Faculty Bibliography

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

Most of the cases of early-onset familial Alzheimer's disease (FAD) are related to missense mutations in the presenilin 1 (PS-1) gene on chromosome 14. Although PS-1 mutations are distributed throughout the entire open reading frame, most mutations are found in transmembrane region II and hydrophilic loop VI encoded by exons 5 and 8, respectively. These two groups of substitutions are associated with an age of onset of 40-43 years for exon 5 and 45-55 years for exon 8, respectively. We have previously described a South American pedigree from Argentina with early-onset FAD (mean age of onset 38.9 +/- 3.9 years) with no mutations in exons 16 and 17 of the beta-protein precursor gene (betaPP770 transcript). Here we report the identification of an A --> T transversion at the first position of codon 146 of PS-1 in these patients. This missense mutation results in a Met --> Leu substitution, as reported for the Italian pedigrees Tor1.1 and FAD4. The significant differences in ages of onset and death among members of generations II-III and IV suggest that other genetic and/or environmental factors may influence disease phenotype in this pedigree

A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation

We screened 703 Australian subjects for an intronic polymorphism in the presenilin-1 (PS-1) gene. PS-1 intronic allele 1 homozygosity was not associated with individuals with early- or late-onset sporadic Alzheimer's disease (EOAD or LOAD). Carriers for the PS-1 intronic allele 1 were also not associated with significantly increased risk for AD regardless of gender. Our results for the Australian population are consistent with those of recent reports for other populations and do not support the conclusion that the PS-1 intronic polymorphism is associated with AD

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

In screening amplified poly(A) mRNA from hippocampal dendrites and growth cones in culture to determine candidates for local translation, we found that select transcription factor mRNAs were present. We hypothesized that synthesis of transcription factor proteins within dendrites would provide a direct signaling pathway between the distal dendrite and the nucleus resulting in modulation of gene expression important for neuronal differentiation. To evaluate this possibility, radiolabeled amplified antisense RNA was used to probe slot blots of transcription factor cDNAs as well as arrayed blots of zinc finger transcription factors. The mRNAs encoding the cAMP response element binding protein (CREB), zif 268, and one putative transcription factor were detected. We expanded upon these results showing that CREB protein is present in dendrites, that translation of CREB mRNA in isolated dendrites is feasible and that CREB protein found in dendrites can interact with the cis-acting cyclic AMP reponse element DNA sequence by using an in situ Southwestern assay. Further, CREB protein in dendrites is not transported to this site from the cell body because fluorescently tagged CREB microperfused into the soma did not diffuse into the dendrites. In addition, CREB protein microperfused into dendrites was rapidly transported to the nucleus, its likely site of bioactivity. Lastly, by using the isolated dendrite system we show that phosphorylation of Ser-133 on CREB protein can occur in isolated dendrites independent of the nucleus. These data provide a regulatory pathway in which transcription factors synthesized and posttranslationally modified in dendrites directly alter gene expression bypassing the integration of signal transduction pathways that converge on the nucleus.

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