OprF functions as a latch to direct Outer Membrane Vesicle release in Pseudomonas aeruginosa.

Bacterial Outer Membrane Vesicles (OMVs) contribute to virulence, competition, immune avoidance and communication. This has led to great interest in how they are formed. To date, investigation has focused almost exclusively on what controls the initiation of OMV biogenesis. Regardless of the mechanism of initiation, all species face a similar challenge before an OMV can be released: How does the OM detach from the underlying peptidoglycan (PG) in regions that will ultimately bulge and then vesiculate? The OmpA family of OM proteins (OprF in P. aeruginosa) is widely conserved and unusually abundant in OMVs across species considering their major role in PG attachment. OmpA homologs also have the interesting ability to adopt both PG-bound (two-domain) and PG-released (one-domain) conformations. Using targeted deletion of the PG-binding domain we showed that loss of cell wall association, and not general membrane destabilization, is responsible for hypervesiculation in OprF-modified strains. We therefore propose that OprF functions as a 'latch', capable of releasing PG in regions destined to become OMVs. To test this hypothesis, we developed a protocol to assess OprF conformation in live cells and purified OMVs. While >90% of OprF proteins exist in the two-domain conformation in the OM of cells, we show that the majority of OprF in OMVs is present in the one-domain conformation. With this work, we take some of the first steps in characterizing late-stage OMV biogenesis and identify a family of proteins whose critical role can be explained by their unique ability to fold into two distinct conformations.

Fingolimod mitigates synaptic deficits and psychosis-like behavior in APP/PSEN1 mice.

Introduction: Current treatments for psychosis in Alzheimer's disease (AD), a syndrome characterized by more rapid deterioration and reduced synaptic protein abundance relative to non-psychotic AD, are inadequate. Fingolimod, a currently US Food and Drug Administration (FDA)-approved pharmacotherapy for multiple sclerosis, alters synaptic protein expression and warrants preclinical appraisal as a candidate pharmacotherapy for psychosis in AD. Methods: Presenilin and amyloid precursor protein transgenic mice (APPswe/PSEN1dE9) and wild-type mice were randomized to fingolimod or saline for 7 days. Psychosis-associated behaviors were quantified by open field testing, pre-pulse inhibition of the acoustic startle response testing, and habituation of the acoustic startle response testing. Synaptic proteins were quantified by liquid chromatography/mass spectrometry in homogenate and postsynaptic density fractions. Results: Fingolimod treatment increased the synaptic protein abundance in cortical homogenates and normalized psychosis-associated behaviors in APPswe/PSEN1dE9 mice relative to saline. Mitochondrial-related proteins were preferentially altered by fingolimod treatment and correlated with improvements in psychosis-associated behaviors. Discussion: Preclinical studies employing complementary psychosis-associated behavioral assessments and proteomic evaluations across multiple AD-related models are warranted to replicate the current study and further investigate fingolimod as a candidate treatment for psychosis in AD.

A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development.

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

TFOS European Ambassador meeting: Unmet needs and future scientific and clinical solutions for ocular surface diseases.

The mission of the Tear Film & Ocular Surface Society (TFOS) is to advance the research, literacy, and educational aspects of the scientific field of the tear film and ocular surface. Fundamental to fulfilling this mission is the TFOS Global Ambassador program. TFOS Ambassadors are dynamic and proactive experts, who help promote TFOS initiatives, such as presenting the conclusions and recommendations of the recent TFOS DEWS II™, throughout the world. They also identify unmet needs, and propose future clinical and scientific solutions, for management of ocular surface diseases in their countries. This meeting report addresses such needs and solutions for 25 European countries, as detailed in the TFOS European Ambassador meeting in Rome, Italy, in September 2019.

Perspective on Systematic Medical Literature Reviews and Meta-Analyses.

PURPOSE: This study sought to identify factors contributing to the inadequacies of systematic reviews and meta-analyses (SRMAs) published in the ophthalmology literature. DESIGN: Perspective. METHODS: Review and synthesis of selective literature, with interpretation and perspective. RESULTS: Although recommendations for the design, conduct, assessment of quality, and risk of bias of systematic reviews have been widely available, some recent publications illustrate a serious potential failing in this domain: inclusion of refuted science, lack of citation of post-publication correspondence and failure to use ≥1 alternative search strategy. CONCLUSIONS: Examples of inadequacies of peer review in medical literature and perpetuation of erroneous science by unfiltered inclusion in subsequent systematic reviews have been identified, and the problem can be traced to authors, peer reviewers, and editors of journals. This perspective identifies and analyzes several possible causes of the problem and recommends some specific corrective actions to improve the quality and accuracy of such reviews.

Synaptic Proteome Compensation and Resilience to Psychosis in Alzheimer's Disease.

OBJECTIVE: The presence of psychosis in Alzheimer's disease denotes a phenotype with more rapid cognitive deterioration than in Alzheimer's disease without psychosis. Discovery of novel pharmacotherapies that engage therapeutic targets for prevention or treatment of Alzheimer's disease with psychosis would benefit from identifying the neurobiology of resilience to psychosis in Alzheimer's disease. The primary objective of this study was to determine whether alterations in the synaptic proteome were associated with resilience to psychotic symptoms in Alzheimer's disease and, if present, were independent of neuropathologic burden. METHOD: Quantitative immunohistochemistry was used to measure multiple neuropathologies in dorsolateral prefrontal cortex from subjects with early and middle-stage Alzheimer's disease who differed in psychosis status. Synaptic proteins were quantified by liquid chromatography-mass spectrometry in gray matter homogenates from these subjects and from neuropathologically unaffected subjects. The synaptic proteome was similarly evaluated in cortical gray matter homogenate and in postsynaptic density fractions from an APPswe/PSEN1dE9 mouse model of amyloidosis with germline reduction in Kalrn, which has been shown to confer resilience to progression of psychosis-associated behaviors relative to APPswe/PSEN1dE9 alone. RESULTS: Subjects resilient to psychotic symptoms in Alzheimer's disease had higher levels of synaptic proteins compared with those with psychosis and unaffected control subjects. Neuropathologic burden predicted less than 20% of the variance in psychosis status and did not account for the synaptic protein level differences between groups. Reduction of Kalrn in APPswe/PSEN1dE9 mice resulted in higher levels of synaptic proteins in cortical homogenate and normalized protein levels in the postsynaptic density. CONCLUSIONS: Accumulation of synaptic proteins, particularly those that are enriched in the postsynaptic density, is associated with resilience to psychosis in Alzheimer's disease. One candidate mechanism for this synaptic proteome compensation is alteration in levels of proteins that facilitate the transport of synaptic proteins to and from the postsynaptic density.

Minnesota Alliance for Nursing Education (MANE): A Unique Multi-institutional Approach to Preparing Nurses for the Future.

AIM: The purpose of this article is to inform nurse educators of an innovative and transformative approach to advancing nursing education through the creation of the Minnesota Alliance for Nursing Education (MANE). BACKGROUND: MANE was formed to answer national appeals to transform nursing education and demands by health care organizations for a more highly educated nursing workforce. METHOD: An action plan was developed with the Minnesota Action Coalition to create a dual admission community college and university eight-semester baccalaureate curriculum that students could complete seamlessly in four years. RESULTS: Admissions to the program have been robust, matriculation rates to upper division course work have exceeded initial goals, and diversity of students is increasing. CONCLUSION: Measures to date show that MANE is a viable and successful program that is surpassing its initial goals.

Kalirin reduction rescues psychosis-associated behavioral deficits in APPswe/PSEN1dE9 transgenic mice.

Psychosis in Alzheimer's disease (AD+P) represents a distinct clinical and neurobiological AD phenotype and is associated with more rapid cognitive decline, higher rates of abnormal behaviors, and increased caregiver burden compared with AD without psychosis. On a molecular level, AD+P is associated with greater reductions in the protein kalirin, a guanine exchange factor which has also been linked to the psychotic disease, schizophrenia. In this study, we sought to determine the molecular and behavioral consequences of kalirin reduction in APPswe/PSEN1dE9 mice. We evaluated mice with and without kalirin reduction during tasks measuring psychosis-associated behaviors and spatial memory. We found that kalirin reduction in APPswe/PSEN1dE9 mice significantly attenuated psychosis-associated behavior at 12 months of age without changing spatial memory performance. The 12-month-old APPswe/PSEN1dE9 mice with reduced kalirin levels also had increased levels of the active, phosphorylated forms of p21 protein (Cdc42/Rac)-activated kinases (PAKs), which function in signaling pathways for maintenance of dendritic spine density, morphology, and function.

Developmental Trajectories of Auditory Cortex Synaptic Structures and Gap-Prepulse Inhibition of Acoustic Startle Between Early Adolescence and Young Adulthood in Mice.

Cortical excitatory and inhibitory synapses are disrupted in schizophrenia, the symptoms of which often emerge during adolescence, when cortical excitatory synapses undergo pruning. In auditory cortex, a brain region implicated in schizophrenia, little is known about the development of excitatory and inhibitory synapses between early adolescence and young adulthood, and how these changes impact auditory cortex function. We used immunohistochemistry and quantitative fluorescence microscopy to quantify dendritic spines and GAD65-expressing inhibitory boutons in auditory cortex of early adolescent, late adolescent, and young adult mice. Numbers of spines decreased between early adolescence and young adulthood, during which time responses increased in an auditory cortex-dependent sensory task, silent gap-prepulse inhibition of the acoustic startle reflex (gap-PPI). Within-bouton GAD65 protein and GAD65-expressing bouton numbers decreased between late adolescence and young adulthood, a delay in onset relative to spine and gap-PPI changes. In mice lacking the spine protein kalirin, there were no significant changes in spine number, within-bouton GAD65 protein, or gap-PPI between adolescence and young adulthood. These results illustrate developmental changes in auditory cortex spines, inhibitory boutons, and auditory cortex function between adolescence and young adulthood, and provide insights into how disrupted adolescent neurodevelopment could contribute to auditory cortex synapse pathology and auditory impairments.

Chronic desipramine treatment alters tyrosine hydroxylase but not norepinephrine transporter immunoreactivity in norepinephrine axons in the rat prefrontal cortex.

Pharmacological blockade of norepinephrine (NE) reuptake is clinically effective in treating several mental disorders. Drugs that bind to the NE transporter (NET) alter both protein levels and activity of NET and also the catecholamine synthetic enzyme tyrosine hydroxylase (TH). We examined the rat prefrontal cortex (PFC) by electron microscopy to determine whether the density and subcellular distribution of immunolabelling for NET and co-localization of NET with TH within individual NE axons were altered by chronic treatment with the selective NE uptake inhibitor desipramine (DMI). Following DMI treatment (21 d, 15 mg/kg.d), NET-immunoreactive (ir) axons were significantly less likely to co-localize TH. This finding is consistent with reports of reduced TH levels and activity in the locus coeruleus after chronic DMI and indicates a reduction of NE synthetic capacity in the PFC. Measures of NET expression and membrane localization, including the number of NET-ir profiles per tissue area sampled, the number of gold particles per NET-ir profile area, and the proportion of gold particles associated with the plasma membrane, were similar in DMI- and vehicle-treated rats. These findings were verified using two different antibodies directed against distinct epitopes of the NET protein. The results suggest that chronic DMI treatment does not reduce NET expression within individual NE axons in vivo or induce an overall translocation of NET protein away from the plasma membrane in the PFC as measured by ultrastructural immunogold labelling. Our findings encourage consideration of possible post-translational mechanisms for regulating NET activity in antidepressant-induced modulation of NE clearance.

Subbarrel patterns of thalamocortical innervation in rat somatosensory cortical barrels: Organization and postnatal development.

Barrel hollows in the posteromedial barrel subfield of adult rat somatosensory cortex typically encompass two or three metabolically and structurally distinct regions, termed subbarrels. We used immunohistochemical staining for vesicular glutamate transporter 2 and the neuronal serotonin transporter, in conjunction with cytochrome oxidase (CO) histochemistry, to investigate the distribution of thalamocortical (TC) axon terminals in relation to subbarrel domains. We found, first, that CO-dark subbarrels are more intensely immunoreactive for thalamocortical terminals than the CO-light clefts that separate them. Second, during the first postnatal week, immunoreactivity for markers of TC terminals is relatively homogeneous throughout the barrel hollow; subbarrel patterns of distribution only become recognizable between P-8 and P-10. These observations extend previous findings that subbarrels denote barrel regions enriched in synaptic contacts. The data also indicate that allocation of TC terminals into subbarrel domains does not occur immediately upon thalamic axon ingrowth. Instead, refinement of TC arbors into subbarrels is a gradual process, the outcome of which is not manifest until the second week of postnatal life.

Subbarrel domains in rat somatosensory (S1) cortex.

We used cytochrome oxidase (CO) histochemistry in conjunction with other histological methods to investigate the histochemoarchitecture of barrel hollows in rat somatosensory cortex. We found that individual large barrels in the posteromedial barrel subfield encompass two or three discrete subbarrel domains. Detailed analysis revealed, further, that subbarrel domains are relatively consistent in size, each having average dimensions that approximate those of large barrels in mouse S1. Unexpectedly, subbarrel domains are organized into a few distinct, repeated patterns. The small barrels in rat anterolateral barrel subfield and all barrel hollows in mouse S1 appear to consist of single CO domains. Subbarrel domains revealed here by CO are columnar entities that correspond with cyto- and myeloarchitectonic inhomogeneities within the barrels and are enriched in thalamocortical axon terminals. The present findings together with existing data indicate that barrels in rat posteromedial barrel subfield are structurally and functionally heterogeneous.

Postnatal development of prefrontal inhibitory circuits and the pathophysiology of cognitive dysfunction in schizophrenia.

The typical appearance of the clinical features of schizophrenia during late adolescence or early adulthood suggests that adolescence-related neurodevelopmental events may contribute to the pathophysiology of this disorder. Here the role that GABA-mediated inhibition in the dorsal lateral prefrontal cortex (DLPFC) plays in regulating working memory, a core cognitive process that matures late and that is disturbed in schizophrenia, is reviewed. Recent studies are summarized that demonstrate (1) that certain pre- and postsynaptic markers of GABA neurotransmission in the monkey DLPFC exhibit striking changes during adolescence, and (2) that these same markers are markedly altered in the DLPFC of subjects with schizophrenia. The implications of these findings for treatment and prevention strategies are discussed.

Cortical connections of the lateral mediodorsal thalamus in cynomolgus monkeys.

The prefrontal cortex has been defined as that cortical territory that has "essential or sustaining" connections with the mediodorsal (MD) nucleus of the thalamus. However, recent studies in the monkey have documented projections from MD to the more caudal, agranular regions of the frontal cortex, suggesting that the connections of MD may be characterized by a breadth of distribution and diversity of functional roles too great to be useful as a unifying and defining feature for a specific cortical territory. In this study, we placed tracer injections in the lateral divisions of MD in cynomolgus monkeys (Macaca fascicularis) to assess the relative proportions of connections devoted to diverse regions of the frontal cortex (FC). Three different patterns of label were observed in the cortex, associated with different locations within lateral MD. We have designated these as the ventrolateral MD-arcuate FC circuit, having most label in areas 8 and 6; the caudoventral MD-dorsomedial FC circuit, having most label in areas 24 and presupplementary motor area (SMA); and the anterodorsal MD-anterior FC circuit, with the most label in areas 9, 46, 12, and 10. Only two of the nine cases injected in lateral MD were predominantly connected with the anterior FC. Thus, particular locales within lateral MD are connected with multiple, functionally diverse cortical regions, including several not classically recognized as "prefrontal" areas. This divergence may distinguish MD-frontocortical and reciprocal corticothalamic pathways from the largely segregated pathways arising from the other thalamic nuclei that are interconnected with the frontal cortex, such as those from the ventrolateral nuclear group.

Postnatal development of parvalbumin- and GABA transporter-immunoreactive axon terminals in monkey prefrontal cortex.

In the primate prefrontal cortex, the axon terminals of the chandelier class of inhibitory local circuit neurons have a distinctive time course of postnatal development. In this study, we sought to determine whether the axon terminals of other classes of local circuit neurons are also refined during postnatal development. We examined postnatal changes in the density of punctate structures immunoreactive for the calcium binding protein parvalbumin, which identifies a subset of gamma-aminobutyric acid (GABA) -containing terminals, in the prefrontal cortex of 35 rhesus monkeys ranging in age from newborn to adult. In area 46, the density of parvalbumin- immunoreactive puncta in the superficial and middle layers was extremely low in the newborn animals, then increased more than 10-fold to adult levels, which were achieved by 3 to 4 years of age. In layer V, a band of labeled puncta present in the newborn animals also increased in density until 3 to 4 years of age. Developmental changes of parvalbumin-immunoreactive puncta in area 9 were similar to those in area 46. In contrast, the density of punctate structures labeled with an antibody against a GABA membrane transporter (GAT-1) did not change across development, suggesting that the number of GABAergic terminals is stable over time, but that the level of parvalbumin protein within the terminals varies. The time course of the observed changes in these parvalbumin-labeled terminals is markedly different from that of parvalbumin-immunoreactive chandelier cell terminal clusters. These findings suggest that morphologically specialized classes of inhibitory interneurons assume prominence within the prefrontal cortical network at different stages of postnatal development.