The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-x L was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43 Q331K and TDP-43 M337V ), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell typeselective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43 Q331K , but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43 Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.A myotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are progressive, adult-onset neurodegenerative diseases with overlapping clinical and pathological features (1-3). ALS is characterized by the selective loss of upper and lower motor neurons, leading to progressive fatal paralysis and muscle atrophy. A large majority (∼90%) of ALS and FTLD-U cases are without a known genetic cause. Importantly, in these sporadic cases, the appearance of ubiquitinated inclusions within the affected neurons of the nervous system characterizes both ALS and FTLD-U patients, suggesting an overlapping mechanism underlying both diseases. Biochemical characterization of brains and spinal cords from ALS and FTLD-U patients identified transactivating response region (TAR) DNA binding protein (TDP-43) as the major protein component of these ubiquitinated inclusions (4, 5). The discovery of ALS-linked mutations in the glycine-rich C-terminal domain of TDP-43 (6-8) demonstrated a pathological role of TDP-43 in both diseases. The subsequent identification of mutations in a structurally and functionally related nucleic acid binding protein, FUS/ TLS (fused in sarcoma/translocated in liposarcoma) (9, 10), further implicated defects in RNA processing in ALS pathogenesis.TDP-43 is a multifunctional nucleic acid binding protein.Within the nervous system, TDP-43 binds to >6,000 pre-mRNAs and affects the levels of ∼600 mRNAs and the splicing patterns of another 950 (11). Structura...

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.Mitochondria play a critical role in the regulation of programmed cell death by sequestering apoptogenic proteins such as cytochrome c, Smac/DIABLO, HtrA2/Omi, endonuclease G, and AIF (13,18,29,80). The release of such factors during apoptosis is regulated by a subclass of Bcl-2 proteins (12,14,63,78), including Bax and Bak. These proteins seem to be in an inactive state in healthy cells, with Bax predominantly found in the cytosol. However, during apoptosis induced by various death stimuli, including DNA damage or trophic factor deprivation, they are activated by a process requiring BH3-only Bcl-2 family members. It is thought that BH3-only proteins either bind and sequester Bcl-2 antiapoptotic proteins (this is the case for Bad and Puma) or bind to and directly activate proapoptotic proteins (tBid for example) (9,11,33,45,65,74). This results in the inactivation of Bcl-2 antiapoptotic proteins and in the oligomerization of Bax and Bak in the mitochondrial outer membrane (MOM) with a concomitant release of apoptogenic factors from the mitochondria (17,21,31,43).How permeabilization of the MOM occurs during apoptosis remains a matter of debate and has been extensively studied (for reviews, see references 4, 49, and 85). Recently, a new model has emerged based on the discovery that mitochondria fragment during cell death (32, 46-48, 61, 86). According to this model, the fission of mitochondria would be necessary for permeabilization of the MOM (3, 57, 83).Nevertheless, it is still not clear whether the fragmentation of mitochondria precedes or follows the release of apoptogenic factors (1,22,26).Mitochondrial fission and fusion are normal and frequent events in healthy cells. The protein machinery that underlies mitochondrial fission has been well characterized and extensively reviewed (53,62). In mammalian cells, at least three proteins, Drp1, hFis1, and MTP18 (75,76), are required for this process. The dynamin-related protein Drp1 is a large cytosolic GTPase that translocates to the mitochondria, where it couples GTP hydrolysis with scission of the mitochondrial tubule (59,67,68). Its recept...