New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples

Loss of huntingtin function complemented by small molecules acting as repressor element 1/neuron restrictive silencer element silencer modulators

Increased levels of the repressor element 1/neuron restrictive silencer element (RE1/NRSE) silencing activity promoter, and a consequent reduction in the transcription of many RE1/NRSE-bearing neuronal genes, including brain-derived neurotrophic factor (BDNF), have been demonstrated in Huntington disease (HD) and represent one possible effector of its selective neuronal vulnerability. Restoring the expression levels of neuronal genes in diseased neurons therefore seems to be an attractive therapeutic approach. To this end, we have developed a cell-based reporter assay for monitoring RE1/NRSE silencing activity and validated it by genetically inactivating the RE1/NRSE or pharmacologically stimulating global transcription. In a pilot compound screen, we identified three closely related structural analogues that up-regulate reporter expression at low nanomolar concentrations, and follow-up studies have shown that they efficaciously increase endogenous BDNF levels in HD cells. Moreover, one of the compounds increases the viability of HD cells. Our findings suggest a new avenue for the development of drugs for HD and other neurodegenerative disorders based on the pharmacological up-regulation of the production of the neuronal survival factor BDNF and of other RE1/NRSE-regulated neuronal gene

Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD

Long-term Quality of Life After Intensive Care Unit Admission (a Single-Center Observational Study)

Patients may experience long-term physical, psychological and cognitive impairment after intensive care unit (ICU) discharge, a condition commonly described as post-intensive care syndrome. The relative contribution of each of these components to long-term quality of life was never investigated.The aim of this study is to identify the type and severity of disability and QoL at the discharge from ICU and up to following 6 months.Material and Methods. All patients (n=218) discharged from a university hospital ICU between April 2016 and July 2017 were eligible. Exclusion criteria included: age <18 years, brain or spinal injury, life expectancy <90 days, and ICU stay <12 hours. The Short Form Health Survey (SF-36), and 5-level EuroQoL-5D (EQ-5D-5L) questionnaires were administered at ICU discharge, and at 30-, 90- and 180-days. We compared patients requiring short-term ICU monitoring (IM, Intensive Monitoring, n=109) or patients requiring ICU treatment (IT, Intensive Treatment, n=109).Results. All dimensions of SF-36 and EQ-5D-5L parameters increased from ICU discharge to 180-days, except for the SF-36 Synthetic index linked to mental health (P=0.08). All EQ-5D-5L parameters improved significantly in the IT group, while only Visual Analog Scale Health Perception improved in the IM group.Conclusion. ICU survivors suffer long-term physical and psychological sequelae. The perception of Quality of Life is reduced after ICU discharge. The psychological and cognitive dimensions were more compromised than physical ones. Patients discharged from the ICU may benefit from specific intensive care follow-up clinics addressing their needs in term of psychological and cognitive support

Долгосрочное качество жизни после лечения в отделении реанимации и интенсивной терапии (одноцентровое обсервационное исследование)

Patients may experience long-term physical, psychological and cognitive impairment after intensive care unit (ICU) discharge, a condition commonly described as post-intensive care syndrome. The relative contribution of each of these components to long-term quality of life was never investigated.The aim of this study is to identify the type and severity of disability and QoL at the discharge from ICU and up to following 6 months.Material and Methods. All patients (n=218) discharged from a university hospital ICU between April 2016 and July 2017 were eligible. Exclusion criteria included: age <18 years, brain or spinal injury, life expectancy <90 days, and ICU stay <12 hours. The Short Form Health Survey (SF-36), and 5-level EuroQoL-5D (EQ-5D-5L) questionnaires were administered at ICU discharge, and at 30-, 90- and 180-days. We compared patients requiring short-term ICU monitoring (IM, Intensive Monitoring, n=109) or patients requiring ICU treatment (IT, Intensive Treatment, n=109).Results. All dimensions of SF-36 and EQ-5D-5L parameters increased from ICU discharge to 180-days, except for the SF-36 Synthetic index linked to mental health (P=0.08). All EQ-5D-5L parameters improved significantly in the IT group, while only Visual Analog Scale Health Perception improved in the IM group.Conclusion. ICU survivors suffer long-term physical and psychological sequelae. The perception of Quality of Life is reduced after ICU discharge. The psychological and cognitive dimensions were more compromised than physical ones. Patients discharged from the ICU may benefit from specific intensive care follow-up clinics addressing their needs in term of psychological and cognitive support.После выписки из отделения интенсивной терапии у пациентов может развиваться «синдром последствий интенсивной терапии», включающий долговременные соматические, психологические и когнитивные нарушения. Относительный вклад данных нарушений в долгосрочное качество жизни пациентов практически не изучен.Цель исследования — определить тип и степень тяжести функциональных расстройств и нарушения качества жизни при выписке из отделения реанимации и интенсивной терапии (ОРИТ) и на протяжении последующих 6 месяцев.Материал и методы. В исследование включили 218 пациентов, выписанных из ОРИТ университетской больницы в период между апрелем 2016 и июлем 2017 гг. Критерии исключения: возраст младше 18 лет, повреждение головного или спинного мозга, ожидаемая продолжительность жизни менее 90 дней, период пребывания в ОРИТ менее 12 часов. Использовали опросники SF-36 (краткая форма оценки здоровья) и 5-уровневый EQ-5D-5L (Европейский опросник оценки качества в 5 областях), которые пациенты заполняли при выписке и через 30, 90 и 180 дней после нее. Проводили сравнение пациентов, которым требовалось кратковременное пребывание в ОРИТ (группа интенсивного наблюдения, n=109), и тех, которые прошли курс лечения в ОРИТ (группа интенсивной терапии, n=109).Результаты. Через 180 дней все показатели опросников SF-36 и EQ-5D-5L повысились по сравнению с моментом выписки, за исключением общего показателя психического здоровья SF-36 (p=0,08). В группе интенсивной терапии значительно улучшились все показатели EQ-5D-5L, в то время как в группе интенсивного наблюдения улучшились только показатели визуально-аналоговой шкалы оценки здоровья данного опросника.Заключение. У пациентов, выписанных из ОРИТ, наблюдали длительные соматические и психологические неблагоприятные последствия. После выписки из ОРИТ снижается оценка собственного качества жизни. Нарушения показателей, характеризующих психологическую и когнитивную сферы, были более выраженным, чем у параметров соматического состояния пациентов. Ситуацию поможет исправить наблюдение и лечение пациентов, выписанных из ОРИТ, в специальных постреанимационных клиниках с упором на необходимость психологической и когнитивной поддержки больных

Transcriptional Activation of REST by Sp1 in Huntington's Disease Models

In Huntington's disease (HD), mutant huntingtin (mHtt) disrupts the normal transcriptional program of disease neurons by altering the function of several gene expression regulators such as Sp1. REST (Repressor Element-1 Silencing Transcription Factor), a key regulator of neuronal differentiation, is also aberrantly activated in HD by a mechanism that remains unclear. Here, we show that the level of REST mRNA is increased in HD mice and in NG108 cells differentiated into neuronal-like cells and expressing a toxic mHtt fragment. Using luciferase reporter gene assay, we delimited the REST promoter regions essential for mHtt-mediated REST upregulation and found that they contain Sp factor binding sites. We provide evidence that Sp1 and Sp3 bind REST promoter and interplay to fine-tune REST transcription. In undifferentiated NG108 cells, Sp1 and Sp3 have antagonistic effect, Sp1 acting as an activator and Sp3 as a repressor. Upon neuronal differentiation, we show that the amount and ratio of Sp1/Sp3 proteins decline, as does REST expression, and that the transcriptional role of Sp3 shifts toward a weak activator. Therefore, our results provide new molecular information to the transcriptional regulation of REST during neuronal differentiation. Importantly, specific knockdown of Sp1 abolishes REST upregulation in NG108 neuronal-like cells expressing mHtt. Our data together with earlier reports suggest that mHtt triggers a pathogenic cascade involving Sp1 activation, which leads to REST upregulation and repression of neuronal genes

Short-Term Striatal Gene Expression Responses to Brain-Derived Neurotrophic Factor Are Dependent on MEK and ERK Activation

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is believed to be an important regulator of striatal neuron survival, differentiation, and plasticity. Moreover, reduction of BDNF delivery to the striatum has been implicated in the pathophysiology of Huntington's disease. Nevertheless, many essential aspects of BDNF responses in striatal neurons remain to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we assessed the relative contributions of multipartite intracellular signaling pathways to the short-term induction of striatal gene expression by BDNF. To identify genes regulated by BDNF in these GABAergic cells, we first used DNA microarrays to quantify their transcriptomic responses following 3 h of BDNF exposure. The signal transduction pathways underlying gene induction were subsequently dissected using pharmacological agents and quantitative real-time PCR. Gene expression responses to BDNF were abolished by inhibitors of TrkB (K252a) and calcium (chelator BAPTA-AM and transient receptor potential cation channel [TRPC] antagonist SKF-96365). Interestingly, inhibitors of mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) and extracellular signal-regulated kinase ERK also blocked the BDNF-mediated induction of all tested BDNF-responsive genes. In contrast, inhibitors of nitric oxide synthase (NOS), phosphotidylinositol-3-kinase (PI3K), and CAMK exhibited less prevalent, gene-specific effects on BDNF-induced RNA expression. At the nuclear level, the activation of both Elk-1 and CREB showed MEK dependence. Importantly, MEK-dependent activation of transcription was shown to be required for BDNF-induced striatal neurite outgrowth, providing evidence for its contribution to striatal neuron plasticity. CONCLUSIONS: These results show that the MEK/ERK pathway is a major mediator of neuronal plasticity and other important BDNF-dependent striatal functions that are fulfilled through the positive regulation of gene expression

SIRT2 Ablation Has No Effect on Tubulin Acetylation in Brain, Cholesterol Biosynthesis or the Progression of Huntington's Disease Phenotypes In Vivo

Huntington's disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. The molecular pathogenesis of HD is complex and many mechanisms and cellular processes have been proposed as potential sites of therapeutic intervention. However, prior to embarking on drug development initiatives, it is essential that therapeutic targets can be validated in mammalian models of HD. Previous studies in invertebrate and cell culture HD models have suggested that inhibition of SIRT2 could have beneficial consequences on disease progression. SIRT2 is a NAD[superscript +]-dependent deacetylase that has been proposed to deacetylate α-tubulin, histone H4 K16 and to regulate cholesterol biogenesis – a pathway which is dysregulated in HD patients and HD mouse models. We have utilized mice in which SIRT2 has been reduced or ablated to further explore the function of SIRT2 and to assess whether SIRT2 loss has a beneficial impact on disease progression in the R6/2 mouse model of HD. Surprisingly we found that reduction or loss of SIRT2 had no effect on the acetylation of α-tubulin or H4K16 or on cholesterol biosynthesis in the brains of wild type mice. Equally, genetic reduction or ablation of SIRT2 had no effect on HD progression as assessed by a battery of physiological and behavioural tests. Furthermore, we observed no change in aggregate load or levels of soluble mutant huntingtin transprotein. Intriguingly, neither the constitutive genetic loss nor acute pharmacological inhibition of SIRT2 affected the expression of cholesterol biosynthesis enzymes in the context of HD. Therefore, we conclude that SIRT2 inhibition does not modify disease progression in the R6/2 mouse model of HD and SIRT2 inhibition should not be prioritised as a therapeutic option for HD.American Parkinson Disease Association, Inc. (Fellowship)Johnson & Johnson. Pharmaceutical Research & Development (Fellowship

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool