Dendritic Protein Research Articles

Inhibition of ERK1/2 or CRMP2 Disrupts Alcohol Memory Reconsolidation and Prevents Relapse in Rats.

Relapse to alcohol abuse, often caused by cue-induced alcohol craving, is a major challenge in alcohol addiction treatment. Therefore, disrupting the cue-alcohol memories can suppress relapse. Upon retrieval, memories transiently destabilize before they reconsolidate in a process that requires protein synthesis. Evidence suggests that the mammalian target of rapamycin complex 1 (mTORC1), governing the translation of a subset of dendritic proteins, is crucial for memory reconsolidation. Here, we explored the involvement of two regulatory pathways of mTORC1, phosphoinositide 3-kinase (PI3K)-AKT and extracellular regulated kinase 1/2 (ERK1/2), in the reconsolidation process in a rat (Wistar) model of alcohol self-administration. We found that retrieval of alcohol memories using an odor-taste cue increased ERK1/2 activation in the amygdala, while the PI3K-AKT pathway remained unaffected. Importantly, ERK1/2 inhibition after alcohol memory retrieval impaired alcohol-memory reconsolidation and led to long-lasting relapse suppression. Attenuation of relapse was also induced by post-retrieval administration of lacosamide, an inhibitor of collapsin response mediator protein-2 (CRMP2)-a translational product of mTORC1. Together, our findings indicate the crucial role of ERK1/2 and CRMP2 in the reconsolidation of alcohol memories, with their inhibition as potential treatment targets for relapse prevention.

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3β and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

Learning and memory require activity-induced changes in dendritic translation, but which mRNAs are involved and how they are regulated are unclear. In this study, to monitor how depolarization impacts local dendritic biology, we employed a dendritically targeted proximity labeling approach followed by crosslinking immunoprecipitation, ribosome profiling and mass spectrometry. Depolarization of primary cortical neurons with KCl or the glutamate agonist DHPG caused rapid reprogramming of dendritic protein expression, where changes in dendritic mRNAs and proteins are weakly correlated. For a subset of pre-localized messages, depolarization increased the translation of upstream open reading frames (uORFs) and their downstream coding sequences, enabling localized production of proteins involved in long-term potentiation, cell signaling and energy metabolism. This activity-dependent translation was accompanied by the phosphorylation and recruitment of the non-canonical translation initiation factor eIF4G2, and the translated uORFs were sufficient to confer depolarization-induced, eIF4G2-dependent translational control. These studies uncovered an unanticipated mechanism by which activity-dependent uORF translational control by eIF4G2 couples activity to local dendritic remodeling.

Putative Cellular Identity of Dystrophic Neurites in FTLD‐TDP Type C

AbstractBackgroundApproximately 90% of individuals diagnosed with the clinical syndrome of semantic variant of primary progressive aphasia (PPA‐S) will present at autopsy with the TDP‐43 type C pathologic form of frontotemporal lobar degeneration (FTLD‐TDP‐C). FTLD‐TDP‐C is primarily characterized by the presence of TDP‐43‐immunoreactive long, thick dystrophic neurites (DNs), which tend to be found in superficial cortical layers, perpendicular to the cortical surface. The cellular identity of long DNs is not yet known. This study aims to understand the neuronal origin of DNs via colocalization of pathologic TDP with axonal and dendritic markers.MethodTwo right‐handed cases with FTLD‐TDP‐C as the sole pathologic diagnosis and a clinical diagnosis of PPA‐S were identified from the Northwestern University Alzheimer’s Disease Research Center (NU‐ADRC) brain bank. Participants were co‐enrolled in the NU PPA program, a longitudinal study of individuals with this dementia syndrome. Paraffin‐embedded sections of right inferior frontal gyrus (IFG) were fluorescently double‐stained immunohistochemically with an antibody to TDP‐43 phosphorylated at Ser 409 / 410 (pTDP) and antibodies to either phosphorylated neurofilament (pNFH) or non‐phosphorylated neurofilament (npNFH) to visualize axons or proximal dendrites, respectively. A digital image of each slide was obtained at 20X magnification using the Olympus VS200 Slide Scanner and analyzed using QuPath software (v.0.4.3).ResultIn both cases, staining revealed that a small amount (∼2%) of total DNs per section colocalize with npNFH (see Figure 1). Both long and short DNs, the latter of which are commonly apparent in other pathologic TDP subtypes (e.g., type A), demonstrated colocalization. By observation, half of colocalized DNs were oriented vertically. No DNs were found to colocalize with pNFH.ConclusionPreliminary findings suggest that DNs in FTLD‐TDP‐C may be remnants of dystrophic dendrites. The vertical orientation of colocalized DNs could reflect the orientation of apical dendrites in superficial layers of cortex. It is possible that DNs that did not colocalize with dendritic or axonal protein may be of distal dendritic origin or may reflect loss of dendritic protein in affected neurons. While it is difficult to determine the pathogenic history of DNs, these observations are promising and warrant future in‐depth investigation.

Age-Dependent Regulation of Dendritic Spine Density and Protein Expression in Mir324 KO Mice.

Dendritic spines are small, dynamic protrusions along the dendrite that comprise more than 90% of excitatory connections in the brain, making them essential sites for neuronal communication. These synaptic sites change throughout the process of development, reducing in density and shifting morphology as synapses are refined. One important class of dendritic spine regulators is microRNA (miRNA), small-noncoding RNAs that post-transcriptionally regulate gene expression. Several studies suggest that miRNA-324-5p regulates dendritic spine formation. In addition, we have previously shown that miR-324-5p plays a role in seizure and long-term potentiation, both of which involve dendritic spine changes. In this study, we aimed to characterize the role of miRNA-324-5p in developmental spine regulation by assessing the effect of Mir324 knockout (KO) on dendritic spine density and expression of a subset of dendritic proteins at select developmental time points. We show that miR-324-5p expression is developmentally regulated and peaks at 4weeks of age. We demonstrate that loss of miR-324-5p expression leads to differential changes in both target protein expression and spine density at different time points during development, disrupting the pattern of spine density changes and leading to a premature loss of dendritic spines in KO mice, which is compensated later. Our findings indicate that miR-324-5p plays a role in synaptic refinement across development. Additionally, our data illustrate the importance of context in the study of miRNA, as regulation by and/or of miRNA can vary dramatically across development and in disease.

Golgi satellites are essential for polysialylation of NCAM and expression of LTP at distal synapses

The complex cytoarchitecture of neurons poses significant challenges for the maturation of synaptic membrane proteins. It is currently unclear whether locally secreted synaptic proteins bypass the Golgi or whether they traffic through Golgi satellites (GSs). Here, we create a transgenic GS reporter mouse line and show that GSs are widely distributed along dendrites and are capable of mature glycosylation, in particular sialylation. We find that polysialylation of locally secreted NCAM takes place at GSs. Accordingly, in mice lacking a component of trans-Golgi network-to-plasma membrane trafficking, we find fewer GSs and significantly reduced PSA-NCAM levels in distal dendrites of CA1 neurons that receive input from the temporoammonic pathway. Induction of long-term potentiation at those, but not more proximal, synapses is severely impaired. We conclude that GSs serve the need for local mature glycosylation of synaptic membrane proteins in distal dendrites and thereby contribute to rapid changes in synaptic strength.

Domain and cell type-specific immunolocalisation of voltage-gated potassium channels in the mouse striatum

Diverse classes of voltage-gated potassium channels (Kv) are integral to the variety of electrical activity patterns that distinguish different classes of neurons in the brain. A feature of their heterogenous expression patterns is the highly precise manner in which specific cell types target their location within functionally specialised sub-cellular domains. Although Kv expression profiles in cortical brain regions are widely reported, their immunolocalisation in sub-cortical areas such as the striatum, and in associated diseases such as Parkinson’s disease (PD), remain less well described. Therefore, the broad aims of this study were to provide a high resolution immunolocalisation analysis of various Kv subtypes within the mouse striatum and assess their potential plasticity in a model of PD. Immunohistochemistry and confocal microscopy revealed that immunoreactivity for Kv1.1, 1.2 and 1.4 overlapped to varying degrees with excitatory and inhibitory axonal marker proteins suggesting these Kv subtypes are targeted to axons innervating striatal medium spiny neurons (MSNs). Immunoreactivity for Kv1.3 strongly overlapped with signal for mitochondrial marker proteins in MSN somata and dendrites. Kv1.5 immunoreactivity was expressed in parvalbumin-immunopositive neurons whereas Kv1.6 was located in cells immunopositive for microglia. Signal for Kv2.1 was concentrated on the somatic and proximal dendritic plasma membrane of MSNs, whilst immunoreactivity for Kv4.2 was targeted to their distal dendritic regions. Finally, striatal Kv2.1 expression, at both the mRNA and protein levels, was decreased in alpha-synuclein overexpressing mice, yet increased in alpha-synuclein knockout mice, compared to wild-type counterparts. The data indicate a variety of Kv expression patterns that are distinctive to the striatum and susceptible to pathology that mirrors PD. Furthermore, these findings advance our understanding of the molecular diversity of various striatal cell types, and potentially have implications for the homeostatic changes of MSN excitability during associated medical conditions such as PD.

A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway.

As a neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.

192-LB: Ductal Epithelial Cells Adopt Transcriptional Features of Tolerogenic Dendritic Cells in Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells destroy insulin-producing beta cells. Disease etiology is dependent on the interplay of multiple heterogeneous cell types in the pancreatic environment; however, technical constraints have thus far impeded our understanding of the initial molecular perturbations that occur during disease progression and a comprehensive identification of the pathogenic cell types in T1D. In particular, it is difficulty to safely biopsy the human pancreas of living donors, and there is significant disease progression and beta cell destruction by the time patients are clinically diagnosed with T1D. To address this, the Human Pancreas Analysis Program (HPAP) collects pancreatic tissues from deceased organ donors diagnosed with T1D and performs comprehensive molecular phenotyping. Using the full spectrum of imaging, genetic, and genomic technologies available through HPAP, we discovered that a subset of pancreatic ductal cells acquires the signature of tolerogenic dendritic cells in an apparent attempt at immune suppression in T1D donors. Single cell RNA-seq revealed a highly significant enrichment of the tolerogenic dendritic cell gene signature in ductal cells of T1D donors, including activation of interferon genes. In addition, via highly multiplexed flow CyTOF, we identified a population of ductal cells expressing dendritic cell proteins that was enriched in pancreata from T1D donors. Finally, we confirmed these results independent of islet culture via analysis of spatial features in pancreatic tissue using imaging mass cytometry. Together, our multi-modal analyses, ranging from transcriptomics to spatial proteomics, suggest that in the setting of disease, ductal cells are transcriptionally similar to tolerogenic ductal cells These results imply a previously unappreciated role of these exocrine cells in modulating immune cell activity in long-term T1D. Disclosure K. H. Kaestner: None. Hpap consortium: n/a. G. Vahedi: None. Funding NIH UC4 DK112217NIH R01CA230800Susan G. Komen CCR185472448 NIH R01HL145754NIH U01DK127768The Burroughs Wellcome FundThe Chan Zuckerberg Initiative, Penn EpigeneticsThe Sloan Foundation

Alantolactone Alleviates Rise in Markers of Periodontitis by Lipopolysaccharide in Human Periodontal Ligament Cells

Alantolactone, a natural sesquiterpene lactone, exerts its antiallergic, antibacterial, antimicrobial, antifungal, anticancer, and neuroprotective effects by virtue of its anti-inflammatory and antioxidant properties. We have extended these observations by examining its effect on markers of periodontitis in lipopolysaccharide treated human periodontal ligament cells. Treatment with lipopolysaccharide induced increase in cell apoptosis and decrease in cell viability that were decreased by alantolactone. Also, alantolactone suppressed lipopolysaccharide induced oxidative stress through upregulation of superoxide dismutase and reduced glutathione and downregulation of malondialdehyde and myeloperoxidase. Moreover, lipopolysaccharideinduced upregulation of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in human periodontal ligament cells was also reduced by alantolactone. Moreover, alantolactone attenuated lipopolysaccharide-induced increase in osteoclast markers, receptor activator of nuclear factor-kappa B ligand, nuclear factor of activated T cells c1, dendritic cell-specific transmembrane protein, and cellular proto-oncogene Fos in RAW 264.7. The increased number of tartrate resistant acid phosphatase-positive RAW 264.7 was also reduced by alantolactone. Finally, alantolactone downregulated levels of p-p65, p-protein kinase, and p-Phosphatidylinositol 3-kinase in lipopolysaccharide treated human periodontal ligament cells. In conclusion, alantolactone reduced osteoclast formation, exerts antioxidant and anti-inflammatory effects in lipopolysaccharide induced human periodontal ligament cells through inactivation of Akt and nuclear factor-kappa B pathways.

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

Exploring Brain Information Storage/Reading for Neuronal Connectivity Using Macromolecular Electrochemical Sensing Motors

Dense electroactive gels of conducting polymers, carbon nanotubes, or graphenes, constituted by multisensing macromolecular electrochemical motors, are presented herein as model materials to get a physicochemical characterization of the opening/closing cycles of ion protein channels in neurons. The conformational contracted energetic states of these macromolecular motors store both long‐term and short‐term information packages. Under a potential cycle, the energy of each ionic pulse generated during the channel opening reads the stored information, which is restored during the channel conformational closing. Simultaneously the motor actuation senses the working physical and chemical energetic conditions, transferring to the ionic pulse energy those sensing information packages. A quantitative description of the stored and read information packages is attained from basic electrochemical, mechanical, and polymeric concepts. Translated to ion channel proteins in brain dendrites, it can describe brain information storage mechanisms and the energetic information packages read and carried through each ionic pulse to the action potential. An unexplored way is opened to try to understand and describe how the different brain parts can use this information to originate brain functions. In parallel, the construction of new electrochemical devices and computers replicating biological functions will provide feedback quantitative information to decipher brain functions.

Connected neurons in multiple neocortical areas, comprising parallel circuits, encode essential information for visual shape learning

Neocortical areas comprised of multiple neuronal circuits which are encoded with innumerable advanced cognitive tasks. Studies focused on neuronal network and synaptic plasticity has hypothesized that every specific neuron and the circuit process the explicit essential information for the specific tasks. However, the structure of these circuits and the involved critical neurons remain to be elucidated. Considering our previous studies, showing the specificity of rat postrhinal cortex comprising specific neuronal circuit for encoding both the learning and recall of shape discrimination through a fast neurotransmitter release from the transduced neurons, here we have demonstrated that postsynaptic neurons in two distinct areas, perirhinal cortex and the ventral temporal association areas are required for the specific visual shape discriminations learning. The constitutively active PKC was delivered into neuronal cells in postrhinal cortex, and the animals were allowed to learn the new shape discriminations, and then the silencing siRNA was delivered into postsynaptic neurons in either perirhinal cortex or ventral temporal association areas, using a novel technology for gene transfer into connected neurons. We observed that expression of the siRNA caused the deficits in visual performance, via blocking the activity in the neurons, as displayed by activity-dependent gene imaging, and also subsequently obstructed the activation of specific signaling pathways required for further learning, and dendritic protein synthesis and CREB. Thus, ratifying the conclusion that the two parallel circuits are both required for the visual shape discrimination learning.

Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

Cryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

Mitochondrial support and local translation of mitochondrial proteins in synaptic plasticity and function.

Complex neural and brain functions are executed through structural and functional alterations of synapses and neurons. Neuronal compartmentalization requires neurons to allocate mitochondria and proteins in a spatiotemporal manner to allow their plasticity, function and homeostasis. Importantly, mitochondria are known to interact with and modulate synaptic activities through their ATP supply, calcium buffering and signaling abilities. Over the years, mitochondrial support and local translation (including mitochondrial proteins) at neuronal sub-compartments and their synaptic specializations have been considered critical for maintaining synaptic plasticity and function. Recently, evidence has shown that late endosomes can serve as sites for local translation of mRNAs crucial for mitochondrial integrity and mitochondrial compartments can fuel plasticity-induced local translation. Indeed, failed mitochondrial homeostasis and subsequent synaptic dysfunction are often intricately linked in the malfunction of the central nervous system in synaptic aging and diseases. In this review, I will discuss the critical role of local translation (including mitochondrial proteins) in dendrites, axons and synapses on neuronal/synaptic plasticity and function.

Regional and organ-level responses to local lung irradiation in sheep

Lung is a dose-limiting organ in radiotherapy. This may limit tumour control when effort is made in planning to limit the likelihood of radiation-induced lung injury (RILI). Understanding the factors that dictate susceptibility to radiation-induced pulmonary fibrosis will aid in the prevention and management of RILI, and may lead to more effective personalized radiotherapy treatment. As the interaction of regional and organ-level responses may shape the chronic consequences of RILI, we sought to characterise both aspects of the response in an ovine model. A defined volume of left pulmonary parenchyma was prescribed 5 fractions of 6 Gy within 14 days while the contralateral lung dose was constrained. Radiographic changes via computed tomography (CT) were documented to define differences in radio-exposed lung relative to non-exposed lung at d21, d63 and d171 (n = 2), and at d21, d147 and d227 (n = 2). Gross and histologic lung changes were evaluated in samples derived at necropsy examination to define the chronic pulmonary response to radiation. Irradiated lung demonstrated reduced radio-density and increased homogeneity as evidenced from texture based radiomic feature analysis, relative to the control lung. At necropsy, the radiation field was readily defined by pallor on the pleural surface, which was also evident on the cut surface of fixed lung specimens. The degree and homogeneity of pallor reflected the sparse presence of erythrocytes in alveolar septal capillaries of radiation-exposed lung. These changes contrasted with dilated and congested microvasculature in the contralateral control lung. Referencing data to measurements made in control lung volumes of sheep experiencing acute RILI indicated that interstitial collagen continues to deposit in the radio-exposed lung field. Overall lung vascularity increased during the chronic response, as evidenced by increased expression of endothelial cell marker (CD31); however, vascularity was consistently decreased in irradiated lung and was negatively correlated with lung collagen. Other organ-level responses included increased expression of alpha smooth muscle actin (ASMA), increased numbers of proliferating cells (Ki67 positive), and cells expressing the dendritic cell-lysosomal associated membrane protein (DC-LAMP) antigen. The chronic response to RILI in this model is effected at both the whole organ and local lung level. Whilst the long-term consequences of exposure to radiation involved the continued deposition of collagen in the radiation field, organ-level responses also included increased vascularization and increased expression of ASMA, Ki67 and DC-LAMP. Interrupting the interplay between these aspects may influence susceptibility to pulmonary fibrosis after radiotherapy. We advocate for the importance of large animal model systems in pursuing these opportunities to target local, organ-level and systemic mechanisms in parallel within the same subject over time.

Upregulation of eIF4E, but not other translation initiation factors, in dendritic spines during memory formation.

Local translation can provide a rapid, spatially targeted supply of new proteins in distal dendrites to support synaptic changes that underlie learning. Learning and memory are especially sensitive to manipulations of translational control mechanisms, particularly those that target the initiation step, and translation initiation at synapses could be a means of maintaining synapse specificity during plasticity. Initiation predominantly occurs via recruitment of ribosomes to the 5' mRNA cap by complexes of eukaryotic initiation factors (eIFs), and the interaction between eIF4E and eIF4G1 is a particularly important target of translational control pathways. Pharmacological inhibition of eIF4E-eIF4G1 binding impairs formation of memory for aversive Pavlovian conditioning as well as the accompanying increase in polyribosomes in the heads of dendritic spines in the lateral amygdala (LA). This is consistent with a role for initiation at synapses in memory formation, but whether eIFs are even present near synapses is unknown. To determine whether dendritic spines contain eIFs and whether eIF distribution is affected by learning, we combined immunolabeling with serial section transmission electron microscopy (ssTEM) volume reconstructions of LA dendrites after Pavlovian conditioning. Labeling for eIF4E, eIF4G1, and eIF2α-another key target of regulation-occurred in roughly half of dendritic spines, but learning effects were only found for eIF4E, which was upregulated in the heads of dendritic spines. Our results support the possibility of regulated translation initiation as a means of synapse-specific protein targeting during learning and are consistent with the model of eIF4E availability as a central point of control.

Small GTPases of the Rab and Arf Families: Key Regulators of Intracellular Trafficking in Neurodegeneration.

Small guanosine triphosphatases (GTPases) of the Rab and Arf families are key regulators of vesicle formation and membrane trafficking. Membrane transport plays an important role in the central nervous system. In this regard, neurons require a constant flow of membranes for the correct distribution of receptors, for the precise composition of proteins and organelles in dendrites and axons, for the continuous exocytosis/endocytosis of synaptic vesicles and for the elimination of dysfunctional proteins. Thus, it is not surprising that Rab and Arf GTPases have been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Both pathologies share characteristics such as the presence of protein aggregates and/or the fragmentation of the Golgi apparatus, hallmarks that have been related to both Rab and Arf GTPases functions. Despite their relationship with neurodegenerative disorders, very few studies have focused on the role of these GTPases in the pathogenesis of neurodegeneration. In this review, we summarize their importance in the onset and progression of Alzheimer’s and Parkinson’s diseases, as well as their emergence as potential therapeutical targets for neurodegeneration.

Cerebrospinal Fluid Concentration of Neurogranin in Hip Fracture Patients with Delirium.

Delirium is associated with an increased risk of incident dementia and accelerated progression of existing cognitive symptoms. Reciprocally, dementia increases the risk of delirium. Cerebrospinal fluid (CSF) concentration of the dendritic protein neurogranin has been shown to increase in early Alzheimer's disease (AD), likely reflecting synaptic dysfunction and/or degeneration. To elucidate the involvement of synaptic dysfunction in delirium pathophysiology, we tested the association between CSF neurogranin concentration and delirium in hip fracture patients with different AD-biomarker profiles, while comparing them to cognitively unimpaired older adults (CUA) and AD patients. The cohort included hip fracture patients with (n = 70) and without delirium (n = 58), CUA undergoing elective surgery (n = 127), and AD patients (n = 46). CSF was collected preoperatively and diagnostically in surgery and AD patients respectively. CSF neurogranin concentrations were analyzed in all samples with an in-house ELISA. Delirium was assessed pre-and postoperatively in hip fracture patients by trained investigators using the Confusion Assessment Method. Hip fracture patients were further stratified based on pre-fracture dementia status, delirium subtype, and AD fluid biomarkers. No association was found between delirium and CSF neurogranin concentration (main analysis: delirium versus no delirium, p = 0.68). Hip fracture patients had lower CSF neurogranin concentration than AD patients (p = 0.001) and CUA (p = 0.035) in age-adjusted sensitivity analyses. The findings suggest that delirium is not associated with increased CSF neurogranin concentration in hip fracture patients, possibly due to advanced neurodegenerative disease and age and/or because synaptic degeneration is not an important pathophysiological process in delirium.

Apremilast inhibits inflammatory osteoclastogenesis.

Psoriatic arthritis (PsA) is associated with bone erosion and inflammation-induced bone loss, which are mediated by osteoclasts (OC) and modulated by inflammatory cytokines. Apremilast (APR) (a selective phosphodiesterase 4 inhibitor) is efficacious in PsA and acts by inhibiting cytokine production. However, there are no direct data informing whether and how APR affects osteoclast formation in humans. Osteoclastogenic cytokine production by activated human peripheral blood mononuclear cells (PBMCs) was measured in the presence and absence of APR. Effects of APR on osteoclast differentiation were tested (i) in co-cultures of activated PBMCs and human CD14+ blood monocytes as well as (ii) in CD14+ blood monocytes stimulated with activated-PBMCs supernatant, TNF or IL-17A. Bone resorption was measured on OsteoAssay plates. Effects of APR on ex vivo osteoclast differentiation were compared in PsA, pre-PsA and psoriasis patients, as well as in healthy controls. APR significantly impaired the expression of key osteoclastogenic cytokines in activated PBMCs. Furthermore, APR dose-dependently and significantly inhibited activated PBMC-driven osteoclast differentiation and ex vivo osteoclast differentiation of PBMCs derived from PsA and pre-PsA patients, but not from psoriasis patients or healthy controls. TNF and IL-17A-enhanced osteoclastogenesis and osteolytic activity of CD14+ blood monocytes from PsA patients was also significantly inhibited by APR. Finally, APR inhibited expression of the key osteoclast fusion protein dendritic cell-specific transmembrane protein. Phosphodiesterase 4 targeting by APR not only inhibits osteoclastogenic cytokine production, but also directly suppresses inflammation-driven osteoclastogenesis. These data provide initial evidence that APR has the potential to provide a direct bone protective effect in PsA.