Tafamidis delays neurological progression comparably across Val30Met and non-Val30Met genotypes in transthyretin familial amyloid polyneuropathy.

BACKGROUND AND PURPOSE: To better characterize the effects of tafamidis in non-Val30Met patients with transthyretin familial amyloid polyneuropathy, this post hoc analysis compared the neurological results from a 12-month, open-label study of non-Val30Met versus Val30Met patients at month 12 from the 18-month, double-blind, placebo-controlled registration study. A baseline covariate adjusted analysis was used to control for differences in baseline neurological severity. METHODS: Neurological function was assessed using the Neuropathy Impairment Score - Lower Limbs (NIS-LL) in three cohorts: Val30Met tafamidis (n = 64), Val30Met placebo (n = 61) and non-Val30Met tafamidis (n = 21). The change in NIS-LL from baseline to month 12 for Val30Met and non-Val30Met tafamidis-treated patients was compared with the change from baseline at month 12 for Val30Met placebo-treated patients using a mixed-effects model for repeated measures (MMRM). RESULTS: The baseline adjusted mean (standard error) change in NIS-LL values at month 12 was similar for Val30Met [1.60 (0.78)] and non-Val30Met [1.62 (1.43)] tafamidis-treated patients and less than that observed in the Val30Met placebo-treated group [4.72 (0.77); P = 0.0055 for Val30Met and P = 0.0592 for non-Val30Met]. Based on the MMRM, the magnitude of change in both tafamidis-treated cohorts was similar across the range of observed baseline NIS-LL values, and was consistently less than that observed in the Val30Met placebo-treated group at month 12. CONCLUSIONS: This baseline-adjusted analysis demonstrated that tafamidis treatment delayed neurological progression comparably in Val30Met and non-Val30Met patients across a range of baseline NIS-LL values. Neurological progression in these two genotype groups may be more similar than previously considered.

Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme in Aplysia neurons.

We have shown previously that the subcellular distribution of a major calmodulin-binding protein is altered under conditions causing increased synthesis of cAMP in Aplysia neurons (Saitoh, T., and J. H. Schwartz, 1983, Proc. Natl. Acad. Sci. USA, 80:6708-6712). We now provide evidence that this Mr 55,000 protein is a subunit of a Ca2+/calmodulin-dependent kinase: (a) both the Mr 55,000 calmodulin-binding protein and kinase activity are loosely attached to the membrane-cytoskeletal complex; (b) both kinase activity and the Mr 55,000 protein are translocated from the membrane-cytoskeleton complex to the cytoplasm under conditions that cause the change in the subcellular distribution of the Mr 55,000 calmodulin-binding protein; and (c) calmodulin-binding activity of the Mr 55,000 protein and the ability to carry out the Ca2+/calmodulin-dependent phosphorylation of synapsin I are purified in parallel. The subcellular localization of the Ca2+/calmodulin-dependent protein kinase appears to be under control of two second messengers: Ca2+ and cAMP. We find that the Mr 55,000 subunit is phosphorylated when the extracted membrane-cytoskeleton complex is incubated with Ca2+, calmodulin, and ATP, with the concomitant release of this phosphorylated peptide from the complex. Previously, we had found that, when translocation occurs in extracts in the presence of cAMP and ATP (but in the absence of Ca2+), there was no detectable phosphorylation of the Mr 55,000 subunit itself. The subcellular distribution of the subunit thus appears to be influenced by (a) cAMP-dependent phosphorylation, which, we infer, modifies some as yet unidentified structural component, causing the release of the enzyme; and (b) Ca2+/calmodulin-dependent phosphorylation of the Mr 55,000 subunit. These studies also suggest that phosphorylation has an important regulatory consequence: during the Ca2+/calmodulin-dependent translocation of the Mr 55,000 subunit, the kinase appears to be activated, becoming independent of added Ca2+/calmodulin.

An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins.

We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica. We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin. Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form.

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction.

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.

Axonal transport of newly synthesized glycoproteins in a single identified neuron of Aplysia californica.

Increasing amounts of glycoprotein synthesized from L-[(3)H]fucose injected into the cell body of R2, an identified Aplysia neuron, were found in the right pleuro-abdominal connective. Autoradiography revealed that the glycoproteins were localized in the axon of R2. Glycoproteins appearing in the axon presumably were synthesized in the cell body, since no significant incorporation was observed when [(3)H]fucose was injected directly into the axon. [(3)H]glycoproteins were detected in the connective after a delay of 1 h after intrasomatic injection. Thereafter, transport from the cell body was rapid, and by 10 h after injection, 45% of the total neuronal [(3)H]glycoprotein had appeared in the axon. By analysing the radioactivity in cell body and connective 4, 10, and 15 h after injection, we found that [(3)H]glycoproteins were transported selectively compared to nonmacromolecular material. Sequential sectioning of the connective revealed that [(3)H]glycoproteins were transported in discrete waves. The population of membrane-associated [(3)H]glycoproteins in the axon differed from that in the cell body. Two of the five somatic components appeared to be transported preferentially. In addition a new component appeared in the axon 10 h after injection.

Axonal transport of [3H]serotonin in an identified neuron of Aplysia californica.

A population of characteristic ellipsoidal dense-core vesicles was identified in axons of the giant cerebral neuron of the mollusc Aplysia. We injected [3H]serotonin into the cell body of this identified serotonergic neuron in the isolated central nervous system in order to study the subcellular components associated with the neurotransmitter. Subcellular fractionation by differential centrifugation indicated that injected serotonin was rapidly taken up into particulate form. [3H]Serotonin appeared in the axons within 2 h after injection, and export continued at a constant rate of 6% of the total in the neuron/h thereafter. The dependence of the total amounts of [3H]serotonin which appeared in the axons in 6 h (export from the cell body) on the amounts injected was consistent with the idea that export is a saturable process, possibly depending on the capacity of somatic vesicles or of some unidentified carrier for serotonin. [3H]Serotonin moved into both major branches of the axon, where it was translocated rapidly. The transmitter, which was shown by autoradiography to be restricted to the axons of the injected cell, was distributed along axons in accumulations of radioactivity; in contrast, its precursor, [5-3H]hydroxytryptophan, moved slowly along axons in a smooth, declining curve, its kinetics consistent with diffusion. Quantitative electron microscope autoradiography revealed that the dense-core vesicles and the cytosol of axons fixed with glutaraldehyde were labeled with [3H]serotonin.

Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia.

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.

Synthesis of glycoproteins in a single identified neuron of Aplysia californica.

Incorporation of L-[(3)H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [(3)H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [(3)H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [(3)H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [(3)H]fucose, gel profiles of particulate fractions labeled with [(14)C]valine were much more complex.

Abundant expression of ras proteins in Aplysia neurons.

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.

Choline acetyltransferase: regional distribution in the abdominal ganglion of Aplysia.

Using a new microassay, we have determined the properties and the regional distribution of choline acetyltransferase in the abdominal ganglion of Aplysia. Enzyme concentrations in homogenates of groups of cells and in single identified cells indicate that neurons which function as neurosecretory cells, and which do not form chemical synapses with other cells or with peripheral structures, have little or no ability to synthesize acetylcholine; neurons which are involved in visceromotor integrations, and which connect with each other or with the periphery, have a substantial concentration of the enzyme.

Molecular biology of learning: modulation of transmitter release.

Until recently, it has been impossible to approach learning with the techniques of cell biology. During the past several years, elementary forms of learning have been analyzed in higher invertebrates. Their nervous systems allow the experimental study of behavioral, neurophysiological, morphological, biochemical, and genetic components of the functional (plastic) changes underlying learning. In this review, we focus primarily on short-term sensitization of the gill and siphon reflex in the marine mollusk, Aplysia californica. Analyses of this form of learning provide direct evidence that protein phosphorylation dependent on cyclic adenosine monophosphate can modulate synaptic action. These studies also suggest how the molecular mechanisms for this short-term form of synaptic plasticity can be extended to explain both long-term memory and classical conditioning.

Membrane glycolipids: regional synthesis and axonal transport in a single identified neuron of Aplysia californica.

Glycolipids moving along an identified axon Aplysia californica were synthesized and incorporated into intracytoplasmic membranes solely in the perikaryon: direct injection of tritiated sugar into the axon revealed no local synthesis or exchange. There was no indication for transfer into axoplasm from glia. Insertion of glycolipids into nascent membranes occurs coordinately with insertion of protein components in the cell body.

Two catalytic subunits of cAMP-dependent protein kinase generated by alternative RNA splicing are expressed in Aplysia neurons.

The amino acid sequences of two catalytic (C) subunits of Aplysia cAMP-dependent protein kinase (cAPK) have been deduced from the nucleotide sequences of cDNAs generated from neuronal poly(A)+ RNA. Both subunits contain 352 residues and are identical except for amino acids 142-183, which differ at 10 out of 42 positions. They derive from alternatively spliced transcripts of a single gene (CAPL) containing two mutually exclusive exon cassettes. CAPL transcripts are present in several classes of identified neurons containing transmitter-sensitive adenylate cyclase, including sensory cells, bag cells, and the left pleural giant cell. Combinatorial expression of the various regulatory (R) and C subunits might produce kinase isoforms with distinct roles in neuronal modulation. Alternatively, holoenzymes with overlapping properties together might contribute to the definition of individual cell types and physiological states.

A regulatory subunit of the cAMP-dependent protein kinase down-regulated in aplysia sensory neurons during long-term sensitization.

Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation. To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI. Immunoblots with antisera raised against the R protein overexpressed in E. coli show that the diminished binding activity, which occurs in long-term facilitation, results from coordinate loss of R protein isoforms. No change was detected in the amount of transcripts for R subunits, suggesting that the down-regulation results from enhanced proteolytic turnover.

Mechanisms for generating the autonomous cAMP-dependent protein kinase required for long-term facilitation in Aplysia.

The formation of a persistently active cAMP-dependent protein kinase (PKA) is critical for establishing long-term synaptic facilitation (LTF) in Aplysia. The injection of bovine catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis-dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin-proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase.

A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis.

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.

Relationship between the rate of H+ transport and pathways of glucose metabolism by turtle urinary bladder.

The urinary bladder of the fresh-water turtle acidifies its contents by actively transporting H(+) ions across the luminal membrane. It is known that the H(+) transport system is dependent upon oxidative metabolism and the substrate glucose; however, the specific biochemical events resulting in H(+) translocation have not been identified. This study examines the relationship between active H(+) transport and a specific oxidative pathway of glucose metabolism, the pentose phosphate shunt. To investigate this relationship the metabolic and transport rates were simultaneously measured under several well-defined conditions. When H(+) transport was inhibited by either the application of an opposing pH gradient or by acetazolamide, glucose metabolism by the pentose phosphate shunt declined. Conversely, stimulation of H(+) transport by either imposing a more favorable pH gradient or by CO(2) addition resulted in an increase in pentose phosphate shunt metabolism. Glycolytic activity, in contrast, was invariant with the maneuvers which altered the rate of H(+) transport. Additional experiments localized pentose phosphate shunt enzyme activity to the mucosal fraction of the bladder which is the cell layer responsible for acid secretion. The finding that the rate of glucose metabolism by the pentose phosphate shunt is related to the rate of H(+) transport suggests but does not prove that the pentose phosphate shunt may be an important metabolic pathway for H(+) transport by the turtle urinary bladder.

Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.

Interleukin 1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells.

The cytokine interleukin 1 (IL-1) inhibits contractile responses in rat aorta by causing endothelium-independent and prolonged activation of soluble guanylate cyclase. The present study tested whether IL-1 activates guanylate cyclase by inducing prolonged production of nitric oxide in cultured rat aortic vascular smooth muscle cells (VSMC). IL-1 induced a marked time-dependent increase in cyclic guanosine monophosphate (cGMP) in VSMC which was significant at 6 h, and increased progressively for up to 36 h. This effect of IL-1 was abolished when protein synthesis was inhibited with cycloheximide or actinomycin D, suggesting that the effect of IL-1 involves new protein synthesis. IL-1-induced cGMP accumulation was inhibited by the soluble guanylate cyclase inhibitors, methylene blue, LY83583, and hemoglobin and by the L-arginine analogue NGmonomethyl-L-arginine (L-NMMA). The inhibitory effect of L-NMMA was reversed by a 10-fold excess of L-arginine, but not by D-arginine. Nitrite, an oxidation product of nitric oxide, accumulated in the media of VSMC incubated with IL-1 for 24 h in the presence of L-arginine, whereas both IL-1-induced cGMP accumulation and nitrite production were attenuated in VSMC incubated in L-arginine-deficient medium. In L-arginine-depleted VSMC, IL-1-induced cGMP accumulation was restored to control levels by a 15-min incubation with L-arginine. These results demonstrate that IL-1 activates guanylate cyclase in rat VSMC by inducing production of nitric oxide via a pathway dependent on extracellular L-arginine.

Effect of dietary fat on sympathetic nervous system activity in the rat.

Previous studies from our laboratory have demonstrated that dietary intake affects the sympathetic nervous system (SNS); carbohydrate intake, in particular, has been shown to stimulate sympathetic activity. The present studies were undertaken to characterize the effect of dietary fat on SNS activity in the rat. Sympathetic activity was assessed by measurement of norepinephrine (NE) turnover in heart, interscapular brown adipose tissue (IBAT), and pancreas and by excretion of NE in the urine. When fed a fat-enriched diet (50% chow, 50% lard), fractional NE turnover in heart (k) increased from 6.3 +/- 0.6% h in ad lib. fed controls to 14.7 +/- 1.3% h in the high-fat group (P less than 0.001); calculated NE turnover rate increased from 24.5 +/- 2.4 ng/heart per h to 36.8 +/- 3.5 (P less than 0.05). Urinary NE excretion more than doubled after 6 d of the same high fat diet (P less than 0.001). Ganglionic blockade produced a greater effect on NE turnover in fat-fed, as compared with chow-fed animals, consistent with increased sympathetic activity in the fat-fed group. When fat absorption was blocked with a bile acid binding resin (cholestyramine), the same high-fat diet did not increase cardiac NE turnover, indicating that fat absorption is required for the stimulatory effect on sympathetic activity. In another series of experiments, in which chow (and hence protein) intake was held constant, the effect of fat and isocaloric sucrose supplements on NE turnover was assessed in heart, IBAT, and pancreas. The caloric value of the supplements was 50, 100, and 335% of the chow in the different experiments. An effect of fat on NE turnover in heart and IBAT was demonstrable at the lowest level of fat supplement. Fat increased pancreatic NE turnover when added in amounts sufficient to double the caloric intake. The stimulatory effect of sucrose and fat on NE turnover in heart and IBAT was similar. These experiments demonstrate that fat increases SNS activity in the rat and that the magnitude of the effect is similar to that of sucrose. The results imply that fat may contribute to dietary thermogenesis in this species.