Amyloid beta-induced neuronal death is bax-dependent but caspase-independent

Fibrillar amyloid beta (Abeta) peptides are major constituents of senile plaques in Alzheimer disease (AD) brain and cause neuronal apoptosis in vitro. Bax and caspase-3 have been implicated in the pathogenesis of AD and are components of a well-defined molecular pathway of neuronal apoptosis. To determine whether Abeta-induced neuronal apoptosis involves bax and/or caspase-3 activation, we examined the effect of Abeta on wild-type, bax-deficient, and caspase-3-deficient telencephalic neurons in vitro. In wild-type cultures, Abeta produced time- and concentration-dependent caspase-3 activation, apoptotic nuclear changes, and neuronal death. These neurotoxic effects of Abeta were not observed in bax-deficient cultures. Caspase-3 deficiency, or pharmacological inhibition of caspase activity, prevented caspase-3 activation and blocked the appearance of apoptotic nuclear features but not Abeta-induced neuronal death. Neither calpain inhibition nor microtubule stabilization with Taxol protected telencephalic neurons from Abeta-induced caspase activation or apoptosis. These results have potential implications regarding the underlying pathophysiology of AD and towards AD treatment strategies.     

In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease.

The mechanism by which cells die in Alzheimer disease (AD) is unknown. Several investigators speculate that much of the cell loss may be due to apoptosis, a highly regulated form of programmed cell death. Caspase-3 is a critical effector of neuronal apoptosis and may be inappropriately activated in AD. To address this possibility, we examined cortical and hippocampal brain sections from AD patients, as well as 2 animal models of AD, for in situ evidence of caspase-3 activation. We report here that senile plaques and neurofibrillary tangles in the AD brain are not associated with caspase-3 activation. Furthermore, amyloid beta (A beta) deposition in the APPsw transgenic mouse model of AD does not result in caspase-3 activation despite the ability of A beta to induce caspase-3 activation and neuronal apoptosis in vitro. AD brain sections do, however, exhibit caspase-3 activation in hippocampal neurons undergoing granulovacuolar degeneration. Our data suggests that caspase-3 does not have a significant role in the widespread neuronal cell death that occurs in AD, but may contribute to the specific loss of hippocampal neurons involved in learning and memory.     

A beta 25-35 induces presynaptic changes in organotypic hippocampal slice cultures

Memory loss in Alzheimer's disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide A beta 25-35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, A beta 25-35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in A beta 25-35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed A beta 25-35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by A beta 25-35 in CA1, CA3 and DG area at concentrations >2.5 microM. In conclusion, A beta 25-35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 microM in organotypic hippocampal slice cultures. These suggest that A beta 25-35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.     

Role of caspase-3 in ethanol-induced developmental neurodegeneration

Acute, transient exposure to ethanol causes a widespread pattern of caspase-3 activation and neuroapoptosis in the developing rodent brain. To determine whether caspase-3 activation is an essential step in ethanol-induced developmental neuroapoptosis, we treated homozygous caspase-3 knockout mice or wild-type mice on postnatal day 7 with an apoptosis-inducing dose of ethanol and examined the brains at appropriate survival times for evidence of apoptotic neurodegeneration. In caspase-3 knockout mice, the cell death process evolved more slowly than in wild-type mice, and morphological changes observed were not those typically associated with apoptosis. However, neuronal cell counts performed 2 weeks post-treatment revealed that the extent of neuron loss was similar in wild-type and caspase-3-deficient mice. We conclude that absence of functional caspase-3 alters the time course and morphological characteristics of the neurodegenerative process but does not prevent ethanol-induced neuron death.     

Chloroquine-induced neuronal cell death is p53 and Bcl-2 family-dependent but caspase-independent

Chloroquine is a lysosomotropic agent that causes marked changes in intracellular protein processing and trafficking and extensive autophagic vacuole formation. Chloroquine may be cytotoxic and has been used as a model of lysosomal-dependent cell death. Recent studies indicate that autophagic cell death may involve Bcl-2 family members and share some features with caspase-dependent apoptotic death. To determine the molecular pathway of chloroquine-induced neuronal cell death, we examined the effects of chloroquine on primary telencephalic neuronal cultures derived from mice with targeted gene disruptions in p53, and various caspase and bcl-2 family members. In wild-type neurons, chloroquine produced concentration- and time-dependent accumulation of autophagosomes, caspase-3 activation, and cell death. Cell death was inhibited by 3-methyladenine, an inhibitor of autophagic vacuole formation, but not by Boc-Asp-FMK (BAF), a broad caspase inhibitor. Targeted gene disruptions of p53 and bax inhibited and bcl-x potentiated chloroquine-induced neuron death. Caspase-9- and caspase-3-deficient neurons were not protected from chloroquine cytotoxicity. These studies indicate that chloroquine activates a regulated cell death pathway that partially overlaps with the apoptotic cascade.     

p35/Cyclin-dependent kinase 5 is required for protection against β-amyloid-induced cell death but not tau phosphorylation by ceramide

Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.     

Prion protein protects against ethanol-induced Bax-mediated cell death in vivo

Prion protein inhibits Bax activation and Bax-mediated cell death in primary cultures of human neurons and in MCF-7 cells. To determine whether prion protein can protect against Bax-mediated cell death in vivo, wild-type, null and prion over-expressing mice were subjected to Bax-dependent ethanol induced neuronal apoptotic cell death and the brains were immunostained for active caspase-3 as a downstream marker of Bax activation. Bax activation occurs in all ethanol-injected mice independent of their genotype. A higher level of cell death is present in ethanol-injected null mice than in wild-type and prion over-expressing mice. We conclude that prion protein protects some, but not all neurons, against Bax-mediated cell death in this experimental paradigm.     

JNK activation is associated with intracellular beta-amyloid accumulation

c-Jun has been implicated in the pathogenesis of Alzheimer's disease (AD), but the upstream cascade leading to c-Jun activation in AD is not known. Activation of c-Jun N-terminal kinase (JNK) is obviously a candidate for the upstream event. We tested this possibility focusing on PS1-linked AD. First, we observed that JNK is actually activated in cerebral neurons of PS1-linked AD patients, using immunohistochemistry and Western blot analyses with anti-activated JNK antibodies. We analyzed the relationship between beta-amyloid (beta A) and JNK activation by using aged transgenic mice overexpressing mutant (M146L) PS1 and human AD brains. The mice showed no neuronal loss but a very few diffuse beta A deposits, corresponding to the early stage of PS1-linked AD brain. Some neurons were reactive for anti-beta A antibodies in the cerebral cortex. Interestingly, JNK activation was observed in neurons showing intracellular beta A immunoreactivity in transgenic mice. Association between intracellular beta A and JNK activation was confirmed in cortical neurons of sporadic and PS1-linked AD patients. Furthermore, introduction of beta A peptides into the primary culture cortical neurons induced JNK activation and cell death. Collectively, these results suggested that intracellular beta A accumulation might trigger JNK activation leading to neuronal death.     

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide

Studies with in-vitro-cultured neurons treated with amyloid-beta (A beta) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca(2+) homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-12, which is activated upon the perturbation of ER Ca(2+) homeostasis, may contribute to A beta toxicity. To elucidate the involvement of deregulation of ER Ca(2+) homeostasis in neuronal death induced by A beta peptides, we have performed a comparative study using the synthetic peptides A beta(25-35) or A beta(1-40) and thapsigargin, a selective inhibitor of Ca(2+) uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 microM) increased the intracellular Ca(2+) levels and activated caspase-3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the A beta peptides (A beta(25-35), 25 microM; A beta(1-40), 0.5 microM), we observed a significant increase in [Ca(2+)](i), in caspase-3-like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca(2+) release through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP(3)R) in A beta neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca(2+) release through RyR or IP(3)R, were able to prevent the increase in [Ca(2+)](i) and the activation of caspase-3 and to protect partially against apoptosis induced by treatment with A beta(25-35) or A beta(1-40). In conclusion, our results demonstrate that the release of Ca(2+) from the ER, mediated by both RyR and IP(3)R, is involved in A beta toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to A beta-induced neuronal death. This study suggests that A beta accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca(2+) homeostasis.     

The enigma of cell death in neurodegenerative disorders

Progressive cell loss in specific neuronal populations is the pathological hallmark of neurodegenerative diseases, but its mechanisms remain unresolved. Apoptotic cell death has been implicated as a major mechanism in Alzheimer disease (AD), Parkinson disease (PD) and other neurodegenerative disorders. However, DNA fragmentation in human brain as a sign of neuronal cell injury is too frequent to account for the continuous loss in these slowly progressive diseases. In a series of autopsy confirmed cases of AD, PD, related disorders, and age-matched controls, DNA fragmentation using the TUNEL method, an array of apoptosis-related proteins (ARP), proto-oncogenes, and activated caspase-3, the key enzyme of late-stage apoptosis, were examined. In AD, a considerable number of hippocampal neurons and glial cells showed DNA fragmentation with a 3- to 6-fold increase related to neurofibrillary tangles and amyloid deposits, but only 1 in 2.600 to 5.600 neurons displayed apoptotic morphology and cytoplasmic immunoreactivity for activated caspase-3, whereas no neurons were labeled in age-matched controls. caspase-3 immunoreactivity was seen in granules of cells with granulovacuolar degeneration, in around 25% co-localized with early cytoplasmic deposition of tau-protein. In progressive supranuclear palsy, only single neurons and several oligodendrocytes in brainstem, some with tau-deposits, were TUNEL-positive and expressed both ARPs and activated caspase-3. In PD, dementia with Lewy bodies, multisystem atrophy (MSA), and corticobasal degeneration, TUNEL-positivity and expression of ARPs or activated caspase-3 were only seen in microglia and oligodendrocytes with cytoplasmic inclusions, but not in neurons. These data provide evidence for extremely rare apoptotic neuronal death in AD and PSP compatible with the progression of neuronal degeneration in these chronic diseases. Apoptosis mainly involves reactive microglia and oligodendroglia, the latter often involved by deposits of insoluble fibrillary proteins, while alternative mechanisms of neuronal death may occur. Susceptible cell populations in a proapoptotic environment show increased vulnerability towards metabolic or other noxious factors, with autophagy as a possible protective mechanism in early stages of programmed cell death. The intracellular cascade leading to cell death still awaits elucidation.     

Amyloid beta peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation.

Amyloid beta peptide (A beta), a 39 to 43 amino acid fragment of the beta-amyloid precursor protein (betaAPP), forms insoluble fibrillar accumulation in neurofibrillary tangles and vascular plaques. A beta has been implicated in neuronal and vascular degeneration in brain regions susceptible to plaque formation because of its cytotoxic effect on neurons and endothelial cells (ECs). The authors used a murine cerebral endothelial cell (CEC) line and primary cultures of bovine CECs to explore the cytotoxic mechanism of A beta. A beta 1-40 and A beta 25-35 peptides caused cell death in a dose-dependent and time-dependent manner. Exposure to either A beta 25-35 or A beta 1-40 at 10 micromol/L for 48 hours caused at least 40% cell death. Cerebral endothelial cell death was characterized by nuclear condensation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage. A beta 25-35 activated both caspase-8 and caspase-3 in murine CECs. zVAD-fmk, a broad-spectrum caspase inhibitor, prevented A beta 25-35-induced increase in caspase-3 activity and CEC death. N-acetyl-cysteine, an antioxidant, also prevented A beta-induced cell death. Together, these findings indicate that A beta-mediated CEC death is an apoptotic process that is characterized by increased oxidative stress, caspase activation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage.     

Fucoidan inhibits cellular and neurotoxic effects of beta-amyloid (A beta) in rat cholinergic basal forebrain neurons

The deposition of beta-amyloid protein (A beta), a 39-43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimer's disease (AD). Seaweeds consumed in Asia contain Fucoidan, a sulfated polysaccharide. Fucoidan has been known to exhibit various biological actions, such as an anti-inflammatory and antioxidant action. In this study, using whole-cell patch clamp recordings we examined the effects of Fucoidan on A beta-induced whole-cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking A beta neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of A beta(25-35) (1 microM) caused a reduction of the whole-cell currents by 16%. Fucoidan, in a dose-dependent manner, blocks the A beta(25-35) reduction of whole-cell currents. Exposure of A beta(25-35) (20 microM) or A beta(1-42) (20 microM) to rat cholinergic basal forebrain cultures for 48 h resulted in 40-60% neuronal death, which was significantly decreased by pretreatment of cultures with Fucoidan (0.1-1.0 microM). Fucoidan also attenuated A beta-induced down-regulation of phosphorylated protein kinase C. A beta(1-42)-induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, A beta activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block A beta-induced reduction in whole-cell currents in basal forebrain neurons and has neuroprotective effects against A beta-induced neurotoxicity in basal forebrain neuronal cultures.     

Apoptotic signals within the basal forebrain cholinergic neurons in Alzheimer's disease

A relatively early and substantial loss of basal forebrain cholinergic neurons is a constant feature of Alzheimer's disease (AD). However, the mechanisms that contribute to the selective vulnerability of these neurons are not fully delineated. In the present series of experiments, we determined the possible contribution of apoptotic processes and other pathologic cascades to the degeneration of the cholinergic neurons of the nucleus basalis of Meynert (NBM) in AD. In contrast to neurons in the frontal cortex which showed prominent DNA fragmentation as detected by the TUNEL method, no DNA fragmentation was observed within the NBM in any of the AD or normal brains. Similarly, immunoreactivity for the apoptotic signals Fas, Fas-ligand, Bax, Bcl-x, caspase-8, caspase-9 and caspase-3 was absent from the NBM of AD and control brains. In contrast, a substantial subpopulation of cholinergic neurons within the NBM in AD displayed prominent immunoreactivity for the apoptotic signal Fas-associated death domain (FADD) in the form of tangles. FADD immunoreactivity was also present in dystrophic neurites. FADD-positive tangle-like structures were localized in neurons which contained immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT) and the low affinity neurotrophin receptor p75NTR. While many of the NBM cholinergic neurons in control brains contained immunoreactivity for the calcium binding protein calbindin-D28K (CB), the NBM neurons in AD displayed a substantial loss of CB immunoreactivity. Importantly, most of FADD-immunoreactive cholinergic neurons were devoid of CB immunoreactivity, and, conversely, most CB-positive cholinergic neurons had no FADD immunoreactivity. FADD immunoreactivity within the basal forebrain was colocalized with phosphorylated tau immunoreactive tangles and dystrophic neurites. In contrast, FADD immunoreactivity did not appear to be related to the primarily diffuse amyloid-beta deposits intermingled between cholinergic neurons in AD NBM. Finally, many CD68-positive microglia were observed surrounding the NBM cholinergic neurons in AD. In conclusion, the findings of the present study indicate that, while the FADD apoptotic signaling pathway may be triggered within the basal forebrain cholinergic neurons in AD, the apoptotic cascade is most likely aborted as no DNA fragmentation was detected and the executioner caspase-3 was not up-regulated within these neurons. The findings also suggest possible relationships between loss of CB, FADD expression and phosphorylation of tau within the basal forebrain cholinergic neurons in AD.     

Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.     

cAMP delays beta-amyloid (25-35) induced cell death in rat cortical neurons

Beta-amyloid (A beta) accumulation is believed to contribute to neuronal cell death in Alzheimer's disease. To understand the role of cAMP in the regulation of A beta induced cell death, we used 8-chlorophenylthio-cAMP (8-CPT-cAMP, a cAMP analog) to raise intracellular cAMP levels. Exposure of rat cortical neurons to A beta(25-35) resulted in a gradual increase in lactate dehydrogenase (LDH) over 48 h, which was preceded by a transient elevation in caspase-3-like activity. In the presence of 8CPT-cAMP, both caspase-3 activity and LDH release was significantly reduced. These data suggest that elevation of intracellular cAMP levels attenuate A beta-induced neurotoxicity and may delay or prevent the onset of A beta-induced neurodegeneration.     

Evidence for involvement of Bax and p53, but not caspases,in radiation-induced cell death of cultured postnatal hippocampal neurons

Bax (a death-promoting member of the bcl-2 gene family), the tumor suppressor gene product p53, and the ICE/ced-3-related proteases (caspases) have all been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation-induced neuronal cell death are poorly understood. In order to further elucidate the molecular mechanisms underlying radiation-induced neuronal cell death, we have examined the ability of ionizing radiation to induce cell death in primary cultured hippocampal neurons obtained from wild-type, p53-deficient and Bax-deficient newborn mice. Survival in neuronal cultures derived from wild-type mice decreased in a dose-dependent manner 24 hr after a single 10 Gy to 30 Gy dose of ionizing radiation. In contrast, neuronal survival in irradiated cultures derived from p53-deficient or Bax-deficient mice was equivalent to that observed in control, nonirradiated cultures. Western blot analyses indicated that neuronal p53 protein levels increased after irradiation in wild-type cells. However, Bax protein levels did not change, indicating that other mechanisms exist for regulating Bax activity. Adenovirus-mediated overexpression of p53 also caused neuronal cell death without increasing Bax protein levels. Irradiation resulted in a significant induction in caspase activity, as measured by increased cleavage of fluorogenic caspase substrates. However, specific inhibitors of caspase activity (zVAD-fmk, zDEVD-fmk and BAF) failed to protect postnatal hippocampal neurons from radiation-induced cell death. Staurosporine (a potent inducer of apoptosis in many cell types) effectively induced neuronal cell death in wild-type, p53-deficient and Bax-deficient hippocampal neurons, indicating that all were competent to undergo programmed cell death. These results demonstrate that both p53 and Bax are necessary for radiation-induced cell death in postnatal cultured hippocampal neurons. The fact that cell death occurred despite caspase inhibition suggests that radiation-induced neuronal cell death may occur in a caspase-independent manner. J. Neurosci. Res. 54:721–733, 1998. © 1998 Wiley-Liss, Inc.     

Caspases, apoptosis, and Alzheimer disease: causation, correlation, and confusion

Extensive neuron loss occurs in Alzheimer disease (AD) brain and some authors have speculated that dysregulation of apoptotic death pathways is etiologically responsible for the disease. Apoptosis is regulated in mammalian cells by a family of cysteine proteases called caspases. At least 7 different caspases (caspases 1, 2, 3, 6, 8, 9, and 12) have been implicated in regulating neuronal cell death in response to amyloid beta (A beta) exposure in vitro, in animal models of neurodegenerative diseases, and in AD brain itself. Despite this seemingly impressive array of data implicating caspases and apoptosis as etiologic factors in AD, the direct involvement of caspase-dependent neuronal apoptosis in AD pathogenesis remains uncertain. Alternative explanations for some findings, contradictory experimental observations, and lack of morphologically convincing apoptotic neurons in the vast majority of AD brains has led to the revised hypothesis that apoptosis-associated molecular events cause neuronal dysfunction in the absence of, or prior to, neuronal death. Unfortunately, this new view renders the term "apoptosis-associated" functionally meaningless since it bears no relationship with apoptotic death and fails to focus scientific investigation on the molecular insults that trigger the "apoptosis-associated" response in AD neurons. On balance, an etiologic role for caspases in AD is far from proven. It remains possible, however, that caspase-dependent neuronal death contributes to AD neuron loss and thus, caspase inhibition offers some hope for extending AD neuron survival so that other agents, targeting upstream events, may delay or reverse primary AD pathology.     

Apoptosis in experimental rabies in bax-deficient mice

The challenge virus standard (CVS) of fixed rabies virus produces a fatal encephalitis in adult and suckling mice after intracerebral inoculation. The infection is associated with apoptotic cell death in brain neurons and increased immunoreactivity to the Bax protein in the hippocampus and cerebral cortex. Five- to 7-day-old bax-deficient mice and their wild-type littermates were inoculated intracerebrally with either CVS or the RV194-2 variant of rabies virus, which is avirulent in adult mice after intracerebral inoculation. The clinical disease was similar with both viruses in bax-deficient and wild-type mice with 100% mortality. CVS produced similar apoptotic changes in bax-deficient and wild-type mice, except that apoptosis was more marked in neurons of the dentate gyrus and cortical neurons in the wild-type mice. After inoculation with RV194-2, the morphologic changes of apoptosis were markedly less severe in the cerebral cortex, hippocampus, and cerebellum of the bax-deficient mice than wild-type mice. However, apoptotic changes were moderate to severe in the brain stem in both wild-type and bax-deficient mice with both viruses. Although apoptotic cell death was much less prominent in bax-deficient mice after inoculation with RV194-2, apoptosis of infected brain stem neurons occurred in this fatal infection. Although the Bax protein plays an important role in modulating rabies virus-induced apoptosis under specific experimental conditions, other modulators are also likely important. Received: 23 November 1998 / Revised, accepted: 27 January 1999     

A novel GSK-3β inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo

Amyloid deposits, neurofibrillary tangles, and neuronal cell death in selectively vulnerable brain regions are the chief hallmarks in Alzheimer's (AD) brains. Glycogen synthase kinase-3 (GSK-3) is one of the key kinases required for AD-type abnormal hyperphosphorylation of tau, which is believed to be a critical event in neurofibrillary tangle formation. GSK-3 has also been recently implicated in amyloid precursor protein (APP) processing/Aβ production, apoptotic cell death, and learning and memory. Thus, GSK-3 inhibition represents a very attractive drug target in AD and other neurodegenerative disorders. To investigate whether GSK-3 inhibition can reduce amyloid and tau pathologies, neuronal cell death and memory deficits in vivo, double transgenic mice coexpressing human mutant APP and tau were treated with a novel non-ATP competitive GSK-3β inhibitor, NP12. Treatment with this thiadiazolidinone compound resulted in lower levels of tau phosphorylation, decreased amyloid deposition and plaque-associated astrocytic proliferation, protection of neurons in the entorhinal cortex and CA1 hippocampal subfield against cell death, and prevention of memory deficits in this transgenic mouse model. These results show that this novel GSK-3 inhibitor has a dual impact on amyloid and tau alterations and, perhaps even more important, on neuronal survival in vivo further suggesting that GSK-3 is a relevant therapeutic target in AD.