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Previous work found evidence that α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors interact with and are functionally regulated by the glycosaminoglycan heparin. The present study tested whether dextran species affect ligand binding, channel kinetics, and calcium permeability of AMPA receptors. Dextran sulfate of 500 kDa markedly reduced high affinity [3H]AMPA binding in solubilized hippocampal membranes. In isolated receptors reconstituted in a lipid bilayer, the same dextran sulfate prolonged the lifetime of open states exhibited by AMPA-induced channel fluctuations. The large polysaccharide further changed the single channel kinetics by increasing the open channel probability five- to sixfold. Such modulation of channel activity corresponded with enhanced levels of calcium influx as shown in hippocampal neurons loaded with Fluo3AM dye. With an exposure time of

Status epilepticus (SE) increases neurogenesis in the subgranular zone (SGZ) of the adult dentate gyrus, but many of the newborn cells die, partly through caspase-induced apoptosis. Here we provide immunohistochemical evidence indicating that the caspase-evoked death of the new neurons involves the mitochondrial but not the death-receptor-mediated pathway. Cytochrome c released from mitochondria was found in a subset of progenitor cell progeny, while Fas ligand and tumor necrosis factor 1 receptor-associated domain as well as the mitochondria-related, caspase-independent apoptosis-inducing factor were not detected. We also show that additional seizures, induced at different stages during neuronal differentiation of progenitor cell progeny following SE, neither potentiate cell death mechanisms in the SGZ nor compromise the survival of the new cells. Thus, we found similar expression of cytochrome c, active caspase-3, caspase-cleaved PARP, and TUNEL/Hoechst-positive DNA fragmentation, as well as numbers of new cells in the SGZ in rats exposed to additional seizures at days 6 and 7 or days 33 and 34 following SE as in control animals only subjected to SE. We propose that the degree of survival of newly generated neurons is determined primarily by the initial SE insult and the ensuing pathology in the tissue environment, whereas spontaneous seizures play a minor role.

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semi-thin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain-cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation.

Multiple lines of evidence implicate lysosomes in a variety of pathogenic events that produce neurodegeneration. Genetic mutations that cause specific enzyme deficiencies account for more than 40 lysosomal storage disorders. These mostly pre-adult diseases are associated with abnormal brain development and mental retardation. Such disorders are characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases including (i) Alzheimer's disease and related tauopathies (ii) Lewy body disorders and synucleinopathies such as Parkinson's disease, and (iii) Huntington's disease and other polyglutamine expansion disorders. Of particular interest for this review is evidence that alterations to the lysosomal system contribute to protein deposits associated with different types of age-related neurodegeneration. Lysosomes are in fact highly susceptible to free radical oxidative stress in the aging brain, leading to the gradual loss of their processing capacity over the lifespan of an individual. Several studies point to this lysosomal disturbance as being involved in amyloidogenic processing, formation of paired helical filaments, and the aggregation of α-synuclein and mutant huntingtin proteins. Most notably, experimentally induced lysosomal dysfunction, both in vitro and in vivo, recapitulates important pathological features of age-related diseases including the link between protein deposition and synaptic loss.

A large proportion of cells that proliferate in the adult dentate gyrus under normal conditions or in response to brain insults exhibit only short-term survival. Here, we sought to determine which cell death pathways are involved in the degeneration of newly formed neurons in the rat dentate gyrus following 2 h of electrically induced status epilepticus. We investigated the role of three families of cysteine proteases, caspases, calpains, and cathepsins, which can all participate in apoptotic cell death. Status epilepticus increased the number of bromodeoxyuridine (BrdU)-positive proliferated cells in the subgranular zone of the dentate gyrus. At the time of maximum cell proliferation, immunohistochemical analyses revealed protein expression of active caspase-cleaved poly (ADP-ribose) polymerase (PARP) in approximately 66% of the BrdU-positive cells, while none of them expressed cathepsin B or the 150-kDa calpain-produced fodrin breakdown product. To evaluate the importance of cysteine proteases in regulating survival of the newly formed neurons, we administered intracerebroventricular infusions of a caspase inhibitor cocktail (zVAD-fmk, zDEVD-fmk and zLEHD-fmk) over a 2-week period, sufficient to allow for neuronal differentiation, starting 1 week after the epileptic insult. Increased numbers of cells double-labelled with BrdU and neuron-specific nuclear protein (NeuN) marker were detected in the subgranular zone and granule cell layer of the caspase inhibitor-treated rats. Our data indicate that caspase-mediated cell death pathways are active in progenitor cell progeny generated by status epilepticus and compromise survival during neuronal differentiation.

Protein deposition is a common event in age-related neurological diseases that are characterized by neuronal dysfunction and eventual cell death. Here, cultured hippocampal slices were infused with the lysosomal disrupter chloroquine to examine the link between abnormal protein processing/deposition and early synaptopathogenesis. Tau species of 55 to 69 kDa increased over several days of treatment with chloroquine, while the protein and message levels of synaptic markers were selectively reduced. Neurons of subfields CA1, CA3, and dentate gyrus accumulated protein deposits recognized by antibodies against paired helical filaments and ubiquitin, and this was accompanied by tubulin fragmentation and deacetylation. The deposition filled the basal pole of pyramidal neurons, encompassing the area of the axon hillock and initial dendritic branching but without causing overt neuronal atrophy. Neurons containing the polar aggregates exhibited severely impaired transport along basal dendrites. Transport capability was also lost along apical dendrites, the opposite direction of deposited material in the basal pole; thus, perpetuating the problem beyond physical blockage must be the associated loss of microtubule integrity. These data indicate that transport failure forms a link between tau deposition and synaptic decline, thus shedding light on how protein aggregation events disrupt synaptic and cognitive functions before the ensuing cellular destruction.

In the brain, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate glutamatergic neurotransmission and, when intensely activated, can induce excitotoxic cell death. In addition to their ionotropic properties, however, AMPA receptors have been functionally coupled to a variety of signal transduction events involving Src-family kinases, G-proteins, and the mitogen-activated protein kinase (MAPK). In the present study, we tested whether AMPA receptors are linked to appropriate signaling events in order to prevent neuronal injury and/or enhance recovery. AMPA stimulation in hippocampal slice cultures caused the selective activation of MAPK through the upstream activator MAPK kinase (MEK). Inhibition of either component of the AMPA receptor--MAPK pathway potentiated cellular damage due to serum deprivation, suggesting that this pathway facilitates compensatory signals in response to injury. Correspondingly, positive modulation of AMPA receptors with the Ampakine 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516) enhanced MAPK activation and reduced the extent of synaptic and neuronal degeneration resulting from excitotoxic episodes. CX516 was neuroprotective when infused into slices either before or after the insult. The Ampakine derivative also elicited neuroprotection in an in vivo model of excitotoxicity as evidenced by reduction in lesion size and preservation of two different types of neurons. Interestingly, the AMPA receptor--MAPK pathway selectively protects against excitotoxicity since enhancing the pathway did not protect against the nonexcitotoxic, slow pathology initiated by lysosomal dysfunction. The results indicate that glutamatergic communication is important for cellular maintenance and that AMPA receptors activate survival signals to counterpoise their own excitotoxic potential.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer's disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5-1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways.

In this unit we describe an overlay brain slice culture assay for studying migration of transgenic neurospheres derived from human embryonic stem cells (hESC). Neuronal progenitor cells were generated from hESC by derivation of embryoid bodies and rosettes. Rosettes were transfected using the PiggyBac transposon system with either control plasmids (GFP) or plasmid encoding a gene important for migration of neuronal progenitor cells, Doublecortin (DCX). Transfected cells were subsequently grown in low-adhesion plates to generate transgenic human neurospheres (t-hNS). Organotypic slice cultures were prepared from postnatal rat forebrain and maintained using the interface method, before transfected t-hNS were overlaid below the cortex of each hemisphere. After 1 to 5 days, forebrain slices were fixed and processed for immunofluorescence. The distance at which cells migrated from the center of neurospheres to the host forebrain tissue was measured using Image J software. This protocol provides details for using the slice culture method for studying migration and integration of human neuronal cells into the host brain tissue.

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not μ-calpain, was facilitated in a dose-dependent manner in vitroby incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of “pathological apoptosis.”

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC50 of 4 μM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location. Synapse 40:154–158, 2001. © 2001 Wiley-Liss, Inc.

Caspase-3 is a major effector protease in several apoptotic pathways, but its role in hypoxic-ischemic (HI) brain injury is incompletely understood. Cerebral HI was induced in 7-day-old rats by unilateral carotid artery ligation and exposure to 7.7% oxygen for 55 min. Caspase-3-like activity was significantly increased at 1 h (208%), peaked at 24 h (2,563%) and was still increased 6 days after HI (169%) in the ipsilateral cerebral cortex. Concomitantly, cleavage of the caspase-3 proform (31/33 kD) was detected on immunoblots, producing 29- and 17-kD fragments. Furthermore, significant degradation of the endogenous caspase-3 substrates inhibitor of caspase-activated DNase (DNA fragmentation factor 45), poly(ADP-ribose) polymerase and fodrin occurred. In conclusion, caspase-3 is activated extensively in the immature brain after HI. The subsequent cleavage of proteins involved in cellular homeostasis and repair may contribute to the process of brain injury.

Adhesion responses triggered by integrin-class matrix receptors have been implicated in the synaptic reorganization events necessary for certain types of neuronal plasticity. Hippocampal slice cultures were used to test whether the related structural transformations elicited by NMDA receptor stimulation are regulated by integrin-type signals. Infusing the slices with NMDA for a short period induced the expected disassembly of the cytoskeletal network, measured with antibodies that selectively recognize spectrin cleavage sites targeted by the protease calpain. Marked levels of the 150-kDa breakdown product (BDP) were produced, whereas concentrations of the parent spectrin were not changed. Interestingly, the calpain cleavage events were attenuated by 60% when integrin-type signaling was disrupted with the antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP). This effect was RGDS-dependent, was largely evident in synapse-dense dendritic areas, particularly in subfield CA1, and was abolished when the NMDA exposure period was >5 min. These findings suggest that only those cytoskeletal alterations associated with brief synaptic activity are regulated by intact contact zones. AMPA-type glutamate receptors also were tested because, like spectrin, they are targets for calpain. Brief NMDA treatment caused a 15% loss of AMPA receptor GluR1 carboxytermini and this modification was augmented to 32% in the presence of GRGDSP. Thus, although blockage of matrix recognition signals decreased spectrin's susceptibility to disassembly, it increased the susceptibility of AMPA receptors to proteolysis. These data indicate that integrin-type signaling complexes are appropriately positioned to govern cytoskeletal reconfiguration while stabilizing the structural nature of AMPA receptors.

RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)–related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.

Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.

Recent work indicates that treatments which block adhesion receptors prevent the stabilization of long term potentiation (LTP). The experiments reported here show that brief stimulation of hippocampal NMDA receptors, a triggering event for LTP induction, results in the extracellular proteolysis of two or more members of the Cell Adhesion Molecule (CAM) family. This effect is rapid, occurs at a consensus serine protease site, and is selective to NMDA receptors. It is also found in vivo after kainic acid induced seizures. Cleavage of adhesive connections could be an early step in the formation of new synaptic configurations.

A critical issue concerning Alzheimer's disease is its selectivity, which leads to cellular degeneration in certain brain areas but not in others, and whether this pathogenic selectivity involves products of the amyloid precursor protein (APP). Here, we show that the amyloid beta protein Abeta1-42 is accumulated gradually and is retained intact by field CA1, but not by other subdivisions, of organotypic hippocampal slice cultures. In contrast, the slightly shorter Abeta1-40 peptide was not sequestered selectively. Sequestration of Abeta1-42 was followed by the build-up of carboxyterminal fragments of the endogenous precursor protein that were identified by immunoprecipitation. Unlike the peptide uptake, this induction appeared to be stochastic at the cellular level. In addition, the APP fragments were distributed more broadly within the CA1 pyramidal neurons than the sequestered Abeta1-42, and they appeared to be localized to synaptic terminals in the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of the subfield CA3. Concentrations of synaptophysin, a presynaptic marker, decreased as the number of neurons producing amyloidogenic species increased. These results indicate that exogenous Abeta1-42 sets into motion a sequence that involves 1) selective uptake of the peptide by vulnerable cells at risk in Alzheimer's disease, 2) markedly enhanced production of amyloidogenic precursor material, and 3) slow deterioration of central synapses.

Native tau isoforms were analyzed in adult mouse brain to determine whether they are differentially distributed and to identify molecular alterations that modify individual isoforms in an age-dependent manner. In general, the distribution profiles of 42-50 kDa tau were distinct from those of larger, hyperphosphorylated species of 55-69 kDa. The hippocampus and neocortex had concentrated levels of 55 kDa tau, and moderate amounts of 62-69 kDa isoforms. The latter species were similarly expressed in thalamic and hindbrain tissue; however, the noncortical regions were uniquely enriched in high molecular weight tau (97-110 kDa). When assessing hippocampal tau across age, increasing levels of 69 kDa tau were found to correlate with a gradual reduction in 42-50 kDa isoforms. Endogenous phosphatase activity induced an opposite correlation, thus supporting the idea that certain isoform conversions that occur with age stem from hyperphosphorylation. Age-related increases in 69 and 97 kDa tau also corresponded to enhanced levels of tau29, a putative tau fragment that exhibited an atypical localization (concentrated in olfactory bulb and hindbrain samples). These findings indicate that phosphorylation and fragmentation events influence tau distribution patterns, and that the former modification may promote the latter. They also raise the possibility that brain regions targeted by Alzheimer disease are distinguished by distinct tau profiles.

Effect of glycine on prelethal and postlethal increases in calpain activity in rat renal proximal tubules. The effect of glycine on hypoxia- and ionomycin-induced increases in calpain activity in rat proximal tubules was determined. Calpain activity was determined both in vitro and in the intact cell using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7- amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC) and Western blotting for calpain-specific spectrin breakdown products (BDP), respectively. At 7.5 minutes of hypoxia (prelethal injury model) there was a significant (10-fold) increase in in vitro calpain activity that was not inhibited by glycine. At 15 minutes of hypoxia (postlethal injury model) there was a further increase in calpain activity that was inhibited by glycine. Normoxic tubules incubated with the calcium ionophore ionomy-cin (5 µM) for two minutes and 10 minutes had a significant increase in calpain activity that was not inhibited by glycine. After 15 minutes of hypoxia in the presence of glycine, there was an increase in calpain-specific spectrin breakdown products (BDP) in both Triton X-100 soluble and cytosolic extracts from proximal tubules. Glycine in concentrations up to 10mM had no direct effect on the in vitro calpain activity of purified calpains. The present study demonstrates that: (1) prelethal increases in calpain activity stimulated by hypoxia and ionomycin treatment are not affected by glycine; (2) calpain-mediated spectrin breakdown during hypoxia occurs in the presence of glycine; (3) glycine does prevent the additional postlethal increase in calpain activity probably by maintaining membrane integrity to calcium influx.

The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino-terminal domain of gephyrin (GNN) and 2) its invariant carboxy-terminus (GNC). Both antibodies recognized an antigen with the expected molecular weight of 93–95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti-GNN was markedly distinct from that labeled by anti-GNC, a difference consistent with carboxy-terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy-terminal gephyrin fragments of 48–50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis. J. Neurosci. Res. 49:381–388, 1997. © 1997 Wiley-Liss, Inc.

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Recently, the neurodegenerative component of multiple sclerosis has come under focus particularly because permanent disability in patients correlates well with neurodegeneration; and observations in both humans and multiple sclerosis animal models highlight neurodegeneration of retinal ganglion cells as an early event. After myelin oligodendrocyte glycoprotein immunization of Brown Norway rats, significant retinal ganglion cell loss precedes the onset of pathologically defined autoimmune optic neuritis. To study the role calcium and calpain activation may play in mediating early degeneration, manganese-enhanced magnetic resonance imaging was used to monitor preclinical calcium elevations in the retina and optic nerve of myelin oligodendrocyte glycoprotein-immunized Brown Norway rats. Calcium elevation correlated with an increase in calpain activation during the induction phase of optic neuritis, as revealed by increased calpain-specific cleavage of spectrin. The relevance of early calpain activation to neurodegeneration during disease induction was addressed by performing treatment studies with the calpain inhibitor calpeptin. Treatment not only reduced calpain activity but also protected retinal ganglion cells from preclinical degeneration. These data indicate that elevation of retinal calcium levels and calpain activation are early events in autoimmune optic neuritis, providing a potential therapeutic target for neuroprotection.

Heparin was found to bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [3H]AMPA binding to detergent-solubilized receptors by 75% (IC50 = 10 micrograms/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [3H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [3H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 micrograms/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

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.

Cultured hippocampal slices retain many in vivo features with regard to circuitry, synaptic plasticity, and pathological responsiveness, while remaining accessible to a variety of experimental manipulations. The present study used ligand binding, immunostaining, and in situ hybridization assays to determine the stability of AMPA- and NMDA-type glutamate receptors and other synaptic proteins in slice cultures obtained from 11 day postnatal rats and maintained in culture for at least 4 weeks. Binding of the glutamate receptor ligands [3H]AMPA and [3H]MK-801 exhibited a small and transient decrease immediately after slice preparation, but the binding levels recovered by culture day (CD) 5-10 and remained stable for at least 30 days in culture. Autoradiographic analyses with both ligands revealed labeling of dendritic fields similar to adult tissue. In addition, slices at CD 10-20 expressed a low to high affinity [3H]AMPA binding ratio that was comparable with that in the adult hippocampus (10:1). AMPA receptor subunits GluR1 and GluR2/3 and an NMDA receptor subunit (NMDAR1) exhibited similar postcutting decreases as that exhibited by the ligand binding levels, followed by stable recovery. The GluR4 AMPA receptor subunit was not evident during the first 10 CDs but slowly reached detectable levels thereafter in some slices. Immunocytochemistry and in situ hybridization techniques revealed adult-like labeling of subunit proteins in dendritic processes and their mRNAs in neuronal cell body layers. Long-term maintenance was evident for other synapse-related proteins, including synaptophysin, neural cell adhesion molecule isoforms (NCAMs), and an AMPA receptor related antigen (GR53), as well as for certain structural and cytoskeletal components (e.g., myelin basic protein, spectrin, microtubule-associated proteins). In summary, following an initial and brief depression, many synaptic components were expressed at steady-state levels in long-term hippocampal slices, thus allowing the use of such a culture system for investigations into mechanisms of brain synapses.

Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak cathepsin B and L inhibitor Z-Phe-Ala-diazomethylketone (PADK) enhances lysosomal cathepsin levels at low concentrations, thereby eliciting protective clearance of PHF-τ and Aβ42 in the hippocampus and other brain regions. Here, a class of positive modulators is established with compounds decoupled from the cathepsin inhibitory properties. We utilized PADK as a departure point to develop nonpeptidic structures with the hydroxyethyl isostere. The first-in-class modulators SD1002 and SD1003 exhibit improved levels of cathepsin up-regulation but almost complete removal of cathepsin inhibitory properties as compared to PADK. Isomers of the lead compound SD1002 were synthesized, and the modulatory activity was determined to be stereoselective. In addition, the lead compound was tested in transgenic mice with results indicating protection against AD-type protein accumulation pathology.

To test whether aging is associated with alterations in the balance of cytoskeletal constituents, the relative concentrations of tau isoforms and seven other structural proteins were compared in the brains of 3–25 month-old mice. A tau species of approx. 63 kDa was substantially increased in the older animals while the levels of ankyrin, talin, spectrin, and actin were differentially decreased. The decrement in ankyrin was evident at earlier ages than that for spectrin. These results suggest that the make-up of the neuronal cytoskeleton changes with age.

The hypothesis that intense stimulation of NMDA receptors activates calpain was tested in long-term cultures of hippocampus. Slices prepared from 10-day-old rats were maintained for periods of up to 6 weeks and then assayed for a stable breakdown product that results from the proteolysis of spectrin by calpain. The breakdown product increased dramatically during the first 24 h after tissue preparation and then decreased to a low level that remained unchanged for weeks. NMDA caused a 2- to 3-fold increase in breakdown product that rose linearly with time (5-30 min) and was blocked by the receptor antagonist MK-801. The effect of NMDA was the same throughout the culture period and was dependent upon the concentration of extracellular calcium with no effect at 2 mM and maximal effect at 4 mM calcium. These results indicate that rapid activation of calpain occurs in undamaged hippocampal neurons following stimulation of NMDA receptors.

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.

Two forms of antisense technology were used to explore the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, Fl, P-57) in the brain. The first method exploited the ability of dividing cell lines to take up and stably integrate recombinant retrovirus vectors. We transfected PC12 cells with a recombinant expression vector containing a human GAP-43 cDNA in reverse (antisense) orientation. Retrieval and characterization of two stable transfectants, AS1 and AS2, expressing high levels of antisense GAP-43 RNA revealed that the level of GAP-43 protein in these cell lines was reduced relative to controls. in the presence of extraceilular calcium, a depolarizing concentration of K + failed to stimulate dopamine release from AS1 and AS2 cells although evoked dopamine release from control cells was normal. Similarly, the calcium ionophore A23189 was ineffective in stimulating dopamine release from the cells expressing GAP-43 antisense cRNA even though it evoked dopamine release from control cells. These data suggest that GAP-43 plays an essential role in calcium-dependent neurotransmitter release in PC12 cells. The antisense GAP-43-transfected PC12 cells displayed processes of normal length when treated with NGF on polylysine or laminin substrates. in contrast, primary rat hippocampal cell cultures plated in the presence of GAP-43 antisense oligonucleotides, in a series of experiments exploiting a second antisense technology, were deficient In process outgrowth. This deficiency was dose-dependent. To venture further into the realm of GAP-43 function in the brain, we have designed a procedure to depress levels of GAP-43 protein in cultured hippocampal slices.

Apoptosis-related mechanisms are important in the pathophysiology of hypoxic–ischemic injury in the neonatal brain. Caspases are the major executioners of apoptosis, but there are a number of upstream players that influence the cell death pathways. The Bcl-2 family proteins are important modulators of mitochondrial permeability, working either to promote or prevent apoptosis. In this study we focused on the anti-apoptotic Bcl-2 protein after neonatal cerebral hypoxia–ischemia (HI) in 8-day-old rats. Bcl-2 translocated to nuclei and accumulated there over the first 24 h of reperfusion after HI, as judged by immunohistochemistry and immuno-electron microscopy. We also found that the total level of Bcl-2 decreased after HI in vivo and after ionophore challenge in cultured human neuroblastoma (IMR-32) cells in vitro. Furthermore, the Bcl-2 reduction was calpain-dependent, because it could be prevented by the calpain inhibitor CX295 both in vivo and in vitro, suggesting cross-talk between excitotoxic and apoptotic mechanisms.

Aims This review posits that fatty acid amide hydrolase (FAAH) inhibition has therapeutic potential against neuropathological states including traumatic brain injury; Alzheimer's, Huntington's, and Parkinson's diseases; and stroke. Main methods This proposition is supported by data from numerous in vitro and in vivo experiments establishing metabolic and pharmacological contexts for the neuroprotective role of the endogenous cannabinoid (“endocannabinoid”) system and selective FAAH inhibitors. Key findings The systems biology of endocannabinoid signaling involves two main cannabinoid receptors, the principal endocannabinoid lipid mediators N -arachidonoylethanolamine (“anandamide”) (AEA) and 2-arachidonoyl glycerol (2-AG), related metabolites, and the proteins involved in endocannabinoid biosynthesis, biotransformation, and transit. The endocannabinoid system is capable of activating distinct signaling pathways on-demand in response to pathogenic events or stimuli, thereby enhancing cell survival and promoting tissue repair. Accumulating data suggest that endocannabinoid system modulation at discrete targets is a promising pharmacotherapeutic strategy for treating various medical conditions. In particular, neuronal injury activates cannabinoid signaling in the central nervous system as an intrinsic neuroprotective response. Indirect potentiation of this salutary response through pharmacological inhibition of FAAH, an endocannabinoid-deactivating enzyme, and consequent activation of signaling pathways downstream from cannabinoid receptors have been shown to promote neuronal maintenance and function. Significance This therapeutic modality has the potential to offer site- and event-specific neuroprotection under conditions where endocannabinoids are being produced as part of a physiological protective mechanism. In contrast, direct application of cannabinoid receptor agonists to the central nervous system may activate CB receptors indiscriminately and invite unwanted psychotrophic effects.

An activated form of calpain I associates with telencephalic membranes in a developmentally regulated fashion during early postnatal ontogeny. During this period, the cytoskeletal component spectrin is available and appears to be differentially susceptible to calpain-mediated cleavage. Lectin blotting techniques demonstrated that the leupeptin-sensitive action of calpain is primarily directed toward large proteins which are glycoconjugate in nature; neuronal cell adhesion molecules are among the glycoproteins whose associations with the telencephalic membranes decrease due to calpain activity. These data suggest that cytoplasmic calpain is translocated to the membrane during early brain development in order to act on the cytoskeletal and adhesive structures responsible in part for neuronal shape and function.

Calcium activated proteolysis of brain spectrin produces characteristic breakdown products (BDPs), the concentrations of which increase markedly in many instances of brain pathology. Results reported here indicate that levels of the BDPs rise with age (3–30 months) in the telencephalon but not in the hindbrain of Balb/c mice. These observations suggest that spectrin breakdown is a pathologic biochemical marker which increases with age in some but not all brain regions.

Slices of hippocampus were incubated with Arg-Gly-Asp (RGD) peptides known to block members of the integrin class of matrix receptors. Though the peptides caused no detectable difference in the amount of long-term potentiation (LTP) expressed in the CA1 field 1-2 min after induction with high frequency stimulation, they did produce a reversible, dose dependent decay of LTP over a period of 40 min. This effect was not obtained with various non-RGD control peptides. These results suggest that stabilization of LTP requires adhesive interactions via specific matrix recognition sites, whereas induction and expression do not.

Immunochemical methods were used to test whether vitronectin receptors exist in synaptosomal membranes (SPMs) and hence are positioned to play a role in synaptic adhesion. Antibodies against the alpha v beta 3 integrin detected proteins in brain homogenates that correspond to conventional integrin subunits. Conversely, these antigens were not found in SPMs prepared from the same brain tissue. The antibodies did, however, express strong immunoreactivity towards a 27 kDa polypeptide that was greatly concentrated in SPMs from major brain regions and that was not found in tissues other than brain. This is an example of an integrin epitope contained in a synaptic polypeptide that is too small to be a conventional matrix receptor, thus, suggesting the possibility that synaptic adhesion involves unusual proteins.

Synaptosomal plasma membranes (SPMs) from adult rat brain tested for fibronectin binding and antigenicity toward antibodies against integrin-type fibronectin receptors. Binding (1-10 pmol mg-1 protein) was considerably greater in SPMs than in homogenates for hippocampus and neocortex but not for brain stem. The tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine blocked up to 90% of the binding indicating that integrin-type receptors are present. Antibodies against two sub-classes of integrins detected high Mr integrin subunits in homogenates but not in SPMs. However, antibodies against hamster ovarian fibronectin receptor reacted intensely with polypeptides of 55,000 and 40,000 Mr that were concentrated 20 to 40-fold in SPMs compared to homogenates. These peptides may constitute a new class of matrix receptor involved in synaptic adhesion.

We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic post-synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post-synaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main post-synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4-7. An engineered deletion construct of GRIP1a/b (GRIP1a4-7), which both contains the aforementioned common region and binds to gephyrin, targets to the post-synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the post-synaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post-synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.

AMPAkines are positive modulators of AMPA receptors, and previous work has shown that these compounds can facilitate synaptic plasticity and improve learning and memory in both animals and humans; thus, their role in the treatment of cognitive impairment is worthy of investigation. In this study, we have utilized an organotypic slice model in which chloroquine-induced lysosomal dysfunction produces many of the pathogenic attributes of Alzheimer's disease. Our previous work demonstrated that synaptic AMPA receptor function is impaired in hippocampal slice cultures exhibiting lysosomal dysfunction leading to protein accumulation. The present study investigated the effect of the AMPAkine CX516 on AMPAR-mediated synaptic transmission as well as the CX516 induced modification of single channel AMPA receptor properties in this organotypic slice-culture model. In whole cell recordings from CA1 pyramidal neurons in chloroquine-treated slices we observed a significant decrease in AMPAR-mediated mEPSC frequency and amplitude indicating synaptic dysfunction. Following application of CX516, these parameters returned to nearly normal levels. Similarly, we report chloroquine-induced impairment of AMPAR single channel properties (decreased probability of opening and mean open time), and significant recovery of these properties following CX516 administration. These results suggest that AMPA receptors may be potential pharmaceutical targets for the treatment of neurodegenerative diseases, and highlights AMPAkines, in particular, as possible therapeutic agents.

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro-Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline-treated rats. Immunohistochemistry and western blot analysis for the calpain-derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus.

Poor survival of cells transplanted into the CNS is a widespread problem and limits their therapeutic potential. Whereas substantial loss of transplanted cells has been described, the extent of acute cell loss has not been quantified previously. To assess the extent and temporal profile of transplanted cell death, and the contributions of necrosis and apoptosis to this cell death following spinal cord injury, different concentrations of Schwann cells (SCs), lentivirally transduced to express green fluorescent protein (GFP), were transplanted into a 1-week-old moderate contusion of the adult rat thoracic spinal cord. In all cases, transplanted cells were present from 10 min to 28 days. There was a 78% reduction in SC number within the first week, with no significant decrease thereafter. Real-time polymerase chain reaction showed a similar 80% reduction in GFP-DNA within the first week, confirming that the decrease in SC number was due to death rather than decreased GFP transgene expression. Cells undergoing necrosis and apoptosis were identified using antibodies against the calpain-mediated fodrin breakdown product and activated caspase 3, respectively, as well as ultrastructurally. Six times more SCs died during the first week after transplantation by necrosis than apoptosis, with the majority of cell death occurring within the first 24 h. The early death of transplanted SCs indicates that factors present, even 1 week after a moderate contusion, are capable of inducing substantial transplanted cell death. Intervention by strategies that limit necrosis and/or apoptosis should be considered for enhancing acute survival of transplanted cells.

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurological injury resulting from birth complications and pre-maturity. Our studies have demonstrated that this injury depletes the subventricular zone (SVZ) of progenitors. In this study, we sought to reveal which cell death pathways are activated within these progenitors after H/I. We found that calpain activity is detected as early as 4 h of reperfusion and is sustained for 48 h, while caspase 3 activation does not occur until 8 h and peaks at 24 h post-insult. Activated calpains and caspase 3 co-localized within precursors situated in the lateral aspects of the SVZ (which coincides with progenitor cell death), whereas neither enzyme was activated in the medial SVZ (which harbors the neural stem cells that are resilient to this insult). These studies reveal targets for neuroprotective agents to protect precursors from cell death towards the goal of restoring normal brain development after H/I.

Endocannabinoids are released in response to pathogenic insults, and inhibitors of endocannabinoid inactivation enhance such on-demand responses that promote cellular protection. Here, AM374 (palmitylsulfonyl fluoride), an irreversible inhibitor of fatty acid amide hydrolase (FAAH), was injected i.p. into rats to test for endocannabinoid enhancement. AM374 caused a prolonged elevation of anandamide levels in several brain regions, including the hippocampus, and resulted in rapid activation of the extracellular signal regulated-kinase/mitogen-activated protein kinase pathway that has been linked to survival. To evaluate the neuroprotective nature of the FAAH inhibitor, we tested AM374 in a seizure model involving rats insulted with kainic acid (KA). AM374 was injected immediately after KA administration, and seizure scores were significantly reduced throughout a 4-h observation period. The KA-induced seizures were associated with calpain-mediated cytoskeletal breakdown, reductions in synaptic markers, and loss of CA1 hippocampal neurons. FAAH inhibition protected against the excitotoxic damage and neuronal loss assessed 48 h postinsult. AM374 also preserved pre- and postsynaptic markers to levels comparable with those found in noninsulted animals, and the synaptic marker preservation strongly correlated with reduced seizure scores. With regard to behavioral deficits in the excitotoxic rats, AM374 produced nearly complete functional protection, significantly improving balance and coordination across different behavioral paradigms. These data indicate that AM374 crosses the blood-brain barrier, enhances endocannabinoid responses in key neuronal circuitries, and protects the brain against excitotoxic damage.

The brains of Alzheimer's disease (AD) patients have large numbers of plaques that contain amyloid beta (Abeta) peptides which are believed to play a pivotal role in AD pathology. Several lines of evidence have established the inhibitory role of Abeta peptides on hippocampal memory encoding, a process that relies heavily on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function. In this study the modulatory effects of the two major Abeta peptides, Abeta(1-40) and Abeta(1-42), on synaptic AMPA receptor function was investigated utilizing the whole cell patch clamp technique and analyses of single channel properties of synaptic AMPA receptors. Bath application of Abeta(1-42) but not Abeta(1-40) reduced both the amplitude and frequency of AMPA receptor mediated excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons by approximately 60% and approximately 45%, respectively, in hippocampal CA1 pyramidal neurons. Furthermore, experiments with single synaptic AMPA receptors reconstituted in artificial lipid bilayers showed that Abeta(1-42) reduced the channel open probability by approximately 42% and channel open time by approximately 65% and increased the close times by several fold. Abeta(1-40), however, did not show such inhibitory effects on single channel properties. Application of the reverse sequence peptide Abeta(42-1) also did not alter the mEPSC or single channel properties. These results suggest that Abeta(1-42) but not Abeta(1-40) closely interacts with and exhibits inhibitory effects on synaptic AMPA receptors and may contribute to the memory impairment observed in AD.