or STAT1 mutations identified. Genetic analysis and familiar VUS have the potential to identify the cause of PIDs. TBX1 transcript and protein expression analysis will contribute to confirming the diagnostic impression.

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

The Impact of Mmu17 Non-Hsa21 Orthologous Genes in the Ts65Dn Mouse Model of Down Syndrome: The Gold Standard Refuted

BackgroundDespite successful preclinical treatment studies to improve neurocognition in the Ts65Dn mouse model of Down syndrome, translation to humans has failed. This raises questions about the appropriateness of the Ts65Dn mouse as the gold standard. We used the novel Ts66Yah mouse that carries an extra chromosome and the identical segmental Mmu16 trisomy as Ts65Dn without the Mmu17 non-Hsa21 orthologous region. MethodsForebrains from embryonic day 18.5 Ts66Yah and Ts65Dn mice, along with euploid littermate controls, were used for gene expression and pathway analyses. Behavioral experiments were performed in neonatal and adult mice. Because male Ts66Yah mice are fertile, parent-of-origin transmission of the extra chromosome was studied. ResultsForty-five protein-coding genes mapped to the Ts65Dn Mmu17 non-Hsa21 orthologous region; 71%–82% are expressed during forebrain development. Several of these genes are uniquely overexpressed in Ts65Dn embryonic forebrain, producing major differences in dysregulated genes and pathways. Despite these differences, the primary Mmu16 trisomic effects were highly conserved in both models, resulting in commonly dysregulated disomic genes and pathways. Delays in motor development, communication, and olfactory spatial memory were present in Ts66Yah but more pronounced in Ts65Dn neonates. Adult Ts66Yah mice showed milder working memory deficits and sex-specific effects in exploratory behavior and spatial hippocampal memory, while long-term memory was preserved. ConclusionsOur findings suggest that triplication of the non-Hsa21 orthologous Mmu17 genes significantly contributes to the phenotype of the Ts65Dn mouse and may explain why preclinical trials that used this model have unsuccessfully translated to human therapies.

Lewy Body-like Pathology and Loss of Dopaminergic Neurons in Midbrain Organoids Derived from Familial Parkinson's Disease Patient.

Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson's disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

Dysregulated systemic metabolism in a Down syndrome mouse model

ObjectiveTrisomy 21 is one of the most complex genetic perturbations compatible with postnatal survival. Dosage imbalance arising from the triplication of genes on human chromosome 21 (Hsa21) affects multiple organ systems. Much of Down syndrome (DS) research, however, has focused on addressing how aneuploidy dysregulates CNS function leading to cognitive deficit. Although obesity, diabetes, and associated sequelae such as fatty liver and dyslipidemia are well documented in the DS population, only limited studies have been conducted to determine how gene dosage imbalance affects whole-body metabolism. Here, we conduct a comprehensive and systematic analysis of key metabolic parameters across different physiological states in the Ts65Dn trisomic mouse model of DS. MethodsTs65Dn mice and euploid littermates were subjected to comprehensive metabolic phenotyping under basal (chow-fed) state and the pathophysiological state of obesity induced by a high-fat diet (HFD). RNA sequencing of liver, skeletal muscle, and two major fat depots were conducted to determine the impact of aneuploidy on tissue transcriptome. Pathway enrichments, gene-centrality, and key driver estimates were performed to provide insights into tissue autonomous and non-autonomous mechanisms contributing to the dysregulation of systemic metabolism. ResultsUnder the basal state, chow-fed Ts65Dn mice of both sexes had elevated locomotor activity and energy expenditure, reduced fasting serum cholesterol levels, and mild glucose intolerance. Sexually dimorphic deterioration in metabolic homeostasis became apparent when mice were challenged with a high-fat diet. While obese Ts65Dn mice of both sexes exhibited dyslipidemia, male mice also showed impaired systemic insulin sensitivity, reduced mitochondrial activity, and elevated fibrotic and inflammatory gene signatures in the liver and adipose tissue. Systems-level analysis highlighted conserved pathways and potential endocrine drivers of adipose-liver crosstalk that contribute to dysregulated glucose and lipid metabolism. ConclusionsA combined alteration in the expression of trisomic and disomic genes in peripheral tissues contribute to metabolic dysregulations in Ts65Dn mice. These data lay the groundwork for understanding the impact of aneuploidy on in vivo metabolism.

Full-length whole-genome sequencing analysis of emerged meropenem-resistant mutants during long-term in vitro exposure to meropenem for borderline meropenem-susceptible carbapenemase-producing and non-carbapenemase-producing Enterobacterales.

Molecular analysis of meropenem-resistant mechanisms in mutants emerging from long-term in vitro meropenem exposure to borderline meropenem-susceptible carbapenemase-producing Enterobacterales (CPE) and non-CPE. Escherichia coli TUM13867 harbouring both blaIMP-6- and blaCTX-M-2-carrying IncN plasmid and Citrobacter koseri TUM13189 with blaCTX-M-2-carrying chromosome were used. Meropenem MIC was 1 mg/L against both strains. Each strain was cultured in the hollow-fibre infection model (HFIM) to approximately 1 × 106 colony formation unit (cfu)/mL, and meropenem 1 g q8h treatment was initiated. Then, changes in total and meropenem-resistant populations were observed for 124 h. Meropenem resistance mechanisms were analysed using full-length whole-genome sequencing (WGS), reverse-transcription quantitative PCR and digital PCR. Meropenem reduced TUM13867 and TUM13189 to approximately 5 and 2 log10 cfu/mL, respectively, at 2 h after initiation, but regrowth was observed at 24 h. The meropenem-resistant mutant emergence frequency at 120 and 124 h was 4.4 × 10-4 for TUM13867 and 7.6 × 10-1 for TUM13189. Meropenem MIC of the mutants derived from TUM13867 (TUM20902) and TUM13189 (TUM20903) increased 4- and 16-fold, respectively. TUM20902, which harboured pMTY20902_IncN plasmid with a 27 505-bp deletion that included blaCTX-M-2, and blaIMP-6 showed 4.21-fold higher levels of transcription than the parental strain. TUM20903 had a 49 316-bp deletion that included ompC and a replicative increase of blaCTX-M-2 to three copies. Molecular analysis including full-length WGS revealed that the resistance mechanisms of meropenem-resistant mutants that emerged during long-term in vitro meropenem exposure were increased blaIMP-6 transcripts in CPE and increased blaCTX-M-2 transcripts due to gene triplication and OmpC loss resulting from ompC deletion in non-CPE.

Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications

Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson’s disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation.