Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1997-01-22
2002-09-10
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S368000, C435S375000
Reexamination Certificate
active
06447988
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of biological assays and more particularly to an in vitro model for brain aging and neuronal ceroid-lipofuscinosis.
BACKGROUND OF THE INVENTION
Aging in neuronal tissue is characterized by a variety of features, including: (1) high concentrations of &bgr;-amyloid in plaque-like structures, (2) high concentrations of ceroid-lipofuscin, a lysosomal pigment that contains lipoidal moieties and emits yellow to greenish autofluorescence under ultraviolet illumination (Brizzee K. R. et al., “The amount and distribution of pigments in neurons and glia of the cerebral cortex.” J. Geront. (1969) 24:127-35; Sohal, R. S. and Wolfe, L. S., “Lipofuscin: Chararacteristics and significance.” In:
Progress in Brain Research
, Swaab et al. (eds), Elsevier Science Publications, 1986, Vol. 70, pp. 171-183.), (3) loss of synaptic proteins, such as synaptophysin, (4) neurofibrillary tangles, (5) abnormal phosphorylation of neurotubule proteins, (6) neuronal atrophy (Bahr, BA et al., “Induction of &bgr;-amyloid containing polypeptides in hippocampus:evidence for a concomitant loss of synaptic proteins and interactions with an excitotoxin”, Exp. Neurol. (1994) 129:81-94.), (7) lipofuscin-filled somatic expansions, known as meganeurites, (Braak, H., “Spindle-shaped appendages of IIIab-pyramidals filled with lipofuscin: A striking pathological change of the senescent human isocortex”, Acta Neuropathol. (1979) 46:197-202.), (8) an increase in the number of perikaryal lysosomes (Brunk, U. and Ericsson, J. L. E. “Electron microscopical studies on rat brain neurons. Localization of acid phosphatase and mode of formation of lipofuscin bodies”, J. Ultrastr. Res. 38 (1972) 38:1-15; Cataldo, A. M. et al., “Properties of the endosomal-lysosomal system in the human central nervous system: Disturbances mark most neurons in populations at risk to degeneration in Alzheimer's disease”, J. Neurosci. (1996) 16:186-99), (9) fragmentation of cytoskeletal proteins (Bahr, B. A., et al., “Spectrin breakdown products increase with age in the telencephalon in mouse brain”, Neurosci. Lett. (1991) 131:237-40), (10) a degeneration of myelinated axons (Peters, A., et al., “The effects of aging on area 46 of the frontal cortex of the rhesus monkey”, Cerebral Cortex (1994) 4:621-35), and (11) cellular processes (neurites) filled with dense lysosomal bodies and vacuoles (Terry et al., “Ultrastructural studies in Alzheimer's presenile dementia.” Am. J. Pathol. (1964) 44: 269-297.) These characteristics, including the production of lipofuscin, are apparent in Alzheimer's disease.
Lipofuscin accumulation is also present in a condition known as neuronal ceroid lipofuscinosis (NCL). NCL is a storage disease that generally becomes evident in early adult life, although it can also occur later in adult life or during childhood. Usually the disease begins with mental deterioration, followed by seizures and ataxia, amongst other symptoms. Central nervous system deterioration with cerebral atrophy is found, and biopsy of the brain reveals the presence of lipofuscin granules. Electron microscopy reveals abnormal inclusions witophin lysosomes in a wide variety of tissues, such as the fibroblast and endothelial cells of the conjunctiva and skin. It is uncertain whether this disorder is a true lysosomal storage disease; single or multiple genetic disorders may be present (Beaudet, A. L. Lysosomal Storage Diseases. In: Harrison's Principles of Internal Medicine, 12th Edition. Wilson, J. D. et al.(eds) McGraw-Hill, Inc. New York, (1991), pg.1854.)
The lack of knowledge about aging and NCL is due in part to the lack of an in vitro model. A genetic model of NCL does exist. Recently, Dunn and his colleagues (Dunn, W A Jr, Raizada, M. K., Vogt, E S, and Brown, E. A. Growth factor-induced neurite growth in primary neuronal cultures of dogs with neuronal ceroid lipofuscinosis. Int. J. Dev. Neurosci. (1994) 12(3): 185-96) established conditions to grow neuronal cells from newborn English setters with NCL. The characteristic inclusion bodies were present in the neurons, and a time-dependent maturation of the inclusion bodies was noted.
Research on the processes underlying brain aging is made difficult by (1) the time span over which the processes act, (2) the complexity of the neural and glial networks involved, (3) the intricate relationships between the wide variety of biochemical and structural changes that occur with aging, and (4) the relative inaccessibility of the brain to prolonged experimental manipulation. The hippocampus is a region of the brain involved in memory encoding and has been shown to exhibit early degeneration in Alzheimer's disease and ischemia, thus it is an excellent area to use in the study of aging (Terry, R. “Interrelations among the lesions of normal and abnormal aging of the brain”, In:
Progress in Brain Research
, Swaab et al. (eds), Elsevier Science Publications, 1986, Vol. 70, pp. 41-7).
Long-term study of hippocampal tissue is possible by maintaining hippocampal slices on porous membranes at the air-medium interface. The long-term hippocampal slice prepared with this method exhibits long-term potentiation, a marker for relatively mature telencephalic connections. The slicesretain or develop many features characteristic of the mature hippocampus including elaborated dendritic spines and pathologic responsiveness, and remain stable for periods of weeks (Stoppini et al. A simple method for organotypic cultures of nervous tissues. J Neurosci Meth. (1991) 57:985-94.; reviewed in Bahr, B. A. Mini-review: Long-term hippocampal slices: a model system for investigating synaptic mechanisms and pathologic processes. J. Neurosci. Res (1995) 42:294-305). Treatment of this type of culture with chloroquine, a lysosomotropic agent, results in the appearance of carboxy-terminal fragments of the amyloid precursor protein (APP), an accumulation of &bgr;-amyloid immunoreactivity, and a loss of synaptophysin, a characteristic protein of the neural synapse, in the hippocampal slices. Pre-treatment with kainic acid made the build-up of &bgr;-amyloid-containing fragments and the loss of synaptophysin more rapid (Bahr, BA et al. Induction of &bgr;-amyloid-containing polypeptides in hippocampus: Evidence for a concomitant loss of synaptic proteins and interactions with an excitotoxin. Exp. Neurol. (1994) 129:81-94).
Other methods used to culture neuronal cells to study aging include (1) conventional in vitro methods used to study long-term plasticity which include rotation-mediated aggregating cell cultures (Chaterjee, S. and Nolder, M. “An aggregate brain cell culture model for studying neuronal degeneration and regeneration” J. Neural Transm. Suppl (1994) 44:47-60) or (2) slice cultures where the slices are incubated in sealed Maximov chambers (Crain, S. M. Neurophysiologic studies in tissue culture. (1976). New York. Raven Press) or (3) slice cultures where the slices are adhered to glass coverslips and rotated in medium-containing tubes for feeding (Gahwiler, B. H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Meth. (1981) 4:329-42; reviewed in Gahwiler, B. H. Organotypic cultures of neural tissue. Trends Neurosci. (1988) 11:4-84-9.).
SUMMARY OF THE INVENTION
An in vitro model of brain aging is provided by treating a brain tissue slice with compounds which interact with the brain and cause the development of characteristics of brain aging. The treatment compounds include: (1) a free radical generator, (2) agents that depress lysosomal enzymes, specifically inhibiting the cathepsins or preferably (3) a combination of (1) and (2). The treatment can be carried out before, after or simultaneous with the addition of a test compound, e.g., a drug. A variety of agents can be used as free radical generators including any metal salts (e.g., iron, cobalt, nickel, copper, manganese, vanadium), hydrogen peroxide, or a mixture of xanthine and xanthine oxidase. Cysteine lysosomal proteases such as cathepsin L, cathepsin B, cathepsin H, cathepsin S
Bednarski Eric
Gall Christine M.
Lynch Gary S.
Ribak Charles E.
Borden Paula A.
Bozicevic Karl
Bozicevic Field & Francis LLP
Parkin J. S.
Scheiner Laurie
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