Faculty Bibliography

A total of 27 subjects began active treatment in this double-blind study comparing the efficacy and safety of trazodone and fluoxetine in geriatric depressed patients, but only 13 completed 6 weeks on study medication. Both agents were effective according to weekly and endpoint analyses, and there was no evidence of significant effects on blood pressure, pulse, or weight. Separate analysis of patients who had received an adequate trial of medication indicated a trend toward relatively more fluoxetine-treated patients meeting clinical criteria for resolved depression

NB2a/dl neuroblastoma cells were exposed to aluminum chloride or aluminum lactate (0.1-1 mM) for 3 and 6 days. Additional cultures were exposed to aluminum salts as the cells were stimulated to elaborate axonal neurites by dibutyryl cyclic AMP. By phase-contrast microscopy, aluminum salts had no effect on the morphology of undifferentiated (NB2a(-] or differentiated (NB2a(+] cells, or on neuritic elaboration and maintenance. Silver straining by the Bielschowsky method, however, demonstrated argyrophilic accumulations in perikarya of many NB2a(-) and NB2a(+) cells treated with aluminum salts. At the ultrastructural level, whorls of intermediate filaments were the most prominent abnormalities in neuronal perikarya. Although phosphorylated high-molecular weight neurofilament subunits (NF-H) are normally detected by immunocytochemical analyses only within axonal neurites of NB2a/dl cells, aluminum salt treatment caused the detection of phosphorylated epitopes of NF-H within perikaryal of NB2a(-) and NB2a(+) cytoskeletons, suggesting that the argyrophilic filamentous accumulations are composed at least partly of phosphorylated NF-H

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 microns downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized.(ABSTRACT TRUNCATED AT 400 WORDS)

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein

Evidence is emerging that calcium-activated neutral proteinases (CANPs) not only participate in intracellular protein turnover but help to regulate the functional reorganization of cytoskeletal proteins in response to calcium and second-messenger stimulation. The high concentration of CANPs in certain neurons has suggested prominent roles for this proteolytic system in neuronal and synaptic function. In addition to acting directly on specific constituents of the cytoplasmic and membrane-associated cytoskeletal networks, CANP may amplify its effects by modulating the activities of protein kinase C and possibly other kinases and phosphatases by limited proteolysis. Given its suspected involvement at the cytoskeleton-membrane interface, calcium-mediated proteolysis is an example of a metabolic process which, if impaired, could provide a unifying basis for the slow progressive development of diverse structural and functional abnormalities within neurons. The multiplicity of mechanisms regulating its activity makes the CANP system a vulnerable target for disruption from various sources. A working hypothesis is advanced that down-regulation (inhibition) of neuronal calcium-mediated proteolysis in Alzheimer's disease is one critical and early step in the development of neurofibrillary degeneration and altered membrane cytoskeleton dynamics, which leads to membrane injury, accumulation of abnormal proteins, and synaptic dysfunction

Prolonged afferent stimulation of the rat dentate gyrus in vivo leads to degeneration only of those cells that lack immunoreactivity for the calcium binding proteins parvalbumin and calbindin. In order to test the hypothesis that calcium binding proteins protect against the effects of prolonged stimulation, intracellular recordings were made in hippocampal slices from cells that lack immunoreactivity for calcium binding proteins. Calcium binding protein-negative cells showed electrophysiological signs of deterioration during prolonged stimulation; cells containing calcium binding protein did not. When neurons without calcium binding proteins were impaled with microelectrodes containing the calcium chelator BAPTA, and BAPTA was allowed to diffuse into the cells, these cells showed no deterioration. These results indicate that, in a complex tissue of the central nervous system, an activity-induced increase in intracellular calcium can trigger processes leading to cell deterioration, and that increasing the calcium binding capacity of a cell decreases its vulnerability to damage

In area CA1 of hippocampus, a subpopulation of gamma-aminobutyric acid (GABA)-containing interneurons that make synaptic contacts on pyramidal cells also contains the neuropeptide, somatostatin. The effects of GABA and somatostatin on hippocampal pyramidal cells have been investigated separately, but it is not known whether an interaction occurs between these co-localized substances. We demonstrate that somatostatin has a potent inhibitory effect on GABA-mediated synaptic potentials which hyperpolarize pyramidal cells. This effect may be relevant to the well-documented epileptogenicity of the hippocampus, as well as the phenomenon of long-term potentiation, which is a well-studied example of synaptic plasticity

The results of several studies have suggested that local circuit neurons, or interneurons, of area CA1 of hippocampus use gamma-aminobutyric acid (GABA) as their neurotransmitter. However, when these cells were labelled by intracellular dye injection, and examined immunocytochemically with antisera raised against GABA, none of the interneurons were immunoreactive. Numerous non-injected interneurons in the same tissue section were clearly immunoreactive. These results suggest that intracellular dyes interfere with immunocytochemical staining of hippocampal interneurons

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo