Phase I First-in-Human Dose Escalation Study of the oral SF3B1 modulator H3B-8800 in myeloid neoplasms.

We conducted a phase I clinical trial of H3B-8800, an oral small molecule that binds Splicing Factor 3B1 (SF3B1), in patients with MDS, CMML, or AML. Among 84 enrolled patients (42 MDS, 4 CMML and 38 AML), 62 were red blood cell (RBC) transfusion dependent at study entry. Dose escalation cohorts examined two once-daily dosing regimens: schedule I (5 days on/9 days off, range of doses studied 1-40 mg, n = 65) and schedule II (21 days on/7 days off, 7-20 mg, n = 19); 27 patients received treatment for ≥180 days. The most common treatment-related, treatment-emergent adverse events included diarrhea, nausea, fatigue, and vomiting. No complete or partial responses meeting IWG criteria were observed; however, RBC transfusion free intervals >56 days were observed in nine patients who were transfusion dependent at study entry (15%). Of 15 MDS patients with missense SF3B1 mutations, five experienced RBC transfusion independence (TI). Elevated pre-treatment expression of aberrant transcripts of Transmembrane Protein 14C (TMEM14C), an SF3B1 splicing target encoding a mitochondrial porphyrin transporter, was observed in MDS patients experiencing RBC TI. In summary, H3B-8800 treatment was associated with mostly low-grade TAEs and induced RBC TI in a biomarker-defined subset of MDS.

Opportunities and Challenges in Implementation of Multiparameter Single Cell Analysis Platforms for Clinical Translation.

The high-content interrogation of single cells with platforms optimized for the multiparameter characterization of cells in liquid and solid biopsy samples can enable characterization of heterogeneous populations of cells ex vivo. Doing so will advance the diagnosis, prognosis, and treatment of cancer and other diseases. However, it is important to understand the unique issues in resolving heterogeneity and variability at the single cell level before navigating the validation and regulatory requirements in order for these technologies to impact patient care. Since 2013, leading experts representing industry, academia, and government have been brought together as part of the Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium to foster the potential of high-content data integration for clinical translation.

Antitumor activity of the polo-like kinase inhibitor, TAK-960, against preclinical models of colorectal cancer.

BACKGROUND: Polo-like kinase 1 (Plk1) is a serine/threonine kinase that is a key regulator of multiple stages of mitotic progression. Plk1 is upregulated in many tumor types including colorectal cancer (CRC) and portends a poor prognosis. TAK-960 is an ATP-competitive Plk1 inhibitor that has demonstrated efficacy across a broad range of cancer cell lines, including CRC. In this study, we investigated the activity of TAK-960 against a large collection of CRC models including 55 cell lines and 18 patient-derived xenografts. METHODS: Fifty-five CRC cell lines and 18 PDX models were exposed to TAK-960 and evaluated for proliferation (IC50) and Tumor Growth Inhibition Index, respectively. Additionally, 2 KRAS wild type and 2 KRAS mutant PDX models were treated with TAK-960 as single agent or in combination with cetuximab or irinotecan. TAK-960 mechanism of action was elucidated through immunoblotting and cell cycle analysis. RESULTS: CRC cell lines demonstrated a variable anti-proliferative response to TAK-960 with IC50 values ranging from 0.001 to > 0.75 µmol/L. Anti-proliferative effects were sustained after removal of drug. Following TAK-960 treatment a highly variable accumulation of mitotic (indicating cell cycle arrest) and apoptotic markers was observed. Cell cycle analysis demonstrated that TAK-960 treatment induced G2/M arrest and polyploidy. Six out of the eighteen PDX models responded to single agent TAK-960 therapy (TGII< 20). The addition of TAK-960 to standard of care chemotherapy resulted in largely additive antitumor effects. CONCLUSION: TAK-960 is an active anti-proliferative agent against CRC cell lines and PDX models. Collectively, these data suggest that TAK-960 may be of therapeutic benefit alone or in combination with other agents, although future work should focus on the development of predictive biomarkers and hypothesis-driven rational combinations.

A First-in-Human, Phase I, Dose-Escalation Study of TAK-117, a Selective PI3Kα Isoform Inhibitor, in Patients with Advanced Solid Malignancies.

Purpose: To evaluate the safety, MTD, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of TAK-117 (MLN1117/INK1117), an investigational PI3Kα-selective inhibitor, in patients with advanced solid tumors.Experimental Design: Seventy-one patients received oral TAK-117 once daily [100-300 mg (n = 24)] or 3 days per week [Monday-Wednesday-Friday (MWF), 200-1,200 mg (n = 27); Monday-Tuesday-Wednesday (MTuW), 200-900 mg (n = 20)], in 21-day cycles. Dose escalation proceeded via a 3 + 3 design.Results: TAK-117 once-daily dosing was associated with dose-limiting grade ≥3 alanine/aspartate aminotransferase (ALT/AST) elevations, resulting in a narrow range of tolerable doses (100-150 mg once daily). With MWF/MTuW dosing, no dose-limiting ALT/AST elevations occurred until the MTD of 900 mg; total weekly dose was 2.6-fold that of 150 mg once daily. Drug-related grade ≥3 adverse events occurred in 25%/22%/35% (including hyperglycemia in 0%/7%/15%) of once-daily/MWF/MTuW patients. TAK-117 (100-1,200 mg) exhibited moderately fast oral absorption, a generally dose proportional increase in exposure, and plasma half-life of approximately 11 hours. Total weekly exposures with 900 mg MWF/MTuW dosing were approximately 4 times greater than with 150 mg once daily. Skin pS6 expression was suppressed at ≥200 mg. There were 3/1/0 partial responses (once daily/MWF/MTuW) and 5/7/5 patients had stable disease lasting ≥3 months (all PIK3CA mutated).Conclusions: Intermittent dosing of TAK-117 had an acceptable safety profile and enabled higher doses and total weekly exposures versus once-daily dosing. Although the potential for TAK-117 as single-agent therapy appears limited, further evaluation in combination approaches for advanced solid tumors is warranted. Clin Cancer Res; 23(17); 5015-23. ©2017 AACR.

An inhibitor of apoptosis protein antagonist T-3256336 potentiates the antitumor efficacy of the Nedd8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924).

Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs. In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNFα-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNFα mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer.

Local apoptosis modulates early mammalian brain development through the elimination of morphogen-producing cells.

Apoptotic cells are observed in the early developing brain. Apoptosis deficiency is proposed to cause brain overgrowth, but here we show that brain malformations in apoptosis-deficient mutants are due to insufficient brain ventricle expansion as a result of uncompleted cranial neural tube closure. Apoptosis eliminates Fgf8-expressing cells in the anterior neural ridge (ANR), which acts as an organizing center of the forebrain by producing FGF8 morphogen. Deficiency of apoptosis leads to the accumulation of undead and nonproliferative cells in the ventral part of the ANR. The undead cells in apoptosis-deficient mutants express Fgf8 continuously, which perturbs gene expression in the ventral forebrain. Thus, apoptosis within a specific subdomain of the ANR is required for correct temporal elimination of an FGF8-producing region within a limited developmental time window, thereby ensuring proper forebrain development.

TAK-960, a novel, orally available, selective inhibitor of polo-like kinase 1, shows broad-spectrum preclinical antitumor activity in multiple dosing regimens.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase involved in key processes during mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and elevated levels of PLK1 have been associated with poor prognosis, making it an attractive target for anticancer therapy. TAK-960 [4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl) benzamide] is a novel, investigational, orally bioavailable, potent, and selective PLK1 inhibitor that has shown activity in several tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1). Consistent with PLK1 inhibition, TAK-960 treatment caused accumulation of G(2)-M cells, aberrant polo mitosis morphology, and increased phosphorylation of histone H3 (pHH3) in vitro and in vivo. TAK-960 inhibited proliferation of multiple cancer cell lines, with mean EC(50) values ranging from 8.4 to 46.9 nmol/L, but not in nondividing normal cells (EC(50) >1,000 nmol/L). The mutation status of TP53 or KRAS and MDR1 expression did not correlate with the potency of TAK-960 in the cell lines tested. In animal models, oral administration of TAK-960 increased pHH3 in a dose-dependent manner and significantly inhibited the growth of HT-29 colorectal cancer xenografts. Treatment with once daily TAK-960 exhibited significant efficacy against multiple tumor xenografts, including an adriamycin/paclitaxel-resistant xenograft model and a disseminated leukemia model. TAK-960 has entered clinical evaluation in patients with advanced cancers.

Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure.

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)-based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.

Maturation of the olfactory sensory neurons by Apaf-1/caspase-9-mediated caspase activity.

Although the apoptotic role of caspases has been largely understood, accumulating evidence in Drosophila suggests that caspases also control other processes than apoptotic cell death. However, how caspases contribute to the development of the mammalian nervous system remains obscure. Here, we provide unique evidence that Apaf-1/caspase-9-mediated caspase signaling regulates the development of olfactory sensory neurons (OSNs), which includes axonal projection, synapse formation, and maturation of these neurons. This caspase signaling leads to a cleavage of Semaphorin 7A, a membrane-anchored semaphorin that is required for the proper axonal projection. Mutant mice deficient for apaf-1 or caspase-9 exhibit misrouted axons, impaired synaptic formation, and defects in the maturation of OSNs without affecting the number of these cells. Our findings suggest that Apaf-1/caspase-9-mediated nonapoptotic caspase signaling is required for the proper neural network formation during olfactory development.

Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury.

Neonatal hypoxic/ischemic (H/I) brain injury causes neurological impairment, including cognitive and motor dysfunction as well as seizures. However, the molecular mechanisms regulating neuron death after H/I injury are poorly defined and remain controversial. Here we show that Atg7, a gene essential for autophagy induction, is a critical mediator of H/I-induced neuron death. Neonatal mice subjected to H/I injury show dramatically increased autophagosome formation and extensive hippocampal neuron death that is regulated by both caspase-3-dependent and -independent execution. Mice deficient in Atg7 show nearly complete protection from both H/I-induced caspase-3 activation and neuron death indicating that Atg7 is critically positioned upstream of multiple neuronal death executioner pathways. Adult H/I brain injury also produces a significant increase in autophagy, but unlike neonatal H/I, neuron death is almost exclusively caspase-3-independent. These data suggest that autophagy plays an essential role in triggering neuronal death execution after H/I injury and Atg7 represents an attractive therapeutic target for minimizing the neurological deficits associated with H/I brain injury.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but it had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

Decreased retinal neuronal cell death in caspase-1 knockout mice.

PURPOSE: To determine whether apoptosis of retinal neurons induced by excessive light exposure and ischemia-reperfusion injury is altered in caspase-1 knockout mice. METHODS: Eight- to 10-week-old caspase-1 knockout mice (Casp1-/-) and wild-type (WT) mice (C57BL/6) were exposed to diffuse, cool, white fluorescent light of 25,000 lux for 2 h. Other mice were subjected to retinal ischemia by increasing the intraocular pressure to 110 mmHg for 45 min. Electroretinograms (ERGs) were recorded before and after the light exposure. TdT-dUTP terminal nick-end labeling (TUNEL) was performed to identify the apoptotic cells after the insults. The inner retinal thickness was measured to evaluate the retinal injury after the ischemia-reperfusion. Expression of caspase-1 protein was studied by immunohistochemical analysis and Western blotting. Caspase-1-like protease activity was determined by a colorimetric tetrapeptide substrate. RESULTS: The morphology of the retina and the amplitudes of the a and b waves of the ERGs of Casp1-/- mice did not differ from those of WT mice. After the light exposure, TUNEL-positive cells were observed in the outer nuclear layer of the WT mice retina. The number of TUNEL-positive photoreceptor nuclei after the light exposure, and the number of nuclei in the inner nuclear layer after the ischemia-reperfusion injury, were significantly less in Casp1-/- mice than in WT mice. There were more caspase-1-positive photoreceptor cells in WT mice after the light injury. The inner retinal layer of Casp1-/- mice was significantly thicker in Casp1-/- mice than in WT mice 2 weeks after the ischemic insult. CONCLUSIONS: Retinal neuronal apoptosis was less prominent in Casp1-/- mice after excessive light exposure and ischemia-reperfusion injury. These data indicate that caspase-1 plays a role in retinal neuronal apoptosis.

The initiator caspase, caspase-10beta, and the BH-3-only molecule, Bid, demonstrate evolutionary conservation in Xenopus of their pro-apoptotic activities in the extrinsic and intrinsic pathways.

Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10beta), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10beta and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10beta and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10beta mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development.

Caspases 3 and 7: key mediators of mitochondrial events of apoptosis.

The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor-mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.

The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection.

Baculovirus inhibitor of apoptosis repeat-containing 1 (Birc1) proteins have homology to several germline-encoded receptors of the innate immune system. However, their function in immune surveillance is not clear. Here we describe a Birc1e-dependent signaling pathway that restricted replication of the intracellular pathogen Legionella pneumophila in mouse macrophages. Translocation of bacterial products into host-cell cytosol was essential for Birc1e-mediated control of bacterial replication. Caspase-1 was required for Birc1e-dependent antibacterial responses ex vivo in macrophages and in a mouse model of Legionnaires' disease. The interleukin 1beta converting enzyme-protease-activating factor was necessary for L. pneumophila growth restriction, but interleukin 1beta was not required. These results establish Birc1e as a nucleotide-binding oligomerization-leucine-rich repeat protein involved in the detection and control of intracellular L. pneumophila.

c-Jun N-terminal kinases mediate reactivation of Akt and cardiomyocyte survival after hypoxic injury in vitro and in vivo.

Akt is a central regulator of cardiomyocyte survival after ischemic injury in vitro and in vivo, but the mechanisms regulating Akt activity in the postischemic cardiomyocyte are not known. Furthermore, although much is known about the detrimental role that the c-Jun N-terminal kinases (JNKs) play in promoting death of cells exposed to various stresses, little is known of the molecular mechanisms by which JNK activation can be protective. We report that JNKs are necessary for the reactivation of Akt after ischemic injury. We identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia, apparently by priming Akt for subsequent phosphorylation by 3-phosphoinositide-dependent protein kinase. The reduction in Akt activity that is induced by JNK inhibition may have significant biological consequences, as we find that JNKs, acting via Akt, are critical determinants of survival in posthypoxic cardiomyocytes in culture. Furthermore, in contrast to selective p38-mitogen-activated protein kinase inhibition, which was cardioprotective in vivo, concurrent inhibition of both JNKs and p38-mitogen-activated protein kinases increased ischemia/reperfusion injury in the heart of the intact rat. These studies demonstrate that reactivation of Akt after resolution of hypoxia and ischemia is regulated by JNKs and suggest that this is likely a central mechanism of the myocyte protective effect of JNKs.

Transforming growth factor beta-dependent sequential activation of Smad, Bim, and caspase-9 mediates physiological apoptosis in gastric epithelial cells.

Transforming growth factor beta (TGF-beta) has been implicated in the maintenance of homeostasis in various organs, including the gastric epithelium. In particular, TGF-beta-induced signaling was shown to be required for the differentiation-associated physiological apoptosis of gastric epithelial cells, but its mechanism has not been well understood. In this study, the molecular mechanism of TGF-beta-induced apoptosis was analyzed in a human gastric epithelial cell line, SNU16, as an in vitro model. Expression of Smad7 and Bcl-X(L), but not viral FLIP, was shown to prevent TGF-beta-induced apoptosis, indicating an exclusive requirement of the activation of Smad signaling pathway and mitochondrial dysfunction followed by activation of caspase-9. In addition, treatment with TGF-beta induced binding of Bim, a proapoptotic Bcl-2 homology domain 3 (BH3)-only protein, to Bcl-X(L), which is dependent on the activation of Smad, and reduction in the expression of Bim by RNA interference decreased the sensitivity to TGF-beta-induced apoptosis. Moreover, we found abnormalities in the gastric epithelium of both Bim and caspase-9 knockout mice; these abnormalities were associated with a defect of physiological apoptosis in gastric epithelial cells. These results indicate for the first time that TGF-beta is involved in the physiological loss of gastric epithelial cells by activating apoptosis mediated by Smad, Bim, and caspase-9.

Notch promotes survival of neural precursor cells via mechanisms distinct from those regulating neurogenesis.

During development of the mammalian brain, many neural precursor cells (NPCs) undergo apoptosis. The regulation of such cell death, however, is poorly understood. We now show that the survival of mouse embryonic NPCs in vitro was increased by culture at a high cell density and that this effect was attributable to activation of Notch signaling. Expression of an active form of Notch1 thus markedly promoted NPC survival. Hes proteins, key effectors of Notch signaling in inhibition of neurogenesis, were not sufficient for the survival-promoting effect of Notch1. This effect of Notch1 required a region of the protein containing the RAM domain and was accompanied by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Moreover, knockdown of these proteins by RNA interference resulted in blockade of the Notch1-induced survival. These results reveal a new function of Notch, the promotion of NPC survival.

Functions of MAP kinases: insights from gene-targeting studies.

Mitogen-activated protein kinases (MAPKs) comprise a family of well-conserved serine/threonine kinases that control a vast array of physiological functions in a number of organisms ranging from yeast to mammals. Recently gene-targeting experiments have shed light on in vivo functions of MAPKs. In particular, embryos deficient in extracellular signal-regulated kinase (ERK) 2 lack mesoderm differentiation and placental angiogenesis. Knockout mice for c-Jun amino-terminal kinases have revealed roles for these kinases in neural apoptosis and activation/differentiation of T cells. Deletion of p38alpha MAPK results in angiogenic defects in the placenta and peripheral vessels. ERK5-deficient embryos are embryonic lethal due to defects in angiogenesis and cardiovascular development. Although these results have provided new insights for MAPK research, development and analysis of conditional knockout mice are required in order to investigate roles of MAPKs, especially, in other biological processes such as disease pathogenesis.

Amyloid beta peptide-induced cerebral neuronal loss is mediated by caspase-3 in vivo.

Amyloid beta peptide (A beta) is widely believed to play a central and etiological role in Alzheimer disease (AD). A beta has been shown to have cytotoxic effects in neural cells, although the mechanism by which it does this is still unclear. To examine the involvement of the apoptotic cascade in A beta-induced cell death, we used mice deficient in caspase-3 (CPP 32), a key protease in this cascade. We microinjected A beta(1-40) into hippocampal regions of the brains of adult mice because AD is an adult-onset disease. We found significant cellular loss in the hippocampal regions of wild-type mice and dramatic rescue of neuronal cell death in caspase-3-deficient mice, with a gene dosage effect. In addition to adult mice, we observed little A beta-induced death of cultured neurons prepared from fetal brains of caspase-3-deficient mice but did observe death of such neurons from wild-type mice. The difference in A beta-induced neuronal death between wild-type and caspase-3-deficient mice was highly significant, indicating that A beta-induced neuronal death is mediated in vivo as well as in vitro by the caspase-3 apoptotic cascade.