Faculty Bibliography

The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation-dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150-microns level and further increased a total of threefold by the 1,200-microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl-terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network

Antibodies to the lysosomal hydrolases, cathepsins B and D and beta-hexosaminidase A, revealed alterations of the endosomal-lysosomal system in neurons of the Alzheimer disease brain, which preceded evident degenerative changes and became marked as atrophy, neurofibrillary pathology, or chromatolysis developed. At the earliest stages of cell atrophy, hydrolase-positive lysosomes accumulated at the basal pole and then massively throughout the perikarya and proximal and proximal dendrites of affected pyramidal neurons in Alzheimer prefrontal cortex and hippocampus, far exceeding the changes of normal aging. Secondary lysosomes as well as tertiary residual bodies (lysosomes/lipofuscin) increased implying stimulated, autophagocytosis and lysosomal system activation. Less affected brain regions, such as the thalamus, displayed similar though less extensive alterations. Certain thalamic neurons exhibited a distinctive lysosome-related abnormality characterized by the presence of cell surface blebs of varying size and number filled with intense hydrolase immunoreactivity. At more advanced stages of degeneration in still intact neurons, hydrolase-positive lipofuscin, particularly in the form of abnormally large aggregates, nearly filled the cytoplasm. Similar lipofuscin aggregates were observed in abundance in the extracellular space following cell lysis and were usually associated with deposits of the beta-amyloid protein. Degenerating neurons and their processes were the major source of these aggregates within senile plaques which contained high concentrations of acid hydrolases. We have shown in previous studies that these lysosomal hydrolases in plaques are enzymatically-active. The persistence of lysosomal structures in the brain parenchyma after neurons have degenerated is a striking and potentially diagnostic feature of Alzheimer disease which has not been observed, to our knowledge, in other degenerative diseases. The lysosomal response in degenerating Alzheimer neurons represents a probable link between an early activation of the lysosomal system in at-risk, normal-appearing neurons and the end-stage contribution of lysosomes to senile plaque formation and emphasizes a slowly progressive disturbance of the lysosomal system throughout the development of Alzheimer disease

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as 'astrogliosis.' Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neurite-promoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and long-term confluent ('aged') neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, 'lesioned' (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite-promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite-promoting factor(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz-type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these proteins, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP- and laminin-enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti-APP and anti-laminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non-conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM-induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ

The lysosomal system has often been considered a prominent morphologic marker of distressed or dying neurons. Lysosomes or their constituent hydrolases have been viewed in different neuropathologic states as either initiators and direct agents of cell death, agents of cellular repair and recompensation, effectors of end-stage cellular dissolution, or autolytic scavengers of cellular debris. Limited data and limitations of methodology often do not allow these potential roles to be discriminated. In all forms of neurodegeneration, it may be presumed that lysosomes ultimately rupture and release various hydrolases that promote cell autolysis during the final stages of cellular disintegration. Beyond this perhaps universal contribution to cell death, the degree to which the lysosomal system may be involved in neurodegenerative states varies considerably. In many conditions, morphologic evidence for activation of the lysosomal system is minimal or undetectable. In other cases, lysosomal activation is evident only when other morphologic signs of cell injury are also present. This level of participation may be viewed as either an attempt by the neuron to compensate for or repair the injury or a late-stage event leading to cell dissolution. The early involvement of the lysosomal system in neurodegeneration occurs most commonly in the form of intraneuronal accumulations of abnormal storage profiles or residual bodies (tertiary lysosomes). Very often the lysosomal involvement can be traced to a primary defect or dysfunction of lysosomal components or to accelerated or abnormal membrane breakdown that leads to the buildup of modified digestion-resistant substrates within lysosomes. Because they are often striking, changes in the lysosomal system are a sensitive morphologic indicator of certain types of metabolic distress; however, whether they reflect a salutary response of a compromised neuron or a mechanism to promote cell death and removal of debris from the brain remains to be established for most conditions. Factors that may influence the lysosomal response during lethal neuronal injury include species differences, stage of neuronal development, duration of injury and pace of cell death. The lysosomal system may be more closely coupled to certain forms of neuronal cell death in lower vertebrate or invertebrate systems than in mammalian systems

Calcium-activated neutral proteinases (CANPs or calpains) are believed to be key enzymes in intracellular signaling cascades and potential mediators of calcium-induced neuronal degeneration. To investigate their involvement in Alzheimer disease, we identified three isoforms of muCANP (calpain I) in human postmortem brain corresponding to an 80-kDa precursor and two autolytically activated isoforms (78 and 76 kDa). As an index of changes in the in vivo activity of muCANP in Alzheimer disease, the ratio of the 76-kDa activated isoform of muCANP to its 80-kDa precursor was measured by immunoassay in selected brain regions from 22 individuals with Alzheimer disease and 18 normal controls. This muCANP activation ratio was elevated 3-fold in the prefrontal cortex from patients with Alzheimer disease but not from patients with Huntington disease. The activation ratio was also significantly elevated, but to a lesser degree, in brain regions where Alzheimer pathology is milder and has not led to overt neuronal degeneration. These findings indicate that muCANP activation is not simply a consequence of cellular degeneration but may be associated with dysfunction in many neurons before gross structural changes occur. The known influences of CANPs on cytoskeleton and membrane dynamics imply that persistent CANP activation may contribute to neurofibrillary pathology and abnormal amyloid precursor protein processing prior to causing synapse loss or cell death in the most vulnerable neuronal populations. Pharmacological modulation of the CANP system may merit consideration as a potential therapeutic strategy in Alzheimer disease

Eleven patients with senile dementia of the Alzheimer type and 11 age-matched control subjects were given the thyrotropin-releasing hormone (TRH) test. The two groups did not differ with respect to peak thyrotropin (TSH) response or TSH levels at baseline, 20, 30, and 45 min after TRH injection. There were significant differences between the groups on Hamilton Depression Rating Scale scores (p < 0.03), although neither group met clinical criteria for depression. Items that were significantly different pertained to depressed mood, loss of interest, loss of insight, suicidal ideation, and obsessional symptoms

We investigated the relative inhibition of purified human mu CANP and mCANP by five cysteine proteinase inhibitors including N-acetyl-Leu-Leu-nor-leucinal (C-I) and N-acetyl-Leu-Leu-methioninal (C-II), calpeptin, E64, and leupeptin. Based on IC50 measurements, calpeptin and C-I were stronger inhibitors by one to two orders of magnitude than C-II, leupeptin or E64. None of the five inhibitors, however, exhibited greater specificity for human mu CANP or mCANP. These results indicate that, although the inhibition of a given cellular event by these compounds may suggest CANP involvement, effects on mu CANP cannot be discriminated from those on mCANP

A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-alpha and -epsilon decreased, and protein kinase C-beta 1 did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase alpha and epsilon in neuritogenesis