Tackling neurodegeneration in vitro with omics: a path towards new targets and drugs.

Drug discovery is a generally inefficient and capital-intensive process. For neurodegenerative diseases (NDDs), the development of novel therapeutics is particularly urgent considering the long list of late-stage drug candidate failures. Although our knowledge on the pathogenic mechanisms driving neurodegeneration is growing, additional efforts are required to achieve a better and ultimately complete understanding of the pathophysiological underpinnings of NDDs. Beyond the etiology of NDDs being heterogeneous and multifactorial, this process is further complicated by the fact that current experimental models only partially recapitulate the major phenotypes observed in humans. In such a scenario, multi-omic approaches have the potential to accelerate the identification of new or repurposed drugs against a multitude of the underlying mechanisms driving NDDs. One major advantage for the implementation of multi-omic approaches in the drug discovery process is that these overarching tools are able to disentangle disease states and model perturbations through the comprehensive characterization of distinct molecular layers (i.e., genome, transcriptome, proteome) up to a single-cell resolution. Because of recent advances increasing their affordability and scalability, the use of omics technologies to drive drug discovery is nascent, but rapidly expanding in the neuroscience field. Combined with increasingly advanced in vitro models, which particularly benefited from the introduction of human iPSCs, multi-omics are shaping a new paradigm in drug discovery for NDDs, from disease characterization to therapeutics prediction and experimental screening. In this review, we discuss examples, main advantages and open challenges in the use of multi-omic approaches for the in vitro discovery of targets and therapies against NDDs.

Chromatin accessibility profiling of targeted cell populations with laser capture microdissection coupled to ATAC-seq.

Spatially resolved omics technologies reveal context-dependent cellular regulatory networks in tissues of interest. Beyond transcriptome analysis, information on epigenetic traits and chromatin accessibility can provide further insights on gene regulation in health and disease. Nevertheless, compared to the enormous advancements in spatial transcriptomics technologies, the field of spatial epigenomics is much younger and still underexplored. In this study, we report laser capture microdissection coupled to ATAC-seq (LCM-ATAC-seq) applied to fresh frozen samples for the spatial characterization of chromatin accessibility. We first demonstrate the efficient use of LCM coupled to in situ tagmentation and evaluate its performance as a function of cell number, microdissected areas, and tissue type. Further, we demonstrate its use for the targeted chromatin accessibility analysis of discrete contiguous or scattered cell populations in tissues via single-nuclei capture based on immunostaining for specific cellular markers.

Preadmission Diet and Zip Code Influences the Pediatric Critical Care Clinical Course for Infants with Severe Respiratory Illness ( N = 187).

We examined preadmission diet and zip code in infants with severe respiratory illness in the pediatric critical care unit. Patients aged 0 to 5 months admitted to the Helen DeVos Children's Hospital from January 2011 to May 2017 ( N = 187), as exclusively formula, exclusively breastfed or mixed diet were included. Formula-fed infants ( n = 88; 47%) clustered to zip codes with lower median incomes (<0.005), used public insurance as their payer type ( p < 0.005), and were prescribed more ranitidine ( p < 0.05) on admission.

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression.

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and ß-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

Pharmacotherapy for methamphetamine/amphetamine use disorder-a systematic review and meta-analysis.

AIMS: Addiction to methamphetamine/amphetamine (MA/A) is a major public health problem. Currently there are no pharmacotherapies for MA/A use disorder that have been approved for use by the US Food and Drug Administration or the European Medicines Agency. We reviewed the effectiveness of pharmacotherapy for MA/A use disorder to assess the quality, publication bias and overall strength of the evidence. METHODS: Systematic review and meta-analysis. We searched multiple data sources (MEDLINE, PsycINFO and Cochrane Library) to April 2019 for systematic reviews (SRs) and randomized controlled trials (RCTs). Included studies recruited adults who had MA/A use disorder; sample sizes ranged from 19 to 229 participants. Outcomes of interest were abstinence, defined as 3 or more consecutive weeks with negative urine drug screens (UDS); overall use, analyzed as the proportion of MA/A negative UDS specimens; and treatment retention. One SR of pharmacotherapies for MA/A use disorder and 17 additional RCTs met our inclusion criteria encompassing 17 different drugs (antidepressants, antipsychotics, psychostimulants, anticonvulsants and opioid antagonists). We combined the findings of trials with comparable interventions and outcome measures in random-effects meta-analyses. We assessed quality, publication bias and the strength of evidence for each outcome using standardized criteria. RESULTS: There was low-strength evidence from two RCTs that methylphenidate may reduce MA/A use: 6.5 versus 2.8% MA/A-negative UDS in one study (n = 34, P = 0.008) and 23 versus 16% in another study (n = 54, P = 0.047). Antidepressants as a class had no statistically significant effect on abstinence or retention on the basis of moderate strength evidence. Studies of anticonvulsants, antipsychotics (aripiprazole), opioid antagonists (naltrexone), varenicline and atomoxetine provided either low-strength or insufficient evidence of no effect on the outcomes of interest. Many of the studies had high or unclear risk of bias. CONCLUSIONS: On the basis of low- to moderate-strength evidence, most medications evaluated for methamphetamine/amphetamine use disorder have not shown a statistically significant benefit. However, there is low-strength evidence that methylphenidate may reduce use.

Pharmacotherapy for Cocaine Use Disorder-a Systematic Review and Meta-analysis.

BACKGROUND: Currently, there are no accepted FDA-approved pharmacotherapies for cocaine use disorder, though numerous medications have been tested in clinical trials. We conducted a systematic review and meta-analysis to better understand the effectiveness of pharmacotherapy for cocaine use disorder. METHODS: We searched multiple data sources (MEDLINE, PsycINFO, and Cochrane Library) through November 2017 for systematic reviews and randomized controlled trials (RCTs) of pharmacological interventions in adults with cocaine use disorder. When possible, we combined the findings of trials with comparable interventions and outcome measures in random-effects meta-analyses. We assessed the risk of bias of individual trials and the strength of evidence for each outcome using standardized criteria. Outcomes included continuous abstinence (3+ consecutive weeks); cocaine use; harms; and study retention. For relapse prevention studies (participants abstinent at baseline), we examined lapse (first cocaine positive or missing UDS) and relapse (two consecutive cocaine positive or missed UDS'). RESULTS: Sixty-six different drugs or drug combinations were studied in seven systematic reviews and 48 RCTs that met inclusion criteria. Antidepressants were the most widely studied drug class (38 RCTs) but appear to have no effect on cocaine use or treatment retention. Increased abstinence was found with bupropion (2 RCTs: RR 1.63, 95% CI 1.02 to 2.59), topiramate (2 RCTs: RR 2.56, 95% CI 1.39 to 4.73), and psychostimulants (14 RCTs: RR 1.36, 95% CI 1.05 to 1.77), though the strength of evidence for these findings was low. We found moderate strength of evidence that antipsychotics improved treatment retention (8 RCTs: RR 1.33, 95% CI 1.03 to 1.75). DISCUSSION: Most of the pharmacotherapies studied were not effective for treating cocaine use disorder. Bupropion, psychostimulants, and topiramate may improve abstinence, and antipsychotics may improve retention. Contingency management and behavioral interventions along with pharmacotherapy should continue to be explored. SR REGISTRATION: Prospero CRD42018085667.

EML proteins in microtubule regulation and human disease.

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy.

Satb2 Is Required for the Development of a Spinal Exteroceptive Microcircuit that Modulates Limb Position.

Motor behaviors such as walking or withdrawing the limb from a painful stimulus rely upon integrative multimodal sensory circuitry to generate appropriate muscle activation patterns. Both the cellular components and the molecular mechanisms that instruct the assembly of the spinal sensorimotor system are poorly understood. Here we characterize the connectivity pattern of a sub-population of lamina V inhibitory sensory relay neurons marked during development by the nuclear matrix and DNA binding factor Satb2 (ISR(Satb2)). ISR(Satb2) neurons receive inputs from multiple streams of sensory information and relay their outputs to motor command layers of the spinal cord. Deletion of the Satb2 transcription factor from ISR(Satb2) neurons perturbs their cellular position, molecular profile, and pre- and post-synaptic connectivity. These alterations are accompanied by abnormal limb hyperflexion responses to mechanical and thermal stimuli and during walking. Thus, Satb2 is a genetic determinant that mediates proper circuit development in a core sensory-to-motor spinal network.

Immunogenicity of Renal Microvascular Endothelial Cells From Genetically Modified Pigs.

BACKGROUND: Disrupting the porcine GGTA1 and CMAH genes [double knockout (DKO)] that produce the gal-α(1,3)-gal and N-glycolylneuraminic acid xenoantigens reduces human antibody binding to porcine peripheral blood mononuclear cells. It is important to examine rejection pathways at an organ-specific level. The object of this study is to evaluate the human preformed antibody reactivity against DKO renal microvascular endothelial cells (RMEC) in vitro. METHODS: Characteristics of DKO RMEC were analyzed using flow cytometry. Human IgG/M binding to primary RMEC, immortalized RMEC (iRMEC), and iRMEC-deficient in B4GALNT2 genes were examined using flow cytometric crossmatch assay. RESULTS: Porcine RMEC expressed gal-α(1,3)-gal, N-glycolylneuraminic acid, and Dolichos biflorus agglutinin glycans recognized by human preexisting antibodies in humans. Antigenicity of DKO RMEC was lower than GGTA1 KO RMEC. The disruption of B4GALNT2 gene in DKO iRMEC further reduced human IgG/IgM binding. CONCLUSIONS: Silencing the porcine GGTA1, CMAH, and B4GALNT2 genes is an effective strategy to reduce human preformed antibody binding to RMEC. Porcine RMEC will be a useful reagent for the further study of xenoimmunology.

Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein.

BACKGROUND: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia. METHODS: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers. RESULTS: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion. CONCLUSIONS: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain.

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection.

BACKGROUND: Manipulating the pig genome to increase compatibility with human biology may facilitate the clinical application of xenotransplantation. Genetic modifications to pig cells have been made by sequential recombination in fetal fibroblasts and liver-derived cells followed by cross-breeding or somatic cell nuclear transfer. The generation of pigs for research or organ donation by these methods is slow, expensive and requires technical expertise. A novel system incorporating the bacterial nuclease Cas9 and single-guide RNA targeting a 20 nucleotide site within a gene can be expressed from a single plasmid leading to a double-strand break and gene disruption. Coexpression of multiple unique single-guide RNA can modify several genetic loci in a single step. We describe a process for increasing the efficiency of selecting cells with multiple genetic modifications. METHODS: We used the CRISPR/Cas system to target the GGTA1, CMAH and putative iGb3S genes in pigs that have been naturally deleted in humans. Cells lacking galactose α-1,3 galactose (α-Gal) were negatively selected by an IB4 lectin/magnetic bead. α-Gal negative multiplexed single-guide RNA-treated cells were used for somatic cell nuclear transfer (SCNT) and transferred to fertile sows. We examined the levels of α-Gal and Neu5Gc expression of 32 day fetuses and piglets and analyzed the targeted genes by DNA sequencing. RESULTS: Liver-derived cells treated with multiple single-guide RNA and selected for an α-Gal null phenotype were significantly more likely to also carry mutations in simultaneously targeted genes. Multiplex single-guide RNA-treated cells used directly for SCNT without further genetic selection produced piglets with deletions in the targeted genes but also created double- and triple-gene KO variations. CRISPR/Cas-treated cells grew normally and yielded normal liters of healthy piglets via somatic cell nuclear transfer. CONCLUSIONS: The CRISPR/Cas system allows targeting of multiple genes in a single reaction with the potential to create pigs of one genetic strain or multiple genetic modifications in a single pregnancy. The application of this phenotypic selection strategy with multiplexed sgRNA and the Cas9 nuclease has accelerated our ability to produce and evaluate pigs important to xenotransplantation.

Identification of a cellular node for motor control pathways.

The rich behavioral repertoire of animals is encoded in the CNS as a set of motorneuron activation patterns, also called 'motor synergies'. However, the neurons that orchestrate these motor programs as well as their cellular properties and connectivity are poorly understood. Here we identify a population of molecularly defined motor synergy encoder (MSE) neurons in the mouse spinal cord that may represent a central node in neural pathways for voluntary and reflexive movement. This population receives direct inputs from the motor cortex and sensory pathways and, in turn, has monosynaptic outputs to spinal motorneurons. Optical stimulation of MSE neurons drove reliable patterns of activity in multiple motor groups, and we found that the evoked motor patterns varied on the basis of the rostrocaudal location of the stimulated MSE. We speculate that these neurons comprise a cellular network for encoding coordinated motor output programs.

Allergic airway disease in mice alters T and B cell responses during an acute respiratory poxvirus infection.

Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8(+) T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8(+) T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4(+) T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism.

Climate change, flooding and the media in Britain.

The weather is a preoccupation of the British public, and flooding has become a salient feature of their experience. Climate science also has important things to say about the prevalence, distribution and dangers of rainfall and flooding, and what we should expect from global warming. This paper looks at British press coverage of flooding, and at the connections made between this theme and climate change. It aims to expose longitudinal patterning, and assess how common the connection is. From here the analysis moves to specific cases of high profile flooding events for a detailed exploration of the tone and tenor, and discursive contours of reports. The results suggest the media's contribution to genuine debate is mixed, that the connection between common forms of reportage and our scientific understanding of the phenomena is often tenuous. The paper explores the implications for the way the politics of climate change plays out.