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Background: Neuroinflammation is ubiquitous in acute stroke and worsens outcome. However, the precise timing of the inflammatory response is unknown, hindering the design of acute anti-inflammatory therapeutic interventions. We sought to identify the onset of the neuroinflammatory cascade using a mobile stroke unit., Methods: The study is a proof-of-concept, cohort investigation of ultra-early blood- and extracellular vesicle-derived markers of neuroinflammation and outcome in acute stroke. Blood was obtained, prehospital, on an mobile stroke unit. Outcomes were biomarker concentrations, modified Rankin Scale score, and National Institutes of Health Stroke Scale score., Results: Forty-one adults were analyzed, including 15 patients treated on the mobile stroke unit between August 2021 and April 2022, and 26 healthy controls to establish biomarker reference levels. Median patient age was 74 (range, 36-97) years, 60% were female, and 80% White. Ten (67%) were diagnosed as stroke, with 8 (53%) confirmed and 2 likely transient ischemic attack or stroke averted by thrombolysis; 5 were stroke mimics. For strokes, median initial National Institutes of Health Stroke Scale score was 11 (range, 4-19) and 6 (75%) received tPA (tissue-type plasminogen activator). Blood was obtained a median of 58 (range, 36-133) minutes after symptom onset. Within 36 minutes after stroke, plasma IL-6 (interleukin-6), neurofilament light chain, UCH-L1 (ubiquitin C-terminal hydrolase L1), and GFAP (glial fibrillary acidic protein) were elevated by as much as 10 times normal. In EVs, MMP-9 (matrix metalloproteinase-9), CXCL4 (chemokine (C-X-C motif) ligand 4), CRP (C-reactive protein), IL-6, OPN (osteopontin), and PECAM1 (platelet and endothelial cell adhesion molecule 1) were elevated. Inflammatory markers increased rapidly in the first 2 hours and continued rising for 24 hours., Conclusions: The neuroinflammatory cascade was found to be activated within 36 to 133 minutes after stroke and progresses rapidly. This is earlier than observed previously in humans and suggests injury from neuroinflammation occurs faster than had been surmised. These findings could inform development of acute immunomodulatory stroke therapies and lead to new diagnostic tools and improved outcomes.

Alzheimer's disease (AD) is responsible for 60%-80% of identified cases of dementia. While the generation and accumulation of amyloid precursor protein (APP) fragments is accepted as a key step in AD pathogenesis, the precise role of these fragments remains poorly understood. To overcome this deficit, we induced the expression of the soluble C-terminal fragment of APP (C99), the rate-limiting peptide for the generation of amyloid fragments, in yeast that contain thermosensitive mutations in genes encoding proteasome subunits. Our previous work with this system demonstrated that these proteasome-deficient yeast cells, expressing C99 when proteasome activity was blunted, generated amyloid fragments similar to those observed in AD patients. We now report the phenotypic repercussions of inducing C99 expression in proteasome-deficient cells. We show increased levels of protein aggregates, cellular stress and chaperone expression, electron-dense accumulations in the nuclear envelope/ER, abnormal DNA condensation, and an induction of apoptosis. Taken together, these findings suggest that the generation of C99 and its associated fragments in yeast cells with compromised proteasomal activity results in phenotypes that may be relevant to the neuropathological processes observed in AD patients. These data also suggest that this yeast model should be useful for testing therapeutics that target AD-associated amyloid, since it allows for the assessment of the reversal of the perturbed cellular physiology observed when degradation pathways are dysfunctional., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Background: Varicella zoster virus (VZV) vasculopathy is characterized by persistent arterial inflammation leading to stroke. Studies show that VZV induces amyloid formation that may aggravate vasculitis. Thus, we determined if VZV central nervous system infection produces amyloid., Methods: Aβ peptides, amylin, and amyloid were measured in cerebrospinal fluid (CSF) from 16 VZV vasculopathy subjects and 36 stroke controls. To determine if infection induced amyloid deposition, mock- and VZV-infected quiescent primary human perineurial cells (qHPNCs), present in vasculature, were analyzed for intracellular amyloidogenic transcripts/proteins and amyloid. Supernatants were assayed for amyloidogenic peptides and ability to induce amyloid formation. To determine amylin's function during infection, amylin was knocked down with small interfering RNA and viral complementary DNA (cDNA) was quantitated., Results: Compared to controls, VZV vasculopathy CSF had increased amyloid that positively correlated with amylin and anti-VZV antibody levels; Aβ40 was reduced and Aβ42 unchanged. Intracellular amylin, Aβ42, and amyloid were seen only in VZV-infected qHPNCs. VZV-infected supernatant formed amyloid fibrils following addition of amyloidogenic peptides. Amylin knockdown decreased viral cDNA., Conclusions: VZV infection increased levels of amyloidogenic peptides and amyloid in CSF and qHPNCs, indicating that VZV-induced amyloid deposition may contribute to persistent arterial inflammation in VZV vasculopathy. In addition, we identified a novel proviral function of amylin., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction: Inflammatory markers have long been observed in the brain, cerebrospinal fluid (CSF), and plasma of Alzheimer's disease (AD) patients, suggesting that inflammation contributes to AD and might be a therapeutic target. However, non-steroidal anti-inflammatory drug trials in AD and mild cognitive impairment (MCI) failed to show benefit. Our previous work seeking to understand why people with the inflammatory disease rheumatoid arthritis are protected from AD found that short-term treatment of transgenic AD mice with the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) led to an increase in activated microglia, a 50% reduction in amyloid load, an increase in synaptic area, and improvement in spatial memory to normal. These results called into question the consensus view that inflammation is solely detrimental in AD. Here, we tested our hypothesis that modulation of the innate immune system might similarly be used to treat AD in humans by investigating the ability of GM-CSF/sargramostim to safely ameliorate AD symptoms/pathology., Methods: A randomized, double-blind, placebo-controlled trial was conducted in mild-to-moderate AD participants (NCT01409915). Treatments (20 participants/group) occurred 5 days/week for 3 weeks plus two follow-up (FU) visits (FU1 at 45 days and FU2 at 90 days) with neurological, neuropsychological, blood biomarker, and imaging assessments., Results: Sargramostim treatment expectedly changed innate immune system markers, with no drug-related serious adverse events or amyloid-related imaging abnormalities. At end of treatment (EOT), the Mini-Mental State Examination score of the sargramostim group increased compared to baseline ( P  = .0074) and compared to placebo ( P  = .0370); the treatment effect persisted at FU1 ( P  = .0272). Plasma markers of amyloid beta (Aβ40 [decreased in AD]) increased 10% ( P  = .0105); plasma markers of neurodegeneration (total tau and UCH-L1) decreased 24% ( P  = .0174) and 42% ( P  = .0019), respectively, after sargramostim treatment compared to placebo., Discussion: The innate immune system is a viable target for therapeutic intervention in AD. An extended treatment trial testing the long-term safety and efficacy of GM-CSF/sargramostim in AD is warranted., Competing Interests: Drs. Potter and Boyd are two of the inventors on several U.S. patents owned by the University of South Florida, but not licensed. As of Feb.1 2021, Dr. Boyd is an employee of Partner Therapeutics., (© 2021 The Authors. Alzheimer's & Dementia: Translational Research & Clinical Interventions published by Wiley Periodicals, Inc. on behalf of Alzheimer's Association.)

Background: Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid., Methods: Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-β [Aβ]) and amyloid, as well as extracellular amylin, Aβ, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aβ42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined., Results: VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aβ, and amyloid. No differences in extracellular amylin, Aβ40, or Aβ42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aβ42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aβ42 aggregation., Conclusions: VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Mutant Tau (MAPT) can lead to frontotemporal lobar degeneration (FTLD). Previous studies associated MAPT mutations and altered function with aneuploidy and chromosome instability in human lymphocytes and in Drosophila development. Here we examine whether FTLD-causing mutations in human MAPT induce aneuploidy and apoptosis in the mammalian brain. First, aneuploidy was found in brain cells from MAPT mutant transgenic mice expressing FTLD mutant human MAPT. Then brain neurons from mice homozygous or heterozygous for the Tau ( Mapt ) null allele were found to exhibit increasing levels of aneuploidy with decreasing Tau gene dosage. To determine whether aneuploidy leads to neurodegeneration in FTLD, we measured aneuploidy and apoptosis in brain cells from patients with MAPT mutations and identified both increased aneuploidy and apoptosis in the same brain neurons and glia. To determine whether there is a direct relationship between MAPT-induced aneuploidy and apoptosis, we expressed FTLD-causing mutant forms of MAPT in karyotypically normal human cells and found that they cause aneuploidy and mitotic spindle defects that then result in apoptosis. Collectively, our findings reveal a neurodegenerative pathway in FTLD-MAPT in which neurons and glia exhibit mitotic spindle abnormalities, chromosome mis-segregation, and aneuploidy, which then lead to apoptosis., (© 2018 Caneus, Granic, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction.

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.

The accumulation and aggregation of fragments of amyloid precursor protein (APP) are central to the development of Alzheimer's disease. The production of the small fragment C99 is thought to form the rate-limiting step in the APP processing pathway, which can lead to the production of the toxic Abeta peptide. It has also been suggested that the proteasome contributes to APP catabolism. While the identities and aggregation propensities of many APP fragments have been studied in vitro, the sequences, structures, and cellular sources of fragments generated in vivo remains poorly elucidated. To better identify the specific APP fragments generated in vivo and to elucidate the role of the proteasome in APP processing, we developed a C99 yeast expression system. Using Zip Tip immunocapture, a specific anti-Abeta antiserum (6E10), and matrix-assisted laser desorption ionization- time of flight mass spectrometry, we identified over one dozen APP-generated peptide fragments in wild-type yeast (PRE1PRE2) and over three dozen unique fragments in proteasome mutant cells (pre1- 1pre2-1) expressing C99. Based on the identities of the immunocaptured species, we propose that defects in proteasome function are compensated by other proteases and that the combination of techniques described here will be invaluable to further delineate the APP processing pathway in vivo.

Protein conformational diseases arise when a cellular protein adopts an aberrant shape that either directly or indirectly alters the physiology of its host cell. Notable conformational diseases include cystic fibrosis, Huntington's disease, the prion-related diseases, Alzheimer's disease, and antitrypsin deficiency. In principle, the severity and progression of conformational diseases can be altered by cellular factors that recognize and attempt to ameliorate the harmful effects of the disease-causing, misshapen protein. To better define the mechanistic underpinnings of cellular factors that mediate quality control, and to understand why a single misfolded protein can impact cell viability, specific proteins that cause each of the diseases listed above have been expressed in a model eukaryote, the yeast Saccharomyces cerevisiae. In this review, we describe what has been learned from these studies, and speculate on future uses of yeast expression systems.

The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation. To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast. Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.

The chemokine receptor CXCR4 is expressed in the embryonic and mature CNS, yet its normal physiological function in neurons remains obscure. Here, we show that its cognate chemokine, stromal cell-derived factor-1 (SDF-1), promotes the survival of cultured embryonic retinal ganglion cell neurons even in the absence of other neurotrophic factors. This survival effect is mediated primarily through a cAMP-dependent pathway that acts through protein kinase A and MAP kinase. Addition of SDF-1 to a human neuronal cell line induces phosphorylation of p44/p42 MAP kinase and GSK3beta. Mouse embryos lacking the CXCR4 receptor have a reduced number of retinal ganglion cells. The ligand of CXCR4, SDF-1, may therefore provide generalized trophic support to neurons during their development and maturation.

APJ is a seven transmembrane domain G-protein-coupled receptor that functions as a coreceptor for some primate immunodeficiency virus strains. The in vivo significance of APJ coreceptor function remains to be elucidated, however, due to the lack of an antibody that can be used to assess APJ expression, and because of the absence of an antibody or ligand that can block APJ coreceptor activity. Therefore, we produced a specific monoclonal antibody (MAb 856) to APJ and found that it detected this receptor in FACS, immunofluorescence, and immunohistochemistry studies. MAb 856 also recognized APJ by Western blot, enabling us to determine that APJ is N-glycosylated. Using this antibody, we correlated APJ expression with coreceptor activity and found that APJ had coreceptor function even at low levels of expression. However, we found that APJ could not be detected by FACS analysis on cell lines commonly used to propagate primate lentiviruses, nor was it expressed on human PBMC cultured under a variety of conditions. We also found that some viral envelope proteins could mediate fusion with APJ-positive, CD4-negative cells, provided that CD4 was added in trans. These findings indicate that in some situations APJ use could render primary cell types susceptible to virus infection, although we have not found any evidence that this occurs. Finally, the peptide ligand for APJ, apelin-13, efficiently blocked APJ coreceptor activity., (Copyright 2000 Academic Press.)

The amyloid beta precursor protein (AbetaPP), which plays a pivotal role in Alzheimer's disease (AD), can exist as either a membrane-bound or soluble protein. The former is cleaved at the level of the plasma membrane to generate the soluble form of the protein (AbetaPP(s)). An alternative pathway exists, however, for the cleavage of AbetaPP to generate a 40-42 amino acid peptide termed amyloid beta (Abeta), either within the lysosomal or the endoplasmic reticulum/Golgi compartments of the cell. In AD, there is an increase in the ratio of the 42 amino acid form of the Abeta peptide (Abeta(42)) to Abeta(40). The Abeta(42) form is the more amyloidogenic form and has an increased potential to form the insoluble amyloid deposits characteristic of AD pathology. Studies on the familial form of the disease, with mutations in AbetaPP or in the presenilin proteins, have confirmed an increase in Abeta(42) generation associated with the early stages of the disease. This review will examine the factors that influence AbetaPP processing, how they may act to modulate the biological effects of AbetaPP(s) and Abeta, and if they provide a viable target for therapeutic intervention to modify the rate of progression of the disease.

Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.

In this study, we have investigated the effect of altered corticosteroid levels on the expression and processing of the amyloid beta precursor protein (A betaPP) and its amyloid precursor-like protein (APLP) homologue in rat brain. Four groups of animals were used in the study: sham operated, adrenalectomised, and adrenalectomised treated with either dexamethasone or aldosterone, with the A betaPP/APLP expression being determined by western blot analysis. While there were no changes in the levels of A betaPP/APLP following adrenalectomy, treatment with dexamethasone, but not aldosterone, resulted in a marked increase in protein expression levels with the level of increase varying between the brain regions examined. Corticosteroids had a more marked effect on the particulate rather than the soluble form of the protein, thus suggesting that elevated glucocorticoids may also be adversely influencing A betaPP/APLP processing.

The amyloid beta precursor protein can exist as both a membrane-bound and a secreted protein, with the former having the potential to generate the amyloid beta peptide present in the neuritic plaques which are characteristic of Alzheimer's disease. In this study, we have used a clone of the AtT20 mouse pituitary cell line which expresses high levels of the amyloid beta precursor protein to characterize the glycosylation state of the secreted and membrane-bound forms of the protein and to examine the role of post-translational modifications in protein processing. Lectin blot analysis of immunoprecipitated amyloid beta precursor protein demonstrated that the soluble form of the protein contains significant amounts of sialic acid, with the lectin staining being reduced in the particulate cellular fractions. Treatment of the cells with mannosidase inhibitors to interfere with the formation of complex-type N-linked glycans resulted in a decrease in secreted amyloid beta precursor protein and an increase in the level of the cellular form of the protein. The increase in amyloid beta precursor protein levels in the cellular fraction was accompanied by an increase in perinuclear staining. Furthermore, cells overexpressing the alpha2,6(N)-sialyltransferase enzyme also demonstrated an increase in amyloid beta precursor protein secretion. These results suggest that the presence of terminal sialic acid residues on complex-type N-glycans may be required for the optimal transport of the amyloid beta precursor protein from the Golgi to the cell membrane with the subsequent cleavage to generate the secreted form of the protein.

Glycoproteins play key roles in the development, structuring, and subsequent functioning of the nervous system. However, the complex glycosylation process is a critical component in the biosynthesis of CNS glycoproteins that may be susceptible to the actions of toxicological agents or may be altered by genetic defects. This review will provide an outline of the complexity of this glycosylation process and of some of the key neural glycoproteins that play particular roles in neural development and in synaptic plasticity in the mature CNS. Finally, the potential of glycoproteins as targets for CNS disorders will be discussed.

Combined sialyltransferase (ST) activities were induced in the HN9 hippocampal cell line following treatment with the synthetic glucocorticoid dexamethasone (dex) for 24 hr. Induction occurred in a dose-dependent manner, with the maximum induction of a 2-fold increase over control enzyme levels occurring at a concentration of 10(-8) M dex. A minimum of 6 hr pretreatment with drug was required before significant induction could be detected and elevated enzyme levels persisted for up to 48 hr post-treatment. The induced form of the enzyme showed an increase in reaction maximum velocity (Vmax) while showing no change in affinity (Km) for the acceptor substrate asialofetuin. The alpha2,6 ST enzyme was demonstrated to be the primary enzyme induced and there was no change in expression of the alpha2,3 ST enzyme. Lectin blot analysis demonstrated an increase in the levels of the alpha2,6-linked cellular sialoglycoproteins and a parallel decrease in the alpha2,3 sialoglycoprotein levels.

Previous studies have demonstrated corticosteroid regulation of sialyltransferase (sialyl-T) enzyme activities in a number of different tissues throughout the body. In this study we examined the regulatory effect of corticosteroids on serum enzyme activity in the rat. The total serum sialyl-T activity was not affected by a decrease in corticosteroid levels following adrenalectomy. However, while there was a significant increase in enzyme activity following dexamethasone treatment, aldosterone had no effect on this parameter. Subsequent examination of individual sialyl-T enzymes demonstrated a slight decrease in alpha2,6 sialyl-T activity following adrenalectomy which was restored to basal levels following dexamethasone treatment. The activity of the alpha2,3 sialyl-T enzyme was not affected by adrenalectomy or dexamethasone treatment, but was stimulated significantly by aldosterone. In general, the levels of serum sialoglycoproteins mirrored well the activities of the appropriate sialyl-T enzymes. These results demonstrate that serum sialyltransferase activity in the rat is under the influence of circulating corticosteroids.

Previous studies have demonstrated sialyltransferase (ST) enzyme activity to be induced in hepatic cells by corticosteroids. In this study, we used the H411e rat hepatoma cell line to further characterise this induction with particular reference to the subsequent changes in the pattern of sialoglycoprotein (SGP) expression. The induction of total ST activity by dexamethasone was concentration dependent with maximum induction occurring 12 h subsequent to drug addition. Western blot analysis demonstrated that the induction was associated with an increase in the expression of the alpha2,6(N) ST enzyme with no change in the expression levels of the alpha2,3(N) enzyme. While the induction resulted in an increase in the reaction velocity (Vmax) of the enzyme for both the sugar donor (CMP-Neu5Ac) and the asialofetuin acceptor protein, there was no significant change in the enzyme affinity (Km) for the substrates, suggestive of either an increase in the expression or efficiency of the existing enzyme(s) rather than an induction of novel ST enzymes. Lectin blot analysis of cellular glycoprotein expression demonstrated no change in the expression patterns of either alpha2,3 or alpha2,6-linked SGP following enzyme induction. These results suggest that the available acceptor sites for the terminal sialic acid group(s) may be fully occupied in the control cells and therefore there are no further sites onto which the sialic acid can be transferred following induction of ST enzyme activity. This may be due to the high basal enzyme levels in the control cells already exhausting endogenous acceptor sites.

In this study we have examined the effects of corticosteroids on both the total sialyltransferase (sialyl-T) activity and on two individual isozymes in neural, hepatic, and renal tissues using an in vivo model system. Rats were adrenalectomised to deplete their endogenous stores of steroid hormones, and some subsequently received steroid replacement with dexamethasone or aldosterone. Adrenalectomy resulted in a significant decrease in total neural sialyl-T activity when compared with sham-operated animals in the four brain regions examined, indicating that total sialyl-T activity is normally under positive corticosteroid control. The subsequent effects of exogenous corticosteroids exhibited regional specificity with the enzyme activities in the cortex, cerebellum, and brainstem being stimulated by both dexamethasone and aldosterone and enzyme activity in the hippocampus being stimulated by aldosterone alone. In general, the changes in total enzyme activity could be attributed to the alpha 2,6 sialyl-T isozyme, although the changes in the cerebellum appeared to coincide with alpha 2,3 sialyl-T activity. In the liver, adrenalectomy resulted in an increase in enzyme activity which was not altered by administration of corticosteroids. There were no changes in total renal sialyl-T activity in any of the four experimental groups although certain changes were observed at the level of individual sialyl-T isozymes. These results demonstrate that sialyl-T activity in certain tissues is under the control of corticosteroids and that this is both a tissue-specific and region-specific effect.

The activities of the sialyltransferase enzymes and the resulting expression of sialoglycoproteins were examined in tumor cells derived from different tissues in order to gain a greater understanding of the factors controlling the cell glycosylation state. Cell-cell contact, which is dependent on cell confluency state, was shown to influence glycosylation in the neurally-derived mouse neuro-2A neuroblastoma and the C6 glioma cell lines. Both showed a relatively high level of cell sialyltransferase activity under sub-confluent conditions with activity decreasing upon the formation of cell-cell contacts associated with confluency. A parallel decrease in the expression of sialoglycoproteins, as determined by lectin blot analysis, was observed under these conditions. In contrast, the H411e hepatoma cell line showed an increase in enzyme activity with confluency with the susceptibility of the enzyme in this cell line to glucocorticoid induction only being detected in sub-confluent cell cultures. The number of trypsinisation cycles of the cells was also shown to affect the enzyme activity of the neuro-2A and C6 cells with an increase in enzyme activity coincident with passage number being observed in the neuro-2A cells, and a decrease in the C6 glioma cell line. Trypsinisation had no effect on enzyme activity in the H411e cells. These results demonstrate that the control of sialyltransferase activity in tumor cells is multifactorial with the tissue of origin playing a key role.

A reliable antemortem serum marker for Alzheimer's disease (AD) would be of great importance for the early detection and subsequent therapeutic management of the disease. We have noted a significant decrease in serum levels of the soluble form of the sialyltransferase enzyme in a group of AD patients when compared with both age-matched elderly (over 60 years) and young (under 60 years) controls. In a population of Down's syndrome patients, who develop AD pathology with increasing age, there was an age-related decrease in serum sialyltransferase activity in patients from 20 to 60 years to approach enzyme levels similar to those observed in the AD group. This significant decrease in serum sialyltransferase levels observed may both prove a useful peripheral early biochemical marker of neurodegeneration and provide an indication of the underlying cellular events that occur during the process of nerve cell death in AD.