Young human alpha synuclein transgenic (BAC-SNCA) mice display sex- and gene-dose-dependent phenotypic disturbances.

Parkinson's disease (PD) is a common neurodegenerative movement disorder, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of aggregated alpha synuclein (aSyn). The disease often presents with early prodromal non-motor symptoms and later motor symptoms. Diagnosing PD based purely on motor symptoms is often too late for successful intervention, as a significant neuronal loss has already occurred. Furthermore, the lower prevalence of PD in females is not well understood, highlighting the need for a better understanding of the interaction between sex and aSyn, the crucial protein for PD pathogenesis. Here, we conducted a comprehensive phenotyping study in 1- to 5-month-old mice overexpressing human aSyn gene (SNCA) in a bacterial artificial chromosome (BAC-SNCA). We demonstrate a SNCA gene-dose-dependent increase of human aSyn and phosphorylated aSyn, as well as a decrease in tyrosine hydroxylase expression in BAC-SNCA mice, with more pronounced effects in male mice. Phosphorylated aSyn was already found in the dorsal motor nucleus of the vagus nerve of 2-month-old mice. This was time-wise associated with significant gait altrations in BAC-SNCA mice as early as 1 and 3 months of age using CatWalk gait analysis. Furthermore, anxiety-related behavioral tests revealed an increase in anxiety levels in male BAC-SNCA mice. Finally, 5-month-old male BAC-SNCA mice exhibited a SNCA gene-dose-dependent elevation in energy expenditure in automated home-cage monitoring. For the first time, these findings describe early-onset, sex- and gene-dose-dependent, aSyn-mediated disturbances in BAC-SNCA mice, providing a model for sex-differences, early-onset neuropathology, and prodromal symptoms of PD.

Transcriptome and proteome associated analysis of flavonoid metabolism in haploid Ginkgo biloba.

Having different number if genome copies affect transcription and metabolite production of plants. This may be due to different gene transcription and protein expression, but the reasons for this remains poorly known. Here we measured flavonoid content in leaves of three haploid and diploid grafted plants of Ginkgo biloba, a model gymnosperm important economically for its flavonoid content. We reported the first combined transcriptomic and proteomic analysis of the difference in flavonoid content in three haploid ginkgos to investigate the effect of haploidy. Haploids had always smaller leaves and flavonoid content than the diploids. The selected haploid had also generally lower gene dosage than the selected diploid, with 1149 up-regulated (46.8 %) and 1309 down-regulated (53.2 %) among 2452 differentially expressed genes (DEGs). Of 686 differentially expressed proteins (DEPs) detected, 289 proteins (42.1 %) were upregulated, and 397 proteins (57.9 %) were downregulated in haploids. A particular attention deserves the downregulation of PAL, PAM, FLS, OMT1 hub genes involved in flavonoid biosynthesis regulation. Our study confirms the trend of haploids to have lower metabolic contents and points that lower flavonoid content in ginkgo monoploids could be due to reduced dosage of the corresponding regulatory genes and downregulation of genes involved in flavonoid synthesis.

Enhancing Cardiomyocyte Transcription Using In Vivo CRISPR/Cas9 Systems.

Endogenous gene activation by programmable transcription factors offers gene-dose-dependent phenotyping of target cells embedded in their in vivo natural tissue environment. Modified CRISPR/Cas9 systems were developed to be used as guide (g) RNA programmable transcriptional activation platforms (CRISPRa) in vitro and in vivo allowing targeted or multiplexed gene activation studies. We specifically developed these tools to be applied in cardiomyocytes providing dCas9VPR expressing mice under the control of the Myosin heavy chain 6 (Myh6) promoter. Here, we describe a protocol for the efficient design and validation of newly identified gRNA for enhancing transcriptional activity of a selected gene of interest. Additionally, we are providing insights into a downstream application in a dCas9VPR expressing mouse model specifically for cardiomyocyte biology.

Deletion of Abi3/Gngt2 influences age-progressive amyloid ß and tau pathologies in distinctive ways.

BACKGROUND: The S209F variant of Abelson Interactor Protein 3 (ABI3) increases risk for Alzheimer's disease (AD), but little is known about its function in relation to AD pathogenesis. METHODS: Here, we use a mouse model that is deficient in Abi3 locus to study how the loss of function of Abi3 impacts two cardinal neuropathological hallmarks of AD-amyloid ß plaques and tau pathology. Our study employs extensive neuropathological and transcriptomic characterization using transgenic mouse models and adeno-associated virus-mediated gene targeting strategies. RESULTS: Analysis of bulk RNAseq data confirmed age-progressive increase in Abi3 levels in rodent models of AD-type amyloidosis and upregulation in AD patients relative to healthy controls. Using RNAscope in situ hybridization, we localized the cellular distribution of Abi3 in mouse and human brains, finding that Abi3 is expressed in both microglial and non-microglial cells. Next, we evaluated Abi3-/- mice and document that both Abi3 and its overlapping gene, Gngt2, are disrupted in these mice. Using multiple transcriptomic datasets, we show that expression of Abi3 and Gngt2 are tightly correlated in rodent models of AD and human brains, suggesting a tight co-expression relationship. RNAseq of the Abi3-Gngt2-/- mice revealed upregulation of Trem2, Plcg2, and Tyrobp, concomitant with induction of an AD-associated neurodegenerative signature, even in the absence of AD-typical neuropathology. In APP mice, loss of Abi3-Gngt2 resulted in a gene dose- and age-dependent reduction in Aß deposition. Additionally, in Abi3-Gngt2-/- mice, expression of a pro-aggregant form of human tau exacerbated tauopathy and astrocytosis. Further, using in vitro culture assays, we show that the AD-associated S209F mutation alters the extent of ABI3 phosphorylation. CONCLUSIONS: These data provide an important experimental framework for understanding the role of Abi3-Gngt2 function and early inflammatory gliosis in AD. Our studies also demonstrate that inflammatory gliosis could have opposing effects on amyloid and tau pathology, highlighting the unpredictability of targeting immune pathways in AD.

Why Females Do Better: The X Chromosomal TLR7 Gene-Dose Effect in COVID-19.

A male sex bias has emerged in the COVID-19 pandemic, fitting to the sex-biased pattern in other viral infections. Males are 2.84 times more often admitted to the ICU and mortality is 1.39 times higher as a result of COVID-19. Various factors play a role in this, and novel studies suggest that the gene-dose of Toll-Like Receptor (TLR) 7 could contribute to the sex-skewed severity. TLR7 is one of the crucial pattern recognition receptors for SARS-CoV-2 ssRNA and the gene-dose effect is caused by X chromosome inactivation (XCI) escape. Female immune cells with TLR7 XCI escape have biallelic TLR7 expression and produce more type 1 interferon (IFN) upon TLR7 stimulation. In COVID-19, TLR7 in plasmacytoid dendritic cells is one of the pattern recognition receptors responsible for IFN production and a delayed IFN response has been associated with immunopathogenesis and mortality. Here, we provide a hypothesis that females may be protected to some extend against severe COVID-19, due to the biallelic TLR7 expression, allowing them to mount a stronger and more protective IFN response early after infection. Studies exploring COVID-19 treatment via the TLR7-mediated IFN pathway should consider this sex difference. Various factors such as age, sex hormones and escape modulation remain to be investigated concerning the TLR7 gene-dose effect.

Serum Concentrations of Cholinesterase Inhibitors in Patients With Alzheimer's Dementia Are Frequently Below the Recommended Levels.

BACKGROUND: Acetylcholinesterase inhibitors (AChE-I) are recommended for the treatment of cognitive symptoms but also of behavioral and psychological symptoms in dementia. They are widely used not only in Alzheimer's disease, but also in other forms of dementia. Efficacy of treatment might depend on serum concentration of the respective AChE-I. OBJECTIVE: In patients with mild to moderate Alzheimer's dementia, we measured serum concentrations of hepatically metabolized donepezil and renally excreted rivastigmine and investigated possible modifiers. Additionally, we looked at correlations between serum concentrations and efficacy for both drugs. METHODS: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR). Allele specific PCR were performed to determine CYP2D6 genotype and gene dose. Clinical efficacy was assessed by changes of the subtest wordlist delayed recall of the Consortium to Establish a Registry for Alzheimer's Disease-Neuropsychological Assessment Battery (CERAD-NAB). RESULTS: Sixty-seven patients treated with a stable dosage of donepezil 10 mg (n=41) or rivastigmine 9.5 mg (n=26) were included. Mean serum concentration of donepezil and rivastigmine were 41.2 and 6.5 ng/ml, respectively. Serum concentrations were below the recommended range in 73% of the subjects in the donepezil group and in 65% of the participants in the rivastigmine group. When applying a dose-related reference, ranges 63% of patients in the donepezil group and 32% in the rivastigmine group had concentrations below the expected range. Gene dose, sex, and duration of treatment significantly predicted donepezil serum concentration (p=0.046, p=0.001, p=0.030 respectively). Only for rivastigmine did the serum concentration significantly contribute to the regression model predicting changes on the subtest word list delayed recall (ß=0.472; p=0.019). CONCLUSIONS: Serum concentrations of about two thirds of the patients were below the recommended range. When not looking at absolute values but at the dose-related reference ranges, these numbers improved but still 32%, respectively 63% of patients had low serum concentrations. High serum concentrations of rivastigmine predicted clinical response to cognition. Therapeutic drug monitoring might help to identify the cause of poor clinical response to cognition and behavioral and psychological symptoms in patients with AChE-I treatment.

PMP22-related disease: A novel splice site acceptor variant and intrafamilial phenotype variability.

PMP22 is the most frequent mutated gene in Charcot-Marie-Tooth disease (CMT) type 1A. Another phenotype, hereditary neuropathy with pressure palsies (HNPP), could be caused by PMP22 mutations. PMP22 encodes a peripheral myelin protein with molecular weight 22-kDa. Various pathomechanisms have been postulated in PMP22-related disease, including dysfunction due to missense mutations, and alteration of a gene dose due to duplication/deletion mutations. We identified a novel PMP22 splice site acceptor variant, c.179-1G>A, in a patient with adult-onset chronic generalized polyneuropathy and two asymptomatic family members. Pathophysiological features of the members mainly overlapped with those reported in HNPP, but broad intrafamilial clinical variations were observed. PMP22 transcripts lacking of exon 4 were produced by the variant, presumably leading to in-frame deletion of 47 amino acids. The variant was also shown to exert effect on dosage of PMP22 mRNA. The complex molecular pathology would lead to the unique clinical and pathophysiological conditions.

HLA-DQ2 homozygosis increases tTGA levels at diagnosis but does not influence the clinical phenotype of coeliac disease: A multicentre study.

BACKGROUND AND PURPOSE: Magnitude of gluten-specific T-cell responses in coeliac disease (CD) might be dependent on HLA-DQ2 gene dose. We aimed to investigate the effects of HLA-DQB1*02 allele dose on clinical outcomes. METHODS: We reviewed the charts of all coeliac patients attending to three Hungarian university clinics after 1997 and included those patients, who (a) were diagnosed with CD, (b) underwent high-resolution HLA typing and (c) were ≥18 years at the time of data collection. HLA typing was performed to determine DQB1*02 allele dose. Patients were divided into risk groups by DQB1*02 allele dose, as follows: high-, intermediate- and low-risk groups corresponded to a double, single and zero doses, respectively. We used ANOVA and Pearson's chi-squared test to explore association between HLA risk and clinical variables. RESULTS: A total of 727 coeliac patients attended the clinics but only 105 (14.4%) patients were eligible for inclusion. High, intermediate and low HLA risk patients comprised 35.3%, 52.3% and 12.3% of the study population, respectively. Double dose of HLA-DQB1*02 was more frequent in patient with high tTGA level (>10 times the upper limit of normal; p = 0.045). Gene dose was not associated with younger age at diagnosis (p = 0.549), gender (p = 0.739), more severe diagnostic histology (p = 0.318), more frequent classical presentation (p = 0.846), anaemia (p = 0.611), metabolic bone disease (p = 0.374), dermatitis herpetiformis (p = 0.381) and autoimmune diseases (p = 0.837). CONCLUSIONS: Our study shows a significant gene dose effect in terms of tTGA level at diagnosis, but no significant association between HLA-DQB1*02 allele dose and the clinical outcomes in CD.

Gene dosage and coexpression with endoplasmic reticulum secretion-associated factors improved the secretory expression of α-galactosidase.

The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications.

Complete or partial reduction of the Met receptor tyrosine kinase in distinct circuits differentially impacts mouse behavior.

BACKGROUND: Our laboratory discovered that the gene encoding the receptor tyrosine kinase, MET, contributes to autism risk. Expression of MET is reduced in human postmortem temporal lobe in autism and Rett Syndrome. Subsequent studies revealed a role for MET in human and mouse functional and structural cortical connectivity. To further understand the contribution of Met to brain development and its impact on behavior, we generated two conditional mouse lines in which Met is deleted from select populations of central nervous system neurons. Mice were then tested to determine the consequences of disrupting Met expression. METHODS: Mating of Emx1 (cre) and Met (fx/fx) mice eliminates receptor signaling from all cells arising from the dorsal pallium. Met (fx/fx) and Nestin (cre) crosses result in receptor signaling elimination from all neural cells. Behavioral tests were performed to assess cognitive, emotional, and social impairments that are observed in multiple neurodevelopmental disorders and that are in part subserved by circuits that express Met. RESULTS: Met (fx/fx) /Emx1 (cre) null mice displayed significant hypoactivity in the activity chamber and in the T-maze despite superior performance on the rotarod. Additionally, these animals showed a deficit in spontaneous alternation. Surprisingly, Met (fx/fx; fx/+) /Nestin (cre) null and heterozygous mice exhibited deficits in contextual fear conditioning, and Met (fx/+) /Nestin (cre) heterozygous mice spent less time in the closed arms of the elevated plus maze. CONCLUSIONS: These data suggest a complex contribution of Met in the development of circuits mediating social, emotional, and cognitive behavior. The impact of disrupting developmental Met expression is dependent upon circuit-specific deletion patterns and levels of receptor activity.

Oncogene amplification as tumor marker in a group of Colombian lung cancer patients / Amplificación oncogénica como marcador tumoral en un grupo de pacientes con cáncer pulmonar

Introduction: In spite of recent treatment advances, lung cancer continues to be the first world cancer related death cause; its mortality associated occupied the fifth place in Colombia in 2004. Complete surgical resection is the therapeutic option with the greatest cure probability, however it results frequently ineffective given the current incapacity in Colombia to an early detection of the disease. This study reports the characterization of a group of 30 lung cancer patients regarding the gene dose (gene copy number) found at the loci corresponding to genes EGFR (erb B1), PIK3CA and C-myc in tumor samples, and compares the results with the dose found in adjacent lung from the same patients. Methods: The gene dose of EGFR (erbB1), PIK3CA, and C-myc were measured by real time PCR in matched tumor and normal lung tissue samples. Results are expressed as the multiplicity of each gene dose with respect to a single copy reference gene. In this case the gene HHB (human hemoglobin). Antiquity of the cases ranged from 5 to 10 years. Results: An increased gene dose for EGFR and PIK3CA was a feature clearly associated to the tumor phenotype of the sample (found in 96 and 100% of the tumors respectively). Quantitative measure of this feature demonstrated for both genes a high sensitivity and specificity for tumor/normal discrimination as confirmed by the ROC analysis. On the other hand, the Spearman test showed a great correlation between EGFR and PIK3CA doses (r=0.75). C-myc was the gene whose dose was less consistently correlated to the tumor phenotype, however most of the patients with amplified C-myc presented distant spread of tumor cells (metastasis) at diagnosis. Conclusion: Quantitative measurement of EGFR, PIK3CA, and C-myc gene dose by real time PCR provides a method for tumor phenotype recognition in DNA samples from lung tissue.

Introducción: A pesar de los avances terapéuticos actuales, el cáncer de pulmón sigue como la primera causa de muerte por cáncer en el mundo, ocupando Colombia el quinto lugar en mortalidad por este tipo de afección en el 2004. La resección quirúrgica total es la alternativa terapéutica con mayores probabilidades de curaciones, pero resulta poco efectiva en el país por la incapacidad actual para detectar tempranamente la enfermedad. Este trabajo informa la caracterización de un grupo de 30 pacientes con cáncer de pulmón con referencia a la dosis génica hallada en los loci correspondientes a los genes EGFR (erb B1), PIK3CA y C-myc en muestras tumorales, comparada con la dosis encontrada en el tejido normal adyacente de los mismos enfermos. Métodos: La dosis génica se midió en cada caso por PCR en tiempo real sobre ADN aislado de tejido tumoral y normal preservado en parafina de cada paciente. Los resultados se expresan como el número de veces que la dosis de cada gen sobrepasa la dosis de un gen de referencia, en este caso el HHB (hemoglobina humana b). El rango de antigüedad de los casos fue de 5 a 10 años. Resultados: Una dosis génica incrementada para los genes EGFR, PIK3CA demostró ser una característica claramente asociada con el fenotipo tumoral (96% y 100% de los tumores respectivamente). La medición cuantitativa de dicho fenómeno demostró en ambos casos gran sensibilidad y especificidad para la discriminación tumor/normal como lo confirma el análisis ROC. Por otro lado, la amplificación simultánea de ambos genes en el mismo paciente fue un hecho observado con alta frecuencia (Spearman=0.75). La dosis de C-myc mostró una asociación menos consistente con el carácter tumoral, sin embargo todos los pacientes con C-myc amplificado presentaron dispersión distante de células tumorales (metástasis).