Brain Injury and Growth Inhibitory Factor (GIF)—a Minireview

Growth inhibitory factor (GIF) is a small (7 kDa), heat-stable, acidic, hydrophilic metallothionein (MT)-like protein. GIF inhibits the neurotrophic activity in Alzheimer's disease (AD) brain extracts on neonatal rat cortical neurons in culture. GIF has been shown to be drastically reduced and down-regulated in AD brains. In neurodegenerative diseases in humans, GIF expression levels are reduced whereas GFAP expression levels are markedly induced in reactive astrocytes. Both GIF and GIF mRNA are present at high levels in reactive astrocytes following acute experimental brain injury. In chronological observations the level of GIF was found to increase more slowly and remain elevated for longer periods than that of glial fibrillary acidic protein (GFAP). These differential patterns and distribution of GIF and GFAP seem to be important in understanding the mechanism of brain tissue repair. The most important point concerning GIF in AD is not simply the decrease in the level of expression throughout the brain, but the drastic decrease in the level of expression in reactive astrocytes around senile plaques in AD. Although what makes the level of GIF decrease drastically in reactive astrocytes in AD is still unknown, supplements of GIF may be effective for AD, based on a review of current evidence. The processes of tissue repair following acute brain injury are considered to be different from those in AD from the viewpoint of reactive astrocytes.     

Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer's disease.

In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimer's disease (AD) brain. Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins. Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes. GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system. The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons. These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions. Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains. The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

Primary structure and gene localization of human prolidase

Complementary DNA clones of prolidase (imidodipeptidase, EC 3.4.13.9) were isolated from human liver and placental cDNA libraries. Two clones named lambda PL21 and lambda PP6 from the liver and placental cDNA libraries, respectively, were analyzed in detail. The first clone, lambda PL21, carried a cDNA insert of 1.7 kilobase pairs and covered all the coding region of human prolidase mRNA. The second clone, lambda PP6, contained a 1.8-kilobase insert with a full-length 3'-untranslated region. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the two clones with the partial amino acid sequence determined by Edman degradation of peptides derived from human erythrocyte prolidase established that both clones code for human prolidase. The amino terminus of the human mature enzyme is blocked and seems to begin with the sequence X-Ala-Ala-Ala. Presumably no processing occurs at the carboxyl terminus. The mature enzyme is composed of 492 residues, corresponding to Mr 54,305. The sequence of prolidase is unique and not similar to any known protein, except for a significant similarity to regions of F1-ATPase alpha and beta subunits from various sources. The gene has been mapped to the short arm of chromosome 19 (19p13.2). Elucidation of the complete amino acid sequence and the gene location of prolidase should provide the basis for understanding structure-function relationships and also inherited disorders caused by deficiency of this metabolically important enzyme.     

Chondroitin Sulfate Proteoglycan Tenascin-R Regulates Glutamate Uptake by Adult Brain Astrocytes

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.     

Lipoprotein Lipase (LPL) is Associated with Neurite Pathology and Its Levels Are Markedly Reduced in the Dentate Gyrus of Alzheimer’s Disease Brains

Lipoprotein lipase (LPL) is involved in regulation of fatty acid metabolism, and facilitates cellular uptake of lipoproteins, lipids and lipid-soluble vitamins. We evaluated LPL distribution in healthy and Alzheimer’s disease (AD) brain tissue and its relative levels in cerebrospinal fluid. LPL immunostaining is widely present in different neuronal subgroups, microglia, astrocytes and oligodendroglia throughout cerebrum, cerebellum and spinal cord. LPL immunoreactivity is also present in leptomeninges, small blood vessels, choroid plexus and ependymal cells, Schwann cells associated with cranial nerves, and in anterior and posterior pituitary. In vitro studies have shown presence of secreted LPL in conditioned media of human cortical neuronal cell line (HCN2) and neuroblastoma cells (SK-N-SH), but not in media of cultured primary human astrocytes. LPL was present in cytoplasmic and nuclear fractions of neuronal cells and astrocytes in vitro. LPL immunoreactivity strongly associates with AD-related pathology, staining diffuse plaques, dystrophic and swollen neurites, possible Hirano bodies and activated glial cells. We observed no staining associated with neurofibrillary tangles or granulovacuolar degeneration. Granule cells of the dentate gyrus and the associated synaptic network showed significantly reduced staining in AD compared to control tissue. LPL was also reduced in AD CSF samples relative to those in controls.     

Regulation of Growth Inhibitory Factor Expression by Epidermal Growth Factor and Interleukin-1β in Cultured Rat Astrocytes

Growth inhibitory factor (GIF) is highly expressed in the CNS under physiological conditions, but its expression is reduced in neurodegenerative diseases, such as Alzheimer's disease. The results of this study show that the levels of GIF and GIF mRNA were not influenced by neuroglial interactions. GIF was highly expressed in confluent astrocytes, but the expression was down-regulated in low-density growing astrocytes. A high level of GIF was not observed in serum-starved low-density cultures. These findings suggest that GIF is a quiescent state-specific protein and that two different mechanisms may exist for the cells to enter the quiescent state. Among interleukin-1beta (IL-1beta), fibroblast growth factor-2, epidermal growth factor (EGF), amyloid beta1-42, and 50% O2, only EGF and IL-1beta altered the level of GIF in confluent astrocytes: EGF increased both GIF mRNA and protein, and IL-1beta decreased GIF mRNA, but did not alter GIF protein. Kinetic analysis of the GIF mRNA level revealed the biphasic regulation of GIF mRNA expression by IL-1beta, i.e., a transient up-regulation followed subsequently by down-regulation, explaining in part the discrepancy between the levels of GIF mRNA and protein in astrocytes treated with IL-1beta.     

Human arylsulfatase B: MOPAC cloning, nucleotide sequence of a full-length cDNA, and regions of amino acid identity with arylsulfatases A and C

cDNAs encoding the human lysosomal hydrolase, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase, EC 3.1.6.1), were isolated from a hepatoma cell cDNA library using an ASB-specific oligonucleotide generated by the MOPAC (mixed oligonucleotide primed amplification of cDNA) technique. To facilitate cDNA cloning, human ASB was purified to apparent homogeneity and a total of 112 amino acid residues were microsequenced from the N-terminus and four internal tryptic peptides of the 47-kDa subunit. Based on the ASB N-terminal amino acid sequence, two oligonucleotide mixtures containing inosines to reduce the mixture complexity were constructed and used as primers to amplify an ASB-specific product from human placental cDNA by the polymerase chain reaction. DNA sequencing of this MOPAC product demonstrated colinearity with 21 N-terminal ASB amino acids. Based on this sequence and on codon usage for the adjacent conserved amino acids in human arylsulfatases A and C, a unique 66-mer was synthesized and used to screen a human hepatoma cell cDNA library. Four putative positive cDNA clones were isolated, and the largest insert (pASB-1) was sequenced in both orientations. The 1834-bp pASB-1 insert had a 1278-bp open reading frame encoding 425 amino acids that was colinear with 85 microsequenced amino acids of the purified enzyme, demonstrating its authenticity. Using the pASB-1 cDNA as a probe, a full-length cDNA clone, pASB-4, was isolated from a human testes library and sequenced in both orientations. pASB-4 had a 2811-bp insert containing a 559-bp 5' untranslated sequence, a 1602-bp open reading frame encoding 533 amino acids (six potential N-glycosylation sites), a 641-bp 3' untranslated sequence, and a 9-bp poly(A) tract. Comparison of the predicted amino acid sequences of arylsulfatases A, B, and C revealed regions of identity, particularly in their N-termini.     

Molecular cloning of a cDNA for a human ADP/ATP carrier which is growth-regulated

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.