Faculty Bibliography

We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm 'axonal window' comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)

Titrations of angiotensin-converting enzyme (ACE; E.C. 3.4.15.1) present in human serum, as well as in homogenates prepared from post-mortem human caudate or mouse (C57BL1/6J) whole brain tissue, were performed with the selective ACE inhibitors, captopril (SQ 14225) and teprotide (SQ 20881). ACE activity present in human serum was more sensitive to inhibition by either inhibitor than the activity present in the brain homogenates. The inhibition curves for the titration of the human serum activity by both inhibitors were sigmoidal while the inhibition curves for the ACE activity present in the brain homogenates were more complex. These results suggest that the brain homogenates contained: at least two species of enzyme activity with properties similar to ACE but with differing affinities for the inhibitors, or substances without ACE activity that are capable of competing with ACE for the binding of the inhibitors. Therefore, measurements of captopril or teprotide-sensitive peptidase activity as well as inhibitor-binding activity may not always reflect ACE concentrations in brain tissue

Angiotensin-converting enzyme (ACE, E.C. 3.4.15.1) has been identified as a normal constituent of human cerebrospinal fluid (CSF). ACE activity in CSF from adult subjects without known neurologic disorder correlated positively (P = 0.002) with age between 50 and 90 years. Patients with moderate degrees of senile dementia of the Alzheimer's type and comparably demented patients with Parkinson's disease or progressive supranuclear palsy exhibited mean levels of ACE activity that were decreased 41, 27 and 53% respectively, compared to the mean level in an age and sex-matched group of neurologically intact individuals. These results raise the possibility that ACE activity in CSF may be an index of neuronal dysfunction in certain central neurodegenerative disorders

Long-term potentiation (LTP) in the hippocampus is a long lasting enhancement of the postsynaptic evoked response following high frequency, repetitive stimulation of afferents. The extracellularly recorded action potential (population spike) can be reversibly blocked, without affecting the extracellularly recorded excitatory postsynaptic potential, by focal application of gamma-aminobutyric acid, tetrodotoxin, or pentobarbital, to the CA1 pyramidal cells of the hippocampal slice. When the population spike is blocked during repetitive stimulation, LTP does not occur. It appears that postsynaptic firing of action potentials during repetitive stimulation is necessary to produce LTP

Long-term potentiation is a long-lasting enhancement of synaptic efficacy following brief, high-frequency, repetitive stimulation of a monosynaptic input. Intracellular recordings have shown that the inhibitory postsynaptic potential changes in amplitude during long-term potentiation. Yet how this may occur is unclear. To test for a possible alteration in postsynaptic sensitivity to the recurrent inhibitory transmitter gamma-aminobutyrate, we have examined the effect of gamma-aminobutyrate, focally applied to the hippocampal CA1 cell-body layer, on the extracellular recorded action potential (population spike). We found that the degree, duration, dose-dependence and time-course of inhibition produced by gamma-aminobutyrate are unchanged during long-term potentiation. This suggests that a change in sensitivity of CA1 pyramidal cells to the transmitter gamma-aminobutyrate is not the reason for the alteration in the inhibitory postsynaptic potential during long-term potentiation

The effects of pressure-applied gamma-aminobutyric acid (GABA) on the soma and dendrites of pyramidal and non-pyramidal neurons of rat visual cortical slices were recorded intracellularly. When applied close to the soma, GABA produced hyperpolarizations and depolarizations, but when GABA was applied more than 250 microns from the soma only depolarizations were recorded. The results suggest that most visual cortical cells respond to GABA and that the responses of pyramidal and non-pyramidal cells to GABA are similar

Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were two- to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP break-down products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFPs by CD comigrated on two-dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskeletal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed

Growing appreciation of the multiple functions of proteolytic enzymes in intracellular protein degradation and post-translational modification, in the release of biologically active macromolecules and peptides from precursors and in cellular protein regulation and quality control has stimulated interest in proteases in neurobiology and neuropathology. In this article, the proteinases and peptidases thus far studied in the human central nervous system are reviewed with respect to their enzymology, anatomical and cytological distributions and contributions to neurological and psychiatric disease states. Though information concerning brain proteases in man is fragmentary, it suffices to establish the importance of these complex systems for advancing knowledge of human cerebral function in health and disease