Preexisting Serum Autoantibodies Against the NMDAR Subunit NR1 Modulate Evolution of Lesion Size in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Recently, we reported high seroprevalence (age-dependent up to >19%) of N-methyl-d-aspartate-receptor subunit NR1 (NMDAR1) autoantibodies in both healthy and neuropsychiatrically ill subjects (N=4236). Neuropsychiatric syndrome relevance was restricted to individuals with compromised blood-brain barrier, for example, apolipoprotein E4 (APOE4) carrier status, both clinically and experimentally. We now hypothesized that these autoantibodies may upon stroke be protective in individuals with hitherto intact blood-brain barrier, but harmful for subjects with chronically compromised blood-brain barrier. METHODS: Of 464 patients admitted with acute ischemic stroke in the middle cerebral artery territory, blood for NMDAR1 autoantibody measurements and APOE4 carrier status as indicator of a preexisting leaky blood-brain barrier was collected within 3 to 5 hours after stroke. Evolution of lesion size (delta day 7-1) in diffusion-weighted magnetic resonance imaging was primary outcome parameter. In subgroups, NMDAR1 autoantibody measurements were repeated on days 2 and 7. RESULTS: Of all 464 patients, 21.6% were NMDAR1 autoantibody-positive (immunoglobulin M, A, or G) and 21% were APOE4 carriers. Patients with magnetic resonance imaging data available on days 1 and 7 (N=384) were divided into 4 groups according to NMDAR1 autoantibody and APOE4 status. Groups were comparable in all stroke-relevant presenting characteristics. The autoantibody+/APOE4- group had a smaller mean delta lesion size compared with the autoantibody-/APOE4- group, suggesting a protective effect of circulating NMDAR1 autoantibodies. In contrast, the autoantibody+/APOE4+ group had the largest mean delta lesion area. NMDAR1 autoantibody serum titers dropped on day 2 and remounted by day 7. CONCLUSIONS: Dependent on blood-brain barrier integrity before an acute ischemic brain injury, preexisting NMDAR1 autoantibodies seem to be beneficial or detrimental.

Channelrhodopsin fluorescent tag replacement for clinical translation of optogenetic hearing restoration.

Sensory restoration by optogenetic neurostimulation provides a promising future alternative to current electrical stimulation approaches. So far, channelrhodopsins (ChRs) typically contain a C-terminal fluorescent protein (FP) tag for visualization that potentially poses an additional risk for clinical translation. Previous work indicated a reduction of optogenetic stimulation efficacy upon FP removal. Here, we further optimized the fast-gating, red-light-activated ChR f-Chrimson to achieve efficient optogenetic stimulation in the absence of the C-terminal FP. Upon FP removal, we observed a massive amplitude reduction of photocurrents in transfected cells in vitro and of optogenetically evoked activity of the adeno-associated virus (AAV) vector-transduced auditory nerve in mice in vivo. Increasing the AAV vector dose restored optogenetically evoked auditory nerve activity but was confounded by neural loss. Of various C-terminal modifications, we found the replacement of the FP by the Kir2.1 trafficking sequence (TSKir2.1) to best restore both photocurrents and optogenetically evoked auditory nerve activity with only mild neural loss few months after dosing. In conclusion, we consider f-Chrimson-TSKir2.1 to be a promising candidate for clinical translation of optogenetic neurostimulation such as by future optical cochlear implants.

RNF20 and RNF40 regulate vitamin D receptor-dependent signaling in inflammatory bowel disease.

Despite the identification of several genetic factors linked to increased susceptibility to inflammatory bowel disease (IBD), underlying molecular mechanisms remain to be elucidated in detail. The ubiquitin ligases RNF20 and RNF40 mediate the monoubiquitination of histone H2B at lysine 120 (H2Bub1) and were shown to play context-dependent roles in the development of inflammation. Here, we aimed to examine the function of the RNF20/RNF40/H2Bub1 axis in intestinal inflammation in IBD patients and mouse models. For this purpose, intestinal sections from IBD patients were immunohistochemically stained for H2Bub1. Rnf20 or Rnf40 were conditionally deleted in the mouse intestine and mice were monitored for inflammation-associated symptoms. Using mRNA-seq and chromatin immunoprecipitation (ChIP)-seq, we analyzed underlying molecular pathways in primary intestinal epithelial cells (IECs) isolated from these animals and confirmed these findings in IBD resection specimens using ChIP-seq.The majority (80%) of IBD patients displayed a loss of H2Bub1 levels in inflamed areas and the intestine-specific deletion of Rnf20 or Rnf40 resulted in spontaneous colorectal inflammation in mice. Consistently, deletion of Rnf20 or Rnf40 promoted IBD-associated gene expression programs, including deregulation of various IBD risk genes in these animals. Further analysis of murine IECs revealed that H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes is RNF20/40-dependent. Finally, these effects were confirmed in a subgroup of Crohn's disease patients which displayed epigenetic and expression changes in RNF20/40-dependent gene signatures. Our findings reveal that loss of H2B monoubiquitination promotes intestinal inflammation via decreased VDR activity thereby identifying RNF20 and RNF40 as critical regulators of IBD.

USP22 exerts tumor-suppressive functions in colorectal cancer by decreasing mTOR activity.

USP22, the deubiquitinating subunit of the SAGA transcriptional cofactor complex, is a member of an 11-gene "death-from-cancer" signature. USP22 has been considered an attractive therapeutic target since high levels of its expression were associated with distant metastasis, poor survival, and high recurrence rates in a wide variety of solid tumors, including colorectal cancer (CRC). We sought to investigate the role of Usp22 during tumorigenesis in vivo using a mouse model for intestinal carcinogenesis with a tissue-specific Usp22 ablation. In addition, we assessed the effects of USP22 depletion in human CRC cells on tumorigenic potential and identified underlying molecular mechanisms. For the first time, we report that USP22 has an unexpected tumor-suppressive function in vivo. Intriguingly, intestine-specific Usp22 deletion exacerbated the tumor phenotype caused by Apc mutation, resulting in significantly decreased survival and higher intestinal tumor incidence. Accordingly, human CRC cells showed increased tumorigenic properties upon USP22 reduction in vitro and in vivo and induced gene expression signatures associated with an unfavorable outcome in CRC patients. Notably, USP22 loss resulted in increased mTOR activity with the tumorigenic properties elicited by the loss of USP22 being reversible by mTOR inhibitor treatment in vitro and in vivo. Here, we demonstrate that USP22 can exert tumor-suppressive functions in CRC where its loss increases CRC burden by modulating mTOR activity. Importantly, our data uncover a tumor- and context-specific role of USP22, suggesting that USP22 expression could serve as a marker for therapeutic stratification of cancer patients.

USP22-dependent HSP90AB1 expression promotes resistance to HSP90 inhibition in mammary and colorectal cancer.

As a member of the 11-gene "death-from-cancer" gene expression signature, overexpression of the Ubiquitin-Specific Protease 22 (USP22) was associated with poor prognosis in various human malignancies. To investigate the function of USP22 in cancer development and progression, we sought to detect common USP22-dependent molecular mechanisms in human colorectal and breast cancer cell lines. We performed mRNA-seq to compare gene expression profiles of various colorectal (SW837, SW480, HCT116) and mammary (HCC1954 and MCF10A) cell lines upon siRNA-mediated knockdown of USP22. Intriguingly, while USP22 depletion had highly heterogeneous effects across the cell lines, all cell lines displayed a common reduction in the expression of Heat Shock Protein 90 Alpha Family Class B Member 1 (HSP90AB1). The downregulation of HSP90AB1 was confirmed at the protein level in these cell lines as well as in colorectal and mammary tumors in mice with tissue-specific Usp22 deletions. Mechanistically, we detected a significant reduction of H3K9ac on the HSP90AB1 gene in USP22-deficient cells. Interestingly, USP22-deficient cells displayed a high dependence on HSP90AB1 expression and diminishing HSP90 activity further using the HSP90 inhibitor Ganetespib resulted in increased therapeutic vulnerability in both colorectal and breast cancer cells in vitro. Accordingly, subcutaneously transplanted CRC cells deficient in USP22 expression displayed increased sensitivity towards Ganetespib treatment in vivo. Together, we discovered that HSP90AB1 is USP22-dependent and that cooperative targeting of USP22 and HSP90 may provide an effective approach to the treatment of colorectal and breast cancer.