Stress proteins in Alzheimer's disease.

Environmental and genetic conditions can cause proteins to misfold or to accumulate abnormally due to impaired clearance. The chaperones which include heat shock proteins, aid survival by preventing protein mis-folding and the formation of cytotoxic protein aggregates. An increasing number of studies point to important roles for molecular chaperones in the biology of neurodegenerative diseases. Heat shock proteins can suppress neurotoxicity in animal models of Parkinson's and polyglutamine diseases, suggesting potential new therapeutic approaches in neurodegenerative disorders associated with abnormal protein folding and toxicity. Recent findings suggest that heat shock proteins can also be neuroprotective in Alzheimer's disease, but this area of research remains largely unexplored. This paper will review the literature related to the role of heat shock proteins in Alzheimer's disease.

Beta-amyloid peptide expression is sufficient for myotube death: implications for human inclusion body myopathy.

Inclusion body myositis (sIBM) is the most common disorder of skeletal muscle in aged humans. It shares biochemical features with Alzheimer's disease, including congophilic deposits, which are immunoreactive for beta-amyloid peptide (Abeta) and C'-terminal betaAPP epitopes. However, the etiology of myofiber loss and the role of intracellular Abeta in IBM is unknown. Here we report correlative evidence for apoptotic cell death in myofibers of IBM patients that exhibit pronounced Abeta deposition. HSV-1-mediated gene transfer of Abeta(42) into cultured C2C12 myotubes resulted in a 12.6-fold increase in dUTP-labeled and condensed nuclei over nonexpressing myotubes (P < 0.05). The C'-terminal betaAPP domain C99 also induced myotube apoptosis, but to a significantly lesser extent than Abeta. Apoptosis specific to Abeta-expressing myotubes was also demonstrated through DNA fragmentation, decreased mitochondrial function and the loss of membrane phospholipid polarity. Myotubes laden with Abeta(42), but not other transgene products, developed cytoplasmic inclusions consisting of fibrillar material. Furthermore, injection of normal mouse gastrocnemius muscle with HSV-encoding Abeta cDNA resulted in TUNEL-positive myofibers with pyknotic nuclei. We conclude that Abeta is sufficient to induce apoptosis in myofibers both in vivo and in vitro and suggest it may contribute to myofiber loss and muscle dysfunction in patients with IBM.

Maintenance of acetylcholine receptor number by neuregulins at the neuromuscular junction in vivo.

ARIA (for acetylcholine receptor-inducing activity), a protein purified on the basis of its ability to stimulate acetylcholine receptor (AChR) synthesis in cultured myotubes, is a member of the neuregulin family and is present at motor endplates. This suggests an important role for neuregulins in mediating the nerve-dependent accumulation of AChRs in the postsynaptic membrane. Nerve-muscle synapses have now been analyzed in neuregulin-deficient animals. Mice that are heterozygous for the deletion of neuregulin isoforms containing an immunoglobulin-like domain are myasthenic. Postsynaptic AChR density is significantly reduced, as judged by the decrease in the mean amplitude of spontaneous miniature endplate potentials and bungarotoxin binding. On the other hand, the mean amplitude of evoked endplate potentials was not decreased, due to an increase in the number of quanta released per impulse, a compensation that has been observed in other myasthenic states. Thus, the density of AChRs in the postsynaptic membrane depends on immunoglobulin-containing neuregulin isoforms throughout the life of the animal.

ARIA: a neuromuscular junction neuregulin.

Motor neurons influence the expression and the distribution of acetylcholine receptors in skeletal muscle. Molecules that mediate this carefully choreographed interaction have recently been identified. One of them, ARIA, is a polypeptide purified from chicken brain on the basis of its ability to stimulate the synthesis of muscle acetylcholine receptors. The predicted amino acid sequence suggests that ARIA is synthesized as a transmembrane precursor protein and that it is a member of a family of ligands that activate receptor tyrosine kinases related to the epidermal growth factor receptor. Certain features of the ligand family (the neuregulins) and their receptors (erbBs) are reviewed. Evidence that ARIA plays an important role at developing and mature neuromuscularjunctions is discussed.

Expression and imprinting of the insulin-like growth factor II gene in neonatal mouse cerebellum.

Insulin-like growth factor II (IGF-II) plays significant roles in the growth and development of mammals through the regulation of mitogenesis and cell survival. Previously, IGF-II mRNA transcripts within the CNS were detected in the choroid plexus and leptomeninges (DeChiara et al., 1991). The objective of this study was to determine the expression pattern of IGF-II mRNA in different cell types of the cerebellum during development. We report here that the IGF-II gene is transcribed in granule and glial cells within the cerebellar parenchyma at various times during the early postnatal period in mice. IGF-II gene expression is further regulated by parent-specific imprinting such that only the paternal IGF-II allele is expressed in granule cells. In contrast, choroid plexus and leptomeninges express IGF-II mRNAs biallelically, indicating that cell type-specific regulation of genomic imprinting occurs within the mammalian CNS.

Differential expression of ARIA isoforms in the rat brain.

ARIA, heregulin, neu differentiation factor, and glial growth factor are members of a new family of growth and differentiation factors whose effects have been assayed on Schwann cells, skeletal muscle cells, and mammary tumor cell lines. To gain insight into their roles in the CNS, we studied the expression of ARIA in the rat brain. We found ARIA mRNA in all cholinergic neurons throughout the CNS, including motor neurons and cells of the medial septal nucleus and the nucleus basalis of Meynert. We also found that ARIA induces tyrosine phosphorylation of a 185 kDa protein in central and peripheral targets of these cholinergic neurons. ARIA mRNA, however, is not restricted to cholinergic neurons, suggesting that it may also play a role at other types of synapses. Its distribution in germinal layers of the telencephalon and cerebellum suggests that it may also play a role in the proliferation and/or migration of neuronal and glial precursor cells.

Pancreatic Reg/pancreatic stone protein (PSP) gene expression does not correlate with beta-cell growth and regeneration in rats.

The Reg/pancreatic stone protein (PSP) gene is postulated to be an important regulator of pancreatic beta-cell growth. To investigate this hypothesis, we analysed the expression of the Reg/PSP gene following a 90% pancreatectomy and after chronic glucose infusion, two well-defined models of pancreatic beta-cell growth. There was a rapid induction of the Reg/PSP gene in the remnant pancreas after a 90% pancreatectomy in rats during the period of marked growth of the exocrine and islet tissue. However, a similar rapid, but smaller, induction of the Reg/PSP gene was observed in sham-operated rats and in non-surgical control rats in which there was no enhanced pancreatic growth. Furthermore, there was no pancreatic Reg/PSP gene induction in a model of selective beta-cell growth, the chronic glucose-infused rat. Thus, it is unlikely that Reg/PSP is a beta-cell specific growth factor, even though the function of this important pancreatic gene is still unknown.

Acetylcholine receptor subunit mRNA changes in burns are different from those seen after denervation: the 1993 Lindberg Award.

Neuromuscular dysfunction of burn trauma is evidenced as muscle weakness and altered sensitivity to neuromuscular relaxants. A biochemical characteristic of the neuromuscular dysfunction is the proliferation and spread of acetylcholine receptors (AChR) throughout the skeletal muscle membrane. Depending on whether the neuromuscular dysfunction is presynaptic, synaptic, or postsynaptic in origin, the transcripts that induce the proliferation of AChR differ. This study, by quantitation of mRNA transcripts of AChR in muscle, attempts to characterize the cause of the neuromuscular dysfunction of burn trauma. Examination of the levels of mRNA encoding alpha, beta, epsilon, gamma, and delta subunits of AChR with northern blot analysis indicate significant (p < 0.03) elevations of beta-subunit mRNA with a trend for increased levels of alpha and delta transcripts. An increase of gamma-subunit mRNA, typical of presynaptic or nerve-mediated neuromuscular dysfunction, was not observed after burns. That neuronal (presynaptic) factors do not cause the neuromuscular dysfunction was confirmed by the lack of elevation of transcripts of myoD and myogenin, which also increase in "denervation states." These findings indicate that the neuromuscular dysfunction of burn trauma is most likely related to synaptic or postsynaptic factors. Further characterization of the cause of the synaptic and postsynaptic neuromuscular changes in burns will have implications for the choice of therapeutic agents to rectify the neuromuscular dysfunction.

Developmental and environmental changes in GAP-43 gene expression in cat visual cortex.

Northern/slot blot analysis was used to determine postnatal developmental and environmentally induced changes in the level of expression of GAP-43 mRNA in visual and frontal cortex. Both structures showed a precipitous decline during the first 5 weeks and a slight further decline to adult levels. Dark rearing resulted in a significant elevation of GAP-43 mRNA which was eliminated by brief visual experience. This effect was specific to visual cortex and did not occur in frontal cortex. The effect also did not occur in normal adult cats placed in prolonged darkness, indicating that GAP-43 mRNA levels are not simply activity dependent and are altered by visual input only during early postnatal life. These results are consistent with a role for GAP-43 in the state of visual cortical plasticity.

Characterization of type I IGF receptor and IGF-I mRNA expression in cultured human and bovine glomerular cells.

Glomerular hypertrophy is reported in several endocrine disorders such as acromegaly and diabetes mellitus, where abnormalities of growth hormone and insulin-like growth factor (IGF-I) have been reported. In the present report, we have cultured bovine and human glomerular endothelial cells, and bovine glomerular epithelial and mesangial cells, and characterized the expression of IGF-I mRNA and its receptor in these cells. High affinity, specific receptors for IGF-I were identified in all three types of cells by radioreceptor assays. Receptor number (Ro) derived by Scatchard analysis revealed an unusually high number of Type I IGF receptors, approx. 1.2 x 10(5) receptors/cell in glomerular endothelial cells. Affinity crosslinking studies and immunoprecipitation with antibodies against the Type I IGF receptor identified the alpha-subunit of the IGF-I receptor as having a molecular mass of 140 kDa. Biologically, IGF-I was more potent than insulin or IGF-II in stimulating DNA synthesis in glomerular endothelial cells. Northern blot analysis showed that glomerular and aortic endothelial cells expressed IGF-1 mRNA of 1.7 kb. In contrast, renal glomeruli showed several IGF-1 mRNAs of 7.5, 1.7 and 1.2 kb. Thus, the demonstration of both a prepondence of Type I IGF receptors coupled with the growth promoting effects of IGF-I in glomerular endothelial and epithelial cells, as well as the local production of IGF-I mRNA suggests that IGF-I serves an important role as an autocrine or paracrine regulator of the growth of renal glomeruli.

Specific, temporally regulated expression of the insulin-like growth factor II gene during muscle cell differentiation.

We have compared the expression of insulin-like growth factor II (IGF-II) messenger RNA (mRNA) to the expression of other mRNAs encoding proteins known to play pivotal roles during the differentiation of continuously cultured, fusing muscle cell lines. These cell lines respond to changes in culture conditions by undergoing a well characterized alteration in gene expression which leads to a change in their phenotype from dividing, mononucleate myoblasts to fused, multinucleate myotubes. The hallmarks of this differentiation program include the induction of myogenic regulatory genes as well as the genes that encode the contractile proteins. We have found that the differentiation of these cells leads to the production of multiple IGF-II transcripts. In one of the cell lines studied, C2C12, IGF-II mRNA levels were rapidly induced during differentiation. Increases in IGF-II mRNA levels preceded the expression of the contractile protein genes but occurred only after the activation of the myogenic regulatory gene myogenin. The same regulated pattern of IGF-II mRNA expression was seen in both rapidly and slowly fusing subclones of this cell line, indicating a requirement for IGF-II at a specific point during muscle differentiation. These results suggest that IGF-II plays an important role during the terminal differentiation of skeletal muscle cells and are consistent with the existence of an autocrine loop through which IGF-II may act to regulate the differentiation process.

Identification of an antisense transcript from the IGF-II locus in mouse.

We have identified a novel antisense RNA transcribed from the insulin-like growth factor-II (IGF-II) locus in mouse. This transcript was identified using probes derived from cloned genomic DNA lying between the insulin II locus and the IGF-II locus. S1 protection assays confirmed that this transcript is transcribed from the strand complementary to the stand encoding IGF-II. A 3.75 kb RNA was consistently detected on northern analysis of mouse tissue using different randomly primed DNA probes generated from this region. S1 nuclease protection analysis identified three exons contained in the transcript. Developmental northern analysis was performed using RNA from embryonic (E) and postnatal (P) tissues of E10, E13, E18, P1, P4, P10, and adult mice. The antisense RNA was most abundant in E13 and E18 mouse and was present in greatest amounts in skull, skeletal muscle, cardiac muscle, and placenta. No signal for this RNA was detected after the fourth day of life in any tissue studied.

ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family.

Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of ARIA cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human here-gulin, ligands for the receptor tyrosine kinase encoded by the neu (c-erbB2, HER2) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptor tyrosine kinases play important roles in this process.

Messenger ribonucleic acid for transforming growth factor-alpha, but not for epidermal growth factor, is expressed in fetal and neonatal mouse brain.

OBJECTIVE: Little is known concerning the presence of transforming growth factor-alpha in fetal and neonatal tissues. Our objective was to analyze messenger ribonucleic acid expression for transforming growth factor-alpha and epidermal growth factor, the structural and biochemical analog of transforming growth factor-alpha in fetal and neonatal murine brain. STUDY DESIGN: Messenger ribonucleic acid was prepared from whole brains of mice from these developmental stages: embryonic days 14, 15, and 17, postnatal days 0, 4, 10, and adult. Polymerase chain reaction was performed on the complementary deoxyribonucleic acid obtained from reverse transcription of messenger ribonucleic acid with transforming growth factor-alpha and epidermal growth factor primers. In addition, ribonuclease protection assay was used to identify transforming growth factor-alpha transcripts. RESULTS: We found messenger ribonucleic acid encoding transforming growth factor-alpha in all stages of development used. Epidermal growth factor messenger ribonucleic acid was not found in any stage. Ribonuclease protection assay confirmed transforming growth factor-alpha transcripts in these tissues. CONCLUSION: Transforming growth factor-alpha may play an autocrine or paracrine role in the differentiation or maintenance of murine fetal and neonatal brains.

Insulin-like growth factor II expression in the developing human brain.

Insulin-like growth factor II (IGF-II) is a polypeptide hormone with insulin-like metabolic activity and neurotrophic activity in vitro that has been implicated in human brain development. In this study, we used northern blot analysis to examine the patterns of IGF-II mRNA expression in selected regions of 18 human brains from cases ranging in age from 20 gestational weeks to 2.5 years (median age 31 gestational weeks). The expression of IGF-II mRNA was widespread throughout the brain from midgestation through the perinatal period. Each region showed a distinct developmental pattern of expression and IGF-II mRNA levels varied considerably between regions. The highest levels of expression at all ages were in leptomeninges and choroid plexus. After two postnatal months, IGF-II mRNA virtually disappeared from parenchymal regions. Beyond the perinatal period, IGF-II expression persisted primarily in choroid plexus. Transcripts of both 6.0 and 4.8 kb were detected in most brain regions. A developmental change in the relative amounts of the two transcripts occurred in choroid plexus, leptomeninges and medulla. The expression of IGF-II mRNA in the brain parenchyma during the last half of gestation correlates with a period of major brain growth and supports the hypothesis that high levels of IGF-II stimulate the proliferation and differentiation of neural cells early in development.

Brief visual experience induces immediate early gene expression in the cat visual cortex.

Brief visual experience causes rapid physiological changes in the visual cortex during early postnatal development. A possible mediator of these effects is the immediate early genes whose protein products are involved in the rapid response of neurons to transsynaptic stimulation. Here we report evidence that the levels of immediate early gene mRNAs in the visual cortex can be altered by manipulating the visual environment. Specifically, we find that brief (1 h) visual experience in dark-reared cats causes dramatic transient inductions of egr1, c-fos, and junB mRNAs in the visual cortex but not in the frontal cortex. Levels of c-jun and c-myc mRNAs are unaffected. These results suggest that select combinatorial interactions of immediate early gene proteins are an important step in the cascade of events through which visually elicited activity controls visual cortical development.

Changes in immediate early gene expression during postnatal development of cat cortex and cerebellum.

Postnatal brain development involves interactions between extracellular signals and preprogrammed genetic events. Immediate early genes (IEGs) are a group of genes that are induced by extracellular signals and their protein products alter transcription by binding regulatory elements in other genes. Using Northern and slot blot analysis of total RNA isolated from visual cortex, frontal cortex, and cerebellum of cats, we have determined the postnatal development patterns of mRNA expression for 5 of these genes, c-fos, erg-1, c-jun, jun-B, and c-myc. Each gene had a distinct developmental pattern of mRNA expression, and for a given gene, these patterns were often different in different brain structures. These results suggest that temporal changes in the combinatorial interaction of different IEGs during early postnatal life are important for normal brain development.