Lysosomal dysfunction results in lamina-specific meganeurite formation but not apoptosis in frontal cortex.

An inhibitor of cathepsins B and L was used to test if lysosomal dysfunction in cultured slices of rat frontal cortex induces pathological features that develop in the human cortex during aging and Alzheimer's disease (AD). Incubation for 6 days with N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD) resulted in a massive proliferation of endosomes-lysosomes in all cortical layers. Slices additionally exposed to a washout of 4 days had numerous meganeurites, blister-like structures in the region of the axon hillock, in layer III but not in other cortical laminae. Meganeurites are a characteristic feature of the human frontal cortex after age 50 and are largely restricted to layer III. Tests for apoptosis were carried out at two intervals following meganeurite formation. TUNEL-labeled neurons were confined to layers II/III on the surface of the slices but there was no evidence for a ZPAD effect. In all, 6 days of lysosomal dysfunction reproduces characteristic effects of normal aging in neocortex without generating some key features of AD.

Selective suppression of cathepsin L results from elevations in lysosomal pH and is followed by proteolysis of tau protein.

Incubation of cultured hippocampal slices with chloroquine, a compound that increases the pH of acidic subcellular organelles, for 10 h reduced the activity of cathepsin L by 83 +/- 0.87% (mean +/- s.e.m.) while only marginally suppressing cathepsin B. This effect was followed within 3 h by an increase in the concentration of mature, single-chain cathepsin D (up 61 +/- 28%). Selective depression of cathepsin L with N-CBZ-L-phenylalanyl-L-phenylalanine-diazomethylketone also resulted in increases in enzymatically active cathepsin D and the delayed appearance of a 29 kDa fragment of the tau protein. These findings demonstrate that the pattern of cathepsin L, B, and D changes found in the aged brain can be reproduced by reducing the acidity of the lysosomal milieu. They also indicate that such pH shifts initiate a sequence of linked disturbances (inactivation of cathepsin L > induction of cathepsin D > aberrant tau proteolysis) likely to play an important role in brain ageing.

Lysosomal dysfunction reduces brain-derived neurotrophic factor expression.

Brain-derived neurotrophic factor (BDNF) expression in hippocampus and cortex is considerably reduced in Alzheimer's disease. The present study tested if lysosomal disturbances, a concomitant of brain aging, impair basal and/or induced expression of BDNF. Cultured hippocampal slices were incubated with N- CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), an inhibitor of cathepsins B and L, for 6 days and processed for in situ hybridization using radiolabeled cRNA probes against BDNF mRNA. Multiple densitometric readings were collected from each of the three principal hippocampal subdivisions. Within-slice averages were substantially lower in the ZPAD-treated group compared to controls. Treatment with the inhibitor did not change average neuron diameter or packing density. Intense stimulation of glutamate receptors with kainate for 30 min (followed by a 90-min recovery period) caused a nearly threefold increase in BDNF mRNA concentrations in the dentate gyrus while having only marginal effects in the other subdivisions. Slice averages of ZPAD-exposed cultures treated with kainate were lower than those of controls exposed to the excitotoxin; however, on a percentage basis, the kainate-induced increase in the dentate gyrus was comparable for the two groups (175 +/- 31 vs 179 +/- 39%). Kainate for 1 h (with a 5-h recovery) affected BDNF mRNA in a manner similar to that found with shorter infusions, i.e., induction in stratum granulosum but not elsewhere, lower overall slice averages with ZPAD treatment, and no evidence that ZPAD blocked the percentage increase in the dentate gyrus. These results provide evidence that lysosomal dysfunction occurring during brain aging could disrupt ongoing BDNF production without substantially impairing the neurotrophin response to intense physiological activity. The first observation suggests a plausible aging sequence leading to pathology while the second may be of interest with regard to possible therapeutics.

Suppression of cathepsins B and L causes a proliferation of lysosomes and the formation of meganeurites in hippocampus.

Cultured hippocampal slices exhibited prominent ultrastructural features of brain aging after exposure to an inhibitor of cathepsins B and L. Six days of treatment with N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD) resulted in a dramatic increase in the number of lysosomes in the perikarya of neurons and glial cells throughout the slices. Furthermore, lysosomes in CA1 and CA3 pyramidal cells were not restricted to the soma but instead were located throughout dendritic processes. Clusters of lysosomes were commonly found within bulging segments of proximal dendrites that were notable for an absence of microtubules and neurofilaments. Although pyknotic nuclei were sometimes encountered, most of the cells in slices exposed to ZPAD for 6 d appeared relatively normal. Slices given 7 d of recovery contained several unique features, compared with those processed immediately after incubation with the inhibitor. Cell bodies of CA1 neurons were largely cleared of the excess lysosomes but had gained fusiform, somatic extensions that were filled with fused lysosomes and related complex, dense bodies. These appendages, similar in form and content to structures previously referred to as "meganeurites," were not observed in CA3 neurons or granule cells. Because meganeurites were often interposed between cell body and axon, they have the potential to interfere with processes requiring axonal transport. It is suggested that inactivation of cathepsins B and L results in a proliferation of lysosomes and that meganeurite generation provides a means of storing residual catabolic organelles. The accumulated material could be eliminated by pinching off the meganeurite but, at least in some cases, this action would result in axotomy. Reduced cathepsin L activity, increased numbers of lysosomes, and the formation of meganeurites are all reported to occur during brain aging; thus, it is possible that the infusion of ZPAD into cultured slices sets in motion a greatly accelerated gerontological sequence.

Cytosolic proteolysis of tau by cathepsin D in hippocampus following suppression of cathepsins B and L.

Incubation of cultured hippocampal slices with an inhibitor [N-CBZ-L-phenylalanyl-L-alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of tau protein and the production of a 29-kDa tau fragment (tau 29). A tau antibody that is sensitive to the phosphorylated state of its epitopes did not recognize tau proteins or the tau 29 fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the tau fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. tau 29 exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal tau-tubulin interactions. Three lines of evidence indicated that ZPAD-induced tau proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co-incubation of cathepsin D and tau at neutral pH resulted in a loss of intact tau proteins and production of a 28-kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD-induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD-induced tau proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The tau fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age-related pathological features.

Translational suppression of calpain blocks long-term potentiation.

Transfection with antisense oligonucleotides was used to reduce calpain 1 activity to approximately 50% of normal values in cultured hippocampal slices. This had no detectable effects on baseline synaptic responses but greatly reduced the incidence and magnitude of long-term potentiation induced with a theta-burst stimulation paradigm. These results suggest that activation of calpain by repetitive bursts of afferent activity, as shown to occur in prior studies, is an essential step in the production of stable increases in synaptic strength.

Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices.

Transfection of cultured hippocampal slices for five days with antisense oligonucleotides directed against mRNA encoding calpain I resulted in an approximately 60% decrease in the amount of caseinolytic activity stimulated by 10 microM calcium. Increases in a single proteolytic fragment of spectrin produced by 10-20 min of NMDA receptor stimulation were substantially (approximately 50%) reduced in antisense treated slices; this effect was not obtained in slices exposed to NMDA for 45 min. Attenuation of NMDA receptor-induced spectrin proteolysis by the antisense oligonucleotides was confirmed in immunoassays using antibodies that recognize multiple spectrin breakdown products and in immunocytochemical experiments with an antibody that detects an individual calpain I-mediated fragment. Translational suppression of calpain I did not detectably affect evoked synaptic responses but markedly improved their recovery from a 15 min infusion of NMDA. These results indicate that spectrin breakdown products provide a useful index of in situ calpain I activity and support the hypothesis that the protease plays a significant role in excitotoxicity.