Viral co-infection, autoimmunity, and CSF HIV antibody profiles in HIV central nervous system escape.

Antiretroviral therapy (ART) suppresses plasma and cerebrospinal fluid (CSF) HIV replication. Neurosymptomatic (NS) CSF escape is a rare exception in which CNS HIV replication occurs in the setting of neurologic impairment. The origins of NS escape are not fully understood. We performed a case-control study of asymptomatic (AS) escape and NS escape subjects with HIV-negative subjects as controls in which we investigated differential immunoreactivity to self-antigens in the CSF of NS escape by employing neuroanatomic CSF immunostaining and massively multiplexed self-antigen serology (PhIP-Seq). Additionally, we utilized pan-viral serology (VirScan) to deeply profile the CSF anti-viral antibody response and metagenomic next-generation sequencing (mNGS) for pathogen detection. We detected Epstein-Barr virus (EBV) DNA more frequently in the CSF of NS escape subjects than in AS escape subjects. Based on immunostaining and PhIP-Seq, there was evidence for increased immunoreactivity against self-antigens in NS escape CSF. Finally, VirScan revealed several immunodominant epitopes that map to the HIV envelope and gag proteins in the CSF of AS and NS escape subjects. Whether these additional inflammatory markers are byproducts of an HIV-driven process or whether they independently contribute to the neuropathogenesis of NS escape will require further study.

Pneumococcal meningitis induces apoptosis in recently postmitotic immature neurons in the dentate gyrus of neonatal rats.

Bacterial meningitis is associated with high rates of morbidity and mortality, despite advances in antibiotic therapy. Meningitis caused by Streptococcus pneumoniae is associated with a particularly high incidence of neurological sequelae including deficits resulting from damage to the hippocampus. Previous studies have documented that in neonatal rats with experimental pneumococcal meningitis, cells in the subgranular layer of the dentate gyrus undergo apoptosis. The aim of the present study was to define in more detail the nature of the dying cells in the dentate gyrus. Using bromodeoxyuridine labeling at different times before infection combined with immunocytochemistry, we identified the vulnerable cells as those which underwent mitosis 6-10 days before infection. A majority of these cells are of neuronal lineage. Thus, immature neuronal cells several days after the last cell division are preferentially triggered into apoptosis during pneumococcal meningitis. The loss of these cells may contribute to the long-lasting impairment of hippocampal function identified in animal models and in humans after bacterial meningitis.

Neuronal production and precursor proliferation defects in the neocortex of mice with loss of function in the canonical Wnt signaling pathway.

To better understand the function of the Wnt pathway in the developing telencephalon, we analyzed neocortical development in low density lipoprotein receptor-related protein (LRP) 6 mutants. LRP6 mutant mice are hypomorphic for the canonical Wnt signaling pathway and have hypoplasia of the developing neocortex. While early telencephalic morphogenesis is largely intact in these mice, probably due to compensation by LRP5, the mutant mice develop a dramatically thinner cortical plate. There is a prominent reduction of neurogenesis leading to a thin cortical plate. Reduced proliferation late in gestation probably also contributes to the hypoplasia. Although there are marked decreases in the numbers of layer 6 and layers 2-4 neurons all laminar identities are generated and there is no evidence of compensatory increases in layer 5 neurons. In addition, LRP6 mutants have partial penetrance of a complex of cortical dysmorphologies resembling those found in patients with developmental forms of epilepsy and mental retardation. These include ventricular and marginal zone heterotopias and cobblestone lissencephaly. This analysis demonstrates that canonical Wnt signaling is required for a diverse array of developmental processes in the neocortex in addition to the previously known roles in regulating precursor proliferation and patterning.

Hippocampal heterotopia with molecular and electrophysiological properties of neocortical neurons.

Cortical malformations resulting from aberrant brain development can be associated with mental retardation, dyslexia, and intractable forms of epilepsy. Despite emerging interest in the pathology and etiology of cortical malformations, little is known about the phenotype of cells within these lesions. In utero exposure to the DNA methylating agent methylazoxymethanol acetate (MAM) during a critical stage in neurodevelopment results in animals with distinct clusters of displaced neurons in hippocampus, i.e. nodular heterotopia. Here we examined the molecular and electrophysiological properties of cells within hippocampal heterotopia using rats exposed to MAM during gestation. Molecular analysis revealed that heterotopic cells do not express mRNA markers normally found in hippocampal pyramidal cells or dentate granule cells (SCIP, Math-2, Prox-1, neuropilin-2). In contrast, Id-2 mRNA, normally abundant in Layer II-III supragranular neocortical neurons but not in CA1 pyramidal neurons, was prominently expressed in hippocampal heterotopia. Current-clamp analysis of the firing properties of heterotopic neurons revealed a striking similarity with supragranular cortical neurons. In particular, both cells were characterized by small hyperpolarizing 'sag' potentials, high input resistance values, slow spike-train afterhyperpolarizations, and the absence of a depolarizing afterpotential. Normotopic CA1 pyramidal neurons (e.g. pyramidal cells with normal lamination adjacent to a heterotopia) in the MAM brain exhibited molecular and electrophysiological properties that were nearly identical to those of age-matched CA1 pyramidal neurons from control rats. We conclude that neuronal heterotopiae in the hippocampus of MAM-exposed rats are comprised of neurons with a Layer II-III supragranular cortex phenotype. The MAM model, therefore, may serve as a useful tool in examination of the factors influencing aberrant brain development and epilepsy.

Plexin-A3 mediates semaphorin signaling and regulates the development of hippocampal axonal projections.

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Here, we show that plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, we provide evidence that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo.

Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus.

In various chemoconvulsant models of human temporal lobe epilepsy, the induction of epileptogenesis by a prolonged period of continuous seizure activity is accompanied by significant changes in hippocampal structure. These changes include an increase in neurogenesis within the proliferative subgranular zone (SGZ) of the dentate gyrus and induction of mossy fiber sprouting in mature dentate granule cells. As dentate granule cell neurogenesis and axon outgrowth are also hallmarks of hippocampal development, we hypothesized that molecules involved in normal development may also play a role in similar changes associated with epileptogenesis. To begin to test this hypothesis, we have analyzed the expression patterns of multiple members of the basic helix-loop-helix (bHLH) family of transcription factors in both normal and epileptic adult rats. bHLH protein expression has been found recently in dentate granule cells at specific developmental stages, and analysis of developmental models suggests specific neural differentiation functions for these molecules. We show that mRNA expression of all seven bHLH family members examined in this study, as well as the divergent homeobox protein Prox1, is present in the adult. Patterns of expression varied considerably between family members, ranging from the limited expression of Mash1 in the neurogenic SGZ of the dentate gyrus to the scattered, widespread profile of Hes5 throughout the dentate gyrus and the hippocampus proper. Moreover, these varied profiles of expression were differentially regulated following status epilepticus, with some increasing (Mash1, Id2), some falling (Hes5, Prox1), and others remaining mostly unchanged (NeuroD/BETA2, NeuroD2/NDRF, Id3, Rath2/Nex1). While the function of these molecules in the adult brain remains to be characterized, our findings support the idea that molecules controlling cell-fate decisions in the developing dentate gyrus are also operative during seizure-induced neurogenesis and plasticity.

Wnt receptors and Wnt inhibitors are expressed in gradients in the developing telencephalon.

The caudomedial margin of the medial pallium, known as the cortical hem, expresses several Wnt genes that have been shown to be crucial for cortical development. We examined the expression of members of the Frizzled (mFz) family of Wnt receptors and the Secreted Frizzled Related Protein (SFRP) family of Wnt inhibitors during telencephalic development. We found that mFz-5 and mFz-8 are specifically expressed in the neocortical neuroepithelium and excluded from the hippocampal neuroepithelium in early telencephalic development, whereas mFz-9 and mFz-10 have expression domains confined to the medial pallium. In addition, SFRP-1 and SFRP-3 are expressed in opposing anterolateral to caudomedial gradients within the telencephalic ventricular zone throughout corticogenesis.

Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS.

Wnt signaling regulates a wide range of developmental processes such as proliferation, cell migration, axon guidance, and cell fate determination. In this report, we studied the expression of secreted frizzled related protein-2 (SFRP-2), which codes for a putative Wnt inhibitor, in the developing nervous system. SFRP-2 is expressed in several discrete neuroepithelial domains, including the diencephalon, the insertion of the eminentia thalami into the caudal telencephalon, and the pallial-subpallial boundary (PSB). We also noted that Wnt-7b expression was similar to SFRP-2 expression. Because many of these structures are disrupted in Pax-6 mutant mice, we examined SFRP-2 and Wnt-7b expression in the forebrains of Pax-6 Sey/Sey mice. We found that Pax-6 mutants lack SFRP-2 expression in the PSB and diencephalon. Interestingly, Pax-6 mutants also lack Wnt-7b expression in the PSB, but Wnt-7b expression in the diencephalon is preserved. Furthermore, in the spinal cord of Pax-6 mutants, SFRP-2 and Wnt-7b expression was greatly reduced. Our results suggest that by virtue of its apposition to Wnt-7b expression, SFRP-2 may modulate its function, particularly at boundaries such as the PSB, and that changes in Wnt signaling contribute to the phenotype of Pax-6 mutants.

An arrow hits the Wnt signaling pathway.

A recent series of papers in Nature shows that low-density lipoprotein receptor-related protein family members play a role in transduction of Wnt signals. These findings, in addition to other work from the past two years, have important implications for the role of Wnts in neural development.

Correlation of clinical and neuroimaging findings in a case of rabies encephalitis.

BACKGROUND: Rabies encephalitis is a feared, virtually uniformly fatal form of central nervous system infection. The incidence of rabies encephalitis in the United States is almost certainly underestimated because of the predominance of bat-borne rabies, which can be spread without traumatic exposure. Because of its rarity in developed countries, rabies encephalitis has been seldom studied with modern imaging techniques. SETTING: University-based teaching hospital. PATIENT: A case of pathologically confirmed rabies encephalitis is presented. Diagnosis of rabies was made by seroconversion testing while the patient was alive and was confirmed postmortem by the presence of rabies antigens and Negri bodies in the brain. The patient had 2 magnetic resonance studies done that showed dramatic abnormalities in the medulla and pons that correlated with features of the neurologic examination and hypothalamic-pituitary abnormalities. RESULT: The patient had a fulminant encephalitic course that ended in death. CONCLUSION: Rabies is an uncommon cause of fatal encephalitis. Anatomic imaging studies such as computed tomographic and magnetic resonance scans have generally been negative in confirmed cases of rabies. We report a case of confirmed rabies with extensive brainstem and hypothalamic-pituitary abnormalities on magnetic resonance imaging. Although these findings are nonspecific, they should raise the clinical suspicion of rabies in the setting of aggressive encephalitis of unclear cause, and appropriate diagnostic tests should be performed.

Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development.

The dentate gyrus of the hippocampus is uniquely organized with a displaced proliferative zone that continues to generate dentate granule cells throughout life. We have analyzed the expression of Notch receptors, Notch ligands, and basic helix-loop-helix (bHLH) genes during dentate gyrus development to determine whether the need to maintain a pool of undifferentiated precursors is reflected in the patterns of expression of these genes. Many of these genes are expressed diffusely throughout the cortical neuroepithelium at embryonic days 16 and 17 in the rat, just preceding the migration of newly born granule cells and dentate precursor cells into the dentate anlage. However, at this time, Mash1, Math3, and Id3 expression are all concentrated in the area that specifically gives rise to granule cells and dentate precursor cells. Two days later, at the time of migration of the first granule cells and dentate precursor cells, cells expressing Mash1 are seen in the migratory route from the subventricular zone to the developing dentate gyrus. Newly born granule cells expressing NeuroD are also present in this migratory pathway. In the first postnatal week, precursor cells expressing Mash1 reside in the dentate hilus, and by the third postnatal week they have largely taken up their final position in the subgranular zone along the hilar side of the dentate granule cell layer. After terminal differentiation, granule cells born in the hilus or the subgranular zone begin to express NeuroD followed by NeuroD2. This study establishes that the expression patterns of bHLH mRNAs evolve during the formation of the dentate gyrus, and the precursor cells resident in the mature dentate gyrus share features with precursor cells found in development. Thus, many of the same mechanisms that are known to regulate cell fate and precursor pool size in other brain regions are likely to be operative in the dentate gyrus at all stages of development.

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy.

BETA2/NeuroD is a homologue of the Drosophila atonal gene that is widely expressed during development in the mammalian brain and pancreas. Although studies in Xenopus suggest that BETA2/NeuroD is involved in cellular differentiation, its function in the mammalian nervous system is unclear. Here we show that mutant mice homozygous for a deletion at the BETA2/NeuroD locus fail to develop a granule cell layer within the dentate gyrus, one of the principal structures of the hippocampal formation. To understand the basis of this abnormality, we analyzed dentate gyrus development by using immunocytochemical markers in BETA2/NeuroD-deficient mice. The early cell populations in the dentate gyrus, including Cajal-Retzius cells and radial glia, are present and appear normally organized. The migration of dentate precursor cells and newly born granule cells from the neuroepithelium to the dentate gyrus remains intact. However, there is a dramatic defect in the proliferation of precursor cells once they reach the dentate and a significant delay in the differentiation of granule cells. This leads to malformation of the dentate granule cell layer and excess cell death. BETA2/NeuroD null mice also exhibit spontaneous limbic seizures associated with electrophysiological evidence of seizure activity in the hippocampus and cortex. These findings thus establish a critical role of BETA2/NeuroD in the development of a specific class of neurons. Furthermore, failure to express BETA2/NeuroD leads to a stereotyped pattern of pathological excitability of the adult central nervous system.

Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.

GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.

Spontaneous intracranial hypotension resulting in stupor caused by diencephalic compression.

A 51-year-old man had a 4-month history of progressive headache and gradual onset of somnolence. MRI suggested spontaneous intracranial hypotension (SIH) with diencephalic compression, but he did not improve after three epidural blood patches. He became alert following intrathecal saline infusion that normalized his CSF pressure. A CSF leak was noted on spinal MRI and confirmed with CT contrast myelography. Surgical ligation of a torn dural root sleeve isolating a ruptured Tarlov's cyst resulted in permanent cure.

Glial cells of the lamprey nervous system contain keratin-like proteins.

Lamprey axons regenerate following spinal cord transection despite the formation of a glial scar. As we were unable to detect a lamprey homologue of glial fibrillary acidic protein (GFAP), a major constituent of astrocytes, we studied the composition of intermediate filament (IF) proteins of lamprey glia. Monoclonal antibodies (mAbs) were raised to lamprey spinal cord cytoskeletal extracts and these mAbs were characterized by using Western blotting and immunocytochemistry. On two-dimensional (2-D) Western blots, five of the mAbs detected three major IF polypeptides in the molecular weight (MW) range of 45-56 kD. Further studies were conducted to determine the relationship between the lamprey glial-specific antigen and other mammalian IF proteins. Antikeratin 8 antibody recognized two of the three polypeptides. Several of the glial-specific mAbs reacted with human keratins 8 and 18 on Western blots. Keratin-like immunoreactivity was found in all parts of the central and peripheral nervous systems in both larval and adult lampreys. The immunocytochemical staining patterns of glial-specific mAbs were indistinguishable on lamprey spinal cord sections. However, on brain sections, two distinct patterns were observed. A subset of mAbs stained only a few glial fibers in the brain, whereas others stained many more brain glia, particularly the ependymal cells. The former group of mAbs recognized only the two lower MW polypeptides on 2-D Western blots, but the latter group of mAbs recognized all three major IF polypeptides. This correlation is supported by the observation that the highest MW IF polypeptide has an increased level of expression in the brain relative to the spinal cord. Thus, in the lamprey, the glial cells of both spinal cord and brain express molecules similar to simple epithelial cytokeratins, but their IFs may contain these keratins in different stoichiometric proportions. The widespread presence in the lamprey of primitive glial cells containing keratin-like intermediate filaments may have significance for the extraordinary ability of lamprey spinal axons to regenerate.

Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides.

The beta-amyloid or beta/A4 peptides that accumulate as filamentous aggregates in the extracellular space of Alzheimer disease (AD) brains are derived from one or more alternatively spliced amyloid precursor proteins (APPs). The more abundant APPs in the central nervous system are the 695-(APP695), 751- (APP751), and 770- (APP770) amino acid isoforms, and each could be the source of beta/A4 peptide that accumulates in the AD brain. It is plausible that altered metabolism of these APPs by central nervous system neurons could lead to the release and deposition of beta/A4 peptide in brain parenchyma. Thus, we examined the expression and processing of the three major brain APPs in nearly pure human neurons (NT2N cells) derived from a teratocarcinoma cell line (NTera2/c1.D1 or NT2 cells) after retinoic acid treatment. NT2N neurons expressed almost exclusively APP695, whereas NT2 cells expressed predominantly APP751/770. Furthermore, the processing of the APPs in NT2N cells was distinct from NT2 and nonneuronal cells. Most significantly, the NT2N neurons but not the NT2 cells constitutively generated intracellular beta/A4 peptide and released it into the culture medium. This work demonstrates the intracellular production of beta/A4 peptide and suggests that cultured NT2N cells may provide a unique model system for understanding the contribution of neurons and APP695 to amyloidogenesis in the AD brain.

NTera 2 cells: a human cell line which displays characteristics expected of a human committed neuronal progenitor cell.

We have identified a human cell line with a phenotype resembling committed CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expressed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule-associated protein (MAP) expressed in human neuroepithelium. NT2/D1 cells also expressed the cell adhesion molecules NCAM and N-cadherin which are thought to be important in cell-cell interactions within the neuroepithelium. These NT2/D1 cells also expressed small amounts of NF-L, alpha-internexin, NF-M, and MAP2c, indicating that they are committed to a neuronal fate. Previous studies have shown that, following RA treatment, a proportion of NT2/D1 cells terminally differentiate into neurons and that this occurs via an asymmetric stem cell mode of differentiation. In light of the identification of the neuroepithelial phenotype of NT2/D1 cells we decided to examine more closely the relationship of in vitro neurogenesis in NT2/D1 cells, during RA treatment to that of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and increased phosphorylation of NF-M. By 10-14 days, these cells began to resemble neurons morphologically, i.e., with rounded cell bodies and processes. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons began to express MAP2b and tau and extinguished their expression of nestin. Recently, we developed a method for obtaining pure cultures of neurons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cells. Given the data obtained in this study and the characterization of the neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons.

Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

Glutamate receptor (GluR) channels are responsible for a number of fundamental properties of the mammalian central nervous system, including nearly all excitatory synaptic transmission, synaptic plasticity, and excitotoxin-mediated neuronal death. Although many human and rodent neuroblast cell lines are available, none has been directly shown to express GluR channels. We report here that cells from the human teratocarcinoma line NT2 are induced by retinoic acid to express neuronal N-methyl-D-aspartate (NMDA) and non-NMDA GluR channels concomitant with their terminal differentiation into neuron-like cells. The molecular and physiologic characteristics of these human GluR channels are nearly identical to those in central nervous system neurons, as demonstrated by PCR and patch clamp recordings, and the cells demonstrate glutamate-induced neurotoxicity.