Ricardo J.S. Viana

Bile acids: regulation of apoptosis by ursodeoxycholic acid

Bile acids are a group of molecular species of acidic steroids with peculiar physical-chemical and biological characteristics. At high concentrations they become toxic to mammalian cells, and their presence is pertinent in the pathogenesis of several liver diseases and colon cancer. Bile acid cytoxicity has been related to membrane damage, but also to nondetergent effects, such as oxidative stress and apoptosis. Strikingly, hydrophilic ursodeoxycholic acid (UDCA), and its taurine-conjugated form (TUDCA), show profound cytoprotective properties. Indeed, these molecules have been described as potent inhibitors of classic pathways of apoptosis, although their precise mode of action remains to be clarified. UDCA, originally used for cholesterol gallstone dissolution, is currently considered the first choice therapy for several forms of cholestatic syndromes. However, the beneficial effects of both UDCA and TUDCA have been tested in other experimental pathological conditions with deregulated levels of apoptosis, including neurological disorders, such as Alzheimers, Parkinsons, and Huntingtons diseases. Here, we review the role of bile acids in modulating the apoptosis process, emphasizing the anti-apoptotic effects of UDCA and TUDCA, as well as their potential use as novel and alternate therapeutic agents for the treatment of apoptosis-related diseases.

Bile acids and apoptosis modulation: an emerging role in experimental Alzheimer's disease

The potential role of apoptosis in Alzheimers disease (AD) has been an area of intense research in recent years. Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA) are endogenous bile acids that act as potent inhibitors of apoptosis. Their therapeutic effects have been tested in many experimental pathological conditions, including neurological disorders, such as AD. TUDCA regulates precise transcriptional and post-transcriptional events that impact mitochondrial function in neurons. TUDCA not only stabilizes the mitochondrial membrane and prevents Bax translocation, inhibiting the release of cytochrome c and the activation of caspases, but also interferes with upstream factors, including cell cycle-related proteins. In addition, TUDCA is capable of inducing survival pathways. Here, we review the role of apoptosis in AD and discuss the therapeutic potential of TUDCA in treating this disease.

Endoplasmic Reticulum Enrollment in Alzheimer’s Disease

Alzheimer’s disease (AD) poses a huge challenge for society and health care worldwide as molecular pathogenesis of the disease is poorly understood and curative treatment does not exist. The mechanisms leading to accelerated neuronal cell death in AD are still largely unknown, but accumulation of misfolded disease-specific proteins has been identified as potentially involved. In the present review, we describe the essential role of endoplasmic reticulum (ER) in AD. Despite the function that mitochondria may play as the central major player in the apoptotic process, accumulating evidence highlights ER as a critical organelle in AD. Stress that impairs ER physiology leads to accumulation of unfolded or misfolded proteins, such as amyloid β (Aβ) peptide, the major component of amyloid plaques. In an attempt to ameliorate the accumulation of unfolded proteins, ER stress triggers a protective cellular mechanism, which includes the unfolded protein response (UPR). However, when activation of the UPR is severe or prolonged enough, the final cellular outcome is pathologic apoptotic cell death. Distinct pathways can be activated in this process, involving stress sensors such as the JNK pathway or ER chaperones such as Bip/GRP94, stress modulators such as Bcl-2 family proteins, or even stress effectors such as caspase-12. Here, we detail the involvement of the ER and associated stress pathways in AD and discuss potential therapeutic strategies targeting ER stress.

Tauroursodeoxycholic acid prevents E22Q Alzheimer’s Aβ toxicity in human cerebral endothelial cells

Abstract.The vasculotropic E22Q mutant of the amyloid-β (Aβ) peptide is associated with hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s) of toxicity and nature of the AβE22Q toxic assemblies are not completely understood. Comparative assessment of structural parameters and cell death mechanisms elicited in primary human cerebral endothelial cells by AβE22Q and wild-type Aβ revealed that only AβE22Q triggered the Bax mitochondrial pathway of apoptosis. AβE22Q neither matched the fast oligomerization kinetics of Aβ42 nor reached its predominant β-sheet structure, achieving a modest degree of oligomerization with a secondary structure that remained a mixture of β and random conformations. The endogenous molecule tauroursodeoxycholic acid (TUDCA) was a strong modulator of AβE22Q-triggered apoptosis but did not significantly change the secondary structures and fibrillogenic propensities of Aβ peptides. These data dissociate the pro-apoptotic properties of Aβ peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing new perspectives for modulation of amyloid toxicity.

Apoptosis in transgenic mice expressing the P301L mutated form of human tau.

The rTg4510 mouse is a tauopathy model, characterized by massive neurodegeneration in Alzheimer’s disease (AD)-relevant cortical and limbic structures, deficits in spatial reference memory, and progression of neurofibrillary tangles (NFT). In this study, we examined the role of apoptosis in neuronal loss and associated tau pathology. The results showed that DNA fragmentation and caspase-3 activation are common in the hippocampus and frontal cortex of young rTg4510 mice. These changes were associated with cleavage of tau into smaller intermediate fragments, which persist with age. Interestingly, active caspase-3 was often co-localized with cleaved tau. In vitro, fibrillar Aβ1–42 resulted in nuclear fragmentation, caspase activation, and caspase-3-induced cleavage of tau. Notably, incubation with the antiapoptotic molecule tauroursodeoxycholic acid abrogated apoptosis-mediated cleavage of tau in rat cortical neurons. In conclusion, caspase-3-cleaved intermediate tau species occurred early in rTg54510 brains and preceded cell loss in Aβ-exposed cultured neurons. These results suggest a potential role of apoptosis in neurodegeneration.

Modulation of Amyloid-β Peptide-Induced Toxicity through Inhibition of JNK Nuclear Localization and Caspase-2 Activation

Amyloid-β (Aβ) peptide- induced neurotoxicity is typically associated with apoptosis. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA), an endogenous anti-apoptotic bile acid, modulates Aβ-induced apoptosis. Here, we investigated stress signaling events triggered by soluble Aβ and further explored alternative pathways of neuroprotection by TUDCA in differentiated rat neuronal-like PC12 cells. Morphologic evaluation of apoptosis confirmed that Aβ-induced nuclear fragmentation was prevented by TUDCA. In addition, Aβ exposure resulted in activation of the early stress c-Jun N-terminal kinase (JNK) pathway, JNK nuclear translocation, and caspase-2 activation. Knock-down experiments of JNK established caspase-2 as a specific downstream target of JNK in Aβ-induced apoptosis. Furthermore, active caspase-2 cleaved golgin-160 and was localized to the Golgi complex. Importantly, TUDCA abrogated Aβ-induced JNK/caspase-2 signaling. In conclusion, we show that JNK is the proximal stress sensor for soluble Aβ-induced toxicity, which translocates to the nucleus, activates caspase-2, and is strongly modulated by TUDCA in PC12 neuronal cells. Active caspase-2 cleaves golgin-160, suggesting caspase-2-dependent transduction of Aβ apoptotic signaling through the Golgi complex. These data provide new information linking apoptotic properties of Aβ peptide to distinct subcellular mechanisms of toxicity. Further characterization of this signaling pathway and exact targets of modulation are likely to provide new perspectives for modulation of amyloid-induced apoptosis by TUDCA.

Naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates: Potent, Non-cytotoxic, Antiapoptotic Agents

Compounds containing a quinone moiety represent an important class of biologically active molecules that are widespread in nature, displaying anticancer, antibacterial, antimalarial, and fungicidal activities. In the course of designing 2,3-disubstituted-1,4-naphthoquinones derivatives as potential cysteine protease inhibitors, two naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates, 1a and 1b, were obtained. The antiapoptotic potential of 1a and 1b was then evaluated and compared to that of naphthoquinone 4. Primary rat hepatocytes were incubated with synthesized naphthoquinone derivatives and then exposed to the apoptotic stimulus camptothecin. Our results indicate that naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a and 1b exerted a potent protective role in camptothecin-induced apoptosis in primary rat hepatocytes. Both 1a and 1b significantly increased cell viability, while reducing nuclear fragmentation, caspase-3, -8 and -9 activation, and cytochrome c release induced by camptothecin. In addition, 1a and 1b were shown to up-regulate Bcl-X(L), a pro-survival member of the Bcl-2 family of proteins, which modulates the mitochondrial pathway of apoptosis. Similar protective effects of quinone derivatives were seen in HuH-7 and PC12 cells incubated with distinct apoptotic stimuli, such as camptothecin, TGF-beta1, or rotenone. Our results suggest that naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a and 1b may act as potent, cytoprotective agents, through modulation of apoptotic pathways.

Amyloid-β peptide-induced secretion of endoplasmic reticulum chaperone glycoprotein GRP94.

Amyloid-β (Aβ) peptide-induced neurotoxicity is typically associated with cell death through mechanisms not entirely understood. Here, we investigated stress signaling events triggered by soluble Aβ in differentiated rat neuronal-like PC12 cells. Morphologic evaluation of apoptosis confirmed that Aβ induced nuclear fragmentation that was prevented by pre-treatment with the antiapoptotic bile acid tauroursodeoxycholic acid (TUDCA). In addition, Aβ exposure triggered an early signaling response by the endoplasmic reticulum (ER) and caspase-12-mediated apoptosis, which, however, was independent of the ER-stress pathway. Furthermore, ER stress markers, including GRP94, ATF-6α, CHOP, and eIF2α, were strongly downregulated by Aβ, independent of protein degradation, and partially restored by TUDCA. Calpain inhibition prevented caspase-12 activation and reduced levels of ATF-6α. Importantly, Aβ-induced GRP94 downregulation was related to protein secretion and partially rescued through inhibition of the secretory pathway by geldanamycin and brefeldin. In conclusion, we showed that the ER is a proximal stress sensor for soluble Aβ-induced toxicity, resulting in caspase-12 activation and cell death in PC12 neuronal cells. Moreover, ER chaperone GRP94 secretion was associated with Aβ-induced apoptotic signaling. These data provide new information linking apoptotic properties of Aβ peptide to distinct subcellular mechanisms of toxicity. Further characterization of this signaling pathway is likely to provide new perspectives for modulation of amyloid-induced apoptosis.

Organelle Stress Sensors and Cell Death Mechanisms in Neurodegenerative Diseases

Neurodegenerative diseases trigger neuronal cell death by a variety of endogenous suicide pathways. Although cell death may occur through highly heterogeneous processes, specific cell organelles and stress sensors have shown promise as potential therapeutic targets. The plasma membrane senses stress through residing receptors, which can directly or indirectly activate apoptosis. Importantly, several events involved in neuronal death also affect mitochondria homeostasis, leading to calcium uptake, opening of the permeability transition pore, and release of apoptogenic factors. In addition, nuclear DNA damage triggers cell death, where p53 is activated to modulate the expression of selected apoptosis target genes. Signaling proteins implicated in apoptosis pathways are enriched at the Golgi complex, including death receptors and the phosphoinositide 3-kinase. Finally, neurodegenerative diseases progress with accumulation of misfolded proteins, deficiently removed by intracellular proteases or chaperones, and transport abnormalities due to disturbance of cytoskeletal organization in degenerating neurons. The challenge is to decode the complex signaling network of inter-organellar crosstalk leading to cell death and identify therapeutic approaches for delaying or preventing neurodegenerative diseases.