The impairment of the ubiquitin-proteasome system (UPS) is thought to be an early event in neurodegeneration, and monitoring UPS alterations might serve as a disease biomarker. Our aim was to establish an alternate method to antibody-based assays for the selective measurement of free monoubiquitin in cerebrospinal fluid (CSF). Free monoubiquitin was measured with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MS/MS) in CSF of patients with Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), behavioral variant of frontotemporal dementia (bvFTD), Creutzfeldt-Jakob disease (CJD), Parkinson's disease (PD), primary progressive aphasia (PPA), and progressive supranuclear palsy (PSP). The LC-MS/MS method showed excellent intra- and interassay precision (4.4-7.4% and 4.9-10.3%) and accuracy (100-107% and 100-106%). CSF ubiquitin concentration was increased compared with that of controls (33.0 ± 9.7 ng/mL) in AD (47.5 ± 13.1 ng/mL, p < 0.05) and CJD patients (171.5 ± 103.5 ng/mL, p < 0.001) but not in other neurodegenerative diseases. Receiver operating characteristic curve (ROC) analysis of AD vs control patients revealed an area under the curve (AUC) of 0.832, and the specificity and sensitivity were 75 and 75%, respectively. ROC analysis of AD and FTLD patients yielded an AUC of 0.776, and the specificity and sensitivity were 53 and 100%, respectively. In conclusion, our LC-MS/MS method may facilitate ubiquitin determination to a broader community and might help to discriminate AD, CJD, and FTLD patients.

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Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2′-methoxyethyl (2′MOE) modifications. Through a screen of 192 2′OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2′OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.

Patients suffering from schizophrenia have been characterized by an apparent lack of theta (around 6 Hz) and gamma (>40 Hz) brain oscillatory activity during task execution. The neurocognitive reasons for these abnormal synchronization patterns, however, remain elusive. Recording the electroencephalogramm (EEG) during a selective visual attention task, the current study investigates whether abnormal brain oscillatory resting-state activity in the theta band might account for a lack of task-related brain oscillatory activity in schizophrenia. EEGs were recorded from 26 patients with schizophrenia and 26 healthy matched controls during rest and during the execution of a selective visual attention task, in which an unexpected object (monkey) appeared on the screen. On a behavioral level, patients were less likely to report perceiving the unexpected event than controls. Controls showed a stronger increase in task-related theta power than patients in prefrontal, parietal, and occipital brain regions. Task-related theta power change differed between patients who perceived, and patients who did not perceive the unexpected event. Moreover, patients showed higher levels of theta power during rest than controls, whereas the absolute theta power values during the selective attention task did not differ between groups. These results suggest that the failure to increase oscillatory activity during a cognitive task can be accounted for by abnormally high oscillatory activity in a resting state. This finding has important implications for future studies examining abnormal brain oscillatory activity in schizophrenia, which usually treat resting-state activity as a baseline for task-related activity.