Sustained progression of neuronal cell death causes brain tissue loss and subsequent functional deficits following stroke or central nervous system trauma and in neurodegenerative diseases. Despite obvious differences in the pathology of these neurological disorders, the underlying delayed neuronal demise is carried out by a common biochemical cell death programme. Mitochondrial membrane permeabilization and subsequent release of apoptotic factors are key mechanisms during this process. Bcl-2 family proteins, e.g. the pro-apoptotic Bid, Bax or Bad and the antiapoptotic Bcl-2, Bcl-XL, play a crucial role in the regulation of this mitochondrial checkpoint in neurons. In particular, cleavage of cytosolic Bid and subsequent mitochondrial translocation have been detected in many paradigms of neuronal cell death related to acute or chronic neurodegeneration. The current review focuses on the emerging role of Bid as an integrating key regulator of the intrinsic death pathway that amplifies caspase-dependent and caspase-independent execution of neuronal apoptosis. Therefore pharmacological inhibition of Bid provides a promising therapeutic strategy in neurological diseases where programmed cell death is prominent.
Degeneration and death of neurons cause the functional deficits evolving after cerebral ischaemia and brain trauma and in chronic neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Although the pathological mechanisms differ significantly between these neurological disorders, a shared biochemical cascade of events appears to carry out the underlying cell death process, which involves disruption of cellular calcium homoeostasis, increased oxidative stress and activation of programmed cell death . Neuronal cell death involves mitochondrial ion permeability changes, release of apoptotic factors from mitochondria, e.g. cytochrome c, Smac/DIABLO [direct IAP (inhibitor of apoptosis protein) binding protein with low pI], Htr2A/Omi or AIF (apoptosis inducing factor), activation of caspases and, finally, condensation and degradation of nuclear DNA. An important regulatory step of this process occurs at mitochondrial membranes where members of the Bcl-2 family of proteins either promote (Bax, Bid, Bad and Bim) or prevent (Bcl-2 and Bcl-XL) membrane permeability transition [2,3]. In particular, Bid cleavage to tBid (truncated Bid) has been identified as a key event that significantly amplifies apoptotic stress at the mitochondrial level in various experimental models of neuronal cell death in vitro and in vivo. Here, we summarize mechanisms of Bid activation, downstream pathways of apoptosis execution and promising therapeutic strategies to interfere with the Bid-dependent cell death that causes the progression of brain damage after acute brain injury and in chronic neurodegeneration.
Causal role for Bid in neuronal cell death
Multiple lines of evidence demonstrate that the regulation of Bcl-2 protein family members is crucial for the maintenance of mitochondrial integrity and hence cell viability after severe stress . The Bcl-2 family consists of two large groups of proteins that either prevent (e.g. Bcl-2, Bcl-XL and Bcl-w) or promote (e.g. Bax, Bad, Bak, Bid and Bim) apoptosis. These proteins can form either homo- or hetero-dimers and thus function either independently or in concert to regulate mitochondrial membrane integrity and apoptosis [2,4]. For example, the antiapoptotic Bcl-2 and Bcl-XL can form heterodimers with Bax or Bak to prevent their apoptogenic activity . In contrast with other cells where Bax or Bak can equally mediate mitochondrial damage, the prerequisite role of Bax in neuronal apoptosis may be explained by the absence of full-length Bak in neurons. In neurons, apoptotic stress can induce mitochondrial outer membrane permeabilization through enhanced Bax protein synthesis, Bad-mediated release of Bax from Bax–Bcl-XL or Bax–Bcl-2 heterodimers, or after interaction of Bax with tBid .
Bid is the only Bcl-2 family member that functions as an agonist of Bax or Bak, and the cleavage of Bid to tBid has been identified as a hallmark of the intrinsic apoptosis pathway that amplifies the cell death programme through mitochondrial dysfunction and the release of apoptotic factors from the mitochondrial intermembrane space [7,8]. Accordingly, dimerization of tBid with Bax and the related mitochondrial membrane pore formation have been suggested to be a key mechanism in various models of neuronal cell death related to neurological disorders such as stroke, brain trauma or Alzheimer's disease [3,9]. For example, Bid-deficient neurons are highly resistant to cell death stimuli including OGD (oxygen–glucose deprivation) in vitro or cerebral ischaemia in vivo [10,11]. More recently, we demonstrated that novel low–molecular-mass inhibitors of Bid, as well as siRNA (small interfering RNA)-mediated Bid protein knockdown, prevented neuronal cell death after OGD or glutamate-mediated excitotoxicity  (Figure 1), and similar protective effects of the Bid inhibitors have been observed after exposure of cultured neurons to amyloid-β peptide. Bid cleavage was also detected after experimental traumatic brain injury , and Bid-deficient mice were protected from secondary brain damage after trauma . In addition, formation of tBid and mitochondrial translocation have been detected in seizure-induced neuronal death  and in human brain tissue after temporal lobe epilepsy . Overall, these results clearly demonstrate a key role for Bid in many experimental models of neurodegeneration with relevance to different neurological diseases where programmed cell death is prominent.
AIF translocation precedes neuronal apoptosis after OGD
Activation of Bid
The proteolytic cleavage of Bid to the truncated form has been proposed as a prerequisite for its pro-apoptotic activity. In contrast with many Bcl-2 family members, the inactive full-length Bid lacks a C-terminal membrane-anchoring segment and therefore usually resides in the cytosol of healthy cells. Mitochondrial translocation, interaction with Bax and pore formation by Bid require removal of an N-terminal autorepression domain . Bid cleavage and activation can be exerted by different proteases, suggesting Bid as a key factor in many paradigms of cell death. For example, activation of the Fas/TNF (tumour necrosis factor) death receptor family and the subsequent activation of caspase 8 is the most prominent pathway that leads to Bid cleavage. Active caspase 8 has been detected in cultured neurons challenged by OGD and in rodent brain tissue after transient cerebral ischaemia [18,19]. In cultured neurons, inhibition of both Fas ligand or TNFα prevented neuronal death after OGD. Further, studies in models of cerebral ischaemia in transgenic mice expressing non-functional Fas receptors suggested that death receptor signalling and subsequent activation of caspase 8 and Bid cleavage mediated the delayed neuronal death after stroke [20,21]. It is noteworthy, however, that death receptor signalling may also indirectly contribute to delayed brain damage after stroke by enhancing inflammatory responses. While a major role for direct activation of death receptors in degenerating neurons after cerebral ischaemia has not been clarified, other mechanisms of caspase 8 activation and Bid cleavage have been proposed. For example, activation of caspase 8 has been identified as a feature of DNA-damage-induced cell death , and DNA damage resulting from oxidative stress after ischaemia/reperfusion may function as an early trigger for delayed neuronal death after stroke . A link between DNA damage and Bid-dependent execution of the cell death programme may be provided by caspase 2, an initiator caspase that is activated after genotoxic stress . In contrast with other initiator caspases, e.g. caspase 8 or caspase 9, caspase 2 is incapable of processing other caspase zymogens but can cleave cytosolic Bid protein [24,25]. However, a causal role for caspase 2 in the observed Bid cleavage after DNA damage is still controversial, since siRNA-mediated caspase 2 silencing did not prevent Bid cleavage after genotoxic stress .
More recent results exposed Bid as an ATM (ataxia telangiectasia mutated) effector in DNA damage response in non-neuronal cells [26,27]. After genotoxic stress ATM can phosphorylate Bid, which then induces either cell-cycle arrest or apoptosis, depending on the severity of the insult. On the other hand, Bid was insensitive to caspase 8 cleavage when phosphorylated by CK1 (casein kinase 1) or protein kinase CK2, whereas inhibition of CK1 and protein kinase CK2 or a mutant of Bid that cannot be phosphorylated accelerated Fas-mediated apoptosis . Therefore it remains to be established whether phosphorylated nuclear Bid functions as a link between DNA-damage response and apoptotic signalling in neuronal cell death.
In addition to caspases, Bid cleavage can be exerted by other proteases, including calpains, granzyme B or lysosomal hydrolases. Calpains belong to a family of calcium-dependent proteases that are activated in many paradigms of delayed neuron death, since the elevation of intracellular calcium levels is a common feature of neuronal apoptosis. Calpain substrates include cytoskeletal proteins, kinases and phosphatases, membrane receptors and transporters that are important for maintenance of cellular ion homoeostasis and cell survival [29,30]. Further, calpains can amplify apoptotic signalling through Bid cleavage and subsequent mitochondrial release of apoptotic factors such as AIF, cytochrome c or Smac/DIABLO [3,31]. Therefore calpains are believed to play important roles in the regulation of apoptosis and necrosis, and calpain inhibitors prevented neuronal cell death in a variety of experimental models .
Granzyme B is a serine protease that is released by activated cytotoxic T-lymphocytes to induce target cell apoptosis. Although granzyme B can directly cleave Bid, and resistance against granzyme B has been documented in Bid-deficient cells , others have found that neither Bid nor Bax is required to exert granzyme B-induced mitochondrial depolarization and cell death, suggesting the activation of alternative routes to cell death . In the aging brain or after acute brain injury, transient oxidative stress may contribute to imperfect lysosomal degradation and autophagy processes which involve the release of lysosomal enzymes. The lysosomal involvement in neuronal apoptosis triggered by oxidative stress is being increasingly recognized and may involve mitochondrial membrane permeabilization either directly through activation of phospholipases or indirectly through cleavage of Bid. For example, the lysosomal cysteine proteases such as cathepsins B, D, H and L have been reported to mediate Bid cleavage, subsequent activation of Bax–Bak by tBid, and mitochondrial release of cytochrome c, AIF and Smac/Diablo .
In addition to the well-established role of tBid in mitochondrial membrane permeabilization and neuronal demise, full-length Bid may exert similar pro-apoptotic activities. For example, a recent study in neurons exposed to glutamate demonstrated translocation of full-length Bid to mitochondria and subsequent collapse of the mitochondrial membrane potential with parallel disruption of the intracellular calcium homoeostasis and rapid nuclear condensation . Similar to paradigms of delayed neuronal death that involve tBid formation, the execution of glutamate-induced apoptosis after mitochondrial translocation of full-length Bid was mediated by caspase-independent mechanisms . A similar role for full-length Bid has been also reported from a model of anoikis in a non-neuronal cell line where Bid translocated to mitochondria and induced MOMP (mitochondrial outer membrane protein) without previous cleavage by caspase 8 or interaction with other Bcl-2 family proteins, such as Bax .
Altogether, compelling evidence locates Bid and its truncated form tBid in the centre of many different routes to apoptotic cell death. Future studies are needed to delineate similarities and differences as well as novelties regarding the mechanisms and the particular role of Bid activation in delayed neuronal cell death after acute brain injury and in chronic degenerative diseases.
The Bid-dependent cell death programme
At the molecular level, previous studies suggested release of cytochrome c and subsequent caspase-dependent execution of apoptosis as the major mechanisms of tBid-mediated neuronal cell death . In addition, damaged mitochondria release pro-apoptotic proteins such as Smac/DIABLO or HtrA2/Omi [3,38], which indirectly promote caspase-dependent apoptotic pathways through binding or degradation of inhibitor of apoptosis proteins respectively. Caspases are a family of cell death proteases that are considered as key executioners of the cell death machinery, and activation of caspases is a well-established biochemical hallmark of neuronal and non-neuronal apoptosis . As outlined above, death receptor-mediated signalling can rapidly activate so-called initiator caspases, such as caspase 8, which then trigger a cell death cascade that may involve Bid cleavage and ultimately lead to the activation of executioner caspases such as caspases 3 and 9 [40,41]. While such zymogenic activation of executioner caspases can occur in a direct manner via the extrinsic apoptosis pathway, neuronal cell death is mediated via intrinsic amplification, which involves Bid cleavage and release of cytochrome c from mitochondria. In the cytosol, cytochrome c binds Apaf-1 (apoptotic protease-activating factor 1) and caspase 9 to form the apoptosome complex, which functions as a catalytic unit and activates large amounts of caspase 3 to execute the apoptotic programme . Therefore the intrinsic apoptosis pathways can significantly boost the effect of activated caspases through Bid-mediated mitochondrial release of cytochrome c.
Previous studies in acute models of neuronal cell death, for example after cerebral ischaemia or brain trauma, indeed demonstrated a protective effect due to inactivation of distinct caspases (e.g. caspase 3, caspase 8 or caspase 9) by peptide caspase inhibitors or in respective caspase knockout mice [19,42]. The therapeutic potential of currently available specific caspase inhibitors for treatment of neurodegenerative diseases, however, is rather limited, because these peptide structures cannot pass the blood–brain barrier. In addition, it is noteworthy that emerging evidence indicates a role for caspases in neuroprotection and central nervous system remodelling [43,44], and caspase inhibition alone may not efficiently preserve degeneration of axons and dendrites, which are important for functional plasticity .
More recently, the importance of cytochrome c release and caspase activation has been supplemented by novel insights into a causal role of caspase-independent death signalling, such as the mitochondrial release of AIF, in delayed neuronal death after cerebral ischaemia [12,46]. AIF is a 67 kDa NADH oxidase flavoprotein located in the mitochondrial intermembrane space with essential functions for optimal oxidative phosphorylation and efficient antioxidant defence [47,48]. In many models of apoptosis, AIF is released from mitochondria and translocates to the nucleus where it induces chromatin condensation and large-scale (>50 kb) DNA fragmentation in a variety of different cell types , including neurons [46,50,51]. After OGD in vitro or after an ischaemic insult in vivo, AIF is rapidly translocated to the nucleus of injured neurons and co-localized with DNA damage and apoptotic nuclear condensation  (Figure 1). It is of note that mitochondrial release of AIF occurred several hours before cytochrome c release and caspase 3 activation, suggesting that AIF is in the first line of cell death signalling after ischaemia. On the other hand, the reduction of AIF protein levels in siRNA-treated cultured neurons or in mice carrying the Hq (harlequin) AIF gene mutation resulted in a significant reduction of neuronal cell death in the respective experimental models of ischaemia by approx. 50%  (Figure 2). These results expose AIF as a promising target for neuroprotective strategies in stroke therapy. Since a small molecule inhibitor for AIF is not yet available, and permanent inhibition of AIF may exert enhanced oxidative stress, the mechanism of AIF release is currently a matter of intensive research. For example, AIF-mediated apoptosis is a major feature downstream of PARP-1 [poly(ADP-ribose) polymerase-1] activation in apoptotic neurons [12,51], but how enhanced PARP activity exerts mitochondrial release of AIF has not been clarified. More recent results further suggested a potential involvement of calpains  and the pro-apoptotic Bcl-2 family proteins BimEL  and tBid  in mitochondrial AIF release in neuronal apoptosis, suggesting that there are multiple routes towards mitochondrial AIF release and nuclear translocation. In particular, pharmacological inhibition of tBid blocked mitochondrial AIF release and neuronal cell death after OGD or glutamate exposure [12,53] (Figure 1). Recent results suggested that Bid-induced MOMP requires enhanced calpain activity to mediate the mitochondrial release of AIF after excitotoxic stress. Like many models of neuronal cell death, glutamate-induced excitotoxicity involves increased intracellular calcium levels and activation of calpains. Both the calpain inhibitor calpeptin or inhibition of mitochondrial membrane permeability by cyclosporin A prevented AIF release after the excitotoxic challenge . These results imply a model of AIF release, where tBid and Bax form a membrane pore that allows entry of activated calpain into the mitochondrial intermembrane space where it cleaves AIF thereby reducing its association with the inner mitochondrial membrane. The AIF cleavage products then leave the mitochondria through the membrane pore and translocate to the nucleus to execute DNA degradation and cell death . This model is well supported by multiple lines of evidence for calpain activation in neuronal apoptosis and corresponding protective effects of calpain inhibitors [29,54]. Along these lines, it is interesting to note that many caspase inhibitors also inhibit calpains and thus downstream caspase-independent death signalling. Such unspecific effects may have caused overestimation of the role of caspases in paradigms where caspase activation only partly contributed to neuronal death, as for example after cerebral ischaemia. It is therefore likely that future studies will reveal an important role for caspase-independent mechanisms, such as Bid-mediated AIF release, in many models of delayed neuronal death relevant for neurodegenerative diseases.
AIF mediates neuronal death after OGD
Small-molecule inhibitors of Bid
As outlined above, Bid is a key member of the Bcl-2 family proteins involved in the control of the apoptotic cascade, leading to neuronal cell death in many experimental models relevant to different neurological disorders. Therefore Bid represents a challenging target for strategies aimed at the development of neuroprotective therapeutics. Recently, Pellecchia and co-workers  developed low-molecular-mass 4-phenylsulfanyl-phenylamine derivatives that are capable of occupying a deep hydrophobic crevice on the surface of Bid. These compounds represent the first small molecules targeting Bid, as shown by their ability to inhibit tBid-induced Smac release, caspase 3 activation and cell death in isolated mitochondria and in cancer cell lines respectively . In neuronal cell cultures, these novel inhibitors attenuated cell death induced by glutamate, OGD or amyloid-β in a dose-dependent manner  (Figure 1). The Bid inhibitors preserved mitochondrial integrity and prevented the activation of caspase 3 as well as nuclear translocation of AIF and DNA condensation after OGD- or glutamate-mediated excitotoxicity . These results confirmed the pivotal role of Bid in mediating mitochondrial membrane permeability and subsequent activation of caspase-dependent and caspase-independent cell death pathways. It should be noted that the protective effect of the Bid inhibitors exceeded the effect of siRNA-mediated AIF gene silencing in models of OGD- or glutamate-induced excitotoxicity, suggesting that blocking Bid activity upstream of mitochondrial damage is highly efficient for neuroprotection. These high-affinity small molecular compounds selectively targeting Bid are not only chemical tools that can be used to elucidate the role of Bid in neurodegenerative disorders, but could serve as lead compounds for further drug development towards therapeutic applications in related animal models.
Bid has been exposed to be a unique pro-apoptotic member of the Bcl-2 family proteins since its activation and/or cleavage to tBid is an early key event that recruits mitochondria to the death machinery, thereby amplifying lethal stress and accelerating the execution phase in many different paradigms of programmed cell death (Figure 3). After Bid activation and mitochondrial translocation, the most prominent downstream mechanisms of Bid-dependent neuronal apoptosis involve disruption of mitochondrial membrane integrity and intracellular calcium homoeostasis and the release of pro-apoptotic mitochondrial factors such as cytochrome c, Smac/DIABLO or AIF, which can execute caspase-dependent or caspase-independent cell death respectively. Therefore Bid emerges as a promising target in diseases where delayed cell death is prominent, including neurodegenerative disorders and acute brain injury.
Activation of Bid and downstream pathways of programmed cell death
International Symposium on Neurodegeneration and Neuroprotection: Independent Meeting held at University of Münster, Germany, 23–27 July 2006. Organized and Edited by S. Klumpp and J. Krieglstein (Münster, Germany).
apoptosis inducing factor
apoptotic protease-activating factor 1
ataxia telangiectasia mutated
casein kinase 1
direct IAP (inhibitor of apoptosis protein)-binding protein with low pI
mitochondrial outer membrane protein
small interfering RNA
tumour necrosis factor