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Recent studies by our group and others have demonstrated that growth hormone (GH) is produced endogenously within the hippocampal formation, a brain structure associated with learning and aspects of emotional experience. Here, we demonstrate that this endogenously produced GH is modulated by age and sex differences and the presence of estrogen. GH mRNA levels were higher in females than males, especially during proestrus, a stage of estrus when estrogen levels are elevated. Moreover, GH expression was increased in ovariectomized females that were treated with estradiol. This increase in GH mRNA in response to estrogen was followed by the appearance of GH protein and was negatively correlated with the expression levels of insulin-like growth factor-I mRNA, suggesting a feedback relationship between insulin-like growth factor-I and GH in the brain. GH mRNA levels were also elevated in primary neuronal cultures exposed to 17-β-estradiol in vitro , further confirming the direct influence of estrogen on GH expression. Finally, exposure to an acute stressful event increased the expression and production of GH in both males and females. However, the stress-induced increase of GH in females depended on the stage of the estrous cycle in which they were exposed to the stressful event. Together, these data further demonstrate that GH is endogenously produced in the adult hippocampal formation, where it is regulated by age, estrogen, and exposure to environmental stimuli. These results suggest that GH may be involved in functions ascribed to the hippocampus, such as learning and the response to stressful experience.

Localized translation in mammalian dendrites may play a role in synaptic plasticity and contribute to the molecular basis for learning and memory. The regulatory mechanisms that control localized translation in neurons are not well understood. We propose a role for microRNAs (miRNAs), a class of noncoding RNAs, as mediators of neuronal translational regulation. We have identified 86 miRNAs expressed in mammalian neurons, of which 40 have not previously been reported. A subset of these miRNAs exhibits temporally regulated expression in cortical cultures. Moreover, all of the miRNAs that were tested cofractionate with polyribosomes, the sites of active translation. These findings indicate that a large, diverse population of miRNAs may function to regulate translation in mammalian neurons.

In a wide range of organisms, double-stranded RNA triggers posttranscriptional gene silencing or RNA interference (RNAi). Small interfering RNAs, the 21-nt double-stranded RNA intermediates of this natural pathway, have became a powerful tool to knock down specific gene expression in mammalian cell lines and potentially will be useful for the analysis of loss-of-function phenotypes. In mammalian primary neuronal cultures, where genetic manipulations are especially difficult, RNAi might be developed into a highly efficacious tool to study the roles of specific genes in neuron development and functioning. Neurons, however, have been considered the most resistant to RNAi. We report here an application of RNAi to postmitotic primary neuronal cultures. Synthetic siRNA can be readily introduced into neurons and effectively inhibit the expression of endogenous and transfected genes.

Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau.

How the β-amyloid peptide (Aβ) is cleaved from its precursor, the amyloid precursor protein (APP), preoccupies a large portion of the community studying Alzheimer’s disease. And for good reason. The Aβ protein, the major component of the amyloid in senile plaques, represents a leading target in the quest for agents to combat Alzheimer’s disease. Its formation involves an unusual proteolytic event within a hydrophobic stretch of amino acids predicted to form an α-helix within the membrane. APP is a type I membrane-spanning protein of 770 residues that secretes its large ectodomain after at least two different types of cleavage. Based on the abundance of the cleavage products, the site most frequently cut is at residue 687, which lies within the Aβ peptide and thus precludes its formation. The activity of the responsible, but elusive, enzyme, dubbed the α-secretase, has the property of recognizing an optimal projection length from the membrane more strongly than any specific sequence determinants (1). The α-secretase cleavage occurs in a post-Golgi compartment, possibly including caveolae (2), and may be a glycosyl-phosphatidylinositol-linked aspartyl protease (3). A small percentage of APP is not cleaved at the α-secretase site, but instead is cleaved at residue 672 of APP (which becomes Asp-1 of Aβ) by an activity dubbed β-secretase, the first step in Alzheimer’s-type amyloidogenesis. Other Aβ species with an elongated or shortened N terminus are known (4). The β-secretase cleavage, once completed, sets the stage for the next cleavage by an activity referred to as γ-secretase. In what is now considered a critical event in the disease pathogenesis, the Aβ peptide is released after cleavage by the γ-secretase either between residues 40 and 41 or between residues 42 and 43. Normally, both forms of the Aβ peptide—Aβ42 and Aβ40—are present, with Aβ40 in great excess of Aβ42. The precise cleavage site of the γ-secretase, and hence the generation of the shorter or longer Aβ peptide, is widely believed to have important pathologic consequences. Compared with Aβ40, Aβ42 deposits earlier in the disease process (5) and assembles more rapidly into filaments in vitro (6). Explanations of the mechanism of Aβ42 vs. Aβ40 cleavage by the γ-secretase are problematic because the two proteolytic sites lie on opposite sides of the predicted α-helical transmembrane domain of APP.

Neurofibrillary tangles and senile plaques are the principal pathological features of Alzheimer disease. Neurofibrillary tangles and the neurites of senile plaques contain paired helical filaments (PHF) that consist of two 10-nm filaments twisted into a double helix. The precursor proteins of PHF are not fully known. To identify these precursors, numerous immunochemical studies have been carried out during the past decade. Two apparently conflicting results have been reported. (i) Some, but not all, monoclonal antibodies to neurofilaments stained neurofibrillary tangles. (ii) Polyclonal antibodies prepared to PHF purified in NaDodSO4 because of their unusual insolubility did not recognize normal proteins, including neurofilaments, on electrophoretic transfer blots of human brain homogenates. These results have been confirmed in several laboratories, including by the use of electron microscopic labeling. Recently, we reported that polyclonal PHF antibodies include antibodies to tau proteins, a family of heat-stable microtubule-associated phosphoproteins, and that antibodies to tau stain Alzheimer neurofibrillary tangles. Those monoclonal neurofilament antibodies that recognize tangles are reported to be directed against phosphorylated epitopes. These facts prompted us to reexamine certain neurofilament monoclonal antibodies that stain neurofibrillary tangles. All monoclonal neurofilament antibodies that stain tangles that we examined, including those initially reported, reacted with tau proteins. Our results suggest that these antibodies react with phosphorylated tau proteins in PHF, not neurofilament proteins, highlighting the problem of using antibodies to phosphorylated protein epitopes in immunochemical studies. Independent evidence for the presence of neurofilament proteins in human paired helical filaments is now required.

The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.