Sex bias in MHC I-associated shaping of the adaptive immune system.

HLA associations, T cell receptor (TCR) repertoire bias, and sex bias have independently been shown for many diseases. While some immunological differences between the sexes have been described, they do not fully explain bias in men toward many infections/cancers, and toward women in autoimmunity. Next-generation TCR variable beta chain (TCRBV) immunosequencing of 824 individuals was evaluated in a multiparametric analysis including HLA-A -B/MHC class I background, TCRBV usage, sex, age, ethnicity, and TCRBV selection/expansion dynamics. We found that HLA-associated shaping of TCRBV usage differed between the sexes. Furthermore, certain TCRBVs were selected and expanded in unison. Correlations between these TCRBV relationships and biochemical similarities in HLA-binding positions were different in CD8 T cells of patients with autoimmune diseases (multiple sclerosis and rheumatoid arthritis) compared with healthy controls. Within patients, men showed higher TCRBV relationship Spearman's rhos in relation to HLA-binding position similarities compared with women. In line with this, CD8 T cells of men with autoimmune diseases also showed higher degrees of TCRBV perturbation compared with women. Concerted selection and expansion of CD8 T cells in patients with autoimmune diseases, but especially in men, appears to be less dependent on high HLA-binding similarity than in CD4 T cells. These findings are consistent with studies attributing autoimmunity to processes of epitope spreading and expansion of low-avidity T cell clones and may have further implications for the interpretation of pathogenic mechanisms of infectious and autoimmune diseases with known HLA associations. Reanalysis of some HLA association studies, separating the data by sex, could be informative.

Evidence for the involvement of gamma delta T cells in the immune response in Rasmussen encephalitis.

BACKGROUND: Rasmussen encephalitis (RE) is a rare neuroinflammatory disease characterized by intractable seizures and progressive atrophy on one side of the cerebrum. Perivascular cuffing and clusters of T cells in the affected cortical hemisphere are indicative of an active cellular immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) and brain-infiltrating lymphocytes (BILs) were isolated from 20 RE surgery specimens by standard methods, and CD3(+) T cell populations were analyzed by flow cytometry. Gamma delta T cell receptor spectratyping was carried out by nested PCR of reversed transcribed RNA extracted from RE brain tissue, followed by high resolution capillary electrophoresis. A MiSeq DNA sequencing platform was used to sequence the third complementarity determining region (CDR3) of δ1 chains. RESULTS: CD3(+) BILs from all of the RE brain specimens comprised both αß and γδ T cells. The median αß:γδ ratio was 1.9 (range 0.58-5.2) compared with a median ratio of 7.7 (range 2.7-40.8) in peripheral blood from the same patients. The αß T cells isolated from brain tissue were predominantly CD8(+), and the majority of γδ T cells were CD4(-) CD8(-). Staining for the early activation marker CD69 showed that a fraction of the αß and γδ T cells in the BILs were activated (median 42%; range 13-91%, and median 47%; range 14-99%, respectively). Spectratyping T cell receptor (TCR) Vδ1-3 chains from 14 of the RE brain tissue specimens indicated that the γδ T cell repertoire was relatively restricted. Sequencing δ1 chain PCR fragments revealed that the same prevalent CDR3 sequences were found in all of the brain specimens. These CDR3 sequences were also detected in brain tissue from 15 focal cortical dysplasia (FCD) cases. CONCLUSION: Neuroinflammation in RE involves both activated αß and γδ T cells. The presence of γδ T cells with identical TCR δ1 chain CDR3 sequences in all of the brain specimens examined suggests that a non-major histocompatibility complex (MHC)-restricted immune response to the same antigen(s) is involved in the etiology of RE. The presence of the same δ1 clones in CD brain implies the involvement of a common inflammatory pathway in both diseases.

Differential expression of interferon-γ and chemokine genes distinguishes Rasmussen encephalitis from cortical dysplasia and provides evidence for an early Th1 immune response.

BACKGROUND: Rasmussen encephalitis (RE) is a rare complex inflammatory disease, primarily seen in young children, that is characterized by severe partial seizures and brain atrophy. Surgery is currently the only effective treatment option. To identify genes specifically associated with the immunopathology in RE, RNA transcripts of genes involved in inflammation and autoimmunity were measured in brain tissue from RE surgeries and compared with those in surgical specimens of cortical dysplasia (CD), a major cause of intractable pediatric epilepsy. METHODS: Quantitative polymerase chain reactions measured the relative expression of 84 genes related to inflammation and autoimmunity in 12 RE specimens and in the reference group of 12 CD surgical specimens. Data were analyzed by consensus clustering using the entire dataset, and by pairwise comparison of gene expression levels between the RE and CD cohorts using the Harrell-Davis distribution-free quantile estimator method. RESULTS: Consensus clustering identified six RE cases that were clearly distinguished from the CD cases and from other RE cases. Pairwise comparison showed that seven mRNAs encoding interferon-γ, CCL5, CCL22, CCL23, CXCL9, CXCL10, and Fas ligand were higher in the RE specimens compared with the CD specimens, whereas the mRNA encoding hypoxanthine-guanine phosphoribosyltransferase was reduced. Interferon-γ, CXCL5, CXCL9 and CXCL10 mRNA levels negatively correlated with time from seizure onset to surgery (P <0.05), whereas CCL23 and Fas ligand transcript levels positively correlated with the degree of tissue destruction and inflammation, respectively (P <0.05), as determined from magnetic resonance imaging (MRI) T2 and FLAIR images. Accumulation of CD4+ lymphocytes in leptomeninges and perivascular spaces was a prominent feature in RE specimens resected within a year of seizure onset. CONCLUSIONS: Active disease is characterized by a Th1 immune response that appears to involve both CD8+ and CD4+ T cells. Our findings suggest therapeutic intervention targeting specific chemokine/chemokine receptors may be useful in early stage RE.

Immune checkpoint blockade induces distinct alterations in the microenvironments of primary and metastatic brain tumors.

In comparison with responses in recurrent glioblastoma (rGBM), the intracranial response of brain metastases (BrM) to immune checkpoint blockade (ICB) is less well studied. Here, we present an integrated single-cell RNA-Seq (scRNA-Seq) study of 19 ICB-naive and 9 ICB-treated BrM samples from our own and published data sets. We compared them with our previously published scRNA-Seq data from rGBM and found that ICB led to more prominent T cell infiltration into BrM than rGBM. These BrM-infiltrating T cells exhibited a tumor-specific phenotype and displayed greater activated/exhausted features. We also used multiplex immunofluorescence and spatial transcriptomics to reveal that ICB reduced a distinct CD206+ macrophage population in the perivascular space, which may modulate T cell entry into BrM. Furthermore, we identified a subset of progenitor exhausted T cells that correlated with longer overall survival in BrM patients. Our study provides a comprehensive immune cellular landscape of ICB's effect on metastatic brain tumors and offers insights into potential strategies for improving ICB efficacy for brain tumor patients.

Clonally Focused Public and Private T Cells in Resected Brain Tissue From Surgeries to Treat Children With Intractable Seizures.

Using a targeted transcriptomics approach, we have analyzed resected brain tissue from a cohort of 53 pediatric epilepsy surgery cases, and have found that there is a spectrum of involvement of both the innate and adaptive immune systems as evidenced by the differential expression of immune-specific genes in the affected brain tissue. The specimens with the highest expression of immune-specific genes were from two Rasmussen encephalitis cases, which is known to be a neuro-immunological disease, but also from tuberous sclerosis complex (TSC), focal cortical dysplasia, and hemimegalencephaly surgery cases. We obtained T cell receptor (TCR) Vß chain sequence data from brain tissue and blood from patients with the highest levels of T cell transcripts. The clonality indices and the frequency of the top 50 Vß clonotypes indicated that T cells in the brain were clonally restricted. The top 50 Vß clonotypes comprised both public and private (patient specific) clonotypes, and the TCR Vß chain third complementarity region (CDR3) of the most abundant public Vß clonotype in each brain sample was strikingly similar to a CDR3 that recognizes an immunodominant epitope in either human cytomegalovirus or Epstein Barr virus, or influenza virus A. We found that the frequency of 14 of the top 50 brain Vß clonotypes from a TSC surgery case had significantly increased in brain tissue removed to control recurrent seizures 11 months after the first surgery. Conversely, we found that the frequency in the blood of 18 of the top 50 brain clonotypes from a second TSC patient, who was seizure free, had significantly decreased 5 months after surgery indicating that T cell clones found in the brain had contracted in the periphery after removal of the brain area associated with seizure activity and inflammation. However, the frequency of a public and a private clonotype significantly increased in the brain after seizures recurred and the patient underwent a second surgery. Combined single cell gene expression and TCR sequencing of brain-infiltrating leukocytes from the second surgery showed that the two clones were CD8 effector T cells, indicating that they are likely to be pathologically relevant.

Evidence for Innate and Adaptive Immune Responses in a Cohort of Intractable Pediatric Epilepsy Surgery Patients.

Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80-95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4- cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR- CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA- HLA-DR+ CD11b- CD16- cells comprised 86-96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αß T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40-66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation.

Shared HLA Class I and II Alleles and Clonally Restricted Public and Private Brain-Infiltrating αß T Cells in a Cohort of Rasmussen Encephalitis Surgery Patients.

Rasmussen encephalitis (RE) is a rare pediatric neuroinflammatory disease characterized by intractable seizures and unilateral brain atrophy. T cell infiltrates in affected brain tissue and the presence of circulating autoantibodies in some RE patients have indicated that RE may be an autoimmune disease. The strongest genetic links to autoimmunity reside in the MHC locus, therefore, we determined the human leukocyte antigen (HLA) class I and class II alleles carried by a cohort of 24 RE surgery cases by targeted in-depth genomic sequencing. Compared with a reference population the allelic frequency of three alleles, DQA1*04:01:01, DQB1*04:02:01, and HLA-C*07:02:01:01 indicated that they might confer susceptibility to the disease. It has been reported that HLA-C*07:02 is a risk factor for Graves disease. Further, eight patients in the study cohort carried HLA-A*03:01:01:01, which has been linked to susceptibility to multiple sclerosis. Four patients carried a combination of three HLA class II alleles that has been linked to type 1 diabetes (DQA1*05:01:01:01~DQB1*02:01:01~DRB1*03:01:01:01), and five patients carried a combination of HLA class II alleles that has been linked to the risk of contracting multiple sclerosis (DQA1*01:02:01:01, DQB1*06:02:01, DRB1*15:01:01:01). We also analyzed the diversity of αß T cells in brain and blood specimens from 14 of these RE surgery cases by sequencing the third complementarity regions (CDR3s) of rearranged T cell receptor ß genes. A total of 31 unique CDR3 sequences accounted for the top 5% of all CDR3 sequences in the 14 brain specimens. Thirteen of these sequences were found in sequencing data from healthy blood donors; the remaining 18 sequences were patient specific. These observations provide evidence for the clonal expansion of public and private T cells in the brain, which might be influenced by the RE patient's HLA haplotype.

Evidence for Resident Memory T Cells in Rasmussen Encephalitis.

Rasmussen encephalitis (RE) is a rare pediatric neuroinflammatory disease of unknown etiology characterized by intractable seizures, and progressive atrophy usually confined to one cerebral hemisphere. Surgical removal or disconnection of the affected cerebral hemisphere is currently the only intervention that effectively stops the seizures. Histopathological evaluation of resected brain tissue has shown that activated brain resident macrophages (microglia) and infiltrating T cells are involved in the inflammatory reaction. Here, we report that T cells isolated from seven RE brain surgery specimens express the resident memory T cell (TRM) marker CD103. CD103 was expressed by >50% of CD8(+) αß T cells and γδ T cells irrespective of the length of time from seizure onset to surgery, which ranged from 0.3 to 8.4 years. Only ~10% of CD4(+) αß were CD103(+), which was consistent with the observation that few CD4(+) T cells are found in RE brain parenchyma. Clusters of T cells in brain parenchyma, which are a characteristic of RE histopathology, stained for CD103. Less than 10% of T cells isolated from brain specimens from eight surgical cases of focal cortical dysplasia (FCD), a condition that is also characterized by intractable seizures, were CD103(+). In contrast to the RE cases, the percent of CD103(+) T cells increased with the length of time from seizure onset to surgery. In sections of brain tissue from the FCD cases, T cells were predominantly found around blood vessels, and did not stain for CD103. The presence of significant numbers of TRM cells in RE brain irrespective of the length of time between clinical presentation and surgical intervention supports the conclusion that a cellular immune response to an as yet unidentified antigen(s) occurs at an early stage of the disease. Reactivated TRM cells may contribute to disease progression.

Photoconvertible fluorescent protein-based live imaging of mitochondrial fusion.

Mitochondria are highly dynamic organelles that undergo fusion and fission on a relatively fast time scale. Here, a straightforward method is described for capturing mitochondrial fusion events in real time using a photoconvertible fluorescent protein and a far-field fluorescence microscope equipped with appropriate image acquisition and analysis software. The Kaede photoconvertible fluorescent protein is tagged with a mitochondrial targeting sequence and delivered to primary neurons by lentiviral transduction, which ensures efficient low copy number transgene insertion, as well as stable transgene expression.

In Rasmussen encephalitis, hemichannels associated with microglial activation are linked to cortical pyramidal neuron coupling: a possible mechanism for cellular hyperexcitability.

AIMS: Rasmussen encephalitis (RE) is a rare but devastating condition, mainly in children, characterized by sustained brain inflammation, atrophy of one cerebral hemisphere, epilepsy, and progressive cognitive deterioration. The etiology of RE-induced seizures associated with the inflammatory process remains unknown. METHODS: Cortical tissue samples from children undergoing surgical resections for the treatment of RE (n = 16) and non-RE (n = 12) were compared using electrophysiological, morphological, and immunohistochemical techniques to examine neuronal properties and the relationship with microglial activation using the specific microglia/macrophage calcium-binding protein, IBA1 in conjunction with connexins and pannexin expression. RESULTS: Compared with non-RE cases, pyramidal neurons from RE cases displayed increased cell capacitance and reduced input resistance. However, neuronal somatic areas were not increased in size. Instead, intracellular injection of biocytin led to increased dye coupling between neurons from RE cases. By Western blot, expression of IBA1 and pannexin was increased while connexin 32 was decreased in RE cases compared with non-RE cases. IBA1 immunostaining overlapped with pannexin and connexin 36 in RE cases. CONCLUSIONS: In RE, these results support the notion that a possible mechanism for cellular hyperexcitability may be related to increased intercellular coupling from pannexin linked to increased microglial activation. Such findings suggest that a possible antiseizure treatment for RE may involve the use of gap junction blockers.

Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

The cytochrome c gene proximal enhancer drives activity-dependent reporter gene expression in hippocampal neurons.

The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP(+) neurons, while the frequency of mEPSCs was not significantly different. Inhibiting neural activity by applying a GABAA receptor agonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity.

Extensive fusion of mitochondria in spinal cord motor neurons.

The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.

Neuromodulation and mitochondrial transport: live imaging in hippocampal neurons over long durations.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations(1-3). This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 123(4). In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP(5). However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity. Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO(2;)/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

HDAC6 regulates mitochondrial transport in hippocampal neurons.

BACKGROUND: Tubulin is a major substrate of the cytoplasmic class II histone deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which promotes transport of cargoes along microtubules. Microtubule-dependent intracellular trafficking may therefore be regulated by modulating the activity of HDAC6. We have shown previously that the neuromodulator serotonin increases mitochondrial movement in hippocampal neurons via the Akt-GSK3beta signaling pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect. Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons. Inhibition of GSK3beta decreased cytoplasmic deacetylase activity and increased tubulin acetylation, whereas blockade of Akt, which phosphorylates and down-regulates GSK3beta, increased cytoplasmic deacetylase activity and decreased tubulin acetylation. Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin acetylation. GSK3beta was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3beta resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3beta phosphorylation site in HDAC6. GSK3beta may therefore regulate HDAC6 activity by phosphorylation. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that HDAC6 plays an important role in the modulation of mitochondrial transport. The link between HDAC6 and GSK3beta, established here, has important implications for our understanding of neurodegenerative disorders. In particular, abnormal mitochondrial transport, which has been observed in such disorders as Alzheimer's disease and Parkinson's disease, could result from the misregulation of HDAC6 by GSK3beta. HDAC6 may therefore constitute an attractive target in the treatment of these disorders.

Dopamine inhibits mitochondrial motility in hippocampal neurons.

BACKGROUND: The trafficking of mitochondria within neurons is a highly regulated process. In an earlier study, we found that serotonin (5-HT), acting through the 5-HT1A receptor subtype, promotes axonal transport of mitochondria in cultured hippocampal neurons by increasing Akt activity, and consequently decreasing glycogen synthase kinase (GSK3beta) activity. This finding suggests a critical role for neuromodulators in the regulation of mitochondrial trafficking in neurons. In the present study, we investigate the effects of a second important neuromodulator, dopamine, on mitochondrial transport in hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that dopamine, like 5-HT, regulates mitochondrial motility in cultured hippocampal neurons through the Akt-GSK3beta signaling cascade. But, in contrast to the stimulatory effect of 5-HT, administration of exogenous dopamine or bromocriptine, a dopamine 2 receptor (D2R) agonist, caused an inhibition of mitochondrial movement. Moreover, pretreatment with bromocriptine blocked the stimulatory effect of 5-HT on mitochondrial movement. Conversely, in cells pretreated with 5-HT, no further increases in movement were observed after administration of haloperidol, a D2R antagonist. In contrast to the effect of the D2R agonist, addition of SKF38393, a dopamine 1 receptor (D1R) agonist, promoted mitochondrial transport, indicating that the inhibitory effect of dopamine was actually the net summation of opposing influences of the two receptor subtypes. The most pronounced effect of dopamine signals was on mitochondria that were already moving directionally. Western blot analysis revealed that treatment with either a D2R agonist or a D1R antagonist decreased Akt activity, and conversely, treatment with either a D2R antagonist or a D1R agonist increased Akt activity. CONCLUSIONS/SIGNIFICANCE: Our observations strongly suggest a role for both dopamine and 5-HT in regulating mitochondrial movement, and indicate that the integrated effects of these two neuromodulators may be important in determining the distribution of energy sources in neurons.

Two isoforms of the cold-inducible mRNA-binding protein RBM3 localize to dendrites and promote translation.

A diverse set of mRNA-binding proteins (BPs) regulate local translation in neurons. However, little is known about the role(s) played by a family of cold-inducible, glycine-rich mRNA-BPs. Unlike neuronal mRNA-BPs characterized thus far, these proteins are induced by hypothermia and are comprised of one RNA recognition motif and an adjacent arginine- and glycine-rich domain. We studied the expression and function of the RNA-binding motif protein 3 (RBM3), a member of this family, in neurons. RBM3 was expressed in multiple brain regions, with the highest levels in cerebellum and olfactory bulb. In dissociated neurons, RBM3 was observed in nuclei and in a heterogeneous population of granules within dendrites. In sucrose gradient assays, RBM3 cofractionated with heavy mRNA granules and multiple components of the translation machinery. Two alternatively spliced RBM3 isoforms that differed by a single arginine residue were identified in neurons; both were post-translationally modified. The variant lacking the spliced arginine exhibited a higher dendritic localization and was the only isoform present in astrocytes. When overexpressed in neuronal cell lines, RBM3 isoforms-enhanced global translation, the formation of active polysomes, and the activation of initiation factors. These data suggest that RBM3 plays a distinctive role in enhancing translation in neurons.

Serotonin stimulates mitochondrial transport in hippocampal neurons.

Axonal transport of mitochondria is critical for proper neuronal function. However, little is known about the extracellular signals that regulate this process. In the present study, we show that the neuromodulator serotonin (5-HT) greatly enhances mitochondrial movement in the axons of rat hippocampal neurons in vitro. Administration of a 5-HT1A receptor antagonist inhibited mitochondrial movement, whereas addition of fluoxetine, a selective serotonin reuptake inhibitor, promoted mitochondrial movement. 5-HT receptors are known to activate the Akt/Protein kinase B pathway. Consistent with this, directional mitochondrial movement was almost completely blocked by a specific Akt inhibitor. Moreover, an inhibitor of glycogen synthase kinase-3beta (GSK3beta), a kinase whose activity is blocked by Akt-mediated phosphorylation, promoted mitochondrial movement. These findings show that 5-HT1A receptor activation stimulates mitochondrial movement in hippocampal neurons by inhibiting GSK3beta activity via Akt. Our findings suggest that 5-HT may mediate the redistribution of energy sources within responsive neurons, a possibility that has significant implications for understanding the global biological effects of this important neuromodulator.

Reduced glioma infiltration in Src-deficient mice.

Malignant brain tumors, such as glioblastoma, are characterized by extensive angiogenesis and permeability of the blood-brain barrier (BBB). The infiltration of glioma cells away from the primary tumor mass is a pathological characteristic of glial tumors. The infiltrating tumor cells represent a significant factor in tumor recurrence following surgical debulking, radiation, and chemotherapy treatments. Vascular endothelial growth factor (VEGF)-mediated vascular permeability (VP) has been associated with the progression of glioma tumor growth and infiltration into surrounding normal brain parenchyma. While VEGF induces a robust VP response in control mice (src+/+ or src+/-), the VP response is blocked in src-/- mice that demonstrate a 'leakage-resistant phenotype' in the brain. We used the Src-deficient mouse model to determine the role of Src in the maintenance of the BBB following orthotopic implantation and growth of glioma cells in the brain. Although solid tumor growth was the same in control and src-/- mice, the infiltrating component of glioma growth was reduced in src-/- mice. Characterization of the expression and localization of the extracellular matrix (ECM) protein fibrinogen was evaluated to determine the effect of a Src-mediated VP defect in the host compartment. These studies indicate that the reduced VP of host brain blood vessels of src-/- mice mediates a reduction in glioma cell invasion in a mouse brain tumor xenograft model.

Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.