Astrocyte expression of aging-associated markers positively correlates with neurodegeneration in the frontal lobe of the rhesus macaque brain.

Introduction: As the population over the age of 65 increases, rates of neurodegenerative disorders and dementias will rise - necessitating further research into the cellular and molecular mechanisms that contribute to brain aging. With the critical importance of astrocytes to neuronal health and functioning, we hypothesized that alterations in astrocyte expression of aging-associated markers p16INK4a (p16) and sirtuin 1 (SIRT1) with age would correlate with increased rates of neurodegeneration, as measured by FluoroJade C (FJC) staining. Methods: To test this hypothesis, 19 rhesus macaques at the Tulane National Primate Research Center were selected based on the following criteria: archival FFPE CNS tissue available to use, no noted neuropathology, and an age range of 5-30 years. Tissues were cut at 5 µm and stained for GFAP, p16, SIRT1, and FJC, followed by whole-slide imaging and HALO® image analysis for percentage of marker-positive cells and relative intensity of each stain. Results: We found the percentage of p16+ cells increases with age in total cells and astrocytes of the frontal (p = 0.0021, p = 0.0012 respectively) and temporal (p = 0.0226, p = 0.0203 respectively) lobes, as well as the relative intensity of p16 staining (frontal lobe: p = 0.0060; temporal lobe: p = 0.0269). For SIRT1, we found no correlation with age except for an increase in the relative intensity of SIRT1 in the temporal lobe (p = 0.0033). There was an increase in neurodegeneration, as measured by the percentage of FJC+ cells in the frontal lobe with age (p = 0.0057), as well as in the relative intensity of FJC staining in the frontal (p = 0.0030) and parietal (p = 0.0481) lobes. Importantly, increased p16 and SIRT1 expression in astrocytes correlated with increasing neurodegeneration in the frontal lobe (p = 0.0009, p = 0.0095 respectively). Discussion: Together, these data suggest that age-associated alterations in astrocytes contribute to neurodegeneration and provide a target for mechanistic studies in the future.

Regulation of pulmonary plasma cell responses during secondary infection with influenza virus.

During secondary infection with influenza virus, plasma cells (PCs) develop within the lung, providing a local source of antibodies. However, the site and mechanisms that regulate this process are poorly defined. Here, we show that while circulating memory B cells entered the lung during rechallenge and were activated within inducible bronchus-associated lymphoid tissues (iBALTs), resident memory B (BRM) cells responded earlier, and their activation occurred in a different niche: directly near infected alveoli. This process required NK cells but was largely independent of CD4 and CD8 T cells. Innate stimuli induced by virus-like particles containing ssRNA triggered BRM cell differentiation in the absence of cognate antigen, suggesting a low threshold of activation. In contrast, expansion of PCs in iBALTs took longer to develop and was critically dependent on CD4 T cells. Our work demonstrates that spatially distinct mechanisms evolved to support pulmonary secondary PC responses, and it reveals a specialized function for BRM cells as guardians of the alveoli.

Two ancient membrane pores mediate mitochondrial-nucleus membrane contact sites.

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

Role of affinity in plasma cell development in the germinal center light zone.

Protective immune responses to many pathogens depend on the development of high-affinity antibody-producing plasma cells (PC) in germinal centers (GCs). Transgenic models suggest that there is a stringent affinity-based barrier to PC development. Whether a similar high-affinity barrier regulates PC development under physiologic circumstances and the nature of the PC fate decision has not been defined precisely. Here, we use a fate-mapping approach to examine the relationship between GC B cells selected to undergo additional rounds of affinity maturation, GC pre-PC, and PC. The data show that initial PC selection overlaps with GC B cell selection, but that the PC compartment accumulates a less diverse and higher affinity collection of antibodies over time. Thus, whereas the GC continues to diversify over time, affinity-based pre-PC selection sieves the GC to enable the accumulation of a more restricted group of high-affinity antibody-secreting PC.

Functional and biochemical characterization of the Toxoplasma gondii succinate dehydrogenase complex.

The mitochondrial electron transport chain (mETC) is a series of membrane embedded enzymatic complexes critical for energy conversion and mitochondrial metabolism. In commonly studied eukaryotes, including humans and animals, complex II, also known as succinate dehydrogenase (SDH), is an essential four-subunit enzyme that acts as an entry point to the mETC, by harvesting electrons from the TCA cycle. Apicomplexa are pathogenic parasites with significant impact on human and animal health. The phylum includes Toxoplasma gondii which can cause fatal infections in immunocompromised people. Most apicomplexans, including Toxoplasma, rely on their mETC for survival, yet SDH remains largely understudied. Previous studies pointed to a divergent apicomplexan SDH with nine subunits proposed for the Toxoplasma complex, compared to four in humans. While two of the nine are homologs of the well-studied SDHA and B, the other seven have no homologs in SDHs of other systems. Moreover, SDHC and D, that anchor SDH to the membrane and participate in substrate bindings, have no homologs in Apicomplexa. Here, we validated five of the seven proposed subunits as bona fide SDH components and demonstrated their importance for SDH assembly and activity. We further find that all five subunits are important for parasite growth, and that disruption of SDH impairs mitochondrial respiration and results in spontaneous initiation of differentiation into bradyzoites. Finally, we provide evidence that the five subunits are membrane bound, consistent with their potential role in membrane anchoring, and we demonstrate that a DY motif in one of them, SDH10, is essential for complex formation and function. Our study confirms the divergent composition of Toxoplasma SDH compared to human, and starts exploring the role of the lineage-specific subunits in SDH function, paving the way for future mechanistic studies.

Continually recruited naïve T cells contribute to the follicular helper and regulatory T cell pools in germinal centers.

Follicular helper T cells (TFH) mediate B cell selection and clonal expansion in germinal centers (GCs), and follicular regulatory T cells (TFR) prevent the emergence of self-reactive B cells and help to extinguish the reaction. Here we show that GC reactions continually recruit T cells from both the naïve conventional and naive thymic regulatory T cell (Treg) repertoires. In the early GC, newly recruited T cells develop into TFH, whereas cells entering during the contraction phase develop into TFR cells that contribute to GC dissolution. The TFR fate decision is associated with decreased antigen availability and is modulated by slow antigen delivery or mRNA vaccination. Thus, invasion of ongoing GCs by newly developing TFH and TFR helps remodel the GC based on antigen availability.

Interferon-γ couples CD8+ T cell avidity and differentiation during infection.

Effective responses to intracellular pathogens are characterized by T cell clones with a broad affinity range for their cognate peptide and diverse functional phenotypes. How T cell clones are selected throughout the response to retain a breadth of avidities remains unclear. Here, we demonstrate that direct sensing of the cytokine IFN-γ by CD8+ T cells coordinates avidity and differentiation during infection. IFN-γ promotes the expansion of low-avidity T cells, allowing them to overcome the selective advantage of high-avidity T cells, whilst reinforcing high-avidity T cell entry into the memory pool, thus reducing the average avidity of the primary response and increasing that of the memory response. IFN-γ in this context is mainly provided by virtual memory T cells, an antigen-inexperienced subset with memory features. Overall, we propose that IFN-γ and virtual memory T cells fulfil a critical immunoregulatory role by enabling the coordination of T cell avidity and fate.

Advances in HIV therapeutics and cure strategies: findings obtained through non-human primate studies.

Human immunodeficiency virus (HIV), the main contributor of the ongoing AIDS epidemic, remains one of the most challenging and complex viruses to target and eradicate due to frequent genome mutation and immune evasion. Despite the development of potent antiretroviral therapies, HIV remains an incurable infection as the virus persists in latent reservoirs throughout the body. To innovate a safe and effective cure strategy for HIV in humans, animal models are needed to better understand viral proliferation, disease progression, and therapeutic response. Nonhuman primates infected with simian immunodeficiency virus (SIV) provide an ideal model to study HIV infection and pathogenesis as they are closely related to humans genetically and express phenotypically similar immune systems. Examining the clinical outcomes of novel treatment strategies within nonhuman primates facilitates our understanding of HIV latency and advances the development of a true cure to HIV.

The mystery of massive mitochondrial complexes: the apicomplexan respiratory chain.

The mitochondrial respiratory chain is an essential pathway in most studied eukaryotes due to its roles in respiration and other pathways that depend on mitochondrial membrane potential. Apicomplexans are unicellular eukaryotes whose members have an impact on global health. The respiratory chain is a drug target for some members of this group, notably the malaria-causing Plasmodium spp. This has motivated studies of the respiratory chain in apicomplexan parasites, primarily Toxoplasma gondii and Plasmodium spp. for which experimental tools are most advanced. Studies of the respiratory complexes in these organisms revealed numerous novel features, including expansion of complex size. The divergence of apicomplexan mitochondria from commonly studied models highlights the diversity of mitochondrial form and function across eukaryotic life.

Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites.

Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication.

Imaging of surface microdomains on individual extracellular vesicles in 3-D.

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single-particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three-dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo-EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated.

Protein lipoylation in mitochondria requires Fe-S cluster assembly factors NFU4 and NFU5.

Plants have evolutionarily conserved NifU (NFU)-domain proteins that are targeted to plastids or mitochondria. "Plastid-type" NFU1, NFU2, and NFU3 in Arabidopsis (Arabidopsis thaliana) play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here, we confirmed that NFU4 and NFU5 are targeted to the mitochondria. The proteins were constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of NFU4 and NFU5 proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes or aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth improved with elevated CO2 treatment. In addition, pyruvate, 2-oxoglutarate, and branched-chain amino acids accumulated in nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, most likely providing Fe-S clusters to lipoyl synthase.

Extracellular Vesicles as a Means of Viral Immune Evasion, CNS Invasion, and Glia-Induced Neurodegeneration.

Extracellular vesicles (EVs) are small, membrane-bound vesicles released by cells as a means of intercellular communication. EVs transfer proteins, nucleic acids, and other biologically relevant molecules from one cell to another. In the context of viral infections, EVs can also contain viruses, viral proteins, and viral nucleic acids. While there is some evidence that the inclusion of viral components within EVs may be part of the host defense, much of the research in this field supports a pro-viral role for EVs. Packaging of viruses within EVs has repeatedly been shown to protect viruses from antibody neutralization while also allowing for their integration into cells otherwise impervious to the virus. EVs also bidirectionally cross the blood-brain barrier (BBB), providing a potential route for peripheral viruses to enter the brain while exiting EVs may serve as valuable biomarkers of neurological disease burden. Within the brain, EVs can alter glial activity, increase neuroinflammation, and induce neurotoxicity. The purpose of this mini-review is to summarize research related to viral manipulation of EV-mediated intercellular communication and how such manipulation may lead to infection of the central nervous system, chronic neuroinflammation, and neurodegeneration.

The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe-S supply in grxs15 mutants.

Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex.

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies.

The Zinc Finger Protein Zbtb18 Represses Expression of Class I Phosphatidylinositol 3-Kinase Subunits and Inhibits Plasma Cell Differentiation.

The PI3K pathway plays a key role in B cell activation and is important for the differentiation of Ab producing plasma cells (PCs). Although much is known about the molecular mechanisms that modulate PI3K signaling in B cells, the transcriptional regulation of PI3K expression is poorly understood. In this study, we identify the zinc finger protein Zbtb18 as a transcriptional repressor that directly binds enhancer/promoter regions of genes encoding class I PI3K regulatory subunits, subsequently limiting their expression, dampening PI3K signaling and suppressing PC responses. Following activation, dividing B cells progressively downregulated Zbtb18, allowing gradual amplification of PI3K signals and enhanced development of PCs. Human Zbtb18 displayed similar expression patterns and function in human B cells, acting to inhibit development of PCs. Furthermore, a number of Zbtb18 mutants identified in cancer patients showed loss of suppressor activity, which was also accompanied by impaired regulation of PI3K genes. Taken together, our study identifies Zbtb18 as a repressor of PC differentiation and reveals its previously unappreciated function as a transcription modulator of the PI3K signaling pathway.

Transcriptional signatures of Zika virus infection in astrocytes.

Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.

NUBPL mitochondrial disease: new patients and review of the genetic and clinical spectrum.

BACKGROUND: The nucleotide binding protein-like (NUBPL) gene was first reported as a cause of mitochondrial complex I deficiency (MIM 613621, 618242) in 2010. To date, only eight patients have been reported with this mitochondrial disorder. Five other patients were recently reported to have NUBPL disease but their clinical picture was different from the first eight patients. Here, we report clinical and genetic findings in five additional patients (four families). METHODS: Whole exome sequencing was used to identify patients with compound heterozygous NUBPL variants. Functional studies included RNA-Seq transcript analyses, missense variant biochemical analyses in a yeast model (Yarrowia lipolytica) and mitochondrial respiration experiments on patient fibroblasts. RESULTS: The previously reported c.815-27T>C branch-site mutation was found in all four families. In prior patients, c.166G>A [p.G56R] was always found in cis with c.815-27T>C, but only two of four families had both variants. The second variant found in trans with c.815-27T>C in each family was: c.311T>C [p.L104P] in three patients, c.693+1G>A in one patient and c.545T>C [p.V182A] in one patient. Complex I function in the yeast model was impacted by p.L104P but not p.V182A. Clinical features include onset of neurological symptoms at 3-18 months, global developmental delay, cerebellar dysfunction (including ataxia, dysarthria, nystagmus and tremor) and spasticity. Brain MRI showed cerebellar atrophy. Mitochondrial function studies on patient fibroblasts showed significantly reduced spare respiratory capacity. CONCLUSION: We report on five new patients with NUBPL disease, adding to the number and phenotypic variability of patients diagnosed worldwide, and review prior reported patients with pathogenic NUBPL variants.

CRISPR based editing of SIV proviral DNA in ART treated non-human primates.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.