Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity
Alexander disease ( AxD ) is a rare and invariably fatal neurological disorder that affects primarily infants and small children , but can also manifest later in life ( Alexander , 1949; Sosunov et al . , 2018; Messing , 2018 ) . Autosomal dominant gain-of-function mutations in GFAP , which encodes glial fibrillary acidic protein ( GFAP ) , cause AxD ( Messing , 2018; Brenner et al . , 2001 ) . GFAP is the major component of the intermediate filament ( IF ) cytoskeleton in astrocytes ( Hol and Pekny , 2015 ) . The accumulation and incorporation of mutant GFAP within cytoplasmic aggregates called Rosenthal fibers ( RFs ) , causes reactive gliosis , leading to secondary injury to neurons and non-neuronal cells ( Olabarria and Goldman , 2017; Wang et al . , 2015; Li et al . , 2018; Jones et al . , 2018 ) . Silencing GFAP via antisense oligonucleotide intervention in vivo eliminates RFs , reverses the stress responses in astrocytes and other cell types , and improves the clinical phenotype in a mouse model of AxD ( Hagemann et al . , 2018 ) . While the utility of GFAP as a key therapeutic target in AxD is clear , the molecular mechanisms for how AxD-associated GFAP missense mutations ( affecting over 70 different residues on GFAP ) lead to defective GFAP proteostasis are not well understood . Deciphering these mechanisms may yield novel interventions , not only for AxD patients , but also for patients with other diseases where IF proteostasis is severely compromised . Normal functioning IFs are stress-bearing structures that organize the cytoplasmic space , scaffold organelles , and orchestrate numerous signaling pathways . In contrast , dysfunctional IFs directly cause or predispose to over 70 tissue-specific or systemic diseases , including neuropathies , myopathies , skin fragility , metabolic dysfunctions , and premature aging ( Omary , 2009; www . interfil . org ) . Disease-associated IF proteins share two key molecular features: abnormal post-translational modifications ( PTMs ) ( Snider and Omary , 2014 ) and pathologic aggregation . The GFAP-rich RF aggregates that are hallmarks of AxD astrocytes bear strong similarities to pathologic aggregates of other IFs , including epidermal keratins ( Coulombe et al . , 1991 ) , simple epithelial keratins ( Nakamichi et al . , 2005 ) , desmin ( Dalakas et al . , 2000 ) , vimentin ( Müller et al . , 2009 ) , neurofilaments ( Zhai et al . , 2007 ) and the nuclear lamins ( Goldman et al . , 2004 ) . There are unique advantages to studying IF proteostasis mechanisms in the context of GFAP because of its restricted cellular expression , homopolymeric assembly mechanism , and because GFAP is the sole genetic cause of AxD as a direct result of its toxic gain-of-function accumulation and aggregation . Like all IF proteins , GFAP contains three functional domains: amino-terminal ‘head’ domain , central α-helical ‘rod’ domain and carboxy-terminal ‘tail’ domain ( Eriksson et al . , 2009 ) . The globular head domain is essential for IF assembly and disassembly , which are regulated by various PTMs , in particular phosphorylation ( Omary et al . , 2006 ) . It was shown previously that phosphorylation of multiple sites in the head domain of GFAP ( Thr-7 , Ser-8 , Ser-13 , Ser-17 and Ser-34 ) regulates filament disassembly during mitosis and GFAP turnover in non-mitotic cells ( Inagaki et al . , 1990; Takemura et al . , 2002a; Inagaki et al . , 1994; Inagaki et al . , 1996 ) . Additionally , phosphorylation of GFAP has been observed after various injuries of the central nervous system ( CNS ) including kainic acid-induced seizures , cold-injury , and hypoxic-ischemic models , where phosphorylated GFAP is expressed in reactive astrocytes ( Valentim et al . , 1999; Takemura et al . , 2002b; Sullivan et al . , 2012 ) . These observations reveal that phosphorylation of GFAP is important for re-organization of the astrocyte IF cytoskeleton and plasticity in response to injury . However , it is not clear if , and how , abnormal GFAP phosphorylation compromises proteostasis and contributes to AxD pathogenesis . Here , we identified a critical phosphorylation site in the GFAP head domain that is selectively and strongly upregulated in the brain tissues of AxD patients who died very young , independently of the position of the disease mutation that they carried . Further , we show that this site-specific phosphorylation promotes GFAP aggregation and is a marker of perinuclear GFAP aggregates associated with deep nuclear invaginations in AxD patient astrocytes , but not in isogenic control astrocytes . Finally , we demonstrate a correlation between site-specific GFAP phosphorylation and caspase cleavage in cells and in post-mortem brain tissue from AxD patients . Although our study does not establish a causal relationship between GFAP phosphorylation and caspase cleavage , we show that caspase-6 is a new marker for the most severe form of human AxD . Collectively , our results reveal a new PTM signature that is associated with defective GFAP proteostasis in the most severe form of AxD . Future interventional studies targeting these PTMs will determine whether they contribute to , or are the consequence of , disease severity .
IFs undergo protein synthesis-independent turnover and re-organization to meet cellular demands ( Robert et al . , 2016 ) . PTMs are key in that process , as they regulate filament polymerization and depolymerization , protein-protein interactions , and oligomerization properties of IF proteins ( Snider and Omary , 2014 ) . Of all known PTMs that regulate IFs , phosphorylation is the most ubiquitous and can facilitate or antagonize other types of PTMs via complex cross-talk mechanisms ( Omary et al . , 2006 ) . We hypothesized that AxD-associated GFAP missense mutations ( Figure 1A ) promote GFAP accumulation and aggregation by dysregulating site-specific phosphorylation . We extracted GFAP from post-mortem brain cortex tissue of 13 AxD patients , representing 10 different mutations ( Supplementary file 1 ) and three non-AxD controls ( Supplementary file 2 ) . GFAP from the insoluble high salt extracts ( HSEs ) , prepared according to the procedure described in Figure 1—figure supplement 1 , was used in phospho-proteomic analysis , revealing 12 unique phosphorylation sites on GFAP in AxD ( Figure 1B–C ) . While the AxD-specific phospho-peptides localized to all three functional domains of GFAP ( head , rod , tail ) , the most abundantly phosphorylated residue was a conserved serine ( Ser13 ) in the head domain ( Figure 1C–D ) . Strikingly , we found that the pSer13-GFAP peptide was selectively elevated in the cortex tissue from AxD patients who died very young ( median age at death = 1 . 7 years; range 0 . 5–14 years ) ( Figure 2A ) . Overall , we did not observe significant phosphorylation of GFAP in the control subjects ( Figure 2—source data 1 ) , or in AxD patients who lived 27–50 years ( median age at death = 38 years ) . Further , immunoblot analysis using a phospho-specific antibody ( KT13 ) ( Sekimata et al . , 1996 ) against pSer13-GFAP validated the mass spectrometry results in the AxD patients ( Figure 2B–C ) . Although there was one notable outlier in each age group ( Figure 2B lanes 3 and 11 ) , our results suggest that pSer13-GFAP is primarily associated with the more aggressive , infantile form of AxD . Furthermore , the differences in phosphorylation were not a result of age , since pSer13 GFAP was generally not present in the brain lysates from non-AxD control subjects , regardless of age ( Figure 2D ) . To determine the functional significance of pSer13 on GFAP filament organization , we analyzed the filament properties of non-phosphorylatable ( S13A ) and phospho-mimic ( S13D and S13E ) GFAP mutants . We optimized a transient over-expression system in the SW13 vimentin-negative adrenocarcinoma cells ( SW13vim- ) for this assay , which resulted in primarily filamentous WT GFAP and insoluble aggregated forms of common AxD mutants of GFAP ( Figure 3—figure supplement 1 ) . Compared to wild-type ( WT ) GFAP , the S13D and S13E mutants assembled primarily into large aggregates , similar to the most common AxD-associated mutant R79H-GFAP ( Figure 3A–B ) . S13A formed mostly filaments , although they appeared shorter compared to WT GFAP . To determine if the phospho-mimic mutation directly promotes aggregation , we compared the assembly properties of purified WT , S13A and S13D GFAP ( Figure 3C ) . Consistent with the phenotype observed in the transfected cells , the S13A mutant formed abnormally short filaments in vitro . In contrast , S13D was completely incapable of filament assembly , forming globular structures that were homogeneous in size and not aggregation-prone . Our results with the phospho-deficient and phospho-mimic mutants reveal that S13 is a key site that regulates the assembly properties of GFAP and that its phosphorylation status may modulate the dynamics between filaments and aggregates . In order to explore the function of this phosphorylation event in a disease-relevant system , we used an in vitro human astrocyte model of AxD . We generated iPSCs using fibroblasts from a young AxD patient and characterized their pluripotency by immunofluorescence staining ( Figure 4A ) . Karyotype analysis showed that there were no chromosomal abnormalities due to the reprogramming process ( Figure 4—figure supplement 1 ) . To generate isogenic control cells , we corrected the heterozygous point mutation in GFAP ( c . 715C > T , p . R239C ) using CRISPR/Cas9 mediated gene editing ( Figure 4B ) . Representative chromatograms are shown for the original patient cells and the isogenic controls ( Figure 4C ) . We also isolated ‘CRISPR control’ clones , which were edited on the wild-type GFAP allele , thereby retaining the AxD-causing mutation and serving as an additional disease control for the gene editing procedure . Similar to the original patient cells , the edited cells were karyotyped and characterized for pluripotency ( Figure 4—figure supplement 1 ) . We confirmed that there were no off-target effects due to the editing procedure ( Supplementary file 3 ) . AxD , CRISPR control , and isogenic control iPSCs were differentiated to astrocytes ( iPSC-astrocytes ) via neural progenitor cells ( NPCs ) , as described in the Materials and methods and shown schematically in Figure 4D . After 54 days in culture , iPSC-astrocytes express classical astrocyte markers ( Zhang et al . , 2014 ) , including alcohol dehydrogenase 1 family member L1 ( ALDH1L1 ) , solute carrier family 1 member 3 ( SLC1A3 ) , excitatory amino acid transporter 2 ( EAAT2 ) , Connexin 43 and GFAP ( Figure 4—figure supplement 2 ) . To assess if our model recapitulated key features of AxD , we analyzed total GFAP expression in the iPSC-astrocytes by immunoblot , and found that GFAP levels were significantly higher in the cells that carried the heterozygous GFAP point mutation ( AxD patient and CRISPR control lines ) relative to the isogenic controls ( Figure 4E–F ) . This is consistent with in vivo observations of GFAP levels in AxD patients ( Jany et al . , 2015 ) and mouse models ( Jany et al . , 2013 ) . In addition , high-molecular-mass ( hmm ) GFAP oligomers were present in the AxD iPSC-astrocytes , similar to what we observed when we ectopically expressed the R239C-GFAP mutant ( Figure 5A ) . Finally , we observed by immunofluorescence staining that the AxD mutant iPSC-astrocytes formed both GFAP filaments and perinuclear aggregates ( Figure 5B ) , whereas the isogenic control iPSC-astrocytes formed only GFAP filaments ( Figure 5C ) . In vivo , GFAP antibodies stain the periphery , while DAPI stains the core of RFs ( Der Perng et al . , 2006; Sosunov et al . , 2017 ) . The in vitro-derived AxD iPSC-astrocytes displayed similar characteristics , with RF-like perinuclear aggregates staining positively for GFAP at their periphery and DAPI in the center ( Figure 5B ) . Next , we determined if pSer13-GFAP was present in the AxD iPSC-astrocytes , similar to what we observed in the human brain tissues . As shown in Figure 6A , pSer13-GFAP signal was detected strongly within the core of the perinuclear GFAP aggregates of AxD iPSC-astrocytes . Somewhat surprisingly , we also observed pSer13-GFAP signal in the isogenic control cells , possibly triggered by the in vitro culture conditions . Nevertheless , unlike AxD astrocytes , in the isogenic control astrocytes pSer13-GFAP organization was filamentous and paralleled that of total GFAP . Therefore , the in vitro iPSC-astrocyte model revealed that , only in the presence of the AxD disease mutation , pSer13-GFAP is incorporated within the core of perinuclear inclusions . While in all AxD cells pSer13 signal was detected in the aggregates , we also observed cells with pSer13-positive diffuse cytoplasmic staining and filaments , likely reflecting different states of the GFAP network ( Figure 6—figure supplement 1 ) . Furthermore , the pSer13-positive GFAP aggregates appeared adjacent to prominent nuclear invaginations ( Figure 6A ) . Nuclear deformations , similar to what we observed in the AxD iPSC-astrocytes , are also present in RF-bearing astrocytes in AxD human brain ( Sosunov et al . , 2017 ) . To determine whether the perinuclear aggregates compromised the nuclear envelope , we examined the AxD iPSC-astrocytes by electron microscopy . While we observed filamentous bundles on the cytoplasmic side of the nuclear invaginations , the nuclear envelope appeared intact ( Figure 6B ) . Thus , pSer13-GFAP marks cytoplasmic GFAP aggregates adjacent to nuclear invaginations . It should be noted that the perinuclear aggregates containing disorganized GFAP filaments are not identical to the electron-dense RFs that are seen in post-mortem patient brain , but that they may reflect an intermediate state of GFAP accumulation . To understand the mechanism for how GFAP phosphorylation may promote GFAP aggregation , we conducted a biochemical analysis of the S13A , S13D and S13E GFAP mutants . In line with our immunofluorescence result ( Figure 3A ) , we observed an increase in high-molecular-mass ~100 kDa GFAP oligomer in the phospho-mimic mutant by immunoblot analysis ( Figure 7A ) . However , more strikingly , we observed increased levels of a cleaved GFAP fragment ( 24 kDa ) in S13D and S13E , which was significantly lower in WT- and S13A-GFAP ( Figure 7A–B ) . Cleavage of GFAP by caspase-6 in vitro generates two fragments of 24 and 26 kDa size ( Chen et al . , 2013 ) . The 24 kDa C-terminal fragment is recognized by the monoclonal GA5 antibody , ( Chen et al . , 2013 ) which was used here . Therefore , we tested the effect of a peptide inhibitor of caspase-6 ( Ac-VEID-CHO ) , and found that it significantly reduced the amount of cleaved S13D-GFAP ( Figure 7C–D ) . Furthermore , we observed augmented cleavage of S13D-GFAP when combined with an AxD-causing mutation ( S13D/R79H double mutant ) , and this was also blocked by the caspase-6 inhibitor ( Figure 7C–D ) . Further analysis of the AxD mutant R79H in the transfection system revealed phosphorylation not only at S13 , but also at nearby Y14 , S16 , and S17 ( Figure 7—figure supplement 1 and Figure 7—source data 1 ) . Of note , mutagenesis of S16 and S17 to non-phosphorylatable alanines reduced both the cleavage and oligomerization of R79H ( Figure 7—figure supplement 1 ) . Phospho-motif analysis revealed that S13 , S16 and S17 are part of a segment in the GFAP head domain that is a potential target for several kinases ( Supplementary file 4 ) . Candidate kinases include casein kinase 2 ( CK2 ) , protein kinase A ( PKA ) , PKC , MAP kinase activated protein kinase 2 ( MAPKAP2 ) , and glycogen synthase kinase 3 ( GSK3 ) . These data suggest that phosphorylation of Ser13 ( and nearby S16/17 ) may promote caspase-6-mediated cleavage of GFAP in the context of AxD mutations . In line with that , we observed increased levels of cleaved GFAP ( upon normalization for total GFAP ) in the AxD iPSC-astrocytes compared to isogenic control astrocytes ( Figure 7E ) , along with intense caspase-6 staining within perinuclear GFAP aggregates in AxD iPSC-astrocytes , but not isogenic control astrocytes ( Figure 7F ) . To determine how blocking GFAP cleavage affects aggregation , we performed site-directed mutagenesis to block cleavage of GFAP at Asp225 . As shown in Figure 8A–B , the D225E mutation reduced cleavage of S13D GFAP by >90% . This resulted in partial rescue of filament structure in S13D , although the D225E mutation on its own caused significant filament bundling and perinuclear structures that resembled large aggregates ( Figure 8C–D ) . We also tested the effect of the caspase-6 inhibitor Ac-VEID-CHO , and found that it reduced both the size of the S13D aggregates ( Figure 8E ) and the presence of ~100 kDa hmm GFAP oligomers ( Figure 8F–G ) . However , similar to the mutagenesis experiment , filament bundles were observed in WT and S13D GFAP treated with Ac-VEID-CHO , suggesting that caspase-6 regulates both aggregation and normal GFAP filament re-organization . Caspase-6 is not expressed highly in the normal human brain , especially after birth ( Godefroy et al . , 2013 ) . Therefore , we wanted to examine its expression in the context of AxD . Using immunoblot analysis of total brain lysates , we found that caspase-6 is expressed in the brain tissue from all 8 AxD patients who died very young , but is essentially undetectable in the patients who survived longer ( Figure 9A ) . To ensure caspase-6 expression is not simply more abundant in young individuals , we compared brain lysates from young and old AxD patients to non-AxD control brains from age-matched individuals , and observed a significant increase in caspase-6 expression selectively in young AxD patients , but not in the other groups ( Figure 9B–C ) . Next we asked whether AxD patients , particularly young AxD patients that exhibit more pSer13-GFAP and caspase-6 expression , also displayed increased GFAP cleavage . To determine the extent of caspase-6-cleaved GFAP in AxD patient brains , we utilized an antibody that specifically recognizes N-terminally caspase-6-cleaved GFAP ( D225 ) ( Chen et al . , 2013 ) . We detected cleaved GFAP in extracts from AxD patient brains , and we observed a significant increase in the amount of D225 signal in young AxD patients , which paralleled the increased pSer13 signal in these samples ( Figure 9D–E ) . In agreement with the biochemical evidence , brain tissues from young AxD patients stained intensely for cleaved GFAP , while the signal was significantly weaker in AxD patients who were older ( Figure 9F; Figure 9—figure supplement 1 ) . The signal was particularly strong around perinuclear areas and surrounded circular structures that stained positive for DAPI ( Figure 9F , bottom panels ) , similar to what we observed in the AxD iPSC-astrocytes . Thus , our results show that caspase-6 expression in AxD patient brain tissue parallels the presence of cleaved GFAP , and both are selectively and significantly elevated in patients who succumbed to the disease very early in life .
Our study reveals that missense mutations , affecting discrete domains on the GFAP molecule , share a common PTM signature that is associated with compromised GFAP proteostasis in the severe form of AxD . Using patient brain tissue and human iPSC-derived AxD astrocytes , we show that head domain phosphorylation promotes defective filament assembly and perinuclear accumulation and incorporation of mutant GFAP within nuclear invaginations . By taking an unbiased mass spectrometry proteomic approach , we were able to identify GFAP phospho-peptides that were selectively elevated in human AxD brain tissue , and subsequently validated these results using a phospho-specific antibody against the most abundant epitope ( pSer13-GFAP ) . We demonstrate the importance of the Ser13 site for GFAP assembly in vitro and in cells . Phospho-mimetic mutation S13D completely abolished the ability of GFAP to form filaments in vitro , without leading to aggregation . In transfected SW13vim- cells , phospho mimic S13D-and S13E-GFAP mutants formed highly abnormal perinuclear aggregates that correlated with increased cleavage of GFAP by caspase-6 . We detected a dramatic increase in caspase-6 expression , in association with Ser13 phosphorylation and cleavage of GFAP , in the brain tissue of AxD patients who succumbed to the disease very early in life . While the N-terminal caspase-6 fragment of GFAP promotes filament aggregation in vitro ( Chen et al . , 2013 ) , presently we do not have direct evidence of cause and effect between caspase-6 cleavage and GFAP aggregation in AxD patient cells . Nevertheless , our current findings provide a basis for exploring PTM-based diagnostic and potential therapeutic strategies in AxD . Our study does not address whether Ser13 phosphorylation directly promotes caspase cleavage of GFAP , or if these two PTMs are independent markers of an increased cellular stress response in AxD . One possibility is that Ser13 phosphorylation destabilizes the filament structure , thereby promoting access of caspase-6 to the rod domain Asp225 residue , where the cleavage occurs . Another likely possibility is that the increased cleavage of GFAP is an indirect result of stress-dependent caspase-6 activation in the more severe form of AxD . This is supported by previous studies showing that AxD mutations promote activation and nuclear accumulation of p53 ( Wang et al . , 2015 ) , which can directly induce caspase-6 expression ( MacLachlan and El-Deiry , 2002 ) . Future studies in AxD iPSC-astrocytes and animal models will be required to determine the timing of GFAP phosphorylation and caspase-6 activation in relationship to GFAP cleavage and aggregation . Given our findings that pSer13-GFAP is enriched in the most aggressive form of AxD , monitoring the levels of this phospho-epitope ( in addition to total GFAP ) in AxD patient cerebrospinal fluid or blood may provide added sensitivity for disease activity ( Jany et al . , 2015 ) . Phosphorylation of Ser13 by protein kinase C and cAMP-dependent protein kinase was initially described in vitro using purified recombinant GFAP ( Inagaki et al . , 1990 ) . In the presence of active kinases , Ser-13 phosphorylation occurred in conjunction with phosphorylation at three additional sites ( Thr-7 , Ser-8 , and Ser-34 ) . Phosphorylation of monomeric GFAP at these sites prevented filament assembly , while phosphorylation of in vitro assembled GFAP filaments led to their disassembly ( Inagaki et al . , 1990 ) . Using the same antibody to pSer13-GFAP that we used in this paper ( clone KT13 ) it was later shown that Aurora-B and Rho-associated kinase phosphorylate GFAP in cultured astrocytoma cells during mitosis ( Inagaki et al . , 1996 ) . This may bear relevance to AxD , since human and mouse AxD astrocytes with RFs display mitotic abnormalities ( Sosunov et al . , 2017 ) . However , it was also shown using knock-in mice with the human GFAP head domain that , in vivo , the distribution of pSer13 localization was not limited to mitotic astrocytes , but that select astrocyte populations within multiple regions were pSer13 positive , such as those in the olfactory bulb , subpial regions , and subventricular zone ( Takemura et al . , 2002b; Hagemann et al . , 2005 ) . Interestingly , the regional distribution of pSer13 largely overlaps with areas that are known to be most enriched in RFs in the AxD mouse model ( Hagemann et al . , 2005 ) . Therefore , this particular phosphorylation event on GFAP may occur during mitosis , or in phenotypically distinct astrocyte populations . This remains to be addressed in the future using the appropriate model systems , as over-expression studies in cancer cell lines ( such as the SW13vim- cells we used here ) may not be truly reflective of the signaling that occurs in astrocytes . In particular , it remains to be resolved whether phosphorylation of GFAP on Ser13 is part of a sequentially priming phosphorylation cascade involving nearby Ser16/17 ( as predicted by the kinase motif analysis ) or if Ser16/17 phosphorylation is unique to the SW13 over-expression system . Importantly , identifying the relevant in vivo kinase ( s ) that phosphorylate GFAP in human AxD may lead to potential novel interventions via kinase inhibition . Caspase-mediated proteolysis of IF proteins is an important mechanism by which the filament networks re-organize during apoptosis . Although multiple effector caspases are capable of cleaving IF proteins , caspase-6 is frequently implicated in cleavage at a conserved motif within the linker L12 region of the rod domain , which results in the generation of two fragments of similar sizes . This was initially demonstrated to be the case for the type I keratins ( Caulín et al . , 1997 ) , and later shown to also occur on vimentin ( Byun et al . , 2001 ) , desmin ( Chen et al . , 2003 ) , A-type lamins ( Ruchaud et al . , 2002 ) , and GFAP ( Hol and Pekny , 2015 ) . Caspase-6 cleavage of GFAP at 222VELD225 in vitro generates an N-terminal 26 kDa fragment and a C-terminal 24 kDa fragment . The N-terminal fragment directly impairs assembly of full-length GFAP and promotes aggregation in vitro ( Chen et al . , 2013 ) . Using a specific antibody recognizing the N-terminal GFAP fragment ( D225 ) , we show here that GFAP cleavage is significantly increased in AxD tissues from patients presenting with an aggressive form of AxD , and that this parallels elevated expression of caspase-6 . This could suggest that misregulation of caspase-6 may contribute to the severity of AxD . However , we were not able to demonstrate in cells that inhibition of caspase-6 , or mutagenesis of the cleavage site on GFAP , can resolve aggregate formation . These results point to a more complex function for caspase-6 , likely involving cytoskeletal remodeling in response to stress . Indeed , caspase-6 upregulation has been reported in other neurodegenerative diseases involving protein aggregation , including Huntington’s Disease ( HD ) and Alzheimer’s Disease ( AD ) ( Albrecht et al . , 2007; Graham et al . , 2010; Guo et al . , 2004 ) . Similar to GFAP , there is a caspase-6 cleavage site on the aggregation-prone proteins in both AD ( amyloid precursor protein ) and HD ( huntingtin ) . Furthermore , in caspase-6 cleavage-resistant genetic mouse models of both HD and AD , neuronal dysfunction and degeneration are rescued ( Graham et al . , 2006; Galvan et al . , 2006; Saganich et al . , 2006 ) . Caspase-6 can promote neurodegeneration via induction of neuronal apoptosis or axon pruning ( Geden et al . , 2019 ) . However , the functions of caspase-6 in astrocytes are not clear . In the context of human AxD it still remains to be determined which astrocyte populations express caspase-6 , and whether it promotes apoptosis or performs a non-apoptotic role , such as sculpting the cytoskeletal architecture in reactive astrocytes . Based on our demonstration that caspase-6 localizes within the perinuclear GFAP inclusions in the AxD iPSC-astrocytes , it is intriguing to speculate that , similar to keratin inclusions in epithelial cells ( Dinsdale et al . , 2004 ) , RFs sequester active caspases away from other cellular substrates and may protect reactive astrocytes from apoptosis . Recently , iPSC-derived patient astrocyte models have emerged as an important system for dissecting the cellular mechanisms in AxD . For example , these novel tools have revealed that AxD astrocytes have defects in the secretory pathway , impaired ATP release , and attenuated calcium waves ( Jones et al . , 2018 ) ; that they inhibit oligodendrocyte precursor cell proliferation ( Li et al . , 2018 ) , providing a potential mechanistic explanation for the degeneration of white matter observed in patients; and that they have defects in mechanotransduction signaling pathways ( Wang et al . , 2018 ) . A novel aspect of the AxD astrocyte cell model that we generated in our study is the perinuclear accumulation of pSer13-GFAP that was associated with prominent nuclear abnormalities . As such , these patient-derived cells replicate a key phenotypic characteristic of RF-bearing AxD astrocytes in vivo , since nuclear invaginations have been described in electron microscopy studies of AxD mouse models and AxD patient cortex ( Sosunov et al . , 2017 ) . Another important parallel is that the GFAP inclusions we observe in the AxD patient astrocytes in vitro stain positive for DAPI , and it was shown that DAPI is a reliable and sensitive marker of RFs in human and mouse brain ( Sosunov et al . , 2017 ) . Therefore patient-derived iPSC-astrocytes provide a unique model system to investigate cytoplasmic-nuclear mechanics in AxD . Invaginations of the nucleus , such as those we observe here , have been described in physiological and pathological states ( Malhas et al . , 2011 ) . Control of nuclear shape is critical for regulation of gene expression and response to mechanotransduction signals ( Uhler and Shivashankar , 2017 ) . The effects of impaired nuclear morphology can be very severe , as evidenced by mutations in lamin A that lead to defective nuclear morphology in Hutchinson-Gilford Progeria Syndrome ( HGPS ) , where patients experience accelerated aging ( Eriksson et al . , 2003 ) . An elegant study combining multiple 3D imaging strategies established a direct link between intermediate filaments , actin and the nuclear envelope within nuclear invaginations , and genetic evidence indicates that filamentous actin may play a role in generating these structures ( Jorgens et al . , 2017; Frost et al . , 2016 ) . It is hypothesized that nuclear invaginations provide localized control of gene expression and nuclear-cytoplasmic transport deep within the nucleus , since they have been found to contain calcium receptors and nuclear pores ( Malhas et al . , 2011 ) . Our study provides the first link between abnormal cytoplasmic PTM processing and perinuclear accumulation of mutant GFAP with nuclear defects , setting the stage to address how nucleo-cytoskeletal coupling is adversely impacted by defective IF proteostasis in AxD and related human diseases .
The following antibodies were used: rabbit anti-GFAP ( DAKO Agilent , Santa Clara , CA , Z0334 ) , rabbit anti-caspase-6 ( Cell Signaling Technologies , Danvers , MA , 9762 ) , rabbit anti-Caspase-6 ( abcam , Cambridge , UK , ab185645 ) , rabbit anti-D225 ( Chen et al . , 2013 ) , mouse anti-GFAP ( Sigma , GA5 ) , mouse anti-pSer13-GFAP ( KT13 [Sekimata et al . , 1996] ) , mouse anti-pan Actin , mouse anti-Tra-1–60 , mouse anti-SSEA4 , rabbit anti-Oct4 , rabbit anti-Sox2 , and Alexa 488- and Alexa 594-congujated goat anti mouse or rabbit antibodies ( Thermo Fisher Scientific , Waltham , MA ) . SW13vim- cells were provided by Dr Bishr Omary and cultured in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin . Authentication of the cell line was done by short tandem repeat ( STR ) profiling by ATCC . Fibroblasts from a male 6 year old type I AxD patient were obtained from the Coriell institute ( Camden , NJ ) . Sanger sequencing was performed to confirm the AxD mutation was present in the cells ( c . 715C > T; p . Arg239Cys ) . The cell lines used tested negative for mycoplasma contamination , as assayed using the Universal Mycoplasma Detection Kit ( ATCC 30–1012K ) . De-identified post-mortem fresh-frozen and fixed AxD patient and control brain tissues were provided by the NIH NeuroBioBank and are described in Supplementary files 1 and 2 . Sample Preparation: HSEs from AxD patient post-mortem brain cortex tissue were prepared as described previously ( Battaglia et al . , 2017; Snider and Omary , 2016 ) and in Figure 1—figure supplement 1 , then subjected to SDS-PAGE followed by Coomassie stain . Bands corresponding to GFAP were excised and the proteins were reduced , alkylated , and in-gel digested with trypsin overnight at 37 °C . Peptides were extracted , desalted with C18 spin columns ( Pierce – Thermo Fisher Scientific ) and dried via vacuum centrifugation . Peptide samples were stored at −80°C until further analysis . LC-MS/MS Analysis: The peptide samples were analyzed by LC/MS/MS using an Easy nLC 1200 coupled to a QExactive HF mass spectrometer ( Thermo Fisher Scientific ) . Samples were injected onto an Easy Spray PepMap C18 column ( 75 μm id ×25 cm , 2 μm particle size ) ( Thermo Fisher Scientific ) and separated over a 1 hr method . The gradient for separation consisted of 5–40% mobile phase B at a 250 nl/min flow rate , where mobile phase A was 0 . 1% formic acid in water and mobile phase B consisted of 0 . 1% formic acid in 80% ACN . The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent fragmentation . Resolution for the precursor scan ( m/z 300–1600 ) was set to 120 , 000 with a target value of 3 × 106 ions . MS/MS scans resolution was set to 15 , 000 with a target value of 1 × 105 ions . The normalized collision energy was set to 27% for HCD . Dynamic exclusion was set to 30 s , peptide match was set to preferred , and precursors with unknown charge or a charge state of 1 and ≥7 were excluded . Data Analysis: Raw data files were processed using Proteome Discoverer version 2 . 1 ( Thermo Fisher Scientific ) . Peak lists were searched against a reviewed Uniprot human database , appended with a common contaminants database , using Sequest . The following parameters were used to identify tryptic peptides for protein identification: 10 ppm precursor ion mass tolerance; 0 . 02 Da product ion mass tolerance; up to two missed trypsin cleavage sites; phosphorylation of Ser , Thr and Tyr were set as variable modifications . The ptmRS node was used to localize the sites of phosphorylation . Peptide false discovery rates ( FDR ) were calculated by the Percolator node using a decoy database search and data were filtered using a 5% FDR cutoff . The peak areas for the identified peptides were extracted and used for relative quantitation across samples . Mutagenesis of GFAP ( Origene , Rockville , MD , in vector CMV6-XL6 ) was performed using the QuikChange II mutagenesis kit ( Agilent ) to generate the designated point mutants . Sanger sequencing of the entire coding sequence of GFAP was performed to confirm the wild-type and mutant sequences . We used established procedures for the purification and in vitro assembly of GFAP ( Perng et al . , 2016 ) . For transfections , lipofectamine 2000 was used according to the supplier instructions ( Invitrogen , Thermo Fisher Scientific , Carlsbad , CA ) , and experiments were performed 20–24 hr after transfection . For immunofluorescence , cells were fixed in methanol at −20°C for 10 min , washed three times in PBS and incubated in blocking solution ( 2 . 5% bovine serum albumin , 2% normal goat serum in PBS ) for 1 hr at room temperature . Primary antibodies were diluted into blocking buffer and incubated overnight at 4°C . The next day , cells were washed 3 times in PBS and incubated with Alexa Fluor-conjugated secondary antibodies diluted into blocking buffer for 1 hr at room temperature . Cells were washed 3 times in PBS , incubated in DAPI for 5 min , washed 3 times and mounted in Fluoromount-G ( SouthernBiotech , Birmingham , AL ) overnight . Cells were imaged on Zeiss 880 confocal laser scanning microscope using a 63x ( 1 . 4 NA ) oil immersion objective ( Zeiss , Jena , Germany ) . High salt extracts ( HSEs ) and triton-X ( TX ) lysates were prepared as previously described ( Battaglia et al . , 2017 ) . Total lysates were prepared by homogenizing 25 mg tissue directly into hot 2X Tris-Glycine SDS Sample Buffer ( Thermo Fisher Scientific ) and heating for 5 min at 95°C . Immunoblotting was performed as previously described ( Trogden et al . , 2018 ) . Briefly , samples were resolved on 4–20% gradient SDS-PAGE gels transferred onto activated polyvinylidene difluoride membranes at 40V overnight . The transferred gels were routinely stained with Coomassie blue and the membranes were blocked in 5% non-fat milk in 0 . 1% tween 20/PBS ( PBST ) . Post-transfer Coomassie-stained gels served as another loading control where the levels of housekeeping protein ( actin ) varied ( Figure 4E ) . For immunoblotting , the membranes were incubated with the appropriate primary antibody diluted in 5% milk/PBST , with the exception of KT13 , which was incubated in 5% bovine serum albumin/PBST for blocking , primary antibodies and secondary antibodies . Antibodies were detected using ECL reagents ( PerkinElmer Life Sciences , Hopkinton , MA ) . For 2D gel analysis , HSEs were dissolved in 2-D starter kit rehydration/sample buffer ( Biorad; 1632106 ) for separation by isoelectric focusing ( IEF ) . Immobilized pH gradient ( IPG ) strips ( Biorad; 11 cm; pH 4–7; 1632015 ) were passively rehydrated in 2-D starter kit rehydration/sample buffer overnight . Cup loading method was employed to load the protein samples in cathode side ( as isoelectric point of GFAP is 5 . 2 ) of the Protean IEF cell tray ( Biorad; 1654020 ) . The IEF separation was done using 72000 vh . After IEF separation the protein samples were further separated based on molecular weight using SDS-PAGE gel by applying constant 90 volts . Skin fibroblasts were reprogrammed under feeder free conditions using Cytotune –iPS 2 . 0 Sendai Reprogramming kit and individual iPSC clones were picked for propagation in culture for 10 passages . To confirm stemness and differentiation capabilities of reprogrammed and edited iPSCs , we used the qPCR based TaqMan human Pluripotent Stem Cell Scorecard Panel ( Thermo Fisher Scientific ) . iPSCs were differentiated into all three germ layers using STEMdiff Trilineage Differentiation Kit ( StemCell Technologies , Vancouver , Canada ) , and a monolayer-based protocol was used to directly differentiate hES cells in parallel into the three germ layers ( ~1 week ) . Non-differentiated and differentiated cells were lysed and total RNA purified using the RNeasy kit ( QIAGEN ) . RNA reverse transcription was performed following the Taqman Scorecard’s manufacture guidelines and the qRT-PCR was carried out using the QuantStudio 7 Flex Real-Time PCR system . The TaqMan PCR assay combines DNA methylation mapping , gene expression profiling , and transcript counting of lineage marker genes ( Bock et al . , 2011 ) . Reprogrammed and edited iPSCs were submitted to a standard G-band analysis consisting of 20 metaphase spreads . The analysis ( carried out by Karyologic Inc ) can identify gender , chromosome number , and detect aberrations that include trisomies , monosomies , deletions , insertions , translocations , duplications , breaks , polyploidy , among others . No abnormalities were found in our cell lines ( Figure 4—figure supplement 2A ) . We used the TrueCut Cas9 Protein V2 , sgRNAs and the Neon Transfection system ( Thermo Fisher Scientific ) to edit iPSCs . The recombinant TrueCut Cas9 V2 was diluted in resuspension buffer R provided in the kit and mixed with 900 ng of sgRNA and 2700 ng of single-stranded donor oligonucleotide , incubated 15 min at room temperature and then a total of 3 × 105 iPSCs were electroporated with the ribonucleoprotein mix . Seventy-two hours after electroporation , cells were dissociated into single cells , diluted , and seeded on Matrigel-coated 96-well plates . Single-cell colonies were selected after two weeks and tested for gene correction . Genomic DNA of single clones was extracted and the gene of interest amplified by PCR using allele specific primers . Sanger sequencing of positive clones demonstrated single or double allele gene correction . Off-target sites within the exons of genes were predicted via selection of the top candidates using the MIT software ( CRISPR . mit . edu ) . The analysis was performed via PCR of 400 bp fragments , which flanked the predicted off-target cut site followed by Sanger sequencing . The chromatograms for edited clones were compared to sequences from the original AxD patient cells . iPSCs were maintained on Matrigel in StemFlex medium ( Thermo Fisher Scientific ) and passaged every 3–4 days with 0 . 5 mM EDTA dissociation solution . iPSCs were differentiated into neural progenitor cells ( NPC ) using an embryoid body ( EB ) protocol . Briefly , iPSCs at 80% confluence were collected , resuspended in Neural Induction Medium ( NIM , StemCell Technologies ) and seeded on one well of an Aggrewell 800 plate ( StemCell Technologies ) at 3 × 106 cells per well . At day five , EBs were seeded on poly-ornithine and laminin ( PLO/LAM ) -coated dishes in NIM . Rosette selection was performed after 12 days using Rosette Selection Reagent ( StemCell Technologies ) . NPCs were expanded for 7 days in Neural Progenitor Medium ( StemCell Technologies ) . NPCs were then differentiated into astrocyte precursors by seeding dissociated single cells at 1 × 105 cells/cm2 density on PLO/LAM dishes in STEMdiff astrocyte differentiation medium ( StemCell Technologies ) . Astrocyte precursors were maintained for 20 days with medium changes every 48 hr and splitting every week with Accutase ( Millipore , Burlington , MA ) . Astrocytes were expanded for up to 120 days in STEMdiff astrocyte maturation medium ( StemCell Technologies ) . AxD iPSC-astrocytes grown on a polystyrene dish were fixed in 2 . 5% glutaraldehyde in 0 . 1M sodium cacodylate buffer , pH 7 . 4 , for one hour at room temperature and stored at 4°C . The cells were washed 3 times in 0 . 1M sodium cacodylate buffer followed by post-fixation in 1% buffered osmium tetroxide for 1 hr . After three washes in deionized water , the cells were dehydrated in ethanol , infiltrated and embedded in situ in PolyBed 812 epoxy resin ( Polysciences , Inc , Warrington , PA ) . The cell monolayer was sectioned en face to the substrate with a diamond knife and Leica UCT Ultramicrotome ( Leica Microsystems , Inc , Buffalo Grove , IL ) . Ultrathin sections ( 70 nm ) were mounted on 200 mesh copper grids and stained with 4% uranyl acetate and lead citrate . The sections were observed and digital images were taken using a JEOL JEM-1230 transmission electron microscope operating at 80kV ( JEOL USA , Inc , Peabody , MA ) equipped with a Gatan Orius SC1000 CCD Digital Camera ( Gatan , Inc , Pleasanton , CA ) .
Alexander disease ( AxD ) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein ( GFAP ) , which supports the structural integrity of astrocytes . Over 70 GFAP missense mutations cause AxD , but the mechanism linking different mutations to disease-relevant phenotypes remains unknown . We used AxD patient brain tissue and induced pluripotent stem cell ( iPSC ) -derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications ( PTMs ) on GFAP . Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young , independently of the mutation they carried . AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations , resembling the hallmark Rosenthal fibers observed in vivo . Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6 . Furthermore , caspase-6 was selectively expressed in young AxD patients , and correlated with the presence of cleaved GFAP . We reveal a novel PTM signature linking different GFAP mutations in infantile AxD .
Alexander disease is a rare and fatal neurodegenerative condition . It is caused by mutations in a gene that is crucial for the structure of astrocytes , a type of brain cell whose role is to support neurons . The gene codes for a protein called GFAP , which is made almost exclusively in astrocytes . In Alexander disease , mutated versions of the gene make GFAP collect in disordered clumps or aggregates , which interfere with the astrocytes’ normal activities . All Alexander disease patients develop GFAP aggregates , but the type of mutation they have in the gene for GFAP does not predict how their illness will progress . The age of onset of disease , for example , can vary between less than one year old to more than 70 years old . Battaglia et al . sought to understand how GFAP aggregates form in the cells of Alexander disease patients . One way that GFAP can be altered in the cell is by a process called phosphorylation . Enzymes called kinases add phosphate groups to GFAP , which can regulate the protein’s activity , stability and interactions with other proteins . Battaglia et al . found high levels of phosphorylation at one specific site in the GFAP protein in people who had very early onset of Alexander disease . This phosphorylation was not related to any particular mutation in the gene for GFAP . An added phosphate group at this location in the protein made GFAP more likely to be broken into two pieces by an enzyme called caspase-6 . One of the breakdown products is already known to play a role in aggregation . Young patients with Alexander disease had high levels of GFAP breakdown products and caspase-6 . The phosphorylated protein and this enzyme were found to accumulate in astrocyte aggregates . The findings provide a basis for investigating new strategies to treat Alexander disease that target phosphorylation – as removing or preventing the addition of phosphate groups can be done with drugs . But before exploring how to do this , it will be necessary to find out which enzyme is responsible for phosphorylating GFAP at this particular position . These studies may also give a broader understanding of other GFAP-like proteins ( called intermediate filament proteins ) , which are involved in over 70 human diseases .