Faculty Bibliography

Calcium-activated neutral proteases (calpains) are regulated by specific endogenous protein inhibitors, the calpastatins, which are widely distributed in mammalian tissues. Calpastatins from different species or in various tissues from the same species exhibit considerable size heterogeneity on sodium dodecyl sulfate (SDS) gels, reflecting both transcriptional and posttranslational regulation. This heterogeneity has complicated previous biochemical characterizations. In this study, we purified bovine brain calpastatin to homogeneity. The inhibitor was purified 2,463-fold from a cytosolic fraction of fresh bovine cerebral cortex by chromatographies on diethylaminoethyl cellulose, Ultrogel AcA44, phenyl-Sepharose, concanavalin A-Sepharose, and Q-Sepharose. The major calpastatin displayed a native molecular mass of 250-300 kDa by gel filtration and was composed of 125-kDa polypeptide chains by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Small amounts of a 68-kDa calpastatin fragment were detected particularly in molecules exhibiting smaller native molecular mass (250 kDa). When electroeluted from SDS gels, the 125- and 68-kDa polypeptides each inhibited calpain. The purified protein was strongly immunoreactive toward antibodies raised against a synthetic peptide, CEKLGEKEETIPPDYR, shown to be a conserved repetitive motif in the calpastatin gene or a recombinant polypeptide corresponding to domains L and 1 of human calpastatin. Calpastatins purified from bovine and human erythrocytes exhibited molecular masses of 78 and 68 kDa, respectively, by SDS-PAGE. Both erythrocyte calpastatins reacted strongly with antibodies against the conserved sequence but not with antibodies raised against domains L and 1 of human calpastatin, indicating that the erythrocyte inhibitors lack these two domains.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the clinical, SPET, immunohistochemical and DNA features of an early-onset familial Alzheimer's disease (FAD) in an Argentine pedigree of South American indian ethnic background. Pedigree spans 5 generations comprising more than 110 biological relatives. Clinical data supported the diagnosis of early onset FAD (mean age at onset 38.9 years) in 10 family members, including 3 with pathological confirmation (mean age at death 48.5). The pattern of transmission suggested autosomal dominant inheritance. Prominent features were mood changes, early language impairment, myoclonus, seizures and cerebellar signs. SPET displayed bilateral frontal, temporo-parietal and cerebellar hypoperfusion in early stages and in an asymptomatic member at risk, suggesting that SPET may have predictive value in this family. Immunohistochemistry showed beta amyloid deposits within neuritic plaques and vessel walls and no anti-PrP immunoreactivity. DNA analysis showed no abnormalities in the beta amyloid precursor protein gene. The identification of additional genetic defects in well characterized independent FAD pedigrees will contribute to the understanding of the pathogenesis of Alzheimer's disease

Lysosomal hydrolases are normally intracellular enzymes but are abundant extracellularly within senile plaques in Alzheimer disease and in other conditions where beta-amyloid accumulates. To examine whether acid hydrolases released from abnormal hydrolase-laden neurons are detectable in CSF, we measured levels of the major aspartic proteinase of lysosomes, cathepsin D (Cat D), in ventricular CSF collected after death from 30 patients with Alzheimer disease, 14 patients with Huntington disease, and seven patients with other neurodegenerative diseases. The levels of Cat D-immunoreactive protein, expressed as micrograms per milliliter of protein, determined by western blot immunoassay using a polyclonal antiserum against human brain Cat D, were more than fourfold higher in the Alzheimer patients than in the other patient groups (p < 0.0005). Cat D activity, assayed separately against [14C]methemoglobin at pH 3.2, was also significantly elevated but less than Cat D content. The lower specific activity of Cat D in Alzheimer CSF therefore indicated that the abnormally accumulated Cat D included a high proportion of inactive enzyme. These results indicate that abnormal Cat D release from affected neurons into the extracellular space is an active, ongoing process in Alzheimer brain. In addition, the levels of this enzyme and possibly other lysosomal hydrolases in CSF may prove to be useful biological markers of Alzheimer disease

In the rat dentate gyrus, pyramidal-shaped cells located on the border of the granule cell layer and the hilus are one of the most common types of gamma-aminobutyric acid (GABA)-immunoreactive neurons. This study describes their electrophysiological characteristics. Membrane properties, patterns of discharge, and synaptic responses were recorded intracellularly from these cells in hippocampal slices. Each cell was identified as pyramidal-shaped by injecting the marker Neurobiotin intracellularly (n = 17). In several respects the membrane properties of the sampled cells were similar to 'fast-spiking' cells (putative inhibitory interneurons) that have been described in other areas of the hippocampus. For example, input resistance was high (mean 91.3 megohms), the membrane time constant was short (mean 7.7 ms), and there was a large afterhyperpolarization following a single action potential (mean 10.5 mV at resting potential). However, the action potentials of most pyramidal-shaped cells were not as brief (mean 1.2 ms total duration) as those of most previously described fast-spiking cells. Many pyramidal-shaped neurons had strong spike frequency adaptation relative to other fast-spiking cells. Although these latter two characteristics were apparent in the majority of the sampled cells, there were exceptional pyramidal-shaped neurons with fast action potentials and weak adaptation, demonstrating the electrophysiological variability of pyramidal-shaped cells. Responses to outer molecular layer stimulation were composed primarily of excitatory postsynaptic potentials (EPSPs) rather than inhibitory postsynaptic potentials (IPSPs), and were usually small (EPSPs evoked at threshold were often less than 2 mV), and brief (less than 30 ms). There was variability, because in a few cells EPSPs evoked at threshold were much larger. However, regardless of EPSP amplitude, suprathreshold stimulation (up to 4 times the threshold stimulus strength) rarely evoked more than one action potential in any cell. The results suggest that stimulation of perforant path axons produces limited excitatory synaptic responses in pyramidal-shaped neurons. This may be one of the reasons why they are relatively resistant to prolonged perforant path stimulation. The pyramidal-shaped neurons located at the base of the granule cell layer have been associated historically with a basket plexus around granule cell somata, and have been called pyramidal 'basket' cells. However, basket-like endings were rare and axon collaterals outside the granule cell layer as the outer molecular layer and the central hilus, and antidromic action potentials could be recorded in some cells in response to weak stimulation of these areas. Taken together with the electrophysiological variability, the results indicate that these cells are physiologically heterogeneous

Calpains (CANPs) are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they activate or alter the regulation of certain enzymes, including key protein kinases and phosphatases, and induce specific cytoskeletal rearrangements, accounting for their suspected involvement in intracellular signaling, vesicular trafficking, and structural stabilization. Calpain activity has been implicated in various aging phenomena, including cataract formation and erythrocyte senescence. Abnormal activation of the large stores of latent calpain in neurons induces cell injury and is believed to underlie neurodegeneration in excitotoxicity, Wallerian degeneration, and certain other neuropathologic states involving abnormal calcium influx. In Alzheimer's disease, we found the ratio of activated calpain I to its latent precursor isoform in neocortex to be threefold higher than that in normal individuals and those with Huntington's or Parkinson's disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimer's disease. The excessive calpain system activation suggested by these findings represents a potential molecular basis for synaptic loss and neuronal cell death in the brain in Alzheimer's disease given the known destructive actions of calpain I and its preferential neuronal and synaptic localization. In surviving cells, persistent calpain activation may also contribute to neurofibrillary pathology and abnormal amyloid precursor protein trafficking/processing through its known actions on protein kinases and the membrane skeleton. The degree of abnormal calpain activation in the brain in Alzheimer's disease strongly correlated with the extent of decline in levels of secreted amyloid precursor protein in brain. Cytoskeletal proteins that are normally good calpain substrates become relatively calpain resistant when they are hyperphosphorylated, which may contribute to their accumulation in neurofibrillary tangles. As a major effector of calcium signals, calpain activity may mirror disturbances in calcium homeostasis and mediate important pathologic consequences of such disturbances