Nde1 is required for heterochromatin compaction and stability in neocortical neurons.

The NDE1 gene encodes a scaffold protein essential for brain development. Although biallelic NDE1 loss of function (LOF) causes microcephaly with profound mental retardation, NDE1 missense mutations and copy number variations are associated with multiple neuropsychiatric disorders. However, the etiology of the diverse phenotypes resulting from NDE1 aberrations remains elusive. Here we demonstrate Nde1 controls neurogenesis through facilitating H4K20 trimethylation-mediated heterochromatin compaction. This mechanism patterns diverse chromatin landscapes and stabilizes constitutive heterochromatin of neocortical neurons. We demonstrate that NDE1 can undergo dynamic liquid-liquid phase separation, partitioning to the nucleus and interacting with pericentromeric and centromeric satellite repeats. Nde1 LOF results in nuclear architecture aberrations and DNA double-strand breaks, as well as instability and derepression of pericentromeric satellite repeats in neocortical neurons. These findings uncover a pivotal role of NDE1/Nde1 in establishing and protecting neuronal heterochromatin. They suggest that heterochromatin instability predisposes a wide range of brain dysfunction.

CARD19 Interacts with Mitochondrial Contact Site and Cristae Organizing System Constituent Proteins and Regulates Cristae Morphology.

CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19-MIC25-MIC60 MICOS subcomplex that regulates cristae morphology.

Tourniquet-induced lower limb ischemia/reperfusion reduces mitochondrial function by decreasing mitochondrial biogenesis in acute kidney injury in mice.

The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet-induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time-dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC-1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat-shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3-I to LC3-II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1-PARK2-mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.

Genetic inactivation of SARM1 axon degeneration pathway improves outcome trajectory after experimental traumatic brain injury based on pathological, radiological, and functional measures.

Traumatic brain injury (TBI) causes chronic symptoms and increased risk of neurodegeneration. Axons in white matter tracts, such as the corpus callosum (CC), are critical components of neural circuits and particularly vulnerable to TBI. Treatments are needed to protect axons from traumatic injury and mitigate post-traumatic neurodegeneration. SARM1 protein is a central driver of axon degeneration through a conserved molecular pathway. Sarm1-/- mice with knockout (KO) of the Sarm1 gene enable genetic proof-of-concept testing of the SARM1 pathway as a therapeutic target. We evaluated Sarm1 deletion effects after TBI using a concussive model that causes traumatic axonal injury and progresses to CC atrophy at 10 weeks, indicating post-traumatic neurodegeneration. Sarm1 wild-type (WT) mice developed significant CC atrophy that was reduced in Sarm1 KO mice. Ultrastructural classification of pathology of individual axons, using electron microscopy, demonstrated that Sarm1 KO preserved more intact axons and reduced damaged or demyelinated axons. Longitudinal MRI studies in live mice identified significantly reduced CC volume after TBI in Sarm1 WT mice that was attenuated in Sarm1 KO mice. MR diffusion tensor imaging detected reduced fractional anisotropy in both genotypes while axial diffusivity remained higher in Sarm1 KO mice. Immunohistochemistry revealed significant attenuation of CC atrophy, myelin loss, and neuroinflammation in Sarm1 KO mice after TBI. Functionally, Sarm1 KO mice exhibited beneficial effects in motor learning and sleep behavior. Based on these findings, Sarm1 inactivation can protect axons and white matter tracts to improve translational outcomes associated with CC atrophy and post-traumatic neurodegeneration.

Gain-of-function mutations in CARD11 promote enhanced aggregation and idiosyncratic signalosome assembly.

BENTA (B cell Expansion with NF-κB and T cell Anergy) is a novel lymphoproliferative disorder caused by germline, gain-of-function (GOF) mutations in the lymphocyte-restricted scaffolding protein CARD11. Similar somatic CARD11 mutations are found in lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL). Normally, antigen receptor (AgR) engagement converts CARD11 into an active conformation that nucleates a signalosome required for IκB kinase (IKK) activation and NF-κB nuclear translocation. However, GOF CARD11 mutants drive constitutive NF-κB activity without AgR stimulation. Here we show that unlike wild-type CARD11, GOF CARD11 mutants can form large, peculiar cytosolic protein aggregates we term mCADS (mutant CARD11 dependent shells). MALT1 and phospho-IKK are reliably colocalized with mCADS, indicative of active signaling. Moreover, endogenous mCADS are detectable in ABC-DLBCL lines harboring similar GOF CARD11 mutations. The unique aggregation potential of GOF CARD11 mutants may represent a novel therapeutic target for treating BENTA or DLBCL.

Sarm1 deletion reduces axon damage, demyelination, and white matter atrophy after experimental traumatic brain injury.

Traumatic brain injury (TBI) often damages axons in white matter tracts and causes corpus callosum (CC) atrophy in chronic TBI patients. Injured axons encounter irreversible damage if transected, or alternatively may maintain continuity and subsequently either recover or degenerate. Secondary mechanisms can cause further axon damage, myelin pathology, and neuroinflammation. Molecular mechanisms regulating the progression of white matter pathology indicate potential therapeutic targets. SARM1 is essential for execution of the conserved axon death pathway. We examined white matter pathology following mild TBI with CC traumatic axonal injury in mice with Sarm1 gene deletion (Sarm1-/-). High resolution ultrastructural analysis at 3 days post-TBI revealed dramatically reduced axon damage in Sarm1-/- mice, as compared to Sarm1+/+ wild-type controls. Sarm1 deletion produced larger axons with thinner myelin, and attenuated TBI induced demyelination, i.e. myelin loss along apparently intact axons. At 6 weeks post-TBI, Sarm1-/- mice had less demyelination and thinner myelin than Sarm1+/+ mice, but axonal protection was no longer observed. We next used Thy1-YFP crosses to assess Sarm1 involvement in white matter neurodegeneration and neuroinflammation at 8 weeks post-TBI, when significant CC atrophy indicates chronic pathology. Thy1-YFP expression demonstrated continued CC axon damage yet absence of overt cortical pathology. Importantly, significant CC atrophy in Thy1-YFP/Sarm1+/+ mice was associated with reduced neurofilament immunolabeling of axons. Both effects were attenuated in Thy1-YFP/Sarm1-/- mice. Surprisingly, Thy1-YFP/Sarm1-/- mice had increased CC astrogliosis. This study demonstrates that Sarm1 inactivation reduces demyelination, and white matter atrophy after TBI, while the post-injury stage impacts when axon protection is effective.

Autophagy and mitochondrial remodelling in mouse mesenchymal stromal cells challenged with Staphylococcus epidermidis.

The bone marrow stroma constitutes the marrow-blood barrier, which sustains immunochemical homoeostasis and protection of the haematopoietic tissue in sequelae of systemic bacterial infections. Under these conditions, the bone marrow stromal cells affected by circulating bacterial pathogens shall elicit the adaptive stress-response mechanisms to maintain integrity of the barrier. The objective of this communication was to demonstrate (i) that in vitro challenge of mesenchymal stromal cells, i.e. colony-forming unit fibroblasts (CFU-F), with Staphylococcus epidermidis can activate the autophagy pathway to execute antibacterial defence response, and (ii) that homoeostatic shift because of the bacteria-induced stress includes the mitochondrial remodelling and sequestration of compromised organelles via mitophagy. Implication of Drp1 and PINK1-PARK2-dependent mechanisms in the mitophagy turnover of the aberrant mitochondria in mesenchymal stromal cells is investigated and discussed.

Components of myelin damage and repair in the progression of white matter pathology after mild traumatic brain injury.

White matter tracts are highly vulnerable to damage from impact-acceleration forces of traumatic brain injury (TBI). Mild TBI is characterized by a low density of traumatic axonal injury, whereas associated myelin pathology is relatively unexplored. We examined the progression of white matter pathology in mice after mild TBI with traumatic axonal injury localized in the corpus callosum. Adult mice received a closed-skull impact and were analyzed from 3 days to 6 weeks post-TBI/sham surgery. At all times post-TBI, electron microscopy revealed degenerating axons distributed among intact fibers in the corpus callosum. Intact axons exhibited significant demyelination at 3 days followed by evidence of remyelination at 1 week. Accordingly, bromodeoxyuridine pulse-chase labeling demonstrated the generation of new oligodendrocytes, identified by myelin proteolipid protein messenger RNA expression, at 3 days post-TBI. Overall oligodendrocyte populations, identified by immunohistochemical staining for CC1 and/or glutathione S-transferase pi, were similar between TBI and sham mice by 2 weeks. Excessively long myelin figures, similar to redundant myelin sheaths, were a significant feature at all post-TBI time points. At 6 weeks post-TBI, microglial activation and astrogliosis were localized to areas of axon and myelin pathology. These studies show that demyelination, remyelination, and excessive myelin are components of white matter degeneration and recovery in mild TBI with traumatic axonal injury.

Adaptive redox response of mesenchymal stromal cells to stimulation with lipopolysaccharide inflammagen: mechanisms of remodeling of tissue barriers in sepsis.

Acute bacterial inflammation is accompanied by excessive release of bacterial toxins and production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox stress. These factors can induce damage to components of tissue barriers, including damage to ubiquitous mesenchymal stromal cells (MSCs), and thus can exacerbate the septic multiple organ dysfunctions. The mechanisms employed by MSCs in order to survive these stress conditions are still poorly understood and require clarification. In this report, we demonstrated that in vitro treatment of MSCs with lipopolysaccharide (LPS) induced inflammatory responses, which included, but not limited to, upregulation of iNOS and release of RNS and ROS. These events triggered in MSCs a cascade of responses driving adaptive remodeling and resistance to a "self-inflicted" oxidative stress. Thus, while MSCs displayed high levels of constitutively present adaptogens, for example, HSP70 and mitochondrial Sirt3, treatment with LPS induced a number of adaptive responses that included induction and nuclear translocation of redox response elements such as NFkB, TRX1, Ref1, Nrf2, FoxO3a, HO1, and activation of autophagy and mitochondrial remodeling. We propose that the above prosurvival pathways activated in MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.

Preserving immunogenicity of lethally irradiated viral and bacterial vaccine epitopes using a radio- protective Mn2+-Peptide complex from Deinococcus.

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

Microglia activation along the corticospinal tract following traumatic brain injury in the rat: a neuroanatomical study.

Traumatic injury to the brain often manifests itself symptomatically and structurally long after the traumatic event. The cellular basis of this complex response is not completely understood. However, we hypothesized that microglia might contribute to the brain-wide process. To test this hypothesis, we employed optical and electron microscopy to study the microglia in rat brains up to 2 months after digitally controlled cortical impact (CCI) to produce traumatic brain injury (TBI). We also used antibodies against ED-1 and Iba-1, respectively, as markers for activated and resting microglia. ED-1 positive microglial cells are observed accompanying the entire corticospinal tract (CST) on the injured side, but not the control, contralateral side of the brain at 2 months. In this case, ED-1 and Iba-1 were observed to co-localize uniquely on the injured side of the brain. At earlier times following CCI, ultrastructural studies reveal that microglial cells have very irregular shapes and have many processes that intermingle with degenerating nerve axons of the CST in the hindbrain pyramids. These cells appear to be engulfing degenerating myelinated axons. The debris within the cells is converted to lipofuscin, the antigen for the ED-1 antibody, and remains in the cell cytoplasm throughout the life of the cell. We conclude, as hypothesized, that microglia are critical cellular components. Based on observed close association with myelin degeneration, interdigitating activated microglia may be contributing to damage control. Finally, based on the close neuroanatomical relationship between the lesioned corticospinal tract and the wide distribution of activated microglia, primary signals from CST neurons per se, may be directing microglial responses along the entire damaged rat neuroaxis. The role of persistent activation of microglia has not been determined.

The oligo-acyl lysyl antimicrobial peptide C12K-2ß12 exhibits a dual mechanism of action and demonstrates strong in vivo efficacy against Helicobacter pylori.

Helicobacter pylori has developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pylori therapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C(12)K-2ß(12), that shows potent in vitro bactericidal activity against H. pylori. Herein, we define the mechanism of action and evaluate the in vivo efficacy of C(12)K-2ß(12) against H. pylori after experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C(12)K-2ß(12) rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C(12)K-2ß(12) can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define the in vivo efficacy of C(12)K-2ß(12), H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C(12)K-2ß(12) 1 day or 1 week postinfection. The efficacy of C(12)K-2ß(12) was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P < 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C(12)K-2ß(12). Overall, our results demonstrate a dual mode of action of C(12)K-2ß(12) against the H. pylori membrane and cytoplasmic components. Moreover, and consistent with the previously reported in vitro efficacy, C(12)K-2ß(12) shows significant in vivo efficacy against H. pylori when used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment of H. pylori infection.

Rostrocaudal analysis of corpus callosum demyelination and axon damage across disease stages refines diffusion tensor imaging correlations with pathological features.

Noninvasive assessment of the progression of axon damage is important for evaluating disease progression and developing neuroprotective interventions in multiple sclerosis patients. We examined the cellular responses correlated with diffusion tensor imaging-derived axial (lambda(parallel)) and radial (lambda(perpendicular)) diffusivity values throughout acute (4 weeks) and chronic (12 weeks) stages of demyelination and after 6 weeks of recovery using the cuprizone demyelination of the corpus callosum model in C57BL/6 and Thy1-YFP-16 mice. The rostrocaudal progression of pathological alterations in the corpus callosum enabled spatially and temporally defined correlations of pathological features with diffusion tensor imaging measurements. During acute demyelination, microglial/macrophage activation was most extensive and axons exhibited swellings, neurofilament dephosphorylation, and reduced diameters. Axial diffusivity values decreased in the acute phase but did not correlate with axonal atrophy during chronic demyelination. In contrast, radial diffusivity increased with the progression of demyelination but did not correlate with myelin loss or astrogliosis. Unlike other animal models with progressive neurodegeneration and axon loss, the acute axon damage did not progress to discontinuity or loss of axons even after a period of chronic demyelination. Correlations of reversible axon pathology, demyelination, microglia/macrophage activation, and astrogliosis with regional axial and radial diffusivity measurements will facilitate the clinical application of diffusion tensor imaging in multiple sclerosis patients.

The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype.

Cells receive signals from the extracellular matrix through receptor-dependent interactions, but they are also influenced by the mechanical properties of the matrix. Although bulk properties of substrates have been shown to affect cell behavior, we show here that nanoscale properties of collagen fibrils also play a significant role in determining cell phenotype. Type I collagen fibrils assembled into thin films provide excellent viewing of cells interacting with individual fibrils. Cells can be observed to extensively manipulate the fibrils, and this behavior seems to result in an incompletely spread stellate morphology and a nonproliferative phenotype that is typical of these cells in collagen gels. We show here that thin films of collagen fibrils can be dehydrated, and when seeded on these dehydrated fibrils, smooth muscle cells spread and proliferate extensively. The dehydrated collagen fibrils appear to be similar to the fully hydrated collagen fibrils in topology and in presentation of beta(1) integrin ligation sites, but they are mechanically stiffer. This decrease in compliance of dehydrated fibrils is seen by a failure of cell movement of dehydrated fibrils compared to their ability to rearrange fully hydrated fibrils and from direct measurements by nanoindentation and quantitative atomic force measurements. We suggest that increase in the nanoscale rigidity of collagen fibrils can cause these cells to assume a proliferative phenotype.

The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa.

We have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of Neurospora crassa and to better understand the role(s) of dynein during hyphal tip growth. Based on video-enhanced DIC light microscopy, the mature, growing hyphae of N. crassa wild type could be divided into four regions according to cytoplasmic organization and behavior: the apical region (I) and three subapical regions (II, III, and IV). A well-defined Spitzenkörper dominated the cytoplasm of region I. In region II, vesicles ( approximately 0.48 micro m diameter) and mitochondria maintained primarily a constant location within the advancing cytoplasm. This region was typically void of nuclei. Vesicles exhibited anterograde and retrograde motility in regions III and IV and followed generally parallel paths along the longitudinal axis of the cell. A small population of mitochondria displayed rapid anterograde and retrograde movements, while most maintained a constant position in the advancing cytoplasm in regions III and IV. Many nuclei occupied the cytoplasm of regions III and IV. In ro-1 hyphae, discrete cytoplasmic regions were not recognized and the motility and/or positioning of vesicles, mitochondria, and nuclei were altered to varying degrees, relative to the wild type cells. Immunofluorescence microscopy revealed that the microtubule cytoskeleton was severely disrupted in ro-1 cells. Transmission electron microscopy of cryofixed cells confirmed that region I of wild-type hyphae contained a Spitzenkörper composed of an aggregation of small apical vesicles that surrounded entirely or partially a central core composed, in part, of microvesicles embedded in a dense granular to fibrillar matrix. The apex of ro-1 the hypha contained a Spitzenkörper with reduced numbers of apical vesicles but maintained a defined central core. Clearly, dynein deficiency in the mutant caused profound perturbation in microtubule organization and function and, consequently, organelle dynamics and positioning. These perturbations impact negatively on the organization and stability of the Spitzenkörper, which, in turn, led to severe reduction in growth rate and altered hyphal morphology.