Faculty Bibliography

The family of calpains (CANP or calcium activated neutral proteases) and their endogenous inhibitor calpastatin have been implicated in many neural functions; however, functional distinctions between the major calpain isoforms, calpain I and II, have not been clearly established. In the present study we analyzed the gene expression patterns for calpain I and II and calpastatin in mouse brain and spinal cord by measuring both their mRNA and protein levels. Our results show that the overall mRNA level measured by competitive reverse transcription polymerase chain reaction for calpain II is 15-fold higher and for calpastatin is three-fold higher than that for calpain I. Overall, both mRNA and protein expression levels for the calpains and calpastatin showed no significant difference between the spinal cord and the brain. The cellular distributions of mRNA for calpain I or calpastatin, measured by in situ hybridization, are relatively uniform throughout the brain. In contrast, calpain II gene expression is selectively higher in certain neuron populations including pyramidal neurons of the hippocampus and the deep neocortical layers, Purkinje cells of cerebellum, and motor neurons of the spinal cord. The motor neurons were the most enriched in calpain message. Motor neurons possessed 10-fold more calpain II mRNA than any other spinal cord cell type. The differential distribution of the two proteases in the brain and the spinal cord at the mRNA level indicates that the two calpain genes are differentially regulated, suggesting that they play different physiological roles in neuronal activities and that they may participate in the pathogenesis of certain regional neurological degenerative diseases

We developed a new approach to study single- and double-stranded DNA breaks during chronic, moderate excitotoxicity resulting from the inhibition of the glutamate transporter in cerebellar granule cell primary cultures. A 24 hr treatment of 2-week-old cultures with L-alpha-amino adipate (LAA), an inhibitor of the cerebellar glutamate uptake transporter, caused a gradual extracellular accumulation of endogenous glutamate that induced reversible morphological change of granule neurons but no neuronal cell death despite sustained, but moderate, elevations of the free intracellular calcium concentrations. Nick translation experiments on isolated nuclei or cells from cerebellar cultures chronically exposed to LAA revealed increased radioactive nucleotide incorporation indicative of DNA nicking. This LAA effect was dose-dependent and suppressed by NMDA receptor antagonists. Cultures treated for 24 hr with LAA and subjected to in situ nick translation showed an intense nuclear labeling of neurons but not glia, which could be abolished by MK801. A similar labeling was also observed in altered nuclei of granule neurons acutely exposed to high glutamate concentrations or undergoing an apoptotic cell death. Although the TUNEL labeling method detected no DNA double-strand breaks in LAA-treated cerebellar cultures, it displayed clear evidence of DNA damage during acute glutamate excitotoxicity or during apoptosis. However, Southern blot analysis of nuclear DNA revealed a DNA laddering only in apoptotic cell death. Our results demonstrate that DNA damage, characterized by DNA single-strand breaks, is an early event in chronic, moderate excitotoxicity. This type of DNA degradation, which appears before any nuclear morphological changes, is distinct from the massive DNA single- and/or double-strand damages observed during acute glutamate excitotoxicity or apoptosis

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system

Specific antibodies and cytochemical markers combined with several imaging and morphometric techniques were used to characterize the endosomal-lysosomal system in mature neurons of the normal human central nervous system and to quantitate changes in its function in Alzheimer's disease. Compartments containing cathespin D (Cat D) and other acid hydrolases included a major subpopulation of mature lysosomes lacking mannose-6-phosphate receptors (MPR) and smaller populations of late endosomes (MPR-positive) and lipofuscin granules (MPR-negative). Antibodies to the pro-isoform of Cat D decorated perinuclear vacuolar compartments corresponding to late endosomes. Neurons and glia contained lysosomes with differing complements of acid hydrolases, implying different processing capabilities. Endosome/lysosome number per unit volume of cytoplasm was relatively well conserved within populations of normal neurons. By contrast, in morphometric analyses of Alzheimer's disease brains, 80-93% of pyramidal cells in the prefrontal cortex (laminae III or V) and hippocampus (CA2, CA3) displayed two- to eightfold higher numbers of hydrolase-positive vacuolar compartments than did corresponding cell populations in age-matched normal brains. Only 5-10% of cerebellar Purkinje cells, a less vulnerable population, showed the same statistically significant elevations. Most affected in these brain regions and in subcortical areas seemed otherwise normal by conventional histological staining and ultrastructural inspection. That both lysosomal and pro-Cat D- and MPR-positive endosomal compartments increased in number demonstrates that the endosomal-lysosomal system is activated markedly in vulnerable neuronal populations of Alzheimer's disease brains and implies that endocytosis or autophagy or both are accelerated persistently at an early stage of cellular compromise, greatly surpassing the degree of activity associated with normal aging. Early activation of the endosomal-lysosomal system represents a biological event potentially linking major etiological factors in Alzheimer's disease, including defective membrane proteins, apolipoprotein E function, and altered amyloid precursor protein processing

Microtubule-associated proteins (MAPs), such as tau, modulate neuronal shape and process outgrowth by influencing the stability and organization of microtubules. The dynamic nature of MAP-microtubule interactions in vivo, however, is poorly understood. Here, we have assessed the stability of these interactions by investigating the synthesis and axoplasmic transport of tau in relation to that of tubulin and other MAPs within retinal ganglion cells of normal adult mice in vivo. Using immunoprecipitation and Western blot analysis with anti-tau monoclonal and polyclonal antibodies, we unequivocally identified in optic axons a family of 50-60 kDa tau isoforms and a second 90-95 KDa tau family, the members of which were shown to contain the domain of tau encoded by exon 4A. To measure the rates of translocation of tau proteins in vivo, we injected mice with 35S-methionine intravitreously and, after 6-30 d, quantitated the radiolabeled tau isoforms immunoprecipitated from eight consecutive 1.1 mm segments of the nerve and optic tract and separated by electrophoresis. Linear regression analysis of protein transport along optic axons showed that the tau isoforms advanced at a rate of 0.2-0.4 mm/d, and other radiolabeled MAPs, identified by their association with taxol-stabilized microtubules, moved three- to fivefold more rapidly. By contrast, tubulins advanced at 0.1-0.2 mm/d, significantly more slowly than tau or other MAPs. These studies establish that tau is not cotransported with tubulin or microtubules, indicating that associations of tau with microtubules within axons are not as stable as previously believed. Our findings also reveal differences among various MAPs in their interactions with microtubules and provide evidence that assembly and reorganization of the microtubule network is an active process even after axons establish connections and fully mature

The repertoire of the lysosomal system extends beyond its function in degrading biologic macromolecules for energy and recycling purposes. Controlled shifts in lysosomal activity help neurons to regulate their cytoplasmic volume and to remodel local cellular domains. Newly identified regulatory controls over targeting to lysosomes and the limited proteolytic actions of 'lysosomal' hydrolases, together with other recent findings, are suggesting potential roles for the endosomal-lysosomal system in modifying functions of specific proteins, acquiring nutrients essential for growth and repair, influencing the output of secretory products, and helping neurons to modulate trophic signals. The prominent involvement of the endosomal-lysosomal system in Alzheimer's disease and other major pathologies has redoubled interest in how this system serves neurons

The mature form of cathepsin D (Cat D), purified to homogeneity from postmortem human brain or mouse brain, behaved as a 42-kDa protein in its native state but revealed additional proteolytic processing under denaturing conditions. Human brain Cat D was composed of a 30-32 kDa heavy chain and a protein doublet consisting of 14 and 15 kDa light chains. Mouse Cat D, which closely resembled the human enzyme in amino acid composition, existed mainly as the uncleaved 42-kDa protein, but up to 40% existed as a complex of 30-32 kDa and 12-14 kDa chains. The 3:1 ratio of light to heavy (30-32 kDa) chains suggested processing of some 30-kDa chains. Cleavage of the 42-kDa chain could not be induced autolytically. Human brain Cat D had a 2-3-fold higher specific activity than the mouse enzyme but shared other properties, including similar biphasic pH optima (peaks at pH 3.30 and 4.2), Km values for methemoglobin and inhibitor profiles. Human Cat D displayed the same polypeptide chain composition when purified from brains differing in postmortem interval (3-28 h). Fresh SH-SY5Y human neuroblastoma cells analyzed on Western blots with anti-Cat D antibodies also displayed only cleaved forms of mature Cat D. Furthermore, brain Cat D isolated from mice stored after death for 5, 15 or 30 h at 25 degrees C contained the same molar ratios of cleaved and uncleaved enzyme found in fresh mouse brain . Cat D activity was stable in human brains with postmortem intervals up to 27 h and stored frozen for up to 3 years. Similarly, total Cat D activity was essentially unchanged in brains of mice subjected to stimulated postmortem conditions for 0.5-4.2 h, although 20% of the total soluble brain protein became insoluble during this postmortem interval. These results demonstrate a remarkable postmortem stability of Cat D and strongly suggest that limited proteolytic cleavage of mature brain Cat D is an in vivo event, the extent of which varies markedly in different species

Addition of 400 microM AlCl3 to the culture medium for 72 h has been previously shown to induce perikaryal whorls of intermediate-sized filaments in intact mouse NB2a/d1 neuroblastoma cells. Immunoblot analyses demonstrated that in vivo treatment of cells with aluminum induced the de novo appearance of extensively phosphorylated NF-H isoforms in cytoskeletons of undifferentiated cells and increased levels of these isoforms in differentiated cells. Neurofilament subunits isolated from intact cells treated with aluminum were resistant to dephosphorylation in vitro by alkaline phosphatase and to in vitro degradation by endogenous calcium-dependent protease(s). These alterations were accompanied by a greater tendency of neurofilaments to form insoluble aggregates after isolation. These findings demonstrate direct effects of aluminum on neurofilament subunits within intact neuronal cells similar to those previously demonstrated following in vitro exposure of isolated neurofilaments to aluminum

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal ('C1') and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity