ubb , a loss - of - function variant of ubiquitin b ( ubb ) , accumulates in neurofibrillary tangles , a pathological hallmark in alzheimer s disease ( ad ) ( van leeuwen et  al . , 1998 ) .
ubb is translated from an aberrant mrna encoding a  + 1 frameshift protein in  which the c - terminal glycine residue required for ubiquitylation is replaced by an extension of 20 amino acids ( dennissen et  al . ,  2010 ) .
the detrimental impact of ubb has been studied in  neuronal cell cultures , transgenic mice , and yeast ( de vrij et  al . , 2001 ;
ubb is a substrate for truncation , ubiquitylation , and proteasomal degradation ( dennissen et  al . , 2011 ; lindsten et  al . , 2002 ;
whereas the ubiquitin - proteasome system ( ups ) can assure the degradation of low levels of ubb , higher levels impair the ups and subvert the homeostatic mechanisms allowing for its elimination ( fischer et  al . , 2009 ; lindsten et  al . , 2002 ;
at high levels , ubb affects mitochondrial dynamics and triggers neuronal cell death ( de vrij et  al . , 2001 ; tan et  al . , 2007 ) through as - yet elusive mechanisms .
yeast is an established model for studying programmed cell death mechanisms that are often shared with animal cells , including the contribution of caspases and mitochondrion - associated cell death proteins , such as cytochrome c ( carmona - gutierrez et  al . , 2010 ) .
yeast models have been used to explore cell killing by neurotoxic proteins , such as parkinson - disease - associated -synuclein , and the outcome could be successfully translated to fly , worm , and murine disease models , as well as to human disease ( braun et  al . , 2010 ; bttner et  al . ,
2013 ) . driven by these premises , we established a yeast cell death model for ubb - triggered neurotoxicity .
our findings revealed that ubb interfered with the ups and triggered the perturbation of the mitochondrion - associated basic amino acid synthesis executing cell death .
the mitochondrion - associated ups subroutine , depending on the aaa - atpase cdc48 and its co - factor vms1 , strongly antagonized ubb cytotoxicity . since vms1 , the human homolog of yeast vms1 ,
co - exists with ubb in neurofibrillary tangles , these data imply a potential pivotal role of the ups at mitochondria in ad .
to investigate whether the introduction of ubb into yeast recapitulates hallmarks of ubb accumulation in neurons , we expressed monomeric ubiquitin b ( ubb ) , ubb , as well as an ubb variant lacking two lysine residues ( k29,48r ) that are important for its ubiquitylation .
when expressing ubb , we detected a discrete immunoreactive band at the size of monomeric ubiquitin ( 9  kda ) , and an immunoreactive smear across a wide range of the immunoblot that corresponds to ubiquitylated proteins ( figure  1a ) .
this smear was not detectable upon transformation with ubb or ubb - k29,48r , reflecting their loss of function .
instead , ubb or ubb - k29,48r were detectable as 12 and 9  kda protein species ( full - length and truncated ubb ; fl - ubb and tubb ) that accumulated over time ( figures 1a , s1a , and s1b ) . in cells expressing ubb , a faint higher molecular weight species corresponding to the size of monoubiquitylated fl - ubb ( 21  kda ) appeared ( figure  1a , flag long exposure , asterisks ) .
consistent with a role of lysines 29 and/or 48 in the ubiquitylation of ubb , this band was absent in cells expressing ubb - k29,48r .
these results suggest that in yeast human ubb ( but not ubb ) can serve as a substrate for ubiquitin ligases and that , like in neurons , ubb is ubiquitylated and truncated .
next , we investigated whether ubb expression results in ups impairment by means of three complementary assays : ( 1 ) the measurement of polyubiquitylated endogenous proteins by immunoblot ; ( 2 ) the assessment of the abundance of transgenic ubiquitin - g76v - gfp , which is a substrate of the ubiquitin - fusion degradation pathway ; and ( 3 ) an enzymatic assay designed to quantify the chymotrypsin - like proteasomal activity .
cells expressing ubb or ubb - k29,48r contained a higher level of polyubiquitylated proteins than cells transformed with vector controls ( figures 1b and 1c ) , suggesting decreased ups - dependent protein turnover .
the steady - state levels of ubiquitin - g76v - gfp were significantly increased upon expression of ubb or ubb - k29,48r ( figure  1d ) .
in contrast , ubb or ubb - k29,48r expression did not reduce chymotrypsin - like proteasomal activities ( figure  s1c ) .
these data suggest that , in yeast like in neurons , ubb expression impairs the ups . however , in yeast ubb does neither directly affect the enzymatic activity of proteasomes , nor is its ubiquitylation essential for ups dysfunction . to assess its effects on the fitness of proliferating cells , we performed growth assays on agar plates and in liquid cultures . as a positive control of cytotoxicity , tdp-43 , a causal factor for motor neuron degeneration , was expressed . in sharp contrast with tdp-43 , ubb and
ubb - k29,48r failed to compromise the growth of cells on agar plates ( figure  2a ) , and in liquid cultures ( figure  2b ) , suggesting that ubb is unable to kill proliferating cells .
next , we studied the effects of ubb or ubb - k29,48r on chronologically aged cultures . for this
, the proportion of viable cells capable of forming a colony ( clonogenicity ) on nutrient - containing  solid medium was studied at different time points following ubb or ubb - k29,48r expression .
consistent with the growth assays , 16  hr ( day 1 ) after ubb or ubb - k29,48r expression cells exhibited a similar clonogenic potential as did  cells expressing vector controls ( figure  2c ) .
in contrast , we  observed a 10% and 25% decrease in clonogenic cell survival when expressing ubb for 2 and 3  days , respectively .
exogenously applied stressors , including acetate and hydrogen peroxide , further enhanced the cytotoxicity of prolonged ubb expression ( figures s2a and s2b , left ) . upon both chronological aging and stress experiments , ubb - k29,48r turned out to be slightly less cytotoxic as compared to ubb ( figures 2c , s2a , and s2b , left ) , suggesting that ubiquitylated ubb is slightly more cytotoxic than ubb .
we next examined whether the ubb - induced loss of clonogenicity correlated with the manifestation of oxidative stress , which can be detected by the intracellular conversion of the reactive oxygen species ( ros)-sensitive stain dihydroethidium ( dhe ) to fluorescent ethidium .
we observed indistinguishable  low levels of oxidative stress after expressing ubb or ubb - k29,48r for 16  hr ( day 1 ) ( figure  2d ) . at later time points ,
the levels of oxidative stress progressively increased in all cultures with chronological aging , and ubb - expressing cells exhibited a mild but significant increase in oxidative stress as compared to vector controls .
thus , upon ubb expression increased markers of oxidative stress coincided with decreased clonogenic cell survival ( cf .
figures 2c , 2d , and s2c ) . when combined with protracted ubb expression , acetate or hydrogen peroxide exacerbated the signs of oxidative stress ( figures s2a and s2b , right ) .
as shown for clonogenic survival , ubb - k29,48r demonstrated slightly decreased levels of oxidative stress upon chronological aging or exogenously applied stress as compared to ubb ( figures 2d , s2a , and s2b ) .
to determine the mode of cell death triggered by the expression of ubb or ubb - k29,48r , we performed double staining with annexin v - fitc and propidium iodide ( pi ) .
annexin v - fitc labels externalized phosphatidylserine that appears on the surface of apoptotic cells , whereas pi is a vital dye that stains cells that have lost plasma membrane integrity during necrosis .
two days after ubb expression the frequencies of early apoptotic ( annexin v - fitc pi ) , late apoptotic or secondary necrotic ( annexin v - fitc pi ) , and necrotic cells ( annexin v - fitc pi ) were increased , as compared with vector controls ( figures 2e and s2d ) .
apoptosis induction by ubb could be confirmed by the terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) that detects fragmentation of nuclear dna ( figures 2f and s2e ) .
consistent with the results obtained from the clonogenic survival and oxidative stress experiments , ubb - k29,48r triggered cell death in a lower number of cells as compared to ubb ( figures 2e and 2f ) .
altogether , these results indicate that the protracted expression of ubb can induce apoptotic and necrotic killing of yeast cells , and that ubiquitylated ubb is a slightly better killer than ubb . to investigate the putative contribution of dysfunctional ups to ubb - triggered cytotoxicity , we measured the cytotoxic potential of ubb in the context of enhanced or suppressed ups .
since full knockout of genes coding for proteasomal subunits is lethal , yeast strains bearing point mutations in one or two proteasomal genes were employed ( heinemeyer et  al . , 1993 ) .
the chymotrypsin - like proteasomal activity was reduced in strains carrying mutant alleles in the proteasomal subunits pre1 and pre2 by > 88% ( figure  3a ) . in these conditions of close - to - complete proteasomal inactivation ,
significantly reduced clonogenic cell survival was only observed in the pre1 - 1 and the pre1 - 1/pre2 - 2 strains upon ubb expression for day 1 , and in the pre1 - 1/pre2 - 2 strain upon ubb expression for day 2 ( figures 3b and s3a ) , as compared to wild - type strain .
one explanation for the increased ubb - triggered cytotoxicity would be that ubb accumulates in these strains due to impaired ubb degradation .
however , we could not observe increased steady - state levels of ubb in these strains ( neither fl - ubb , nor tubb , nor ubiquitylated fl - ubb ) ( figures s3d  s3f ; data not shown ) .
thus , although severe proteasomal inactivation can increase ubb - triggered cell death , there is no strict correlation between the loss of proteasomal capacity on the one hand , and the increase in ubb - triggered cytotoxicity or the increase in the steady - state levels of ubb on the other hand .
next , we measured ubb - induced cytotoxicity in knockout strains lacking selective ups genes , including ( 1 ) ubi4 encoding ubiquitin ( finley et  al . , 1987 ) , ( 2 ) rpn4 encoding a major transcriptional ups activator ( mannhaupt et  al .
, 1999 ) , ( 3 ) ubr2 encoding the e3 ligase responsible for rpn4 degradation ( kruegel et  al . , 2011 ) , ( 4 ) yuh1 encoding the ubiquitin protease that cleaves fl - ubb into tubb ( dennissen et  al . , 2011 ) , and ( 5 ) ubp6 encoding a deubiquitinase , which can be inhibited by extended ubiquitin proteins ( krutauz et  al . , 2014 ) .
only rpn4 deletion significantly impaired the chymotrypsin - like proteasomal capacity of the cells ( figure  3c ) .
notably , upon comparable fl - ubb steady - state levels ( figures s3 g and s3i ) , ubb - triggered cytotoxicity was significantly increased in ubi4 as compared to rpn4 upon both stressed und unstressed conditions ( figures 3d and s3b ) , although the proteasomal capacity was lower in rpn4 as compared to ubi4 ( figure  3c ) .
these data propose that the ratio of mutant ubiquitin ( ubb ) to wild - type ubiquitin ( encoded by ubi4 ) is more relevant for determining ubb - triggered cytotoxicity than the proteasomal capacity . upon stressed conditions ,
ubb - triggered cytotoxicity was markedly increased in yuh1 as compared to wild - type cells upon comparable fl - ubb steady - state levels ( figures 3d , s3b , s3h , and s3i ) .
these data suggest that ubb truncation is a putative protective event , for instance , as part of a mechanism to degrade excessive ubb . in ubp6
as compared to wild - type cells ubb - triggered cytotoxicity was unaltered upon comparable fl - ubb steady - state levels ( figures 3d , s3b , s3 g , and s3i ) , suggesting that ubp6 activity is not protective against the accumulation of the extended ubiquitin ubb .
ubb - triggered cytotoxicity was significantly relieved in ubr2 cells upon stressed conditions ( figures 3d and s3b ) , in which rpn4 is stabilized and consequently the ups activity is increased ( kruegel et  al . , 2011 ) .
consistently , rpn4 expression , which also leads to increased ups activities ( figure  3e ) , was protective for ubb - expressing wild - type cells ( figures 3f and s3c ) but not for cells lacking ubi4 ( figure  s3c ) .
in both cases , the protective effect can not be explained by decreased steady - state levels of ubb ( figures s3 g and s3i  s3k ) .
these data show that increasing ups capacity is protective for ubb - expressing cells , but not by affecting the turnover of ubb itself but rather by interrupting the lethal signaling cascade triggered by ubb .
oxidative stress and mitochondrial impairment are hallmarks of neurotoxin - elicited death in yeast and neurons ( braun , 2012 ; debattisti and scorrano , 2013 ) .
therefore , we analyzed whether oxidative stress , which occurred starting by day 2 of ubb expression ( figure  4a ) , correlated with mitochondrial impairment .
two days after inducing ubb expression , the mitochondrial network was fragmented in both ubb - expressing cells , as well as in cells carrying vector controls ( data not shown ) , which is typical for stationary phase cultures .
however , after shifting these cultures to fresh growth medium ( which represses ubb expression ) the recovery of the mitochondrial network was significantly compromised in cultures transformed with ubb - encoding constructs as compared with vector controls ( figures 4b and s4a ) .
we further tested for mitochondrial impairment by measuring the cellular oxygen consumption , the mitochondrial membrane potential , and the atp levels in cells expressing ubb for days 2 and 3 . whereas the cellular oxygen consumption and mitochondrial membrane potential were significantly increased by day 3 in ( surviving ) cells expressing ubb ( figures 4c and 4d ) , the cellular atp levels were significantly decreased by days 2 and 3 ( figure  4e ) .
these data hint at hyperactive mitochondria , which are incapable to prevent a metabolic crisis in ubb - expressing cells . in yeast ,
alterations in the cytochrome bc1 complex of the mitochondrial respiratory chain may contribute to the loss of respiratory capacity and the production of lethal ros ( diaz et  al .
for instance , loss of the rieske iron - sulfur protein rip1 , a key component of the cytochrome bc1 complex , results in increased ros generation and  mitochondrial dysfunction ( diaz et  al . , 2012 ) .
indeed , the  cellular level of rip1 was markedly decreased by days 2 and 3 upon ubb expression as compared with vector controls ( figures s4b and s4c ) .
consistently , rip1 and cytochrome c were depleted in the mitochondrial fraction of ubb - expressing cells ( figures 4f and 4 g ) . these data
further hint at  a major ubb - induced mitochondrial dysfunction , in which  the respiratory chain is impaired ( depletion of rip1 and cytochrome c ) , leading to the production of ros ( for which cellular oxygen is needed ) , and the decline of cellular atp levels .
hyperpolarization of mitochondria may precede mitochondrion - dependent yeast death ( eisenberg et  al . , 2007 ) ; therefore , we expressed ubb in strains deleted for genes encoding a range of mitochondrial cell death proteins , including the yeast bh3-only protein ( ybh3 ) that translocates to mitochondria to mediate their permeabilization , and several potentially cytotoxic proteins that can be released from mitochondria such as apoptosis - inducing factor 1 ( aif1 ) , endonuclease g ( nuc1 ) , and the two cytochrome c isoforms ( cyc1 , cyc7 ) .
deletion of nuc1 resulted in a paradoxical increase in ubb - triggered cytotoxicity , and loss of ybh3 did not have any effect upon both stressed and unstressed conditions ( figures 4h and s4d ) .
in contrast , ubb - mediated cytotoxicity was significantly decreased in strains depleted from isoform 2 of cytochrome c ( cyc7 ) upon stressed conditions ( figures 4h and s4d ) .
the steady - state levels of ubb were not decreased in the cyc7 as compared to wild - type strain ( figure  s4f ) , and this strain maintained a normal state of respiratory competence ( presumably due to the presence of the cytochrome c isoform 1 cyc1 ) ( figure  s4i ) , excluding trivial explanations for the cytoprotective action of cyc7 .
thus , our data suggest the implication of mitochondria in ubb - triggered cell death .
next , we tested for a possible role of the unfolded protein response ( upr ) and the er in ubb - triggered cytotoxicity and expressed ubb for 2  days in cells lacking the upr kinase ire1 and its downstream target hac1 , as well as in cells lacking the er cell death protease kex1 ( which executes cell death in which mitochondria play a pivotal role [ hauptmann and lehle , 2008 ] ) . upon stress ,
ubb - triggered cytotoxicity was relieved in ire1 and kex1 but not in hac1 cells ( figures 4i and s4e ) , under conditions where the steady - state levels of ubb were comparable ( figures s4 g and s4h ) .
these data suggest for an implication of the er in ubb - triggered cytotoxicity , but , due to the lack of rescue in the hac1 cells , a critical involvement of the upr is unlikely .
next , we performed quantitative proteomic analyses of crude mitochondria after  stable isotope labeling by amino acids in cell culture  ( silac ) .
this approach led to the identification of 16 proteins whose abundance was significantly altered ( increased for ten or decreased for six proteins ) upon ubb expression ( figure  5a ; table s2 ) . among the proteins with established mitochondrial localization , three were enzymes participating in amino acid metabolism , namely , put1 ( involved in proline degradation ) , arg5,6 , and arg8 ( involved in arginine and  ornithine biosynthesis ) . in addition , ubb induced the accumulation of the cytosolic enzyme lys1 ( involved in lysine biosynthesis ) , an increase in the motor protein myo3 and the ( putative ) peroxisomal proteins gpd1 and str3 , in crude mitochondria . upon acetate stress , deletion of the arg5,6 , arg8 , and
the  lys1 genes restored the clonogenic potential of ubb - expressing cells , whereas the deletion of all other genes  had no effect ( figures 5b and s5a ) .
these data point to a hitherto unexpected involvement of the biosynthesis of basic amino acids  ( arginine , ornithine , and lysine ) in ubb - triggered cytotoxicity . to challenge this hypothesis
, we measured the cellular steady - state levels of arginine , ornithine , and lysine in cultures expressing ubb ( figure  5c ) . indeed
, we observed a marked increase in the cellular levels of all three basic amino acids , in particular , ornithine , upon ubb accumulation .
to weigh the contribution of arginine and ornithine ( as opposed to their metabolic intermediates ) to ubb cytotoxicity , we measured ros production upon ubb expression in strains depleted from the arginine and ornithine biosynthetic enzymes ( figure  5d ) .
depletion of all enzymes operating upstream of cytosolic ornithine ( arg2 , arg5,6 , arg7 , and ort1 ) significantly relieved ubb - triggered cytotoxicity both in unstressed and acetate - stressed conditions ( figures 5e and s5b ) .
in contrast , none of the enzymes downstream of cytosolic ornithine ( arg3 , arg1 , and arg4 , which are needed for the conversion of ornithine into arginine ) were required for the cytotoxic action of ubb .
notably , all tested enzymes operating upstream of cytosolic ornithine are mitochondrion - associated ( ljungdahl and daignan - fornier , 2012 ) .
therefore , we concluded that ubb triggers the mitochondrion - associated biosynthesis of ornithine , leading to increased cytosolic levels of ornithine ( and its product arginine ) .
if this model is true , increasing cytosolic levels of either ornithine or arginine ( which can easily be interconverted into each other ) should recover the cytotoxic effect of ubb in strains with interrupted mitochondrion - associated biosynthesis of ornithine .
therefore , we measured ubb - triggered cell death in the strain depleted for the mitochondrial protein ort1 in growth media with increasing concentrations of arginine and ornithine , respectively .
it turned out that ort1 cells were not able to efficiently uptake ornithine from the growth media , because the severe growth deficit of the ort1 strain in growth media lacking arginine could not be relieved by increasing concentrations of ornithine in the growth media ( data not shown ) .
in contrast , ort1 cells grew well in the presence of arginine in growth media lacking ornithine ( data not shown ) , demonstrating the efficient cellular uptake of arginine .
as expected , yeast cells lacking ort1 were protected from ubb - triggered cell death upon moderate concentrations of arginine ( 30 and 50  mg / l ) in the growth media ( figure  5f ) .
in contrast , elevated concentrations of arginine in the growth media ( 150 and 300  mg / l ) recovered the cytotoxic effect of ubb ( figure  5f ) , substantiating the decisive role of increased cellular levels of arginine ( and cytosolic ornithine ) in executing ubb - triggered cell death . in order to address the role of cellular levels of lysine
, we measured ubb - triggered cell death in the strain depleted from lys1 in growth media with increasing concentrations of lysine .
whereas deletion of lys1 relieved ubb - triggered cytotoxicity as compared to wild - type strain ( figure  5b ) , increasing the lysine concentrations did not promote cytotoxicity in the lys1 strain ( figure  5 g ) . thus
, in contrast to arginine / ornithine the cellular lysine level appears to be negligible in accelerating ubb - triggered cell death .
the aforementioned data incriminate mitochondria and the ups  in the execution of ubb - triggered cytotoxicity , notably because of the protective impact of the removal of mitochondrial enzymes involved in basic amino acid synthesis and the overexpression of the transcriptional ups activator rpn4 . among the known rpn4 targets are the conserved aaa - atpase cdc48 and its cofactor npl4 ( bosis et  al . , 2010 ) .
cdc48 and npl4 are involved in the ups , and determined by their cofactor vms1 , regulate mitochondrion - associated protein degradation ( heo et  al . ,
driven by these premises , we evaluated the involvement of the cdc48/vms1/npl4-dependent ups pathway to ubb - triggered cytotoxicity .
for this , we measured ubb - triggered cytotoxicity in normal and acetate - stressed conditions in strains expressing increased levels of wild - type cdc48 or the pro - apoptotic cdc48-s565 g variant ( madeo et  al . , 1997 ) , which is characterized by decreased vms1 binding and mitochondrion - associated degradation ( heo et  al . , 2010 ) .
we also determined the cytotoxicity of ubb in strains depleted from the cdc48 cofactors npl4 and vms1 , or overexpressing vms1 .
ubb - triggered cytotoxicity was markedly attenuated in cultures expressing increased levels of wild - type cdc48 , as compared to cells expressing cdc48-s565 g or controls with endogenous cdc48 only ( figures 6a and s6a ) .
ubb - triggered cytotoxicity was significantly increased in cultures depleted from npl4 under non - stressed conditions ( figures 6b and s6b ) .
depletion of vms1 resulted in a marked elevation in cytotoxicity upon stress ( figures 6c and s6c ) , while overexpression of vms1 significantly protected against ubb upon acetate stress , as measured by the clonogenic approach ( figures 6d and s6d ) .
high levels of vms1 also protected from cell death  and oxidative stress induced by ubb expression ( figures 6e and 6f ) .
notably , high amounts of cdc48 and cdc48-s565 g resulted in markedly decreased steady - state levels of ubb ( figures s6e and s6f ) , whereas neither the deletion of vms1 , nor its overexpression had an effect on the cellular ubb amounts ( figures s6g  s6j ) .
these data point to a protective role of vms1 , which is independent from ubb degradation , potentially by improving the quality control at mitochondria .
in contrast , the beneficial role of high amounts of cdc48 could be due to both increased vms1-independent ubb degradation and improved vms1-dependent mitochondrial quality control . in order to address whether elevated vms1 levels prevent from ubb - triggered mitochondrial impairment , we measured the cellular oxygen consumption , the mitochondrial membrane potential , and the cellular atp levels in cells expressing ubb upon endogenous or elevated amounts of vms1 ( figures 6g6i ) . whereas the cellular oxygen consumption and the mitochondrial membrane potential were significantly decreased by day 3 and days 2 and 3 , respectively ,
cellular atp levels were significantly increased by day 2 upon high amounts of vms1 . in other words ,
high amounts of vms1 reverted the mitochondrial damage induced by high levels of ubb ( see figures 4c4e ) . in a next step , we used silac technology to comparatively assess alterations of the mitochondrial proteome between ubb - expressing cells with endogenous and high levels of vms1 ( figure  6j ; table s3 ) .
we observed that among the 16 proteins whose abundance levels were altered by ubb as compared with the vector control ( figure  5a ; table s2 ) , ten were no more altered upon expression of both ubb and vms1 ( figure  6j , blue - labeled proteins ) .
among these ten proteins , which were particularly stringently associated with the cytopathic activity of ubb , the basic amino acid synthesis enzymes arg5,6 , arg8 , and lys1 were significantly decreased in ubb - expressing cells upon high levels of vms1 , as compared to endogenous vms1 levels .
consistently , vms1 overexpression blunted the ubb - mediated increase in the steady - state levels of arginine , ornithine , and lysine ( figure  6k , see figure  5c ) .
these data point to a pivotal role of the vms1-dependent mitochondrial ups activity in avoiding the ubb - triggered lethal overproduction of basic amino acids .
pathological hallmarks include intracellular neurofibrillary tangles comprising aberrant forms of the microtubule - associated protein tau , ubb , and the mitochondrial outer membrane voltage - dependent anion channel 1 ( vdac1 ) ( reddy , 2013 ; van leeuwen et  al . , 1998 ) .
immunohistochemistry revealed expression of vms1 , the human homolog of yeast vms1 , in pyramidal cells within the hippocampi from ad patients and aged non - demented controls ( figure  7a , all arrows ; tables s5 and s6 ) .
vms1 stained structures reminiscent of tau pathology , including tangle - like ( yellow arrows ) and neuropil thread - like structures ( blue arrows ) , as well as other cellular staining patterns ( green arrows ) , were observed in samples from ad patients , and aged  non - demented controls with tau pathology .
we also observed these tangle - like and thread - like staining patterns when analyzing the sections for aberrant tau , ubb , and vdac1 ( figure  s7 ) .
immunohistochemistry of consecutive paraffin sections from the hippocampi of ad patients ( figure  7b ) confirmed the identification of pyramidal cells with intracellular tangle - like structures , which co - stained for aberrant tau and vms1 ( figure  7c , violet arrows ) , and for ubb , vms1 , and vdac1 ( figure  7d , orange arrows ) .
these data suggest that vms1 is a component of neurofibrillary tangles comprising aberrant tau , ubb , and vdac1 , underscoring a potential role of the cdc48/vms1 complex in ubb - mediated ad progression .
we established a yeast model for dissecting cell death mechanisms triggered by ubb , in which mitochondria play a pivotal role in the execution of cell death ( see the supplemental discussion ) .
ubb triggers neuronal apoptosis accompanied by reduced mitochondrial movement ( tan et  al . , 2007 ) , and mitochondrial impairment likely contributes to ad ( rodolfo et  al . , 2010 ) .
thus , our yeast model corroborates features of cell - death - relevant mitochondrion dysfunctions found in ad neurons .
yeast strains expressing ubb accumulated the basic amino acids arginine , ornithine , and lysine .
deletion of mitochondrion - associated enzymes involved in their synthesis abolished ubb - triggered cell killing , which could be recovered by increasing concentrations of arginine in the growth media .
the accumulation of basic amino acids may trigger mitochondrial damage and cell death in mammalian cells and in yeast ( almeida et  al . , 2007 ; bicz et  al . ,
for instance , increased production of nitric oxide from arginine executes yeast apoptosis ( almeida et  al . , 2007 ) and
increased levels of polyamines , which are produced from ornithine , may impair neuronal ion channel activities ( inoue et  al . , 2013 ) .
the levels of arginine , ornithine , and/or their polyamine products were altered in aged human brains and in brains from ad patients ( inoue et  al . , 2013 ; liu et  al . , 2014 ; rushaidhi et  al . ,
the results of these studies are controversial , and it remains not yet clear whether the observed alterations are cause or consequence of ad . despite that , our data suggest that perturbed basic amino acid  synthesis is a decisive event triggering mitochondrion - dependent cell death upon ubb accumulation in yeast .
further studies aiming at analyzing the role of arginine / ornithine metabolism during aging or ad progression should consider a potential pivotal contribution of ups and mitochondrial dysfunctions . we observed that ubb accumulation impaired the ups , and that the ups activity , in turn , determined ubb cytotoxicity .
yeast cultures that were depleted from ubiquitin ( ubi4 ) were highly vulnerable to ubb .
in contrast , yeast cultures in which the ups was stimulated by the  transcriptional activator rpn4 were insensitive to ubb , but not in cells lacking the ubiquitin gene ubi4 .
extended ubiquitin variants have been proposed to be specific inhibitors of the deubiquitinase ubp6 in yeast ( krutauz et  al . , 2014 ) . since the ubb - triggered cytotoxicity was unaltered in a strain deleted for ubp6 as compared to wild - type strain
, our data suggest that the lethal effect of the extended ubiquitin ubb does not essentially depend on ubp6 .
it is tempting to speculate that the ratio of mutant ( ubb ) to wild - type ubiquitin determines ubb - triggered cytotoxicity with ubb as a competitive inhibitor of wild - type ubiquitin , affecting numerous ubiquitin - regulated cellular processes .
we established that elevated amounts of cdc48 or its cofactor vms1 conferred tolerance against ubb expression .
more specifically , vms1 overexpression relieved the ubb - triggered mitochondrial damage and accumulation of the basic amino acids arginine , ornithine , and lysine .
the cdc48/vms1 complex enables the degradation of mitochondrion - associated proteins ( heo et  al . , 2010 ) . whereas under normal conditions , this complex is predominantly cytosolic , vms1 recruits cdc48 to the mitochondrial outer membrane upon stress , presumably with the scope of improving the local quality of proteins .
our data suggest that cdc48/vms1-mediated processes can prevent the ubb - triggered lethal derangement of mitochondria .
in one possible scenario , cdc48/vms1 might remove protein junk from the mitochondrial outer membrane .
alternatively , cdc48/vms1 might specifically prevent the accumulation of arginine , ornithine , and lysine , through regulation of the turnover of the enzymes arg5,6 , arg8 , and lys1 , which are pivotal for their synthesis .
whereas the activity of the cytoplasmic enzyme lys1 could be regulated by its degradation , the activities of the mitochondrion - associated arg5,6 and arg8 could be controlled by preventing their import into mitochondria via ubiquitylation and proteasomal degradation .
lys1 , arg5,6 , and arg8 are known targets for ubiquitylation ( xu et  al . , 2009 ) , and
the ups regulates the import of mitochondrial intermembrane space proteins ( bragoszewski et  al . , 2013 ; harbauer et  al . ,
it is tempting to speculate for a ups - dependent regulation of the import of the mitochondrial matrix proteins arg5,6 and arg8 .
further studies are needed to address the influence of ups ( dys)function on the turnover of these and other mitochondrial proteins .
this is important because recent studies demonstrated that ups dysfunction can lead to mitochondrial dysfunction and vice versa ( livnat - levanon et  al . , 2014 ; maharjan et  al . , 2014 ; segref et  al . ,
2014 ) , and our data revealed the unexpected link between ubb - triggered ups dysfunction and the accumulation of functional enzymes in the mitochondrial matrix leading to potentially cytotoxic accumulation of basic amino acids .
human vms1 and mitochondrial vdac1 co - existed with ubb in neurofibrillary tangles of ad patients and aged non - demented controls with tau pathology .
ubb accumulates and the number of neurofibrillary tangles and damaged mitochondria markedly increase during ad progression ( dennissen et  al . , 2010 ; rodolfo et  al . , 2010
we propose that vms1-dependent mitochondrial quality control might retard the ad - associated neuronal dysfunction , which is elicited by the accumulation of both aberrant tau and ubb .
yeast expression constructs , strains , and growth conditions were described in the supplemental experimental procedures .
, cultures were treated for 4  hr with acetate . for stable isotope labeling ( silac ) , cells expressing vector controls , ubb , or ubb and vms1 were grown in media supplemented either with lys0 and arg0 ( normal isotopes ) , or with lys4 and arg6 , or with lys8 and arg10 ( heavy isotopes , silantes ) .
briefly , growth deficits upon expression of proteins of interest on solid or liquid media , as compared to vector controls , suggest for cytotoxic effects of these proteins on ( growing ) yeast cells . for clonogenic assays , 500 cells from liquid yeast cultures expressing proteins of interest or vector controls , respectively ,
were plated on agar plates , on which expression is repressed . the number of colonies ( colony forming units [ cfus ] ) formed after 2  days of incubation correlates with the fitness of the culture .
oxidative stress was determined by measuring the conversion of dihydroethidium ( dhe , sigma - aldrich ) to the red fluorescent ethidium applying a fluorescence plate reader or a flow cytometer .
propidium iodide ( pi , sigma - aldrich ) in cells that have lost their plasma membrane integrity using a flow cytometer .
annexin v / pi co - staining ( annexin v - fluos staining kit , roche applied science ) for discriminating early and late apoptosis , as well as necrosis , and terminal deoxynucleotidyl transferase dutp nick end labeling ( tunel ) for measuring apoptosis ( in situ cell death detection kit , roche applied science ) were performed by flow cytometry .
oxygen consumption of stationary yeast cultures was analyzed using the firesting optical oxygen sensor system ( pyro science ) .
mitochondrial membrane potential was assessed with flow cytometry after staining cells with tetramethylrhodamine methyl ester ( tmrm , molecular probes , life technologies ) , a fluorescent dye that accumulates within mitochondria dependent on their membrane potential . to determine the atp level of yeast cultures
atp was measured using the atp determination kit ( molecular probes , life technologies ) .
this assay is based on an atp - dependent reaction of recombinant firefly luciferase , which induces bioluminescence of its substrate d - luciferin and is directly correlated with the atp content .
see the supplemental experimental procedures for details . for determining the level of polyubiquitylated proteins in cellular extracts , immunoblots of cellular extracts
were incubated with an ubiquitin - specific antibody and immunosignals were quantified with imagej 1.47 m. for measuring the turnover of ups substrates , the ubiquitin - fusion protein ubiquitin - g76v - gfp was co - expressed with ubb or vector controls .
gfp fluorescence ( relative fluorescence units [ rfus ] ) and optical densities ( od600 ) were determined using the fluostar omega plate reader .
rfu was normalized to od600 , in order to determine the level of ubiquitin - gfp fusion proteins per culture .
measurement of chymotrypsin - like proteasomal activities were performed using the fluostar omega plate reader , applying the luminescence - based proteasome - glo cell - based assay ( promega ) .
yeast cultures were incubated in expression media ( scgal ) for the indicated time points .
cell extracts were generated by pre - treating yeast pellets in naoh followed by heating in sds lysis buffer .
protein extracts were separated on tricine - sds polyacrylamide gels , transferred on pvdf membranes , and incubated with primary and secondary antibodies coupled to horseradish peroxidase .
immunoblot quantification was done with the gel analysis method in imagej 1.47 m. see the supplemental experimental procedures for details .
crude mitochondrial extracts were taken up in sds lysis buffer , thawed , reduced with dtt , and alkylated using iodoacetamide ( sigma - aldrich ) .
protein mixtures were separated by sds - page using bis - tris gels ( nupage , invitrogen ) .
the gel lanes were cut into slices , which were in - gel digested with trypsin ( promega ) , and the resulting peptide mixtures were processed on stage tips .
mass spectrometry was performed on a ltq orbitrap xl mass spectrometer ( thermo fisher scientific ) coupled to an eksigent nanolc - ultra .
electro spray high - resolution mass spectrometry ( ip - rp - lc / hrms ) .
lc / ms measurements were normalized to the total number of cells of each sample . see the supplemental experimental procedures for details .
experiments with human materials were in accordance with the local ethical committees at the universities of bayreuth ( germany ) and maastricht ( the netherlands ) .
postmortem tissues of hippocampi from ad patients and non - demented controls were obtained from the netherlands brain bank ( table s6 ) as paraffin sections . for immunohistochemistry
, sections were deparaffinated , incubated with primary antibodies against the indicated proteins , and with biotin - coupled secondary antibodies followed by the avidin - biotin - peroxidase complex .
 summaryneuronal accumulation of ubb+1 , a frameshift variant of ubiquitin b , is a hallmark of alzheimer s disease ( ad ) . 
 how ubb+1 contributes to neuronal dysfunction remains elusive . here 
 , we show that in brain regions of ad patients with neurofibrillary tangles ubb+1 co - exists with vms1 , the mitochondrion - specific component of the ubiquitin - proteasome system ( ups ) . 
 expression of ubb+1 in yeast disturbs the ups , leading to mitochondrial stress and apoptosis . 
 inhibiting ups activity exacerbates while stimulating ups by the transcription activator rpn4 reduces ubb+1-triggered cytotoxicity . 
 high levels of the rpn4 target protein cdc48 and its cofactor vms1 are sufficient to relieve programmed cell death . 
 we identified the ubb+1-induced enhancement of the basic amino acids arginine , ornithine , and lysine at  mitochondria as a decisive toxic event , which can be reversed by cdc48/vms1-mediated proteolysis . 
 the fact that ad - induced cellular dysfunctions can be avoided by ups activity at mitochondria  has  potentially far - reaching pathophysiological implications . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions