Mitofusin 2 Protects Cerebellar Granule Neurons against Injury-induced Cell Death

Abstract

Of the GTPases involved in the regulation of the fusion machinery, mitofusin 2 (Mfn2) plays an important role in the nervous system as point mutations of this isoform are associated with Charcot Marie Tooth neuropathy. Here, we investigate whether Mfn2 plays a role in the regulation of neuronal injury. We first examine mitochondrial dynamics following different modes of injury in cerebellar granule neurons. We demonstrate that neurons exposed to DNA damage or oxidative stress exhibit extensive mitochondrial fission, an early event preceding neuronal loss. The extent of mitochondrial fragmentation and remodeling is variable and depends on the mode and the severity of the death stimuli. Interestingly, whereas mitofusin 2 loss of function significantly induces cell death in the absence of any cell death stimuli, expression of mitofusin 2 prevents cell death following DNA damage, oxidative stress, and K ؉ deprivation induced apoptosis. More importantly, whereas wild-type Mfn2 and the hydrolysis-deficient mutant of Mfn2 (Mfn2 RasG12V ) function equally to promote fusion and lengthening of mitochondria, the activated Mfn2 RasG12V mutant shows a significant increase in the protection of neurons against cell death and release of proapoptotic factor cytochrome c. These findings highlight a signaling role for Mfn2 in the regulation of apoptosis that extends beyond its role in mitochondrial fusion.It has been recently demonstrated that the apoptotic program includes the regulated induction of mitochondrial fragmentation (1, 2). In addition, it has been shown that the rate of mitochondrial fusion is reduced early in the apoptotic program (3, 4), which together with the activation of the fission machinery leads to a morphological shift of the mitochondria to the fragmented state. The regulatory mechanisms and functional importance of these events during death remain unclear. For example, it is not clear whether the inhibition of mitochondrial fusion is an essential step in apoptosis or if fragmentation is promoted mainly because of the increase in fission. In addition, it has been shown that the loss of either Drp1 or hFis1 delayed, but did not block cell death, questioning the importance of the mitochondria morphological shift in the apoptotic cascade (5, 6). In this context, there is an emerging emphasis on the examination of mitochondrial fusion in the context of cell death.Mitochondrial fusion is regulated by at least three essential GTPases, the outer membrane-anchored proteins mitofusin 1 (Mfn1), 3 and mitofusin 2 (Mfn2) along with the intermembrane space GTPase, Opa1 (7,8). Although the functions of Mfn2 overlap with Mfn1 in the process of mitochondrial fusion (9), there are clear distinctions between these two GTPases. Perhaps most informative of their distinct function is their biochemical difference in nucleotide binding and hydrolysis properties, where Mfn1 has a faster GTPase hydrolysis rate and higher affinity for nucleotide relative to Mfn2 (10, 11). In addition, Mfn1, but not Mfn2, has been shown to geneticall...

Mitochondrial fission/fusion dynamics and apoptosis

“…In other studies overexpression of Mfn2 and the rat homologue Fzo1 reduced cytochrome c release and apoptosis (Jahani-Asl et al, 2007;Sugioka et al, 2004) while in a fourth study, overexpression of Mfn1, Mfn2 or Opa1 had no effect on cytochrome c release or apoptosis (Sheridan et al, 2008). Importantly, the authors of the latter study demonstrated that cells displaying fused phenotypes still lost cytochrome c in response to pro-apoptotic stimuli, indicating that Bax/Bakdependent pore formation can still occur in fused mitochondria.…”

“…Within a similar time frame, mitochondria fragment from filamentous tubules into numerous small punctate particles (Frank et al, 2001;Gao et al, 2001;Jahani-Asl et al, 2007;Lee et al, 2004;Sheridan et al, 2008; Figure 2). These fragmented mitochondria often collapse from an extended network covering the majority of the cell, into a clustered perinuclear…”

Mitochondrial respiratory chain dysfunction: Implications in neurodegeneration

“…Accordingly, over-expression of proteins involved in mitochondrial fusion prevented mitochondrial fragmentation and cell death in cerebellar granule neurons treated with H 2 O 2 (Jahani- Asl et al, 2007;Jahani-Asl et al, 2011). The mitonetwork response to oxidative stress seemed to be dose-dependent, as higher concentrations or higher exposure times to H 2 O 2 increased the mitochondrial fragmentation extent and cell death rates Jahani-Asl et al, 2007;Jendrach et al, 2008). In agreement, lower H 2 O 2 doses induced mitochondrial hyperfusion associated to decreased Fis1 expression levels and augmented Mfn1 and Mfn2 .…”

“…Different cultured cells, including neurons, under oxidative stress due to NO or H 2 O 2 treatment showed extensive mitochondrial network fragmentation and increased cell death (Barsoum et al, 2006;Yoon et al, 2006;Jahani-Asl et al, 2007;Jendrach et al, 2008). Although the precise mechanisms by which oxidative stress modulates mitochondrial dynamics are not well understood, some authors suggested that ROS could modulate the expression levels and post-traslational modifications of the proteins involved in mitochondrial dynamics.…”

“…In this regard, decreased fission and fusion events due to an altered expression of the FIS1, DRP1 and MFN1 mRNAs after H 2 O 2 exposure were described (Jendrach et al, 2008). Accordingly, over-expression of proteins involved in mitochondrial fusion prevented mitochondrial fragmentation and cell death in cerebellar granule neurons treated with H 2 O 2 (Jahani- Asl et al, 2007;Jahani-Asl et al, 2011). The mitonetwork response to oxidative stress seemed to be dose-dependent, as higher concentrations or higher exposure times to H 2 O 2 increased the mitochondrial fragmentation extent and cell death rates Jahani-Asl et al, 2007;Jendrach et al, 2008).…”