Introduction
Frontotemporal dementia (FTD) is the second most common form of presenile dementia after Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease. Although originally regarded as distinct diseases, FTD and ALS are now known to form a continuum with significant proportions of FTD patients displaying clinical features of ALS, and ALS patients showing FTD like symptoms [
30]. Aside from this clinical overlap, FTD and ALS also display pathogenic similarities. Thus, mutations in the same genes including
TARDBP (encoding TDP43),
FUS and
C9orf72 cause dominantly inherited familial forms of FTD and ALS, and abnormal accumulations of TDP43 in affected neurons are hallmark pathologies of both diseases [
30].
There are no cures for either FTD or ALS. Many current therapeutic approaches involve correcting damaged physiological processes but the broad range of damage seen in FTD/ALS renders this complex. Thus, damage to mitochondria, the ER including activation of the unfolded protein response, Ca
2+ signaling, lipid metabolism, autophagy, axonal transport, synaptic function and inflammatory responses are all features of FTD/ALS [
32,
33,
39]. The biological conundrum is how so many diverse cellular functions are collectively damaged; the therapeutic challenge is selecting which of these damaged functions to prioritise in drug discovery programmes.
Recently, some attention has focussed on signaling between the ER and mitochondria in FTD/ALS [
32,
33,
39]. ER membranes form close contacts with mitochondria and this enables the two organelles to communicate with each other and so respond in an orchestrated fashion to changes in cellular physiology. These regions of ER are termed mitochondria-associated ER membranes. The two primary functions of ER-mitochondria signaling are to synthesise some major phospholipids and to facilitate IP3 receptor delivery of Ca
2+ from ER stores to mitochondria. This delivery is essential for mitochondrial ATP production since dehydrogenases in the tricarboxylic acid cycle are Ca
2+-dependent. ER-mitochondria signaling thus regulates both lipid and bioenergetic linked functions such as synaptic activity, autophagy, ER stress and axonal transport, all functions that are damaged in FTD/ALS [
6,
32,
39]. A number of studies have now shown that several FTD/ALS linked familial genetic insults disrupt ER-mitochondria communications. These include mutant
TARDBP,
FUS,
C9orf72, MAPT (encoding Tau),
SIGMAR1 (encoding the Sigma1 receptor) and
SOD1 (encoding Cu/Zn superoxide dismutase-1) [
3,
8,
7,
18,
20,
29,
42,
47‐
50,
57].
The mechanisms by which ER membranes form contacts with mitochondria to permit inter-organelle signaling are not properly understood but it is generally agreed that it involves “tethering proteins” that serve to recruit and scaffold ER in close proximity to mitochondria [
6,
32,
39]. One well characterised tether involves an interaction between the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) and the outer mitochondrial membrane protein, protein tyrosine phosphatase interacting protein 51 (PTPIP51) (also known as regulator of microtubule dynamics-3 and family with sequence similarity 82 member A2) [
10,
47]. The VAPB-PTPIP51 tethers regulate a number of ER-mitochondria signaling functions including IP3 receptor delivery of Ca
2+ from ER stores to mitochondria, mitochondrial ATP production, autophagy, phospholipid synthesis and synaptic activity [
10,
14,
18‐
17,
40,
43,
47,
48,
58]. Moreover, familial FTD/ALS linked mutant
TARDBP,
FUS and
C9orf72 have all been shown to disrupt the VAPB-PTPIP51 interaction [
18,
47,
48]. Most recently, the VAPB-PTPIP51 interaction has been shown to be disrupted in affected motor neurons in post-mortem human ALS spinal cord [
21].
The VAPB-PTPIP51 tethering proteins thus regulate many of the diverse functions that are perturbed in FTD/ALS and are themselves disrupted by at least three disease associated genetic insults. Correcting disrupted VAPB-PTPIP51 tethering therefore represents a novel target for therapy and one that may positively impact on a number of downstream functions that are damaged in FTD/ALS. However, at present there is little evidence to support this notion.
Here we address this issue by investigating how enhancing ER-mitochondria signaling by expression of VAPB or PTPIP51 affects TDP43 induced damage to ER-mitochondria Ca
2+ exchange and associated synaptic defects. We chose to study TDP43 since abnormal TDP43 accumulations are a hallmark pathology of FTD/ALS and because mutations in
TARDBP cause dominantly inherited familial forms of FTD/ALS; defective TDP43 metabolism is therefore believed to be central to most forms of FTD/ALS [
30,
51]. Expression of both wild-type and familial FTD/ALS mutants of TDP43 have been shown to disrupt the VAPB-PTPIP51 interaction, reduce ER-mitochondria contacts, inhibit IP3 receptor delivery of Ca
2+ to mitochondria and to activate GSK3β to similar extents [
47]. This is in line with the phenotypes seen in transgenic mice expressing wild-type or FTD/ALS mutant TDP43 which exhibit similar aggressive disease onset and progression [
52]. Such findings reinforce the role of VAPB-PTPIP51 tethering in FTD/ALS; clearly, if only mutant TDP43 disrupted the VAPB-PTPIP51 tethers it would mean that disease seen in wild-type TDP43 mice could not involve damage to the tethers.
We show that enhancing ER-mitochondria signaling via expression of VAPB or PTPIP51 alleviates mutant TDP43 induced damage to IP3 receptor delivery of Ca
2+ to mitochondria and associated synaptic defects. We also show that UDCA, an FDA approved drug linked to FTD/ALS therapy but whose mechanism of action is unclear [
19,
59], stimulates VAPB-PTPIP51 binding and corrects TDP43 linked damage to the VAPB-PTPIP51 interaction and IP3 receptor delivery of Ca
2+ to mitochondria. Finally, we show that UDCA inhibits GSK3β which is a known mediator of TDP43 toxicity and which regulates VAPB-PTPIP51 binding [
41,
46,
47].
Materials and methods
Plasmids and lentivirus
Myc-tagged human VAPB, HA-tagged human PTPIP51 and control plasmid containing
Escherichia coli chloramphenicol acetyltransferase in pCI-neo, and EGFP-tagged TDP43-Q331K and TDP43-A382T in pEGFP-C1 were all as described previously [
47]. VAPB and PTPIP51 NanoBit plasmids were generated by cloning VAPB into pBiT1.1-N[TK/LgBiT] and pBiT2.1-N[TK/SmBiT], and PTPIP51 into pBiT1.1-C[TK/LgBiT] and pBiT2.1-C[TK/SmBiT] vectors (Promega) so as to create N-terminal fused Large/Small-BiT VAPB and C-terminal fused Large/Small-BiT PTPIP51 plasmids. Bicistronic vectors containing Myc-tagged TDP43-IRES-EGFP and HA-tagged VAPB and PTPIP51 were synthesised by GenScript. For creation of lentivirus, TDP43-IRES-EGFP, VAPB-HA and HA-PTPIP51 fragments were amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and cloned into lentiviral backbone vector pRRLSIN.cPPT.PGK-GFP.WPRE using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Primers used for amplifications were: 5’-tctagaggatccaccggtcgGTCGACCCACCATGGAGC-3’ and 5’-tgattgtcgacgcggccgctGCGGCCGCTTACTTGTAC-3’ for TDP43-IRES-EGFP; 5’-tctagaggatccaccggtcgCCACCATGTACCCATACG-3’ and 5’-tgattgtcgacgcggccgctCTACAAGGCAATCTTCCC-3’ for VAPB-HA; 5’-tctagaggatccaccggtcgCCACCATGTCTAGACTGG-3’ and 5’-tgattgtcgacgcggccgctTTAAGCGTAATCTGGAACATC-3’ for HA-PTPIP51. For TDP43 vectors, Q331K and A382T mutations were then introduced using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) using primers 5’-GGCAGCACTAAAGAGCAGTTGG-3’ and 5’-TGGGCGGCAGCCATCATG-3’ (TDP43-Q331K) and 5’-TTCTGGTGCAACAATTGGTTG-3’ and 5’-TTAGAGCCACTATAAGAGTTATTTC-3’ (TDP43-A382T). Lentiviruses were prepared as described and viral titer determined using a qPCR Lentivirus Titer Kit (ABM) [
2].
Antibodies and other reagents
Rabbit and rat antibodies to VAPB and PTPIP51 were as described and used at 1:200 for PLAs [
10]. Mouse anti-VDAC1 was from Abcam (ab14734, RRID:AB 443,084) and rabbit anti-IP3 receptor type-1 was from Synaptic Systems (117,003, RRID:AB 2,619,787) and were used for PLAs at 1:200 and 1:100 respectively. Chicken anti-β-Tubulin Isotype III antibody was from Millipore (AB9354, RRID:AB 570,918) and used at 1:500 for immunostaining. Mouse anti-HA epitope-tag (6F2) and mouse anti-Myc epitope tag (9B11) antibodies were from Cell Signaling (H9658, RRID:AB 260,092; 2276 RRID:AB 331,783) and used at 1:500 for immunostaining and 1:1000 for immunoblots. Chicken anti-microtubule-associated protein-2 (MAP2) was from Genetex (GTX82661, RRID:AB 11,172,558) and used for immunostaining at 1:500. Rabbit anti-EGFP was from Invitrogen (A-11,122, RRID:AB 221,569) and used at 1:500. Rabbit anti-serine-9 phosphorylated GSK3β was from Cell Signaling (5558, RRID:AB 10,013,750) and used at 1:100. Species specific goat and donkey anti-mouse, anti-rabbit and anti-chicken Igs coupled to AlexaFluor-488, − 555, -594 or -647 were from Invitrogen and Jackson ImmunoResearch Labs.
Cell culture, transfection and viral transduction
SH-SY5Y cells and human embryonic kidney-293 (HEK293) cells were obtained from European Collection of Cell Cultures. HEK293 cells were grown in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose and SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium/F12 (1:1) containing 3.15 g/l glucose (Gibco). Both media were supplemented with 10% (v/v) foetal bovine serum, Glutamax, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Gibco). Cells were transfected with plasmids using Lipofectamine 2000 (ratio 1 µg DNA: 2 µl Lipofectamine 2000) according to the manufacturer’s instructions (Invitrogen). SH-SY5Y cells were analysed 36 h post-transfection. Primary rat cortical neurons were obtained from embryonic day 18 rat embryos, plated on poly-D-lysine coated glass coverslips (Marienfeld) and cultured in neurobasal medium containing B27 supplement, GlutaMAX, 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco). For FM 4–64 release assays, neurons were transfected with Lipofectamine 2000 as described above at DIV7 or 8 and analysed 24 h post transfection. This timing is line with other studies of synaptic vesicle release using FM4-64 in cortical and hippocampal neurons including those that analysed FTD/ALS linked genes [
26,
44]. For dendritic spine analysis, neurons were transduced with lentiviral vectors on DIV12 and analysed on DIV15; dendritic spines display a mature phenotype at this age [
17]. For UDCA treatment, neurons were transfected at DIV7 and treated 24 h later with UDCA (obtained from Sigma and dissolved in DMSO) or DMSO vehicle for 24 h.
In line with previous studies, we selected cells for analyses that express relatively low levels of transfected proteins (as judged by fluorescent signal) so as to avoid any possible artefacts produced by high levels of expression [
1,
34,
36,
55‐
53].
FM 4–64 synaptic vesicle release assays and analyses of dendritic spines
FM 4–64 release assays were performed essentially as described [
13,
17,
25]. Briefly, 24 h post transfection, neurons were washed in external solution comprising 145 mM NaCl, 2 mM KCl, 5 mM NaHCO
3, 1 mM MgCl
2, 2.5 mM CaCl
2, 10 mM glucose in 10 mM HEPES buffer pH 7.25 and then incubated in external solution containing 5 µM FM 4–64 (Invitrogen), 50 µM DL-2-amino-5-phosphonovaleric acid (D-AP5) and 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Tocris) for 2 min. FM 4–64 loading was stimulated by replacement of external solution with high K
+ load external solution (97 mM NaCl, 50 mM KCl, 5 mM NaHCO
3, 1 mM MgCl
2, 2.5 mM CaCl
2, 10 mM glucose, 50 mM AP5, 10 mM CDQX in 10 mM HEPES, pH 7.25) containing 5 µM FM 4–64 for 2 min. Cells were subsequently incubated for 10 min in 5 µM FM 4–64 dye in external solution to regain baseline followed by washing in external solution to remove excess dye. Synaptic vesicle FM 4–64 release was stimulated by incubation with high K
+ release external solution containing 31.5 mM NaCl, 90 mM KCl, 5 mM NaHCO
3, 1 mM MgCl
2, 2.5 mM CaCl
2, 10 mM glucose, 50 µM AP5, 10 µM CDQX in 10 mM HEPES, pH 7.25). FM 4–64 dye release was monitored by time-lapse as described [
17] using a Nikon Eclipse Ti-2 microscope equipped with an Intenslight C-HGFI light source, CFI Apo Lambda S 60x/1.40 objective, TiND6 PFS-S Perfect Focus Unit and an Andor TuCam camera adapter system with EGFP/DsRed dual filter sets (Chroma Technology) and two Andor Neo sCMOD cameras; temperature was maintained at 37 °C using a microscope incubation chamber (Solent Scientific). Recordings were obtained at 5 s time-lapse intervals using Nikon NIS-Elements AR software and analyses performed using ImageJ. Synaptic vesicle release was expressed as fluorescence signals after KCl treatment relative to the average baseline prior to treatment (F/F0).
Dendritic spine densities were quantified as described previously [
17]. Briefly, cells were fixed in 4% paraformaldehyde and following immunostaining, z-plane images with 0.2 μm intervals were captured using a Nikon Eclipse Ti-E Inverted microscope with 100 × 1.49 NA CFI objective and an Andor iXon EMCCD camera equipped with Visitech iSIM Super Resolution System as described [
17,
18,
37]. Spine densities were quantified using Neuronlucida TM software (MBF Bioscence, VT USA).
SDS-PAGE and immunoblotting
Cells were prepared for SDS-PAGE and analysed by immunoblotting essentially as described previously [
35]. Briefly, cells were washed with phosphate buffered saline (PBS) and harvested by scraping into ice-cold radioimmunoprecipitation (RIPA) lysis buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS in 50 mM Tris-HCl pH 8 with protease inhibitors (Complete, Roche). SDS-PAGE sample buffer was then added and samples heated at 96 ºC for 5 min. Samples were separated on 10% gels using a Mini-PROTEAN 2 gel electrophoresis systems (Bio-Rad) with a discontinuous buffer system. Separated proteins were transferred to BioTrace NT nitrocellulose membrane (0.2 μm pore size; Pall Corporation) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) for 1.5 h. Membranes were blocked with Tris-HCl buffered saline (TBS) containing 5% (w/v) milk powder and 0.1% (v/v) Tween-20 for 1 h. Membranes were probed with primary antibodies in blocking buffers supplemented with 0.1% (v/v) Tween-20 (TBS/Tween-20), washed in TBS/Tween-20 and incubated with IRDye-conjugated secondary antibodies in wash buffer and proteins visualised using an Odyssey CLx near infrared imaging system (Li-Cor Biosciences).
Calcium measurements
Mitochondrial Ca
2+ levels were measured with Rhod-2 AM (Invitrogen) essentially as described previously [
10,
16,
37,
40,
47,
48]. Briefly, SH-SY5Y cells were plated on coverslips (Marienfeld) and 24 h post transfection, selected for analyses via EGFP signal. Cells were loaded with 2 µM Rhod-2 AM in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO
3, 1 mM MgCl
2, 2.5 mM CaCl
2, 10 mM glucose, 10 mM Na-HEPES pH 7.25) in the presence of 0.02% (v/v) Pluronic-F27 (Invitrogen) for 15 min. After removing dye, cells were washed with external solution for 15 min at 37 °C, mounted in a Ludin imaging chamber (Life Imaging Systems, Basel, Switzerland) and kept under constant perfusion in external solution (0.5 ml/min) using an Ismatec REGLO peristaltic pump (IDEX Corporation, Glattbrugg, Switzerland). To invoke Ca
2+ efflux from ER stores and measure changes in Ca
2+ levels, 100 µM oxotremorine-M (Tocris) was applied in external solution. Rhod-2 AM images were recorded in time-lapse mode (2 s interval, 100 ms exposure) using a Nikon Eclipse Ti-2 microscope driven by NIS Elements AR software and equipped with Intenslight C-HGFI light source, CFI Plan Fluor 40x/1.4 NA objective, Andor Neo scientific complementary metal-oxide-semiconductor camera (Andor Technology). Filter sets were from Chroma Technology. Data were analysed with NIS Elements AR software. Mitochondrial and Ca
2+ levels were expressed as fluorescence signals after oxotremorine-M treatment relative to average baseline before oxotremorine-M application (F/F0).
Proximity ligation assays and immunostaining
Proximity Ligation Assays (PLAs) to detect IP3R1-VDAC1 or VAPB-PTPIP51 interactions were performed as described previously using Duolink In Situ Orange Kits (Sigma) according to manufacturer’s instructions [
17,
18,
35]. After amplification steps, samples were immunostained with β-tubulin III (SHSY5Y cells) or MAP2 (neurons) to confirm neural identity.
To detect serine-9 phosphorylated GSK3β, cells were fixed in 4% paraformaldehyde, washed three times in Tris-HCl buffered saline (TBS), permeabilised with 0.1% Triton X-100 in TBS for 15 min and blocked in 5% bovine serum albumin (BSA) in TBS for 1 h. Cells were then labelled with primary antibody diluted in 1% BSA in TBS overnight at 4 °C, washed three times with 0.01% Triton X-100/TBS, incubated with secondary antibodies diluted in TBS, washed three times with TBS and mounted in aqueous mounting medium with DAPI (Abcam). Z-plane images with 0.3 μm intervals were captured using a Nikon Eclipse Ti-E Inverted microscope with CFI Apo Lambda S 60x/1.40 objective and an Andor iXon EMCCD camera equipped with Visitech iSIM Super Resolution System. Mean cytoplasmic fluorescent intensities were quantified using ImageJ.
NanoBiT assays
NanoBiT assays were performed on SH-SY5Y cells cultured in white opaque 96-well plates at initial density of 30,000 cells/well. Assays were performed in living cells according to the manufacturerµ’s instruction (Promega). Briefly, cells were transfected with the 100 ng NanoBiT plasmids as described above and 24 h later, treated with vehicle (DMSO) or UDCA for 24 h. Medium was replaced with 100 µl OptiMEM medium without phenol red (Gibco) and NanoBiT luminescence signals obtained by addition of 25 µl reconstituted NanoGlo Live Assay substrate to each well and quantified using a GloMax Navigator luminometer for 15 min (0.5s integration time/well).
GSK3β ELISAs
ELISAs for quantifying total and serine-9 phosphorylated GSK3β (CST7265C PathScan® Total GSK3β Sandwich ELISA; CST7311C PathScan® Phospho-GSK3β (Ser9) sandwich ELISA) were obtained from Cell Signaling Technology. UDCA and vehicle (DMSO) treated neurons were processed according to the manufacturer’s instructions. Briefly, neurons were washed with PBS and lysed in kit cell lysis buffer (CST9803) containing 1 mM phenylmethylsulfonyl fluoride, protease and phosphatase inhibitors (Complete, Roche). Protein concentrations were determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and an equal amount of total protein (0.15 mg/ml for phospho-GSK3β and 0.045 mg/ml for total GSK3β ELISAs) in 100 µl kit supplied sample buffer loaded in duplicates to the ELISA microwells. Following a 2 h incubation at 37 °C, wells were washed in kit supplied wash buffer and 100 µl of detection antibody added and samples incubated for 1 h at 37 °C. After washing, 100 µl of reconstituted HRP-linked secondary antibody was added to the wells and incubated for 30 min at 37 °C, followed by incubation with 3,3′,5,5′-Tetramethylbenzidine substrate for 10 min at 37 °C. Upon addition of stop solution absorbance was read at 450 nm using a CLARIOstar multiplate reader (BMG LabTech).
Statistical analyses
Statistical analysis was performed using Excel (Microsoft Corporation) and Prism software (version 9.3.1; GraphPad Software Inc.). Details are described in the Figure legends.
Discussion
Communications between ER and mitochondria regulate many of the physiological processes that are damaged in FTD/ALS and studies from multiple research groups have now shown that ER-mitochondria signaling is disrupted by a number of FTD/ALS associated genetic insults. These include both wild-type and mutant TDP43 and FUS, mutant SOD1, mutant
C9orf72-derived dipeptide repeat polypeptides (DPRs) and Sigma-1 loss of function mutations [
3,
7,
8,
18,
20,
29,
42,
47,
48,
50,
57]. VAPB and PTPIP51 are ER-mitochondria tethering proteins and for TDP43, FUS and
C9orf72 DPRs, this disruption to ER-mitochondria signaling involves breaking of the VAPB-PTPIP51 tethers [
18,
47,
48]. This effect of wild-type and mutant TDP43 and FUS, and
C9orf72 DPRs on the VAPB-PTPIP51 interaction in turn disrupts ER-mitochondria contacts and IP3 receptor delivery of Ca
2+ to mitochondria [
18,
47,
48].
A primary function of ER-mitochondria signaling is to facilitate IP3 receptor mediated delivery of Ca
2+ to mitochondria [
6,
32,
39]. Thus, loss of VAPB and/or PTPIP51 inhibits whereas VAPB/PTPIP51 overexpression stimulates IP3 receptor delivery of Ca
2+ to mitochondria [
10,
16,
37,
47]. This delivery is essential for synaptic transmission and the VAPB-PTPIP51 tethers have been shown to regulate both pre- and post-synaptic function [
17,
24].
Damage to synaptic function is a defining feature of FTD/ALS and this includes mutant TDP43 linked disease [
4,
12]. Here we tested whether overexpression of VAPB or PTPIP51 to enhance ER-mitochondria signaling can correct TDP43 induced damage to ER-mitochondria Ca
2+ exchange and linked synaptic function. Wild-type and four different FTD/ALS mutants of TDP43 including the two used in this study have been shown to disrupt the VAPB-PTPIP51 interaction and linked functions in neuronal cells and transgenic mice using quantitative assays, and expression of VAPB and PTPIP51 has been shown to stimulate ER-mitochondria signaling [
47].
We studied VAPB or PTPIP51 expression alone as combined expression leads to a dramatic reorganisation of ER to envelop mitochondria, especially in higher level expressing cells [
47]. This can lead to mitochondrial Ca
2+ overload which is a signal for opening of the transition pore and apoptosis [
6,
31,
32,
39]. We show that VAPB/PTPIP51 expression rescues mutant TDP43 damage to the IP3 receptor-VDAC1 channel, to IP3 receptor delivery of Ca
2+ to mitochondria and to both synaptic vesicle release and dendritic spine numbers. This is the first demonstration that enhancing VAPB-PTPIP51 function to stimulate ER-mitochondria signaling can correct TDP43 linked damage to mitochondrial Ca
2+ delivery and synaptic function.
We also show that UDCA, an FDA approved drug that is linked to FTD/ALS treatment, stimulates VAPB-PTPIP51 binding and corrects mutant TDP43 induced damage to both the VAPB-PTPIP51 interaction and ER-mitochondria Ca
2+ delivery. Clinical trials are underway with UDCA and related compounds for ALS, Alzheimer’s disease and Parkinson’s disease but its protective method of action is unclear; it has been linked to protection against mitochondrial damage, ER stress, apoptosis, autophagy and inflammation in these neurodegenerative diseases [
19,
38,
59]. These damaged functions are all regulated by ER-mitochondria signaling [
6,
32,
39]. The mechanisms by which TDP43, FUS and mutant
C9orf72 DPRs disrupt VAPB-PTPIP51 binding are also not properly understood but involve activation of GSK3β [
18,
47,
48]. We also show that UDCA inhibits GSK3β and rescues mutant TDP43 activation of GSK3β. Interestingly, others have presented evidence that UDCA inhibits GSK3β [
11]. Thus, the beneficial effects of UDCA in FTD/ALS and other neurodegenerative diseases may involve at least in part, correction to damaged VAPB-PTPIP51 tethering and ER-mitochondria signaling via inhibition of GSK3β.
The mechanisms by which activation of GSK3β disrupts VAPB-PTPIP51 binding are not clear but one obvious hypothesis involves GSK3β phosphorylation of VAPB and/or PTPIP51; phosphorylation is a known mechanism for regulating protein-protein interactions. Both VAPB and PTPIP51 are heavily phosphorylated proteins (human VAPB contains 21 and PTPIP51 27 phosphorylation sites (see
https://www.phosphosite.org/homeAction.action) but any GSK3β targeted residues have not as yet been identified. A proper testing of this hypothesis with therefore require the formal identification and subsequent experimental manipulation of such sites. Interestingly, whilst VAPB and PTPIP51 are enriched at ER-mitochondria contact sites, VAPB localises to all ER regions and PTPIP51 throughout the outer mitochondrial membrane [
10,
47]. One possibility is that the inability of VAPB and PTPIP51 to interact is these non-contact regions is linked to their phosphorylation status. Over expression VAPB and PTPIP51 to enhance ER-mitochondria signaling such as we show here, may permit binding by increasing the levels of non-GSK3β phosphorylated VAPB and PTPIP51 that is competent to interact.
Whatever the precise mechanism, our findings reported here support the notion that targeting the VAPB-PTPIP51 tethers has therapeutic potential for FTD/ALS. Moreover, there is evidence that the VAPB-PTPIP51 interaction is also disrupted in Alzheimer’s disease and Parkinson’s disease; notably TDP43 pathology is a feature of both these diseases [
28,
40,
45]. This includes VAPB-PTPIP51 disruption in induced pluripotent stem cell derived dopaminergic neurons from familial Parkinson’s disease patients carrying pathogenic
SNCA (α-synuclein) triplications and in affected neurons in post-mortem Alzheimer’s disease brain [
28,
40]. In these post-mortem Alzheimer’s disease cases, disruption to the VAPB-PTPIP51 interaction occurs early in disease arguing that it contributes to the pathogenic process in a primary fashion and is not just some epiphenomena [
28]. Thus, correcting damaged VAPB-PTPIP51 tethers may also be beneficial for these other age-related neurodegenerative diseases.
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