Faculty Bibliography

Neurons are faced with the formidable challenge of having to assemble most of their cytoskeleton at axonal sites far removed from the protein synthetic machinery in the perikaryon. Their achievement seems all the more impressive now that new evidence is showing that the cytoskeleton may vary markedly in size and composition along the axon and exhibit striking regional specializations. Further complexity is contributed to this structure by a growing assortment of cytoskeleton-associated proteins that cross-link the various fibrous elements and stabilize cytoskeletal architecture. Much of the dynamic behavior of cytoskeletal proteins and polymers in axons is locally controlled. This regulation involves, in part, a system of protein kinases and phosphatases modulated by both intercellular and intracellular signals. Conceptual models of slow axonal transport have evolved to accommodate these new findings

Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.

CNS neurons exhibit a profound, maturation-dependent increase in the vulnerability to injury. Little is, however, known about the cellular mechanisms involved. This study investigated the developmental influence on the ability to recover the resting concentration of free cytoplasmic Ca2+ ([Ca2+]i) following stimulation with 100 microM glutamate in hippocampal and cerebellar granule cells in culture. Primary neurons were exposed to glutamate for either 1 min or 10 min. Hippocampal neurons were evaluated at 7, 12-14, and 17-19 days in vitro (DIV), and cerebellar granule cells were tested at 8-9 or 15-16 DIV. In hippocampal neurons, either an increased age in culture or longer drug exposure were both associated with less efficient [Ca2+]i recovery. Additionally, for both 1-min and 10-min drug exposure, increased age in culture was the primary determinant of the development of secondary [Ca2+]i destabilization followed by a very variable recovery patterns. Similar to hippocampal neurons, older cerebellar granule cells also recovered less efficiently from glutamate-mediated [Ca2+]i rise. The difference in the extent of recovery was not directly influenced by the magnitude of the [Ca2+]i rise, since cerebellar granule cells recovered from both high or low [Ca2+]i rise with similar kinetic profiles. Overall, the results presented in this study implicate the age in culture as an important influencing factor of both the less efficient recovery from glutamate-induced Ca2+ load and the development of secondary [Ca2+]i destabilizations. The progressive, maturation-dependent, decrease in the ability to recover from Ca2+ load might represent a potentially important mechanism contributing to the increased vulnerability of fully developed neurons to injury

We report on the neuropathological examinations of a 74-year-old woman with Alzheimer's disease (AD) and of her 47-year-old nondemented daughter. The brain of the mother showed fully developed pathological changes of AD. By contrast, the brain of the daughter revealed only perineuronal deposition of diffuse amyloid in cerebral cortex and striking abnormalities of the endosomal-lysosomal system, without neurofibrillary, glial, or microglial changes. These observations suggest that amyloid deposition and endosomal-lysosomal changes are early events in late-onset AD and that they may precede the onset of dementia by several decades

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grunberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756-760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins

Once presumed to be relatively uniform, the axonal cytoskeleton can vary markedly in size and composition along its length. New studies emphasize the interactiveness of neurofilaments and identify a family of cytoskeletal proteins that may cross-link the various cytoskeletal polymers of the axon, and anchor this network to the membrane skeleton. These and other findings support a model of the axonal cytoskeleton as a stationary but dynamic structure. Current evidence continues to support the possibility that axonally transported polymers/oligomers and/or monomers may serve as precursors to the cytoskeleton in different situations. Although the motors for slow transport of cytoskeletal proteins remain elusive, possible candidates are emerging

Administration of glutamate (100 microM) to primary cultures of rat hippocampal neurons for 1 h led to calpain I activation as determined by monitoring the extent of spectrin breakdown with the antibodies designed to specifically recognize the calpain I-mediated spectrin breakdown products. Based on the studies with subtype selective antagonists of glutamate receptors, glutamate caused calpain I activation specifically through the activation of the NMDA receptor. In parallel experiments, the magnitude and the temporal profiles of Ca2+ rise were determined by Fura-2 microfluorimetry. Ca2+ influx through voltage-sensitive Ca2+ channels, even though leading to substantial Ca2+ rise, did not by itself activate calpain I. These results indicate that for calpain I activation, the source of Ca1+ influx is more important than the magnitude of Ca2+ rise. Glutamate-mediated calpain I activation was fully blocked by preincubation (30 min) of the cultures with calpain inhibitor I, calpain inhibitor II, or calpeptin (all 10 microM). The presence of calpain inhibitors did not, however, in any way ameliorate the massive excitotoxicity resulting from 16 h exposure to glutamate, indicating that calpain I activation and excitotoxicity are not causally related events. Similarly, preincubation with any of the tested calpain inhibitors was detrimental to the clearance of neuritic from a 10-min exposure to glutamate. Additionally, the presence of calpain inhibitors was detrimental to the clearance of neuritic varicosities resulting from a short-term sublethal exposure to glutamate, suggesting that a physiological level of calpain I activation might actually play an important homeostatic role in the restoration of normal cytoskeletal organization

Formalin fixation, the chemical process in which formaldehyde binds to cells and tissues, is widely used to preserve human brain specimens from autolytic decomposition. Ultrastructure of cellular and mitochondrial membranes is markedly altered by vesiculation, but this does not interfere with diagnostic evaluation of neurohistology by light microscopy. Serious difficulties are encountered, however, when immunocytochemical staining is attempted. Antigens that are immunoreactive in unfixed frozen sections and protein extracts appear to be concealed or destroyed in formalin-fixed tissues. In dilute aqueous solution, formaldehyde is in equilibrium with methylene glycol and its polymeric hydrates, the balance by far in favor of methylene glyco. Carbonylic formaldehyde is a reactive electrophilic species well known for crosslinking functional groups in tissue proteins, nucleic acids, and polysaccharides. Some of its methylene crosslinks are readily hydrolyzed. Others are stable and irreversible. During immunostaining reactions, intra- and inter-molecular links between macromolecules limit antibody permeation of tissue sections, alter protein secondary structure, and reduce accessibility of antigenic determinants . Accordingly, immunoreactivity is diminished for many antigens. Tissues are rapidly penetrated by methylene glycol, but formaldehyde binding to cellular constituents is relatively slow, increasing progressively until equilibrium is reached. In addition, prolonged storage in formalin may result in acidification of human brain specimens. Low pH favors dissociation of methylene glycol into formaldehyde, further reducing both classical staining and antigen detectability. Various procedures have been devised to counter the antigen masking effects of formaldehyde. Examples include pretreatment of tissue sections with proteases, formic acid, or ultrasound. Recently, heating of mounted sections in ionic salt solution by microwave energy was found to restore many antigens. Theory and practice of microwave antigen retrieval are covered extensively in the handbook Microwave Cookbook for Microscopists. A concise overview of microwave methods in the neurosciences has been published, and clinical applications have been reviewed. In this context, it should be noted that fresh tissues may be stabilized for immunocytochemistry by reversible, non-chemical binding processes such as cryosectioning after microwave treatment and freeze-drying. Thus, it may be possible to enhance immunostaining for some antigens by microwave irradiation of unfixed as well as fixed specimens. Parameters to be optimized for microwave retrieval of specific antigens include temperature, irradiation time, tissue buffer composition, salt concentration, and pH. Temperature, irradiation time, and pH are key variables. With this in mind, an optimal method was developed for retrieval of a wide variety of antigens in human brain tissues. Typical microwave protocols employ elevated temperatures that may reach 100 degrees C, where denaturation causes irreversible uncoiling and disruption of protein secondary and tertiary structures. Under these conditions, stable covalent bonds securing methylene crosslinks between polypeptides remain intact, but more reactive links formed by Schiff bases may be hydrolyzed. Resultant conformational changes presumably expose buried loops of continuous amino acids and protruding regions, increasing accessibility of their epitopes. Protein denaturation seems to be a reasonable explanation for the effects of microwaves on antigen retrieval. This idea is supported by the observation that denaturing solutions such as 6 M urea increase immunoreactivity of some antigens. Still, the molecular basis of these effects remains unresolved, in part due to the complex chemistry of formaldehyde reactions with tissue constituents. Indeed, some methylene bridges between similar groups such as NH2 and NH may be hydrolyzed by washing fixed tissues in distilled wa

The calcium-activated neutral proteases (CANP, calpains) have been implicated in both acute and chronic neurodegenerative processes. In the present study, we analyzed the in situ mRNA expression of calpain I and II and their endogenous inhibitor, calpastatin, in the motor neuron degeneration (Mnd) mutant mouse, which exhibits progressive dysfunction of the spinal cord and brain. As the disease progresses, the mutants show increasingly pronounced motor abnormalities which coincide with swelling of the spinal motor neurons, neocortex, hippocampal CA regions and cerebellar Purkinje cells. In situ hybridization studies show that the Mnd mice have a significantly higher level of calpain I, calpain II and calpastatin than the congenic controls in the following brain regions and cell types: hippocampal CA3 region, pyramidal cells, cerebellar Purkinje cells and spinal cord motor neurons. However, no differences in calpain or calpastatin mRNA levels are observed in glial and cerebellar granule cells of Mnd and control mice. Western blots and competitive RT-PCR analyses of brain and spinal cord homogenates are confirmative. Such altered gene expression in specific cell types of brain and spinal cord suggests the involvement of the calpain/calpastatin system