Sherry Bursztajn

Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: Evidence for early up-regulation of the endosomal-lysosomal system

In Alzheimers disease brains, more than 90% of pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D (Cat D) mRNA by in situ hybridization compared with neurologically normal controls. Most of these cells appeared histologically normal. The less vulnerable nonpyramidal neuron population in lamina IV had relatively normal message levels. Neuronal populations expressing more Cat D mRNA also displayed quantitatively increased Cat D immunoreactive protein. Cat D mRNA expression was only moderately increased in astrocytes. Degenerating neurons exhibited intense immunoreactivity but lowered Cat D mRNA levels. The upregulation of Cat D synthesis and accumulation of hydrolase-laden lysosomes indicate an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes. These abnormalities also represent a basis for altered regulation of amyloid precursor protein processing.

Overexpression in Neurons of Human Presenilin-1 or a Presenilin-1 Familial Alzheimer Disease Mutant Does Not Enhance Apoptosis

Programmed cell death, or apoptosis, has been implicated in Alzheimer’s disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer’s disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

Degradation of the acetylcholine receptor in cultured muscle cells: Selective inhibitors and the fate of undegraded receptors

Abstract To learn more about the pathway for degradation of an intrinsic membrane protein, we studied in cultured chick myotubes the effects of certain protease inhibitors and chloroquine (an inhibitor of lysosomal function) on degradation of the acetylcholine receptor measured with the specific ligand 125 I-α-bungarotoxin. Leupeptin, chymostatin, anti-pain and chloroquine decreased by 2–10 fold the rate of degradation of the acetylcholine receptor- 125 I-α-bungarotoxin complex to 125 I-tyrosine (p 125 I-α-bungarotoxin and then incubated with leupeptin or chloroquine contained more radioactive protein than control cells. This material co-electrophoresed with bungarotoxin on sodium dodecylsulfate-urea-polyacrylamide gels. Thus myotubes exposed to these inhibitors seemed to accumulate undegraded bungarotoxin. They did not, however, contain more acetylcholine receptors on their surface. Instead, the inhibitor-treated cells accumulate toxin and receptors at some internal site. Thus treatment with such inhibitors does not appear to be a useful approach to the therapy of myasthenia gravis. The additional 125 I-toxin found in cells incubated with leupeptin or chloroquine was less accessible to exogenous protease than the toxin bound to control cells and was more resistant to extraction by Triton X-100. Since internalization of the receptor continued in the presence of these inhibitors, this process must not be coupled tightly to subsequent proteolysis. Measurement of receptors within cells not exposed to 125 I-α-bungarotoxin showed that incubation of myotubes with leupeptin or chloroquine for 48 hr increased the number of internal bungarotoxin-binding sites 2–11 fold (p

Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells

Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of alpha 4 and beta 2 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the alpha 7 subunit as shown by [125I]alpha-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for alpha 3, alpha 4, alpha 5, alpha 7, beta 2 and beta 4 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]alpha-bungarotoxin binding, immunocytochemistry and PCR. Maximum Ca2+ influx elicited by nicotine, and partly sensitive to alpha-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells.

Regional differences in gene expression for calcium activated neutral proteases (calpains) and their endogenous inhibitor calpastatin in mouse brain and spinal cord

The family of calpains (CANP or calcium activated neutral proteases) and their endogenous inhibitor calpastatin have been implicated in many neural functions; however, functional distinctions between the major calpain isoforms, calpain I and II, have not been clearly established. In the present study we analyzed the gene expression patterns for calpain I and II and calpastatin in mouse brain and spinal cord by measuring both their mRNA and protein levels. Our results show that the overall mRNA level measured by competitive reverse transcription polymerase chain reaction for calpain II is 15-fold higher and for calpastatin is three-fold higher than that for calpain I. Overall, both mRNA and protein expression levels for the calpains and calpastatin showed no significant difference between the spinal cord and the brain. The cellular distributions of mRNA for calpain I or calpastatin, measured by in situ hybridization, are relatively uniform throughout the brain. In contrast, calpain II gene expression is selectively higher in certain neuron populations including pyramidal neurons of the hippocampus and the deep neocortical layers, Purkinje cells of cerebellum, and motor neurons of the spinal cord. The motor neurons were the most enriched in calpain message. Motor neurons possessed 10-fold more calpain II mRNA than any other spinal cord cell type. The differential distribution of the two proteases in the brain and the spinal cord at the mRNA level indicates that the two calpain genes are differentially regulated, suggesting that they play different physiological roles in neuronal activities and that they may participate in the pathogenesis of certain regional neurological degenerative diseases.

Altered gene expression for calpain/calpastatin system in motor neuron degeneration (Mnd) mutant mouse brain and spinal cord

The calcium-activated neutral proteases (CANP, calpains) have been implicated in both acute and chronic neurodegenerative processes. In the present study, we analyzed the in situ mRNA expression of calpain I and II and their endogenous inhibitor, calpastatin, in the motor neuron degeneration (Mnd) mutant mouse, which exhibits progressive dysfunction of the spinal cord and brain. As the disease progresses, the mutants show increasingly pronounced motor abnormalities which coincide with swelling of the spinal motor neurons, neocortex, hippocampal CA regions and cerebellar Purkinje cells. In situ hybridization studies show that the Mnd mice have a significantly higher level of calpain I, calpain II and calpastatin than the congenic controls in the following brain regions and cell types: hippocampal CA3 region, pyramidal cells, cerebellar Purkinje cells and spinal cord motor neurons. However, no differences in calpain or calpastatin mRNA levels are observed in glial and cerebellar granule cells of Mnd and control mice. Western blots and competitive RT-PCR analyses of brain and spinal cord homogenates are confirmative. Such altered gene expression in specific cell types of brain and spinal cord suggests the involvement of the calpain/calpastatin system.

Differential expression and co-assembly of NMDA zeta 1 and epsilon subunits in the mouse cerebellum during postnatal development.

The differential distribution of NMDA receptor subunit mRNAs in the developing mammalian cerebellum has been previously described. In this study, we investigated the temporal expression of NMDA receptor proteins in the postnatal murine cerebellum using antibodies specific for the NMDA zeta 1 and NMDA epsilon subunits. Our results showed a gradual increase during the first three weeks of life in the relative amount of NMDA zeta 1 and NMDA epsilon 1 proteins. In contrast, NMDA epsilon 2 increased transiently during this period, reaching a maximum around postnatal day 9 and decreasing thereafter to nearly undetectable levels by the end of the third week of life. The level of NMDA epsilon 3 increased dramatically between postnatal days 9 and 15 and thereafter remained constant. Immunoprecipitation of native proteins revealed that a large fraction of NMDA epsilon 2 was associated with NMDA zeta 1 and epsilon 1. At later developmental stages, NMDA epsilon 3 was predominantly assembled with NMDA zeta 1 but not with NMDA epsilon 1 or NMDA epsilon 2. These results demonstrate that NMDA receptor subtypes, formed by the assembly of different NMDA epsilon subunits with NMDA zeta 1, are sequentially expressed in the developing mouse cerebellum. The time course of their expression suggest, that these NMDA receptor subtypes may contribute to specific aspects of granule cell differentiation in the cerebellum.

Expression of the α4 neuronal nicotinic acetylcholine receptor subunit in the developing mouse hippocampus

Neurotransmitters such as acetylcholine can control neuritogenesis of hippocampal cells. The timing of its receptors expression consequently may influence synaptogenesis and neuronal activity in the developing hippocampus. We investigated the mRNA expression of the nicotinic acetylcholine‐gated ion channel receptor (nAChR) α4 subunit in the embryonic and postnatal hippocampal formation. Although its expression level is low in the adult hippocampus, this protein consitutes the major nAChR subunit in the central nervous system. We carried out in‐situ hybridization experiments to determine whether or not the α4 AChR subunit mRNA distributions show evidence of regional and developmental regulation during hippocampal maturation. Our studies reveal that α4 AChR mRNA expression was low at the embryonic stage, but increased transiently during postnatal development reaching a maximum during the second week of life and decreasing thereafter, to a minimum at adulthood. In hippocampal regions, the peak values of α4 AChR expression were between 400 and 800% of adult α4 messenger levels. In the postnatal hippocampus, most of the cells from the pyramidal layer of the CA3 and CA2 areas displayed a strong hybridization signal for the α4 AChR subunit. In the hilus and the CA1 regions, the localization of the α4 transcripts seemed to be restricted to some interneurons and pyramidal cells, respectively. Moderate and uniform in‐situ hybridization signals were observed in granular cells from the dentate gyrus. The transient profile of α4 expression suggests that nAChRs may participate in the early postnatal maturation of hippocampal circuity.

Plasticity of NMDA Receptor Expression During Mouse Cerebellar Granule Cell Development

A period of hypersensitivity to N‐methyl‐d‐aspartate (NMDA) has been described during the early development of different types of neuron. Since activation of NMDA receptors can also induce rapid neuron death, the hypersensitivity to NMDA may be tightly controlled. In the present study we show that mouse cerebellar granule neurons become transiently hypersensitive to NMDA between days 10 and 14 after plating in a culture medium containing 30 mM K+. The NMDA sensitivity is higher when cells are cultured in the presence of an NMDA receptor antagonist [30 mM K+ plus 100 μM 3‐((±)‐2‐carboxypiperazine‐4‐yl)‐propyl‐1‐phosphonic acid (CPP)], and no hypersensitivity is observed when cells are cultured in the continuous presence of NMDA (12.5 mM K+ plus 100 μM NMDA). The high NMDA sensitivity in control cells is associated with a higher density of NMDA receptors than that measured in NMDA‐treated cells, suggesting that the sensitivity to NMDA may be partly controlled by activity‐dependent NMDA receptor down‐regulation. We also examined the level of NMDA‐ζ1 mRNA and found no correlation between this parameter and the transient pattern of NMDA sensitivity. Such NMDA receptor plasticity may be of importance in the central nervous system, protecting developing cells from excitotoxicity at critical developmental stages.

Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function

SummaryTreatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 μm (mean ± SEM) compared with 2 ± 0.12 μm after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 μm, p < 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.