Cognitive deficits associated with a recently reported familial neurodegenerative disease: familial encephalopathy with neuroserpin inclusion bodies.

BACKGROUND: We recently discovered an autosomal dominant disease causing a progressive dementia. The disease is caused by a point mutation in the gene coding for the serine protease inhibitor (ie, serpin) neuroserpin. The mutation results in an unstable neuroserpin protein that readily aggregates into intraneuronal inclusions that we identify as Collins bodies. The bodies are distributed throughout the cerebral hemispheres but are significantly more numerous in the cortex and the substantia nigra. We have named the disease familial encephalopathy with neuroserpin inclusion bodies (FENIB). OBJECTIVES: To describe the cognitive and neurophysiological changes exhibited by individuals with FENIB and to correlate the phenotypic expression of the disease with the neuropathological findings. DESIGN: Multiple case studies using neuropsychological assessment, electroencephalography (EEG), magnetic resonance imaging (MRI), and single-photon emission computed tomographic (SPECT) studies of family members were performed. Using these measures, we also compared family members in whom the mutation is present with family members in whom the mutation was absent to control for nonspecific familial factors. SUBJECTS: Nine individuals (5 women, aged 31-64 years; 4 men, aged 43-67 years) from 2 generations of family members related to the first reliably identified individual with symptoms of this disease. Symptoms, by self-report and reports of other family members, ranged from asymptomatic to severe dementia. Six of the 9 individuals carried the disease mutation. RESULTS: All subjects with the mutation demonstrated some cognitive changes, with the greatest demonstrated by subjects older than 40 years. The changes included restricted attention, concentration, and response regulation functions, reduced controlled oral fluency (word-list generation), and restricted visuospatial organization. In general, recall memory was not as affected as other cognitive domains. The most severely affected subject demonstrated global dementia with prominent frontal lobe features. Findings on SPECT showed anomalies limited to frontal areas in the less affected subjects and more global, patchy areas of hypoperfusion in the more severely affected subjects. The 3 oldest and most affected subjects demonstrated slowing on EEG findings. The MRI findings were noncontributory except in the 2 most severe cases, which showed global cortical atrophy. CONCLUSIONS: Cognitive changes in mildly to moderately affected subjects were characterized by deficits in frontal and frontal-subcortical area-dependent processes. Continued progressive deterioration of cerebral functions with relative sparing of recall memory suggests a unique dementia associated with this disease.

Neuroserpin mutation S52R causes neuroserpin accumulation in neurons and is associated with progressive myoclonus epilepsy.

Mutations in the Neuroserpin gene have been reported to cause familial presenile dementia. We describe a new family in which the S52R Neuroserpin mutation is associated with progressive myoclonus epilepsy in 2 siblings. The proband presented myoclonus and epilepsy at age 24, his brother and mother presented a similar disorder when they were 25. A clinical diagnosis of progressive myoclonus epilepsy was made on the proband and his brother. Skin and liver biopsies did not reveal the presence of cytological alterations in the proband. His neurological status worsened over the subsequent 19 yr during which he became demented and had uncontrollable seizures. He died at 43 yr of age from aspiration pneumonia. Neuropathologically, eosinophilic bodies, which were positive for periodic acid-Schiff and immunoreactive with antibodies against human neuroserpin, were present in the perikarya and cell processes of the neurons. They were found in large numbers in the cerebral cortex and substantia nigra and to a lesser extent, in most subcortical gray areas, spinal cord, and dorsal root ganglia. By electron microscopy, the intracytoplasmic bodies were contained within the membranes of the rough endoplasmic reticulum. Occasionally neuroserpin immunopositivity was seen throughout the cytoplasm, even without the presence of well-defined bodies. Our study characterizes for the first time the neuropathologic phenotype associated with hereditary progressive myoclonus epilepsy caused by the S52R Neuroserpin mutation.

Familial encephalopathy with neuroserpin inclusion bodies.

We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State-a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity-familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.

Familial dementia caused by polymerization of mutant neuroserpin.

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

Evidence that the organic cation/H+ exchanger in the brush border membrane of dog kidney is a 41-kDa protein.

Organic cation (OC+)/H+ exchangers are found in several mammalian tissues and in numerous organisms. In the kidney OC+/H+ exchange activity is localized to the brush border membrane of the proximal tubule cells of the nephron and is believed to be responsible for the efflux of numerous xenobiotics from the tubule cell into the tubular fluid. The objective of the present study was to identify the OC+/H+ exchanger in brush border membrane vesicles isolated from dog kidney by photoaffinity labeling. The results show that [3H]azidopine is an ideal photoaffinity labeling reagent; in the dark it binds reversibly, but irreversibly after photoactivation. The photoaffinity labeling reaction is efficient, specific and sensitive. Our findings are consistent with the conclusions that a 41-kDa protein is the exchanger and that it is present at a concentration of 780 +/- 140 fmol/mg membrane protein (n = 4). A 49-kDa protein is labeled to some extent as well. The relationship between the 41- and 49-kDa proteins has not been resolved.

Comparisons of oxidative metabolism and reductive capacity in sulfonamide-tolerant and -intolerant patients with human immunodeficiency virus.

Hypersensitivity reactions to trimethoprim/sulfamethoxazole occur with a high frequency in human immunodeficiency virus (HIV)-infected patients. This study tested whether differences in oxidative metabolism and plasma reductive capacity correlate with sulfonamide intolerance in patients with HIV. Eighteen stable outpatients with HIV were prospectively studied. Nine patients had documented histories of hypersensitivity reactions to trimethoprim/sulfamethoxazole and nine did not. Urinary caffeine metabolite ratios assessed the activity of two oxidative enzymatic pathways: cytochrome P-450 1A2 (demethylation) and 8-hydroxylation. Plasma cyst(e)ine was used as a measure of reductive capacity. The trimethoprim/sulfamethoxazole-intolerant group showed greater rates of 8-hydroxylation, lower rates of demethylation, and lower cyst(e)ine levels. The results of this pilot study extend previous observations of differences in oxidative metabolism and reductive capacity that exist within the population of HIV-infected individuals. In addition, these findings lay the groundwork for future interventional studies that could use agents to inhibit sulfonamide oxidation and increase reductive capacity in sulfonamide-intolerant patients with HIV when rechallenged with trimethoprim/sulfamethoxazole.

Photoaffinity labeling of the organic cation/H+ exchanger in renal brush border membrane vesicles.

The brush border membrane of the proximal tubule contains two efflux pathways for organic cations from the cell to the tubular fluid: a P-glycoprotein and an organic cation/H+ exchanger. There is evidence that they transport many of the same substrates. Their structural relatedness is unknown and is the subject of this report. The experimental approach was to identify the exchanger with photoaffinity labeling reagents. The rationale was that if the P-glycoprotein and the organic cation/H+ exchanger transport many of the same substrates, then they might be photoaffinity labeled by the same reagents. [125I]Iodoarylazidoprazosin and [3H]azidopine are two reagents, which have been used, to photoaffinity label the P-glycoprotein. We found that several polypeptides were photolabeled in a time- and concentration-dependent manner. The photoincorporation into only two of these polypeptides (41 and 28 kDa) was blocked extensively by the presence of known substrates for the exchanger. The photoaffinity labeling of only the 41-kDa polypeptide was affected by treatment with the chemical reagents, N-ethylmaleimide and dithiothreitol, which are known to affect the exchanger reaction. The findings are consistent with the interpretation that a 41-kDa polypeptide is, or is a component of, the exchanger.

Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells.

The capability of cyclosporin to produce direct injury to primary proximal tubular renal cells was studied. These cells, when grown on Millicell inserts, retain the functional polarity of the proximal tubule, i.e., generate a transepithelial pH gradient (apical compartment acidic) that is reversibly blocked by amiloride addition only if it is added to the apical compartment. Administration of ouabain to the basal compartment also blocks the generation of the transepithelial pH gradient. Additionally, the cells were more responsive to parathyroid hormone (PTH), a proximal tubule characteristic, than to arginine vasopressin (AVP), a distal tubule characteristic. The following substances were tested for their effect on the capacity of these cells to generate a pH gradient: Sandimmune, the commercial form of cyclosporin A; the free form of the drug; Cremophor EL, the vehicle used in the commercial preparation; and ethanol, the vehicle used to dissolve the free form. Sandimmune, at 25-50 microM, inhibited the generation of the pH gradient within 24 h. Surprisingly, Cremophor also blocked the development of a pH gradient, although somewhat less effectively. In contrast, 10 microM cyclosporin, regardless of the form tested, had no effect for up to 96 h. These findings show that cyclosporin, in the form of Sandimmune, has a direct toxic effect on these cells; they also suggest that the vehicle, Cremophor, may contribute to the well-established nephrotoxicity of cyclosporin A.

Transepithelial acidification by cultures of rabbit proximal tubules grown on filters.

Transepithelial acidification in the proximal tubule occurs by the simultaneous actions of the Na(+)-H+ exchanger in the brush border and the basolateral Na(+)-HCO3- cotransporter. The presence of these systems has been demonstrated for cultured cells; however, their contributions to the transepithelial movement of acid equivalents has not been confirmed in monolayers. To examine transepithelial acidification by intact cells, tubules were grown on membrane filters. Confluent cultures developed a transepithelial pH gradient within 6 h by decreasing the pH of medium in the apical chamber (6.66 +/- 0.03) while raising the basolateral pH to 7.40 +/- 0.02. Cells maintained on plastic did not acidify the medium during this time. Amiloride (10-100 microM) inhibited development of the gradient only when placed in the top chamber. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10-100 microM), which inhibits basolateral Na(+)-HCO3- cotransport, decreased the gradient only when added to the bottom. These results demonstrate that cultured proximal tubule cells can develop a transepithelial pH gradient and that the polarized distribution of the transport systems is maintained in vitro.

Gentamicin and verapamil compete for a common transport mechanism in renal brush border membrane vesicles.

The effects of gentamicin, an aminoglycoside antibiotic, and verapamil, a calcium channel blocker, on the transport systems in canine renal brush border membrane vesicles were examined. They were tested for their effects on the transport of either a prototypic organic cation, N1-[3H]methylnicotinamide or a prototypic organic anion p-[3H]aminohippurate. They were classified as a substrate for either transport system if the following two criteria were fulfilled: 1) cis inhibition and 2) trans stimulation with concentrative uptake of the prototypic ion. Both gentamicin and verapamil cis inhibited the uptake of the prototypic organic cation N1[3H]methylnicotinamide, with Ki values of 450 and 5 microM, respectively. Similarly, both drugs trans-stimulated N1-methylnicotinamide influx and produced concentrative uptake over the equilibrium value. The results demonstrate that gentamicin and verapamil are substrates for the renal organic cation transport system. We postulate that high-affinity substrates should protect against gentamicin-induced nephrotoxicity.

Operational modes of the organic anion exchanger in canine renal brush-border membrane vesicles.

This study delineates the various operational modes catalyzed by the organic anion exchanger present in the canine renal brush-border membrane. The experiments examined the carrier-mediated effects of various organic and inorganic anions on the transport of either p-[3H]aminohippuric acid ([3H]PAH) or 36Cl-. [3H]PAH countertransport was significantly stimulated by PAH, urate, Cl-, Br-, HCO3-, and by a pH gradient. This pH stimulation remained in the absence of HCO3- (i.e., under N2), implying PAH-OH- exchange. Furosemide, bumetanide, penicillin, and probenecid inhibited countertransport of [3H]PAH. Likewise, the above anions produced cis inhibition of [3H]PAH transport. The cis and trans effects of SO4(-2) and formate were minimal. 36Cl- countertransport was stimulated by PAH, Cl-, Br-, HCO3-, formate, and by a pH gradient that was effective even in the absence of HCO3- (i.e., under N2), implying Cl- -OH- exchange. Cl- -OH- and Cl- -Cl- exchange was inhibited by PAH. In each instance, the trans-stimulation of 36Cl- efflux was insensitive to maneuvers that created an inside-positive membrane potential, demonstrating electroneutral mediated exchange. We conclude that the organic anion transporter can operate in three distinct exchange modes: organic-organic, organic-inorganic, and inorganic-inorganic.

Gentamicin-induced increases in cytosolic calcium in pig kidney cells (LLC-PK1).

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were tested as a model system for assessing the role of calcium in gentamicin-induced nephrotoxicity. Cell viability was evaluated by a vital dye exclusion procedure, and intracellular free calcium [Ca2+]i was measured employing Fura-2 fluorescence. Exposing cell suspensions (10(6)/ml) to concentrations of the drug, which had no apparent effect on viability, produced a rapid and prolonged increase in intracellular [Ca2+]. The perturbation of calcium homeostasis could be blocked by the addition of mepiperphenidol, an inhibitor of the organic cation transport system. We propose that LLC-PK1 cells are an appropriate model to study drug-induced nephrotoxicity. Gentamicin disrupts calcium homeostasis and causes plasma membrane alterations. Since mepiperphenidol blocked the gentamicin-induced Ca2+ increases, the data suggest that aminoglycosides enter the cell via the organic cation transporter.

Mechanism of ochratoxin A transport in kidney.

The effect of the fungal metabolite (mycotoxin) Ochratoxin A (OTA) on the transport of p-amino[3H]hippurate (PAH), a prototypic organic anion, was examined in renal brush border (BBMV) and basolateral membrane vesicles (BLMV). OTA was as effective an inhibitor of PAH uptake in both membranes as probenecid. The dose response curves for OTA in BBMV and BLMV gave IC50 values of 20 +/- 6 and 32 +/- 7 microM, respectively. The effect was specific since the transport of the organic cation N1-methylnicotinamide was not affected. The phenomenon of counterflow was studied to establish that OTA is translocated. OTA produced trans stimulation of PAH transport in both BBMV and BLMV, demonstrating that OTA is transported across both these membranes. The data suggest that OTA interacts with the PAH transport system in both BBMV and BLMV. We conclude that OTA transport in the kidney is mediated via the renal organic anion transport system.

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles.

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.

Proton-coupled organic cation transport in renal brush-border membrane vesicles.

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H+ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294-298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N1-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [3H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the Km and increases the Vmax for NMN transport.

Cl(-)-HCO3- exchange in rat renal basolateral membrane vesicles.

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.

Effect of parathyroid hormone on gentamicin plasma membrane binding and tissue accumulation.

Reportedly, the initiating event in the renal uptake of gentamicin is its binding to anionic, plasma membrane phospholipids. Because parathyroid hormone is known to affect phospholipid metabolism, the plasma membrane binding and tissue accumulation of gentamicin were examined as a function of the parathyroid hormone status of the animal. The experiments were conducted by evaluating the parameters in isolated brush border and basolateral membranes from control, hyper- and hypoparathyroid rats. Scatchard analysis revealed that [125I]gentamicin bound with equal affinity to either membrane to a single class of noninteracting sites. The basolateral membrane had more binding sites than did the brush border, 28 +/- 0.5 vs. 1.8 +/- 0.3 nmol/mg of protein, respectively. Neither the affinity constants nor the number of binding sites were affected by the parathyroid hormone status of the donor animal. On the other hand, in the hypoparathyroid state the amount and the rate of gentamicin accumulation were less than in the hyperparathyroid state. The difference in accumulation cannot be explained on the basis of a change in the number or affinity of the putative receptor. Therefore, the alteration must reflect some difference subsequent to the binding site.

Organic cation secretion in flounder renal tissue.

In the winter flounder, Pseudopleuronectes americanus, renal clearance experiments showed that the model organic cations, tetraethylammonium (TEA) and N'-methylnicotinamide (NMN), were strongly secreted; organic cation-to-polyethylene glycol (glomerular filtration rate marker) clearance ratios averaged 130 and 30, respectively. TEA uptake by isolated renal tubular masses was concentrative and saturable. Transport was inhibited by competitor organic cations and reduced by exposure to NaCN,2,4-dinitrophenol, ouabain, and HgCl2. Organic anions did not reduce TEA uptake. NMN was the poorest inhibitor of TEA uptake of all the organic cations tested. In addition, the rate of NMN uptake was slower than that of TEA, and the steady-state tissue-to-medium ratio was lower (5 for NMN vs. 10 for TEA; both at 25 microM). The data show the presence of an organic cation secretory system in flounder tissue that resembles the mammalian systems in several respects.

N,N'-dicyclohexylcarbodiimide inactivates organic cation transport in renal brush border membranes.

The molecular mechanism of the electroneutral organic cation/H+ antiporter in renal brush border membrane vesicles was studied utilizing the prototypic organic cation N1-methylnicotinamide. The hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD), inactivated organic cation transport irreversibly with an IC50 of 2.6 microM at pH 7.5 and 40 nM at pH 6.0. On the other hand, the hydrophilic reagents, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, did not affect organic cation transport. Substrate did not affect the rate of the DCCD inactivation which followed pseudo-first-order-kinetics. A double logarithmic plot of the apparent rate constants vs. the DCCD concentration gave a straight line with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of DCCD inactivates one carboxylate group per active transport unit and that the carboxylate group is critical for transport.