Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen

Angiotensin produced systemically or locally in tissues such as the brain plays an important role in the regulation of blood pressure and in the development of hypertension. We have established transgenic rats [TGR(ASrAOGEN)] expressing an antisense RNA against angiotensinogen mRNA specifically in the brain. In these animals, the brain angiotensinogen level is reduced by more than 90% and the drinking response to intracerebroventricular renin infusions is decreased markedly compared with control rats. Blood pressure of transgenic rats is lowered by 8 mmHg (1 mmHg = 133 Pa) compared with control rats. Crossbreeding of TGR(ASrAOGEN) with a hypertensive transgenic rat strain exhibiting elevated angiotensin II levels in tissues results in a marked attenuation of the hypertensive phenotype. Moreover, TGR(ASrAOGEN) exhibit a diabetes insipidus-like syndrome producing an increased amount of urine with decreased osmolarity. The observed reduction in plasma vasopressin by 35% may mediate these phenotypes of TGR(ASrAOGEN). This new animal model presenting long-term and tissue-specific down-regulation of angiotensinogen corroborates the functional significance of local angiotensin production in the brain for the central regulation of blood pressure and for the pathogenesis of hypertension.

Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBα and Δ isoforms and to an overexpression of the CREBβ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBα, β, and Δ) inactivated. In contrast to the CREBαΔ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the αβ lineage, which is not affected in CREBαΔ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the αβ T cell lineage. In contrast γδ T cell differentiation is normal in CREB mutant mice.

Dehydroepiandrosterone: A potential signalling molecule for neocortical organization during development

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) are the most abundant steroids produced by the human adrenal, but no receptors have been identified for these steroids, and no function for them has been established, other than as precursors for sex steroid synthesis. DHEA and DHEAS are found in brains from many species, and we have shown that enzymes crucial for their synthesis, especially P450c17 (17α-hydroxylase/c17,20 lyase), are expressed in a developmentally regulated, region-specific fashion in the developing rodent brain. One region of embryonic expression of P450c17, the neocortical subplate, has been postulated to play a role in guiding cortical projections to their appropriate targets. We therefore determined if products of P450c17 activity, DHEA and DHEAS, regulated the motility and/or growth of neocortical neurons. In primary cultures of mouse embryonic neocortical neurons, DHEA increased the length of neurites containing the axonal marker Tau-1, and the incidence of varicosities and basket-like process formations in a dose-dependent fashion. These effects could be seen at concentrations normally found in the brain. By contrast, DHEAS had no effect on Tau-1 axonal neurites but increased the length of neurites containing the dendritic marker microtubule-associated protein-2. DHEA rapidly increased free intracellular calcium via activation of N-methyl-d-aspartate (NMDA) receptors. These studies provide evidence of mechanisms by which DHEA and DHEAS exert biological actions, show that they have specific functions other than as sex steroid precursors, mediate their effects via non-classic steroid hormone receptors, and suggest that their developmentally regulated synthesis in vivo may play crucial and different roles in organizing the neocortex.

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCɛ expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCɛ expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities

Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic (5-HT) neurons in the central nervous system. However, the role of endogenous BDNF in the development and function of these neurons has not been established in vivo because of the early postnatal lethality of BDNF null mice. In the present study, we use heterozygous BDNF+/− mice that have a normal life span and show that these animals develop enhanced intermale aggressiveness and hyperphagia accompanied by significant weight gain in early adulthood; these behavioral abnormalities are known to correlate with 5-HT dysfunction. Forebrain 5-HT levels and fiber density in BDNF+/− mice are normal at an early age but undergo premature age-associated decrements. However, young adult BDNF+/− mice show a blunted c-fos induction by the specific serotonin releaser-uptake inhibitor dexfenfluramine and alterations in the expression of several 5-HT receptors in the cortex, hippocampus, and hypothalamus. The heightened aggressiveness can be ameliorated by the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that endogenous BDNF is critical for the normal development and function of central 5-HT neurons and for the elaboration of behaviors that depend on these nerve cells. Therefore, BDNF+/− mice may provide a useful model to study human psychiatric disorders attributed to dysfunction of serotonergic neurons.

Conserved biological function between Pax-2 and Pax-5 in midbrain and cerebellum development: Evidence from targeted mutations

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15–201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87–89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally, we show that one allele of Pax-2, but not Pax-5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice.

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.

Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer’s disease

To determine whether pathogenic mutations in mtDNA are involved in phenotypic expression of Alzheimer’s disease (AD), the transfer of mtDNA from elderly patients with AD into mtDNA-less (ρ0) HeLa cells was carried out by fusion of platelets or synaptosomal fractions of autopsied brain tissues with ρ0 HeLa cells. The results showed that mtDNA in postmortem brain tissue survives for a long time without degradation and could be rescued in ρ0 HeLa cells. Next, the cybrid clones repopulated with exogenously imported mtDNA from patients with AD were used for examination of respiratory enzyme activity and transfer of mtDNA with the pathogenic mutations that induce mitochondrial dysfunction. The presence of the mutated mtDNA was restricted to brain tissues and their cybrid clones that formed with synaptosomes as mtDNA donors, whereas no cybrid clones that isolated with platelets as mtDNA donors had detectable mutated mtDNA. However, biochemical analyses showed that all cybrid clones with mtDNA imported from platelets or brain tissues of patients with AD restored mitochondrial respiration activity to almost the same levels as those of cybrid clones with mtDNA from age-matched normal controls, suggesting functional integrity of mtDNA in both platelets and brain tissues of elderly patients with AD. These observations warrant the reassessment of the conventional concept that the accumulation of pathogenic mutations in mtDNA throughout the aging process is responsible for the decrease of mitochondrial respiration capacity with age and with the development of age-associated neurodegenerative diseases.

Insulin-like growth factor 1 regulates developing brain glucose metabolism

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-d- [1-14C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1−/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-d- [1-14C]glucose is significantly reduced in synaptosomes prepared from Igf1−/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1−/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in Igf1−/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.

Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: Increased susceptibility of females

Apolipoprotein E (apoE) mediates the redistribution of lipids among cells and is expressed at highest levels in brain and liver. Human apoE exists in three major isoforms encoded by distinct alleles (ɛ2, ɛ3, and ɛ4). Compared with APOE ɛ2 and ɛ3, APOE ɛ4 increases the risk of cognitive impairments, lowers the age of onset of Alzheimer’s disease (AD), and decreases the response to AD treatments. Besides age, inheritance of the APOE ɛ4 allele is the most important known risk factor for the development of sporadic AD, the most common form of this illness. Although numerous hypotheses have been advanced, it remains unclear how APOE ɛ4 might affect cognition and increase AD risk. To assess the effects of distinct human apoE isoforms on the brain, we have used the neuron-specific enolase (NSE) promoter to express human apoE3 or apoE4 at similar levels in neurons of transgenic mice lacking endogenous mouse apoE. Compared with NSE-apoE3 mice and wild-type controls, NSE-apoE4 mice showed impairments in learning a water maze task and in vertical exploratory behavior that increased with age and were seen primarily in females. These findings demonstrate that human apoE isoforms have differential effects on brain function in vivo and that the susceptibility to apoE4-induced deficits is critically influenced by age and gender. These results could be pertinent to cognitive impairments observed in human APOE ɛ4 carriers. NSE-apoE mice and similar models may facilitate the preclinical assessment of treatments for apoE-related cognitive deficits.

Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization

Lissencephaly is a severe brain malformation in humans. To study the function of the gene mutated in lissencephaly (LIS1), we deleted the first coding exon from the mouse Lis1 gene. The deletion resulted in a shorter protein (sLIS1) that initiates from the second methionine, a unique situation because most LIS1 mutations result in a null allele. This mutation mimics a mutation described in one lissencephaly patient with a milder phenotype. Homozygotes are early lethal, although heterozygotes are viable and fertile. Most strikingly, the morphology of cortical neurons and radial glia is aberrant in the developing cortex, and the neurons migrate more slowly. This is the first demonstration, to our knowledge, of a cellular abnormality in the migrating neurons after Lis1 mutation. Moreover, cortical plate splitting and thalomocortical innervation are also abnormal. Biochemically, the mutant protein is not capable of dimerization, and enzymatic activity is elevated in the embryos, thus a demonstration of the in vivo role of LIS1 as a subunit of PAF-AH. This mutation allows us to determine a hierarchy of functions that are sensitive to LIS1 dosage, thus promoting our understanding of the role of LIS1 in the developing cortex.

Refractoriness to melatonin occurs independently at multiple brain sites in Siberian hamsters

The mid-winter development of refractoriness to melatonin (Mel) triggers recrudescence of the atrophied reproductive apparatus of rodents. As a consequence, over-wintering animals become reproductively competent just before the onset of spring conditions favorable for breeding. The neural target tissues that cease to respond to winter Mel signals have not been identified. We now report that the suprachiasmatic nucleus of the hypothalamus, which contains the principal circadian clock, and the reuniens and paraventricular nuclei of the thalamus, each independently becomes refractory to melatonin. Small implants of Mel that were left in place for 40 wk and that act locally on these brain nuclei, induced testicular regression within 6 wk in male Siberian hamsters; 12 wk later Mel implants no longer suppressed reproduction and gonadal recrudescence ensued. Hamsters that were then given a systemic Mel infusion s.c. immediately initiated a second gonadal regression, implying that neurons at each site become refractory to Mel without compromising responsiveness of other Mel target tissues. Refractoriness occurs locally and independently at each neural target tissue, rather than in a separate “refractoriness” substrate. Restricted, target-specific actions of Mel are consistent with the independent regulation by day length of the several behavioral and physiological traits that vary seasonally in mammals.

The neural development and organization of letter recognition: Evidence from functional neuroimaging, computational modeling, and behavioral studies

Although much of the brain’s functional organization is genetically predetermined, it appears that some noninnate functions can come to depend on dedicated and segregated neural tissue. In this paper, we describe a series of experiments that have investigated the neural development and organization of one such noninnate function: letter recognition. Functional neuroimaging demonstrates that letter and digit recognition depend on different neural substrates in some literate adults. How could the processing of two stimulus categories that are distinguished solely by cultural conventions become segregated in the brain? One possibility is that correlation-based learning in the brain leads to a spatial organization in cortex that reflects the temporal and spatial clustering of letters with letters in the environment. Simulations confirm that environmental co-occurrence does indeed lead to spatial localization in a neural network that uses correlation-based learning. Furthermore, behavioral studies confirm one critical prediction of this co-occurrence hypothesis, namely, that subjects exposed to a visual environment in which letters and digits occur together rather than separately (postal workers who process letters and digits together in Canadian postal codes) do indeed show less behavioral evidence for segregated letter and digit processing.

Auditory neuroscience: Development, transduction, and integration

Hearing underlies our ability to locate sound sources in the environment, our appreciation of music, and our ability to communicate. Participants in the National Academy of Sciences colloquium on Auditory Neuroscience: Development, Transduction, and Integration presented research results bearing on four key issues in auditory research. How does the complex inner ear develop? How does the cochlea transduce sounds into electrical signals? How does the brain's ability to compute the location of a sound source develop? How does the forebrain analyze complex sounds, particularly species-specific communications? This article provides an introduction to the papers stemming from the meeting.