Microbiome profiles are associated with cognitive functioning in 45-month-old children.

Prenatal, perinatal, and postnatal factors have been shown to shape neurobiological functioning and alter the risk for mental disorders later in life. The gut microbiome is established early in life, and interacts with the brain via the brain-immune-gut axis. However, little is known about how the microbiome relates to early-life cognitive functioning in children. The present study, where the fecal microbiome of 380 children was characterized using 16S rDNA and metagenomic sequencing aimed to investigate the association between the microbiota and cognitive functioning of children at the age of 45 months measured with the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-III). Overall the microbiome profile showed a significant association with cognitive functioning. A strong correlation was found between cognitive functioning and the relative abundance of an unidentified genus of the family Enterobacteriaceae. Follow-up mediation analyses revealed significant mediation effects of the level of this genus on the association of maternal smoking during pregnancy and current cigarette smoking with cognitive function. Metagenomic sequencing of a subset of these samples indicated that the identified genus was most closely related to Enterobacter asburiae. Analysis of metabolic potential showed a nominally significant association of cognitive functioning with the microbial norspermidine biosynthesis pathway. Our results indicate that alteration of the gut microflora is associated with cognitive functioning in childhood. Furthermore, they suggest that the altered microflora might interact with other environmental factors such as maternal cigarette smoking. Interventions directed at altering the microbiome should be explored in terms of improving cognitive functioning in young children.

The oropharyngeal microbiome is altered in individuals with schizophrenia and mania.

It is being increasingly recognized that human mucosal surfaces are not sterile but are colonized with microorganisms collectively known as the microbiome. The microbiome can alter brain functioning in humans and animals by way of a series of interactions operative in the brain-immune-gut interactome. We characterized the oropharyngeal microbiome in 316 individuals, including 121 with schizophrenia, 62 with mania, 48 with major depressive disorder, and 85 controls without a psychiatric disorder. We found that the oropharyngeal microflora of individuals with schizophrenia and individuals with mania differed from controls in composition and abundance as measured by the weighted UniFrac distance (both p < .003 adjusted for covariates and multiple comparisons). This measure in individuals with major depressive disorder did not differ from that of controls. We also identified five bacterial taxa which differed among the diagnostic groups. Three of the taxa, Neisseria subflava, Weeksellaceae, and Prevotella, were decreased in individuals with schizophrenia or mania as compared to controls, while Streptococci was increased in these groups. One taxa, Schlegelella, was only found in individuals with mania. Neisseria subflava was also positively associated with cognitive functioning as measured by the Repeatable Battery for the Assessment of Neuropsychological Status. There were no taxa significantly altered in individuals with major depression. Individuals with schizophrenia and mania have altered compositions of the oropharyngeal microbiome. An understanding of the biology of the microbiome and its effect on the brain might lead to new insights into the pathogenesis, and ultimately, the prevention and treatment of these disorders.

Long-term, stable differentiation of human embryonic stem cell-derived neural precursors grafted into the adult mammalian neostriatum.

Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic AMP-regulated phosphoprotein. Graft-derived neurons formed mature synapses with host postsynaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin(+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches.

Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord.

BACKGROUND: Effective treatments for degenerative and traumatic diseases of the nervous system are not currently available. The support or replacement of injured neurons with neural grafts, already an established approach in experimental therapeutics, has been recently invigorated with the addition of neural and embryonic stem-derived precursors as inexhaustible, self-propagating alternatives to fetal tissues. The adult spinal cord, i.e., the site of common devastating injuries and motor neuron disease, has been an especially challenging target for stem cell therapies. In most cases, neural stem cell (NSC) transplants have shown either poor differentiation or a preferential choice of glial lineages. METHODS AND FINDINGS: In the present investigation, we grafted NSCs from human fetal spinal cord grown in monolayer into the lumbar cord of normal or injured adult nude rats and observed large-scale differentiation of these cells into neurons that formed axons and synapses and established extensive contacts with host motor neurons. Spinal cord microenvironment appeared to influence fate choice, with centrally located cells taking on a predominant neuronal path, and cells located under the pia membrane persisting as NSCs or presenting with astrocytic phenotypes. Slightly fewer than one-tenth of grafted neurons differentiated into oligodendrocytes. The presence of lesions increased the frequency of astrocytic phenotypes in the white matter. CONCLUSIONS: NSC grafts can show substantial neuronal differentiation in the normal and injured adult spinal cord with good potential of integration into host neural circuits. In view of recent similar findings from other laboratories, the extent of neuronal differentiation observed here disputes the notion of a spinal cord that is constitutively unfavorable to neuronal repair. Restoration of spinal cord circuitry in traumatic and degenerative diseases may be more realistic than previously thought, although major challenges remain, especially with respect to the establishment of neuromuscular connections.

Human neural stem cell grafts ameliorate motor neuron disease in SOD-1 transgenic rats.

BACKGROUND: Experimental therapeutics for degenerative and traumatic diseases of the nervous system have been recently enriched with the addition of neural stem cells (NSCs) as alternatives to fetal tissues for cell replacement. Neurodegenerative diseases present the additional problem that cell death signals may interfere with the viability of grafted cells. The adult spinal cord raises further challenges for NSC differentiation because of lack of intrinsic developmental potential and the negative outcomes of several prior attempts. METHOD: NSCs from human fetal spinal cord were grafted into the lumbar cord of SOD1 G93A rats. The differentiation fate of grafted NSCs and their effects on motor neuron number, locomotor performance, disease onset, and survival trends/longevity were assessed. Trophic mechanisms of observed clinical effects were explored with molecular and cellular methodologies. RESULT: Human NSCs showed extensive differentiation into neurons that formed synaptic contacts with host nerve cells and expressed and released glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor. NSC grafts delayed the onset and progression of the fulminant motor neuron disease typical of the rat SOD1 G93A model and extended the lifespan of these animals by more than 10 days, despite the restricted grafting schedule that was limited to the lumbar protuberance. CONCLUSION: NSC grafts can survive well in a neurodegenerative environment and exert powerful clinical effects; at least a portion of these effects may be related to the ability of these grafts to express and release motor neuron growth factors delivered to host motor neurons via graft-host connections.

Furin cleavage of the HIV-1 Tat protein.

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.

Effects of virus burden and chemokine expression on immunity to SHIV in nonhuman primates.

HIV-1 vaccine candidates are designed to elicit Type 1 immune responses, including cytotoxic T cells and neutralizing antibodies. The type of immune response is influenced by many factors, including the levels of antigen expression and production of cytokines or chemokines; we designed a nonhuman primate study to evaluate the influence of these factors on protective immunity. Recombinant SHIV were engineered to express macrophage inflammatory protein-1 alpha (MIP-1alpha), regulated upon activation, normal T-cell expressed and secreted (RANTES), or Lymphotactin (Ltn) in place of nef in SHIV(89.6) (SHIV(89.6-MIP-1), SHIV(89.6-RANTES), SHIV(89.6-Ltn)). The parental virus SHIV(89.6) was included because it replicates to higher titer while still not causing disease. Control groups included animals that received a recombinant SHIV with a truncated chemokine construct (SHIV(89.6-dLtn)) and unvaccinated macaques. After pathogenic challenge with SHIV(89.6pd), animals from groups that received recombinant (nef-deleted) viruses had peak viremia levels three orders of magnitude lower than unvaccinated controls and increased survival times. Animals that received the original SHIV(89.6) (nef+) were highly resistant to both intrarectal and intravenous challenge with SHIV(89.6PD), and showed no signs of disease. There were no differences in survival times comparing unvaccinated and SHIV(89.6-dLtn) (control) groups, indicating that nef deleted viruses did not provide durable protection in this model. Strongest protection was seen in animals with the highest replicating virus (SHIV(89.6)), and the lower effect on survival after SHIV(89.6) nef-deleted vaccination, likely reflects differences in replication capacity. The protective effect of nef-deleted virus was partly restored by expressing Type 1 chemokines to augment viral immunity.

Targeted knock-down of neuronal nitric oxide synthase expression in basal forebrain with RNA interference.

Nitric oxide (NO) is a gas messenger with diverse physiological roles in the nervous system, from modulation of synaptic plasticity and neurogenesis to the mediation of neuronal death. NO production in the brain is catalyzed by three isoforms of NO synthase (NOS) including neuronal NOS (nNOS), inducible NOS and endothelial NOS. In this report, we demonstrate a method for in vitro and in vivo silencing of nNOS using RNAi strategies. Because of their efficiency in infecting postmitotic cells like neurons, lentiviral vectors were used as nNOS shRNA carriers. Of the siRNA sequences screened, one corresponding to exon 10 of the rat nNOS specifically and efficiently inhibited nNOS expression at the mRNA and protein level. In vitro experiments using rat cortical neurons showed the general efficacy of shRNA vectors in silencing constitutively expressed nNOS. To demonstrate the anatomical specificity of nNOS silencing in vivo, vectors were used to selectively knock-down the endogenous nNOS expression in cortical GABAergic interneurons of rat piriform cortex. Our findings show that the method reported here can achieve stable and highly effective nNOS suppression in an anatomically defined brain region. The ability of our nNOS silencing vectors to effectively and precisely silence nNOS expression shows their value as research tools for further studies of the role of nNOS in specific brain circuits. Furthermore, our findings raise the possibility for future considerations of lentiviral strategies as therapies for diseases of the nervous system involving NO neurotoxic cascades.

Tat-neutralizing antibodies in vaccinated macaques.

The human immunodeficiency virus Tat protein is essential for virus replication and is a candidate vaccine antigen. Macaques immunized with Tat or chemically modified Tat toxoid having the same clade B sequence developed strong antibody responses. We compared these antisera for their abilities to recognize diverse Tat sequences. An overlapping peptide array covering three clade B and two clade C Tat sequences was constructed to help identify reactive linear epitopes. Sera from Tat-immunized macaques were broadly cross-reactive with clade B and clade C sequences but recognized a clade B-specific epitope in the basic domain. Sera from Tat toxoid-immunized macaques had a more restricted pattern of recognition, reacting mainly with clade B and with only one clade B basic domain sequence, which included the rare amino acids RPPQ at positions 57 to 60. Monoclonal antibodies against the amino terminus or the domain RPPQ sequence blocked Tat uptake into T cells and neutralized Tat in a cell-based transactivation assay. Macaques immunized with Tat or Tat toxoid proteins varied in their responses to minor epitopes, but all developed a strong response to the amino terminus, and antisera were capable of neutralizing Tat in a transactivation assay.

Sequence variation within the dominant amino terminus epitope affects antibody binding and neutralization of human immunodeficiency virus type 1 Tat protein.

Tat is among the required regulatory genes of human immunodeficiency virus type 1 (HIV-1). Tat functions both within infected cells as a transcription factor and as an extracellular factor that binds and alters bystander cells. Some functions of extracellular Tat can be neutralized by immune serum or monoclonal antibodies. In order to understand the antibody response to Tat, we are defining antibody epitopes and the effects of natural Tat sequence variation on antibody recognition. The dominant Tat epitope in macaque sera is within the first 15 amino acids of the protein amino terminus. Together with a subdominant response to amino acids 57 to 60, these two regions account for most of the macaque response to linear Tat epitopes and both regions are also sites for the binding of neutralizing antibodies. However, the dominant and subdominant epitope sequences differ among virus strains, and this natural variation can preclude antibody binding and Tat neutralization. We also examined serum samples from 31 HIV-positive individuals that contained Tat binding antibodies; 23 of the 31 sera recognized the amino terminus peptide. Similar to binding in macaques, human antibody binding to the amino terminus was affected by variations at positions 7 and 12, sequences that are distinct for clade B compared to other viral clades. Tat-neutralizing antibodies to the dominant amino terminus epitope are affected by HIV clade variation.