C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Development of a sensitive trial-ready poly(GP) CSF biomarker assay for C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis.

OBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.

Glycans modify mesenchymal stem cell differentiation to impact on the function of resulting osteoblasts.

Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both N- and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts 3 weeks later. In contrast, inhibition of N-glycan processing in MSCs altered differentiation to enhance the mineralization capacity of the osteoblasts. The effect of N-glycans on MSC differentiation was mediated by the phosphoinositide-3-kinase (PI3K)/Akt pathway owing to reduced Akt phosphorylation. Interestingly, by inhibiting PI3K during the first 2 days of osteogenesis, we were able to phenocopy the effect of inhibiting N-glycan processing. Thus, glycan processing provides another layer of regulation that can modulate the functional outcome of differentiation. Glycan processing can thereby offer a novel set of targets for many therapeutically attractive processes.

Proteome-Wide Analysis of Cysteine Reactivity during Effector-Triggered Immunity.

A surge in the accumulation of oxidants generates shifts in the cellular redox potential during early stages of plant infection with pathogens and activation of effector-triggered immunity (ETI). The redoxome, defined as the proteome-wide oxidative modifications of proteins caused by oxidants, has a well-known impact on stress responses in metazoans. However, the identity of proteins and the residues sensitive to oxidation during the plant immune response remain largely unknown. Previous studies of the thimet oligopeptidases TOP1 and TOP2 placed them in the salicylic acid dependent branch of ETI, with a current model wherein TOPs sustain interconnected organellar and cytosolic pathways that modulate the oxidative burst and development of cell death. Herein, we characterized the ETI redoxomes in Arabidopsis (Arabidopsis thaliana) wild-type Col-0 and top1top2 mutant plants using a differential alkylation-based enrichment technique coupled with label-free mass spectrometry-based quantification. We identified cysteines sensitive to oxidation in a wide range of protein families at multiple time points after pathogen infection. Differences were detected between Col-0 and top1top2 redoxomes regarding the identity and number of oxidized cysteines, and the amplitude of time-dependent fluctuations in protein oxidation. Our results support a determining role for TOPs in maintaining the proper level and dynamics of proteome oxidation during ETI. This study significantly expands the repertoire of oxidation-sensitive plant proteins and can guide future mechanistic studies.

Early microgliosis precedes neuronal loss and behavioural impairment in mice with a frontotemporal dementia-causing CHMP2B mutation.

Frontotemporal dementia (FTD)-causing mutations in the CHMP2B gene lead to the generation of mutant C-terminally truncated CHMP2B. We report that transgenic mice expressing endogenous levels of mutant CHMP2B developed late-onset brain volume loss associated with frank neuronal loss and FTD-like changes in social behaviour. These data are the first to show neurodegeneration in mice expressing mutant CHMP2B and indicate that our mouse model is able to recapitulate neurodegenerative changes observed in FTD. Neuroinflammation has been increasingly implicated in neurodegeneration, including FTD. Therefore, we investigated neuroinflammation in our CHMP2B mutant mice. We observed very early microglial proliferation that develops into a clear pro-inflammatory phenotype at late stages. Importantly, we also observed a similar inflammatory profile in CHMP2B patient frontal cortex. Aberrant microglial function has also been implicated in FTD caused by GRN, MAPT and C9orf72 mutations. The presence of early microglial changes in our CHMP2B mutant mice indicates neuroinflammation may be a contributing factor to the neurodegeneration observed in FTD.

C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A.

A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity.

Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

A randomized controlled trial of two interventions to increase colorectal cancer screening among Hispanics on the Texas-Mexico border.

BACKGROUND: Colorectal cancer (CRC) is the second and third leading cause of cancer death for Hispanic men and women, respectively. CRC can be prevented if precursors are detected early and removed and can be successfully treated if discovered early. While one-on-one interventions for increasing CRC screening (CRCS) are recommended, few studies specifically assess the effectiveness of lay health worker (LHW) approaches using different educational materials. PURPOSE: To develop and evaluate the effectiveness of two LHW-delivered CRCS interventions known as Vale la Pena (VLP; "It's Worth It!") on increasing CRCS among Hispanics. DESIGN: The study design was a cluster randomized controlled trial with two treatment arms. SETTING/PARTICIPANTS: Six hundred and sixty five Hispanics 50 years and older were recruited from 24 colonias (neighborhoods) in the Lower Rio Grande Valley of the Texas-Mexico border. INTERVENTION: The interventions were a small media print intervention (SMPI) (including DVD and flipchart), and a tailored interactive multimedia intervention (TIMI) delivered on tablet computers. A no intervention group served as the comparison group. Data were collected between 2007 and 2009 and analyzed between 2009 and 2013. MAIN OUTCOME MEASURES: Measures assessed CRCS behavior, self-efficacy, knowledge, and other psychosocial constructs related to CRCS and targeted through VLP. RESULTS: Among participants reached for follow-up, 18.9 % in the SMPI group, 13.3 % in the TIMI group, and 11.9 % in the comparison group completed CRCS. Intent-to-treat analysis showed that 13.6 % in the SMPI group, 10.2 % in the TIMI group, and 10.8 % in the comparison group completed CRCS. These differences were not statistically significant. CONCLUSION: Results indicated that there are no significant differences in CRCS uptake between groups.

Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling.

We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method "filter-aided N-glycan separation" and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples.

Key informant interviews with coordinators of special events conducted to increase cancer screening in the United States.

Special events such as health fairs, cultural festivals and charity runs are commonly employed in the community to increase cancer screening; however, little is known about their effectiveness. The purpose of this study is to assess the activities, screening outcomes, barriers and recommendations of special events to increase breast, cervical and colorectal cancer screening. In-depth interviews were conducted nationally with 51 coordinators of events in June to September 2012. Health fairs and screening days were the most common events conducted, primarily for breast cancer education. Goals were to increase awareness of cancer screening and reach special populations. Evidence-based Community Guide strategies to increase cancer screening employed were: small media, reducing structural barriers, one-on-one education or group education. For each event that provided screening on-site or through referral, a mean of 35 breast, 28 cervical and 19 colorectal cancer screenings were reported. Coordinators made recommendations for further evaluation of special events, and most plan to conduct another special event. These data are novel and provide baseline documentation of activities and recommendations for a commonly used community-based cancer screening intervention that lacks evidence of effectiveness. Additional research to better understand the use of special events for increasing cancer screening is warranted.

AMIGAS: a multicity, multicomponent cervical cancer prevention trial among Mexican American women.

BACKGROUND: Considerable efforts have been undertaken in the United States to reduce cervical cancer incidence and mortality by increasing screening; however, disparities in screening rates continue to exist among certain racial and ethnic minority groups. The objective of the current study was to determine the effectiveness of a lay health worker-delivered intervention-AMIGAS (Ayudando a las Mujeres con Informacion, Guia, y Amor para su Salud [helping women with information, guidance, and love for their health])-to increase Papanicolaou (Pap) test screening among 3 populations of women of Mexican origin. METHODS: Six hundred thirteen women of Mexican origin in 3 treatment sites were randomized among 4 study arms: the full AMIGAS program with a video and a flip chart (n = 151), the AMIGAS program without the video (n = 154), the AMIGAS program without the flip chart (n = 155), and a usual care control group (n = 153). Six months after enrollment, women were surveyed and reported whether or not they had been screened. RESULTS: Women in any of the intervention arms were statistically significantly more likely to report being screened than those in the usual care group in both an intent-to-treat analysis and a per-protocol analysis. In the intent-to-treat analysis, 25% of women in the control group and 52% in the full AMIGAS program group reported having had Pap tests (P < .001); in the per-protocol analysis, the percentages were 29% and 62%, respectively (P < .001). CONCLUSIONS: AMIGAS was effective in increasing Pap test screening among women of Mexican descent when used in a 1-to-1 setting. Future research should compare the 1-on-1 intervention with the group-based intervention.

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

Assessment of the nuclear pore dilating agent trans-cyclohexane-1,2-diol in differentiated airway epithelium.

BACKGROUND: The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. METHODS: We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). RESULTS: TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. CONCLUSIONS: Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications.

Referrals for suspected hematologic malignancy: a survey of primary care physicians.

Little is known about referrals from primary care providers (PCPs) for suspected hematologic malignancies, including their clinical triggers and frequency. A random sample of 190 Massachusetts PCPs were presented with a vignette concerning a patient with a new finding of moderate anemia, asked how they would respond, and then asked what they would do if the patient returned with persistent anemia plus one additional sign or symptom. We also asked about referral behaviors for suspected hematologic malignancies during the prior year. A total of 134 (70.5%) PCPs responded. At first anemia presentation,only 3.8% reported referring to hematology. The development of a second sign or symptom yielded higher referral rates: pancytopenia 588.7%, leukopenia 5 63.9%, thrombocytopenia 5 63.9%, lymphadenopathy 5 42.9%, leukocytosis 5 37.6%, night sweats 5 25.6%, and weight loss 5 23.3%. The median yearly number (interquartile range) of patients PCPs reported suspecting of having hematologic malignancy was 5 (3, 10), and the median formally referred was 5 (3, 10). We conclude that anemia plus signs and symptoms suggestive of myelodysplasia or leukemia (compared with those suggestive of lymphoma) are more likely to prompt hematology referral. In addition, given their rarity,the numbe

Small media and client reminders for colorectal cancer screening: current use and gap areas in CDC's Colorectal Cancer Control Program.

INTRODUCTION: CDC's Colorectal Cancer Control Program (CRCCP) funds 25 states and 4 tribal organizations to promote and increase colorectal cancer screening population-wide. The CRCCP grantees must use evidence-based strategies from the Guide to Community Preventive Services, including small media and client reminders. METHODS: To assess the existing resources and needs to promote colorectal cancer screening, we conducted 2 web-based surveys of CRCCP grantees and their community partners. Survey 1 sought to identify priority populations, the number and quality of existing colorectal cancer resources for different population subgroups, and the types of small media and client reminder they were most interested in using. Survey 2 assessed screening messages that were used in the past or might be used in the future, needs for non-English-language information, and preferences for screening-related terminology. RESULTS: In survey 1 (n = 125 from 26 CRCCPs), most respondents (83%) indicated they currently had some information resources for promoting screening but were widely dissatisfied with the quality and number of these resources. They reported the greatest need for resources targeting rural populations (62% of respondents), men (53%), and Hispanics (45%). In survey 2 (n = 57 from 25 CRCCPs), respondents indicated they were most likely to promote colorectal cancer screening using messages that emphasized family (95%), role models (85%), or busy lives (83%), and least likely to use messages based on faith (26%), embarrassment (25%), or fear (22%). Nearly all (85%) indicated a need for resources in languages other than English; 16 different languages were mentioned, most commonly Spanish. CONCLUSION: These findings provide the first picture of CRCCP information resources and interests, and point to specific gaps that must be addressed to help increase screening.

Cognitive testing of human papillomavirus vaccine survey items for parents of adolescent girls.

OBJECTIVE: Many studies have been conducted to understand what factors are associated with human papillomavirus (HPV) vaccine acceptability and completion of the 3-dose vaccination series, but few have examined whether people understand the survey items used to assess these relationships. Through a multisite collaborative effort, we developed and cognitively tested survey items that represent constructs known to affect vaccine acceptability and completion. MATERIALS AND METHODS: Investigators from 7 research centers in the United States used cognitive interviewing techniques and in-person and telephone interviews to test 21 items. Four rounds of testing, revising, and retesting were conducted among racially and ethnically diverse parents (n = 62) of girls between the ages of 9 and 17 years. RESULTS: The final survey contained 20 items on attitudes and beliefs relevant to HPV vaccine. Some parents misinterpreted statements about hypothetical vaccine harms as statements of fact. Others were unwilling to answer items about perceived disease likelihood and perceived vaccine effectiveness, because they said the items seemed to have a "right" answer that they did not know. On the basis of these and other findings from cognitive testing, we revised the wording of 14 questions to improve clarity and comprehension. We also revised instructions, response options, and item order. CONCLUSIONS: Cognitive testing of HPV vaccine survey items revealed important differences between intended and ascribed item meaning by participants. Use of the tested survey questions presented here may increase measurement validity and researchers' ability to compare findings across studies and populations. Additional testing using quantitative methods can help to further validate these items.

Navigating the road ahead: public health challenges and the interactive systems framework for dissemination and implementation.

Public health is currently faced with an array of critical challenges and disconnects. Research and evaluation have identified a number of evidence-based strategies for effecting behavior change at individual, group, organizational, and environmental levels, all of which hold promise for leading to substantial reductions in morbidity and mortality, and increased quality of life. Unfortunately, there is huge variability across the public health system in awareness of the value of using evidence to inform decision making, let alone in capacity to locate, assess, compare, select, justify, adapt, implement, and evaluate evidence-based strategies, or to participate in building the evidence base for practice-based innovations. As a result, many communities may not be benefitting from research-tested and practice-based strategies that could help them to meet their public health goals more efficiently and effectively. CDC's Interactive Systems Framework for Dissemination and Implementation (ISF), released in 2008, was designed to help close this gap between research and practice. This commentary identifies the ways in which the ISF framework is useful in addressing the research practice gap; revisits the elements of the framework that have continued to guide research and practice in fruitful ways; and highlights areas that need further development to meet current public health challenges.

An organizing framework for translation in public health: the Knowledge to Action Framework.

A priority for the Centers for Disease Control and Prevention (CDC) is translating scientific knowledge into action to improve the public's health. No area has a more pressing need for translation than the prevention and control of chronic diseases. Staff from CDC's National Center for Chronic Disease Prevention and Health Promotion worked across disciplines and content areas to develop an organizing framework to describe and depict the high-level processes necessary to move from discovery into action through translation of evidence-based programs, practices, or policies. The Knowledge to Action (K2A) Framework identifies 3 phases (research, translation, and institutionalization) and the decision points, interactions, and supporting structures within the phases that are necessary to move knowledge to sustainable action. Evaluation undergirds the entire K2A process. Development of the K2A Framework highlighted the importance of planning for translation, attending to supporting structures, and evaluating the public health impact of our efforts.

Research and comprehensive cancer control coalitions.

The goal of cancer control research is "to generate basic knowledge about how to monitor and change individual and collective behavior and to ensure that knowledge is translated into practice and policy rapidly, effectively, and efficiently" (Division of Cancer Control and Population Sciences in Cancer control framework and synthese rationale, 2010). Research activities span the cancer control continuum from prevention to early detection and diagnosis through treatment and survivorship (Division of Cancer Control and Population Sciences in Cancer control framework and synthese rationale, 2010). While significant advancements have been made in understanding, preventing and treating cancer in the past few decades, these benefits have yielded disproportionate results in cancer morbidity and mortality across various socioeconomic and racial/ethnic subgroups (Ozols et al in J Clin Oncol, 25(1):146-1622, 2007). It has been a high priority since the beginning of the Comprehensive Cancer Control (CCC) movement to utilize research in the development and implementation of cancer plans in the states, tribes and tribal organizations, territories and US Pacific Island Jurisdictions. Nevertheless, dissemination and implementation of research in coalition activities has been challenging for many programs. Lessons learned from programs and coalitions in the implementation and evaluation of CCC activities, as well as resources provided by national partners, can assist coalitions with the translation of research into practice.

Relax, Don't RAN Translate It.

The (GGGGCC)n repeat expansion in C9orf72, which is the most common cause of frontotemporal dementia and amyotrophic lateral sclerosis, is translated through repeat-associated non-AUG (RAN) translation. In this issue of Neuron, Cheng et al. (2019) report that the helicase DDX3X, which unwinds (or relaxes) RNA, suppresses RAN translation and toxicity.