Genetic evidence that brain-derived neurotrophic factor mediates competitive interactions between individual cortical neurons

Brain-derived neurotrophic factor (BDNF) is a secreted protein important for development and function of neocortical circuitry. Although it is well established that BDNF contributes to the sculpting of dendrite structure and modulation of synapse strength, the range and directionality of BDNF signaling underlying these functions are incompletely understood. To gain insights into the role of BDNF at the level of individual neurons, we tested the cell-autonomous requirements for Bdnf in visual cortical layer 2/3 neurons. We found that the number of functional Bdnf alleles a neuron carries relative to the prevailing genotype determines its density of dendritic spines, the structures at which most excitatory synapses are made. This requirement for Bdnf exists both during postnatal development and in adulthood, suggesting that the amount of BDNF a neuron is capable of producing determines its success in ongoing competition in the environment of the neocortex. Our results suggest that BDNF may perform a long-sought function for a secreted growth factor in mediating the competitive events that shape individual neurons and their circuits.     

Axonal transport of a subclass of tau proteins: evidence for the regional differentiation of microtubules in neurons.

Tubulin, the major constituent of microtubules, is anterogradely transported within the axon as part of slow component a (SCa; 0.2-1.0 mm/day). This raises the possibility that the microtubule-associated proteins (MAPs) may be transported at the same rate. To examine this question, the high molecular weight and tau MAPs obtained from whole brain preparations of microtubules were compared with the proteins of SCa in guinea pig retinal ganglion cell axons by using phosphocellulose chromatography and one- and two-dimensional polyacrylamide gel electrophoresis. Only two of the tau proteins were found to be cotransported with axonal tubulin, although four tau and two high molecular weight MAPs were synthesized in the retina. This result suggests either that the retinal ganglion cell synthesizes only those two tau proteins or that it synthesizes several of the MAPs, but commits to axonal transport just two of the tau proteins. In either case, these observations are consistent with the transport of intact microtubules and demonstrate that axonal microtubules represent a distinct subset of brain microtubules. Such a distinction may be related to unique properties of the axonal cytoskeleton.     

Peripheral S-phase T cells in HIV disease have a central memory phenotype and rarely have evidence of recent T cell receptor engagement

Heightened proliferation and death of T lymphocytes may play a key role in human immunodeficiency virus (HIV) pathogenesis; however, the mechanism that mediates this effect and the phenotype of the proliferating T cells have not been clearly determined. We assessed S-phase cell frequencies and phenotype by ex vivo bromodeoxyuridine incorporation and flow-cytometric analysis in a group of 35 HIV-infected individuals. Frequencies of S-phase T cells were increased in HIV disease and were related to plasma HIV RNA levels but not to CD4 cell, total T cell, or total lymphocyte counts. S-phase cells were phenotypically defined as "central memory" cells (CD45RO+CD62L+CCR7+). Although activated (CD38+), S-phase cells lacked CD69 expression, rarely expressed CD25, and were not overrepresented among HIV-specific cells, as might have been expected if these cells had recently been activated by HIV antigens. Thus, in HIV infection, central memory T cells may be highly susceptible to bystander mechanisms of immune activation, leading to S-phase entry.     

Frailty in older adults: evidence for a phenotype

BACKGROUND: Frailty is considered highly prevalent in old age and to confer high risk for falls, disability, hospitalization, and mortality. Frailty has been considered synonymous with disability, comorbidity, and other characteristics, but it is recognized that it may have a biologic basis and be a distinct clinical syndrome. A standardized definition has not yet been established. METHODS: To develop and operationalize a phenotype of frailty in older adults and assess concurrent and predictive validity, the study used data from the Cardiovascular Health Study. Participants were 5,317 men and women 65 years and older (4,735 from an original cohort recruited in 1989-90 and 582 from an African American cohort recruited in 1992-93). Both cohorts received almost identical baseline evaluations and 7 and 4 years of follow-up, respectively, with annual examinations and surveillance for outcomes including incident disease, hospitalization, falls, disability, and mortality. RESULTS: Frailty was defined as a clinical syndrome in which three or more of the following criteria were present: unintentional weight loss (10 lbs in past year), self-reported exhaustion, weakness (grip strength), slow walking speed, and low physical activity. The overall prevalence of frailty in this community-dwelling population was 6.9%; it increased with age and was greater in women than men. Four-year incidence was 7.2%. Frailty was associated with being African American, having lower education and income, poorer health, and having higher rates of comorbid chronic diseases and disability. There was overlap, but not concordance, in the cooccurrence of frailty, comorbidity, and disability. This frailty phenotype was independently predictive (over 3 years) of incident falls, worsening mobility or ADL disability, hospitalization, and death, with hazard ratios ranging from 1.82 to 4.46, unadjusted, and 1.29-2.24, adjusted for a number of health, disease, and social characteristics predictive of 5-year mortality. Intermediate frailty status, as indicated by the presence of one or two criteria, showed intermediate risk of these outcomes as well as increased risk of becoming frail over 3-4 years of follow-up (odds ratios for incident frailty = 4.51 unadjusted and 2.63 adjusted for covariates, compared to those with no frailty criteria at baseline). CONCLUSIONS: This study provides a potential standardized definition for frailty in community-dwelling older adults and offers concurrent and predictive validity for the definition. It also finds that there is an intermediate stage identifying those at high risk of frailty. Finally, it provides evidence that frailty is not synonymous with either comorbidity or disability, but comorbidity is an etiologic risk factor for, and disability is an outcome of, frailty. This provides a potential basis for clinical assessment for those who are frail or at risk, and for future research to develop interventions for frailty based on a standardized ascertainment of frailty.     

Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons.

A diacylglycerol kinase cDNA was isolated from a rat brain cDNA library. This cDNA encoded an 801-amino acid protein of 90,287 Da. This 90-kDa diacylglycerol kinase showed 58% identity in deduced amino acid sequence with a previously isolated rat 80-kDa diacylglycerol kinase. EF-hand motifs, cysteine-rich zinc-finger-like sequences, and putative ATP-binding sites were all conserved between the two kinase species. However, mRNA encoding the 90-kDa kinase was confined to restricted neuronal populations such as the caudate-putamen, the accumbens nucleus, and the olfactory tubercle. Further, the 90-kDa kinase was found to exhibit high phosphorylation activity for long-chain diacylglycerols and was mainly associated with the membrane fraction when the cDNA was transfected into COS-7 cells.     

Subset of early radial glial progenitors that contribute to the development of callosal neurons is absent from avian brain

The classical view of mammalian cortical development suggests that pyramidal neurons are generated in a temporal sequence, with all radial glial cells (RGCs) contributing to both lower and upper neocortical layers. A recent opposing proposal suggests there is a subgroup of fate-restricted RGCs in the early neocortex, which generates only upper-layer neurons. Little is known about the existence of fate restriction of homologous progenitors in other vertebrate species. We investigated the lineage of selected Emx2⁺ [vertebrate homeobox gene related to Drosophila empty spiracles (ems)] RGCs in mouse neocortex and chick forebrain and found evidence for both sequential and fate-restricted programs only in mouse, indicating that these complementary populations coexist in the developing mammalian but not avian brain. Among a large population of sequentially programmed RGCs in the mouse brain, a subset of self-renewing progenitors lack neurogenic potential during the earliest phase of corticogenesis. After a considerable delay, these progenitors generate callosal upper-layer neurons and glia. On the other hand, we found no homologous delayed population in any sectors of the chick forebrain. This finding suggests that neurogenic delay of selected RGCs may be unique to mammals and possibly associated with the evolution of the corpus callosum.Several neural progenitor subtypes have been identified in the developing mammalian neocortex (1–4). At the onset of neurogenesis, multipotent proliferative radial glial cells (RGCs) reside at the ventricular surface. RGC divisions can be “self-renewing,” a symmetrical division in which two identical RGCs are generated; “direct neurogenic,” generating a neuroblast and a multipotent progenitor; or “indirect neurogenic,” which gives rise to an intermediate progenitor that migrates to divide in the subventricular zone. The timing of these events is crucial for cell number and fate determination (5–7) and vary among different mammalian species. Importantly, very little is known about the homologous populations of RGCs in other vertebrate species and about their role in cortical evolution.Observations made from Golgi-stained material (4, 8), birth-dating (5, 9), and lineage-tracing analysis (10–14) contributed to the formation of the “sequential” hypothesis. Accordingly, each RGC in the early brain generates most cortical cell types by sequential mitoses (15). Long-range subcortical projection neurons of the deep layers are generated first, followed by the callosal projection neurons of the upper layers, and finally the glial cells (16).However, recent genetic fate mapping of selected lineages has revealed a subpopulation of early RGCs expressing Cux2, which only gives rise to Satb2-positive upper-layer cortical neurons (17). This finding suggests that the early neurogenic brain may contain several types of cortical progenitors, each of which constrained to generating certain subpopulations of neurons, known as the “restricted progenitor hypothesis” (18). The existence of these restricted progenitors has been challenged (1–4, 19). Early clonal analysis performed by retroviral-mediated gene transfer also led to differing interpretations (12, 14, 20).Interestingly, these alternative interpretations diverge in the origin of callosal upper-layer cortical neurons. The appearance of neurons with callosal projections in the upper layers is considered an important event in the evolution of the mammalian neocortex, as there is no corpus callosum in sauropsid brains (21). Based on selected gene expression patterns, Suzuki et al. suggested that upper-layer–like cortical neurons are situated in a different sector of chick pallium with respect to the position of lower-layer–like neurons (22). Their birth-dating studies demonstrated that upper-layer–like neurons are generated in a biased fashion from spatially restricted progenitors (15). They also suggest that the potential to adopt the sequential pattern of neurogenesis remains, but is suppressed in vivo. To resolve these issues, further comparative clonal studies of single progenitors are needed (23, 24).Here we investigate the lineage of selected Emx2⁺ [vertebrate homeobox gene related to Drosophila empty spiracles (ems)] RGCs in the mammalian pallium. We describe a subset of progenitors that lack neurogenic potential during early neocortical development. After self-renewing and transit-amplifying mitoses, these RGC progenitors exclusively give rise to callosal upper-layer neurons and glia. We therefore suggest the temporal and spatial coexistence of restricted and sequential RGC progenitors in the mammalian cortical ventricular zone. Additionally, we observed no homologous heterochronic behavior in this population within chick forebrain, suggesting the neurogenic delay of some RGCs is a mammal-specific attribute, possibly related to the evolution of neurons involved in interhemispheric dorsal pallial communications. Our study establishes the existence, time frame, and lineage of these early Emx2⁺ RGC progenitor population fate restricted to the upper layers of mammalian cortex but absent in the avian brain.     

Differentiation of human T-lymphoid leukemia cells into cells that have a suppressor phenotype is induced by phorbol 12-myristate 13-acetate.

Treatment of cultured human T-lymphoid (CEM) leukemia cells with nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) resulted in a reduction in cell growth and in the acquisition of a surface antigenic pattern that is common to both suppressor and cytotoxic T lymphocytes. This antigenic pattern was detected by OKT monoclonal antibodies. PMA treatment did not cause the expression of a cytotoxic function but rather induced the expression of a suppressor cell marker. This marker was characterized by the ability of the treated CEM cells to suppress [3H]thymidine incorporation into phytohemagglutinin-activated peripheral blood lymphocytes. After 4 days of treatment of CEM cells from either cloned or the parental cell population with 16 nM PMA, 71-98% of the cells expressed reactivity with OKT3 and OKT8 antibodies whereas reactivity with OKT4 and OKT6 was detected in less than or equal to 1-8% of the cells. The CEM cells can be divided into five groups based on the antigenic patterns of cells from randomly isolated clones. The cells from four of these groups were characterized by either low or high reactivity with each of the four OKT antibodies. The antigenic pattern of the fifth group resembled that of the parent CEM cells. The acquisition of reactivity with the OKT3 antibody in the CEM cells after PMA treatment was dependent on both time and dose and did not require cell replication. Acquisition of reactivity with OKT3 antibody also occurred after treatment with phorbol 12,13-dibutyrate but not after treatment with phorbol 13-monoacetate, phorbol 12,13-diacetate, or dimethyl sulfoxide. These results indicate that treatment of CEM cells with PMA and related agents can cause the cells to express a phenotype that resembles that of a mature suppressor T lymphocyte.     

Properties of postganglionic sympathetic neurons with axons in phrenic nerve.

The aim of the study was to test the reflex and resting properties of postganglionic sympathetic neurons with axons located in the right phrenic nerve. The experiments have been performed on chloralose-anesthetized cats with both vago-aortic nerves cut. The somata or the postganglionic sympathetic neurons were located in the stellate ganglion. Axons of these neurons passed through the upper and lower phrenic nerve roots and through the phrenic nerve itself. The presence of cardiac and respiratory rhythmicities was detected in the activity of the phrenic postganglionic sympathetic neurons. Hyperventilation, which abolished burst discharges of the phrenic nerve, decreased the sympathetic activity by 14%. Systemic hypoxia (ventilating the animals for 2 min with 8% O2 in N2) increased the sympathetic activity threefold. The results of our experiments suggest that axons of the sympathetic neurons located in the right phrenic nerve could possibly be diaphragmatic muscle vasoconstrictors.     

Arrhythmogenic right ventricular cardiomyopathy: a 'final common pathway' that defines clinical phenotype.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C)is a primary heart muscle disease with distinct characteristics.ARVD/C predominantly affects the right ventricle (RV), withRV dilation and thinning due to fibrofatty infiltration of theventricular myocardium, and ultimately depressed systolic functionleading to right heart failure or biventricular failure.¹ Earlyin its clinical course, ARVD/C typically presents with ventriculararrhythmias (usually with a left bundle branch pattern), syncope,or sudden cardiac death.² Tragically, this clinical scenariocommonly occurs in young, healthy, athletic individuals. A setof clinical criteria, known as the ‘Task Force Criteria’,first described by McKenna et al.³ in 1994 and later modifiedfor inclusion of family members,⁴ utilizes     

Inheritance of combined hyperlipoproteinemia: evidence for a new lipoprotein phenotype

There are patients whose serum contains elevated levels of low density and very low density lipoproteins (LDL and VLDL). These lipoproteins exhibit normal beta and prebeta electrophoretic mobility. To ascertain whether these patterns are genetic traits, lipoproteins have been quantified by preparative ultracentrifugation in the members of three kindreds with familial hyperlipoproteinemia and in one set of monozygotic twins. Elevations in both LDL and VLDL levels followed a heritable pattern. Among alternative genetic interpretations, the most plausible is that the combined pattern results from the chance combination of two separate genetic determinants, one for elevated LDL levels and another for elevated VLDL levels, with modification by other genetic factors. The genotype for elevated LDL levels is not likely to be identical with that of familial hyperbeta lipoproteinemia (type II), since a low frequency of isolated elevations in LDL levels in these kindreds and absence of the abnormality in childhood members who were at risk are incompatible features. Subjects with combined hyperlipoproteinemia manifested characteristics that are usually prevalent in type IV kindreds; that is, impaired glucose tolerance, obesity and hyperuricemia, whereas clinical characteristics of the familial type II disorder, notably xanthomas, were absent. Ischemic heart disease seemed to be associated with the combined pattern primarily when concurrent abnormalities of glucose tolerance were present, suggesting synergy between disturbed carbohydrate and lipoprotein metabolism in the genesis of coronary artery insufficiency in these subjects.     

Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation.

The ubiquitous plasma membrane Na+/H+ exchanger (termed NHE1) is activated by diverse hormonal signals, with the notable exception of hormones acting through cAMP as second messenger. Therefore, the Na+/H+ exchanger found in the nucleated trout red cell is of particular interest since it is activated by catecholamines, forskolin, and cAMP analogues. We report here that a cloned cDNA encoding the red cell exchanger restores functional Na+/H+ activity when transfected into Na+/H+ antiporter-deficient fibroblasts (i.e., it regulates intracellular pH in a Na-dependent and amiloride-sensitive manner). This red cell exchanger represents an additional form of Na+/H+ exchanger (termed beta NHE), which is characterized by a specific cytoplasmic domain involved in activation by the cAMP-dependent signaling pathway. After transfection in the same cellular context, beta NHE, but not NHE1, is activated by cAMP or by hormones that increase cAMP levels. Comparison of the amino acid sequences of exchangers shows that beta NHE, but not NHE1, contains two clustered consensus motifs for phosphorylation by a cAMP-dependent protein kinase (protein kinase A; PKA). A deletion mutant devoid of the C-terminal region of the cytoplasmic loop containing the two PKA sites restores Na+/H+ activity but is no longer activated by cAMP analogues or catecholamines. In red blood cells, the Na+/H+ exchanger is also activated by another pathway involving protein kinase C (PKC). Expression of beta NHE in fibroblasts shows that these two independent signaling pathways impinge on two distinct domains of the exchanger. The cytoplasmic segment containing PKA consensus sites, which is crucial for cAMP activation, is unnecessary for stimulation by PKC activators.     

Catecholaminergic axons innervate LH-releasing hormone immunoreactive neurons of the human diencephalon.

Catecholamines have been shown to modulate gonadal functions via interactions with hypothalamic LH-releasing hormone (LHRH)-synthesizing neurons. To reveal the morphological background of this phenomenon, the distribution of LHRH neurons and tyrosine hydroxylase (TH)-immunoreactive (IR), catecholaminergic structures were mapped in the human diencephalon. First, the location of LHRH and TH-IR neuronal elements was analyzed, and then the relationship between the two different systems was examined. The LHRH-IR cell bodies were mainly present in the medial preoptic and infundibular areas. The TH-IR perikarya were located in the periventricular, paraventricular, and supraoptic hypothalamic nuclei and also in the median eminence. The TH-IR fibers were numerous in septal, infundibular, periventricular, and lateral hypothalamic regions. The brown, diaminobenzidine-labeled LHRH-containing perikarya were found to receive black, silver-intensified, TH-positive axon terminals in the infundibular and medial preoptic areas. However, in the preoptic and caudal parts of the diencephalon, only a few juxtapositions were noted. The present results indicate that hormone released from diencephalic LHRH-IR neurons in humans may be influenced by the central catecholaminergic system via direct synaptic mechanisms.     

Sodium channels in dendrites of rat cortical pyramidal neurons.

The voltage-dependent properties that have been directly demonstrated in Purkinje cell and hippocampal pyramidal cell dendrites play an important role in the integrative capacities of these neurons. By contrast, the properties of neocortical pyramidal cell dendritic membranes have been more difficult to assess. Active dendritic conductances near sites of synaptic input would have an important effect on the input-output characteristics of these neurons. In the experiments reported here, we obtained direct evidence for the existence of voltage-dependent Na+ channels on the dendrites of neocortical neurons by using cell-attached patch and whole cell recordings from acutely isolated rat neocortical pyramidal cells. The qualitative and quantitative properties of dendritic and somatic currents were indistinguishable. Insofar as Na+ currents are concerned, the soma and primary apical dendrite can be considered as one relatively uniform compartment. Similar dendritic Na+ currents on dendrites in mature neurons would play an important role in determining the integrative properties of these cortical units.     

Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc.

We have characterized a simple method that uses 65ZnCl2 to detect zinc-binding proteins that have been immobilized on nitrocellulose. Conditions have been identified that permit the detection of as little as 1 microgram of some zinc-binding proteins. The specificity of the binding is indicated by the ability of other divalent metal ions to compete with 65Zn(II) in this assay. We have used this technique to provide evidence that the nucleic acid-binding gag protein of retroviruses also binds zinc. This technique can be applied to biological mixtures of proteins and may be used in proteolytic mapping studies to identify protein fragments that have zinc-binding activity.     

Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity

The aromatase-knockout (ArKO) mouse provides a useful model to examine the role that estrogens play in development and homeostasis in mammals. Lacking a functional Cyp19 gene, which encodes aromatase, the ArKO mouse cannot synthesize endogenous estrogens. We examined the adipose depots of male and female ArKO mice, observing that these animals progressively accumulate significantly more intraabdominal adipose tissue than their wild-type (WT) littermates, reflected in increased adipocyte volume at gonadal and infrarenal sites. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared with WT controls, as were elevated insulin levels, although blood glucose levels were unchanged. Associated with these changes, a striking accumulation of lipid droplets was observed in the livers of ArKO animals. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.     

Mistargeting hippocampal axons by expression of a truncated Eph receptor

Topographic mapping of axon terminals is a general principle of neural architecture that underlies the interconnections among many neural structures. The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been implicated in the formation of topographic projection maps. We show that multiple Eph receptors and ligands are expressed in the hippocampus and its major subcortical projection target, the lateral septum, and that expression of a truncated Eph receptor in the mouse brain results in a pronounced alteration of the hippocamposeptal topographic map. Our observations provide strong support for a critical role of Eph family guidance factors in regulating ontogeny of hippocampal projections.     

Dominant negative protein kinase mutations that confer a G1 arrest phenotype.

The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle. We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28. Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants. The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein. DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases. These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function. By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component. Suppressors that may encode this other component have been isolated on high-copy-number plasmids.