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. 2016 Apr 27;11:33.
doi: 10.1186/s13024-016-0096-1.

Tau Mutant A152T, a Risk Factor for FTD/PSP, Induces Neuronal Dysfunction and Reduced Lifespan Independently of Aggregation in a C. Elegans Tauopathy Model

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Free PMC article

Tau Mutant A152T, a Risk Factor for FTD/PSP, Induces Neuronal Dysfunction and Reduced Lifespan Independently of Aggregation in a C. Elegans Tauopathy Model

Ghulam Jeelani Pir et al. Mol Neurodegener. .
Free PMC article

Abstract

Background: A certain number of mutations in the Microtubule-Associated Protein Tau (MAPT) gene have been identified in individuals with high risk to develop neurodegenerative diseases, collectively called tauopathies. The mutation A152TMAPT was recently identified in patients diagnosed with frontotemporal spectrum disorders, including Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD), Corticobasal Degeneration (CBD), and Alzheimer disease (AD). The A152TMAPT mutation is unusual since it lies within the N-terminal region of Tau protein, far outside the repeat domain that is responsible for physiological Tau-microtubule interactions and pathological Tau aggregation. How A152TMAPT causes neurodegeneration remains elusive.

Results: To understand the pathological consequences of this mutation, here we present a new Caenorhabditis elegans model expressing the mutant A152TMAPT in neurons. While expression of full-length wild-type human tau (Tau(wt), 2N4R) in C. elegans neurons induces a progressive mild uncoordinated locomotion in a dose-dependent manner, mutant tau (Tau(A152T), 2N4R) induces a severe paralysis accompanied by acute neuronal dysfunction. Mutant Tau(A152T) worms display morphological changes in neurons reminiscent of neuronal aging and a shortened life-span. Moreover, mutant A152T overexpressing neurons show mislocalization of pre-synaptic proteins as well as distorted mitochondrial distribution and trafficking. Strikingly, mutant tau-transgenic worms do not accumulate insoluble tau aggregates, although soluble oligomeric tau was detected. In addition, the full-length A152T-tau remains in a pathological conformation that accounts for its toxicity. Moreover, the N-terminal region of tau is not toxic per se, despite the fact that it harbours the A152T mutation, but requires the C-terminal region including the repeat domain to move into the neuronal processes in order to execute the pathology.

Conclusion: In summary, we show that the mutant Tau(A152T) induces neuronal dysfunction, morphological alterations in neurons akin to aging phenotype and reduced life-span independently of aggregation. This comprehensive description of the pathology due to Tau(A152T) opens up multiple possibilities to identify cellular targets involved in the Tau-dependent pathology for a potential therapeutic intervention.

Keywords: AD, Alzheimer disease; CBD, Corticobasal degeneration; FTD, Frontotemporal dementia; PSP, Progressive supranuclear palsy.

Figures

Fig. 1
Fig. 1
Mutant human TauAT causes severe paralysis in C. elegans. a Bar diagram of Tau (2N4R, 441 residues, largest isoform in human CNS) with 2 inserts (N1, N2 near the N-terminus) and 4 repeats (R1-R4 in the C-terminal half) used to generate tau-transgenic lines. The point mutation A152T lies outside of the repeat domain in the proline rich region. The pan-neuronal promoter (Psnb-1) drives the expression of human tau cDNA in the C. elegans nervous system and the 3′UTR aids in tau expression. b Total worm lysates from synchronized day-3 old adults analyzed for Tau by western blotting using pan-tau antibody K9JA. Two independently integrated strains expressing Tau at comparably low and high levels were selected from each transgene: wild-type htau40 (Tauwt-lo and Tauwt-hi) and mutant htau40A152T (TauAT-lo and TauAT-hi). Tubulin serves as internal control. c Quantification of total tau in wild-type Tau lines (Tauwt-lo and Tauwt-hi) and mutant Tau-A152T lines (TauAT-lo and TauAT-hi). Error bars denote SEM. One-way ANOVA with Tukey’s test was applied for multiple comparisons (ns, non-significant, *P < 0.05, **P < 0.01). d Immunochemistry of whole worms with pan-tau K9JA antibody shows tau staining in the nervous system. Top panels depict nerve ring ganglion, bottom panels show ventral cord region. e Synchronized day-1 old adults were placed onto the centre of NGM plates freshly spotted with E. coli OP-50 and photographed after 10 min. Both TauAT-lo and TauAT-hi worms show strong paralytic phenotype apparent by their coiled body shape and lack of tracks on the E. coli lawn. The corresponding lines expressing wild-type human tau (Tauwt-lo and Tauwt-hi) possess a mild uncoordinated phenotype and are able to crawl away from the point of origin. f Thrashing assay of synchronized day-1 old adults in liquid. Control worms (non-tg control or worms expressing GFP in the pharynx as transformation marker only) display a high frequency of ~50 thrashes/30s. Worms expressing Tauwt display a dose-dependent motor impairment with reduced thrashing frequency (~30/30s for Tauwt -lo, ~20/30s for Tauwt -hi). Motor impairment is very severe in the worms expressing mutant TauAT (both -lo and -hi, ~2/30s). One-way ANOVA with Tukey’s test was applied for multiple comparisons. Error bars denote SEM, n  30. (ns., non-significant, *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 2
Fig. 2
Mutant TauAT leads to substantial damage in GABAergic motor neurons. Punc-25::gfp reporter that labels GABAergic inhibitory neurons with GFP, was crossed with respective tau-transgenic worms to visualize these neurons. a Cartoon depicting the GABAergic inhibitory motor system in an adult worm. b-f depict representative maximum intensity projections (MIP) of day-1 old adults of the transgenes. For whole worm MIPs, see Additional file 11. Abnormalities seen as gaps in the GABAergic inhibitory neurons are highlighted by arrowheads. GABAergic neurons show normal connectivity in non-tg reporter worms, both dorsal and ventral nerve cords are intact (b). Expression of Tauwt produces dose dependent abnormalities in GABAergic neurons, with Tauwt -hi neurons (d) accumulating more damage than Tauwt -lo (c). However, mutant TauAT expression leads to severe abnormalities in the form of gaps in the dorsal and ventral nerve cords, both TauAT-lo (e) and TauAT-hi (f). One of the striking features of mutant TauAT worms is the absence of stretches of dorsal cord (bracketed areas), not found in non-tg worms or Tauwt worms. See Table 1 for detailed quantitative analysis
Fig. 3
Fig. 3
Mutant TauAT induces morphological changes in mechanosensory neurons early in the adulthood and reduces life-span. a Cartoon depicting a normal healthy neuron and a neuron showing morphological changes associated with aging like sprouting, non-specific branching and bending. b Mutant TauAT worms show neuronal abnormalities reminiscent of aging neurons. Pmec-4::gfp reporter that expresses GFP in mechanosensory neurons, was crossed into tau-transgenic worms and neurons visualized for morphological abnormalities at the days indicated. Note the soma outgrowth (yellow arrowhead) and bending of neuronal process (white arrow) in TauAT-lo at day 1. At this age, Tauwt (both -lo and -hi) show normal morphology and do not differ from non-tg. Only the incidence of an infrequent posterior extension (white arrow heads) increases in the Tauwt worms (to 30 % compared with 10 % in non-tg at day 1), whereas in TauAT-lo this posterior extension occurs in almost 100 % of the animals. c The severity of the phenotype increases with age. Soma outgrowths visible in TauAT-lo animals at day 1 grow and undergo further branching with age (yellow double arrowhead). The volume marker GFP accumulates in beaded structures in the posterior extension in TauAT-lo worms and small outgrowths emanate from posterior extension (white double arrowhead). These beaded structures could represent starting points of new branches, and were previously shown to be associated with mitochondria [24]. d Quantification of animals with gross non-specific neuronal abnormalities (bends and branches) at two time points, day 1 and day 3. Error bars denote mean ± SEM, n ≥ 20. **P ≤ 0.01, ***P ≤ 0.001. Paired t-test with unequal variance was used for comparison. e Representative survival curves of tau-transgenic animals, non-tg serves as control. Mantel-Cox log-rank test was performed to determine the statistical significance for the worm life-span. (for P-values see Table 2)
Fig. 4
Fig. 4
Mutant TauAT worms show aberrant localization of presynaptic components in mechanosensory neurons. a Schematic representation of presynaptic cargo distribution in a normal healthy mechanosensory neuron and a neuron in an aged animal. b Day-1 old worms visualized after crossing them into vdEx262:[Pmec-4::mCherry::rab-3] transgene that expresses mCherry fused to synaptic vesicle associated RAB-3 in mechanosensory neurons. Tauwt-lo and Tauwt-hi show a similar distribution as non-tg reporter strain. TauAT-lo worms show accumulation of mCherry::RAB-3 in the end neuron (yellow arrowhead), cell body (CB, white arrowhead) and posterior neurite (white arrow). By contrast, the mid-neuron of TauAT-lo shows less puncta (6 ± 3 measured per 40 μm length) than non-tg (15 ± 5) and Tauwt-lo (17 ± 6) worms. On day 3, mislocalization of mCherry::RAB-3 worsens in TauAT-lo worms, whereas Tauwt-lo and Tauwt-hi start accumulating mCherry:RAB-3 puncta in the distal axon and posterior neurite (see Additional file 12)
Fig. 5
Fig. 5
Mutant TauAT worms show presynaptic protein mislocalization in dendrites of PVD neurons and neurotransmission defects. a Schematic representation of a PVD neuron with normal localization of mCherry::RAB-3 (pre-synaptic marker, red dots) and a PVD neuron with altered localization. The branched morphology represents the dendritic compartment; the unbranched ventral process is the axon. b Representative images of dendrites and axons at day 1 and day 3 of the mentioned transgenes. In non-tg and Tauwt worms (-lo and -hi), mCherry::RAB-3 localizes in the axonal compartment and is excluded from the dendrites in young adults (day 1). By contrast, TauAT-lo worms show mCherry::RAB-3 mislocalized to the dendrites (yellow arrowheads) and a reduced distribution in the axons. With age, Tauwt (-lo and -hi) also show slight mislocalization to the dendritic compartments (yellow arrowheads). Yellow dotted areas correspond to the dendrite not visible in non-tg, Tauwt-lo and Tauwt-hi worms at day1. c Quantification of the fractions of animals with mislocalized presynaptic mCherry::RAB-3 puncta in tau-transgenes at day 1 (d1) and day 3 (d3). Non-tg worms serve as control. Error bars denote mean ± SEM, n ≥ 20. **P ≤ 0.01, ***P ≤ 0.001. Paired t-test with unequal variance was used for comparison. d Time-dependent paralysis induced by aldicarb (acetylcholine esterase inhibitor). Data represents the percentage of worms (mean ± SEM) still able to move on 1 mM aldicarb plates after being touched, as a function of time. Non-tg and Tauwt-lo worms are highly sensitive (bottom curves, grey and olive). Tauwt-hi, TauAT-lo and TauAT-hi worms show some resistance (blue, ochre, green curves, resp.). Strongly resistant lev-1 (AChR mutant carrying the e211 allele) and mildly resistant rab-3 (Ras GTPase mutant carrying the js49 allele) are used as additional controls (black and red curve, resp). After applying two-way ANOVA with Bonferroni correction, P < 0.01 at time points 45 and 180 min, whereas P < 0.001 at time points 90 and 135 min is obtained for Tauwt-hi, TauAT-lo and TauAT-hi against non-tg. n = 20 animals, three independent repetitions. e Time-dependent paralysis induced by levamisole (acetylcholine receptor agonist). Data represents the percentage of worms (mean ± SEM) still able to move on 0.2 mM levamisole plates after being touched, as a function of time. All the four tau-lines were as sensitive to levamisole as non-tg. Strongly resistant lev-1 (e211) is shown as an additional control. n = 20 animals, three independent repetitions
Fig. 6
Fig. 6
Mutant TauAT worms display abnormal distribution of mitochondria. a Schematic representation of DA9 motor neuron displaying a normal and an abnormal patterning of mitochondria in different compartments (blue dots). b Day-1 old adult worms of the tau-transgenes (left panel) visualized after crossing them into the wyEx2709 [Pitr-1::TOM-20 1-54aa ::yfp] reporter that highlights YFP-tagged mitochondria in DA9 motor neurons. Magnified images of the region corresponding to the proximal axon (boxed area in the schematic) are shown to visualize the faint mitochondrial particles (yellow arrowheads). Mitochondrial particles in day-3 old adult worms of the transgenes (right panel). Areas in white marked by asterisk shows body autofluorescence. c, d, and e Quantification of the average number of mitochondria in different regions of DA9 neurons designated as proximal axon (boxed region), distal axon (away from boxed region) and dendrite. TauAT-lo animals exhibit fewer mitochondria than non-tg and Tauwt worms in all the specified compartments of the neuron at day 1 and day 3. Error bars show mean ± SEM, n ≥ 20. **P ≤ 0.01, ***P ≤ 0.001. Paired t-test with unequal variance was used for comparison
Fig. 7
Fig. 7
Time-resolved imaging of mitochondria reveals deficits in the trafficking machinery of mutant TauAT animals. a Representative kymographs showing the nature of particle movements in different worm lines. Mitochondria in non-tg, Tauwt-lo and Tauwt-hi worms show long range movements (in the range 8–15 μm) compared to TauAT-lo where mitochondria exhibit short and oscillatory movements (<5 μm). Quantitation of number of mitochondrial trafficking events in proximal (b) and mid region (c) of mechanosensory neuron in day 1 and day 3 adults. TauAT-lo animals show significant reduction in mitochondrial trafficking compared to non-tg and Tauwt-lo both at day 1 and day 3. However, Tauwt showed dose dependent decreasing trends of trafficking events in both the regions of the neuron. Error bars show mean ± SEM, n ≥ 10. **P ≤ 0.001. Paired t-test with unequal variance was used for comparison
Fig. 8
Fig. 8
Mutant TauAT does not aggregate, adopts a pathological conformation and anti-aggregation compounds do not rescue the paralysis. a Sequential extraction of tau from aged animals (mixed stage consisting worms of mostly > 3 days old). CK10 line transgenic for htauV337M (1N4R) accumulates detergent insoluble tau (insoluble aggregated tau); while no insoluble tau is detected in Tauwt or TauAT lines. 2N4R-tau (single arrow) in Tauwt and TauAT lines migrates slower than the 1N4R-tau (double arrow) in CK10 worms. With age, more Tau accumulates in the detergent soluble fraction (representing Tau bound to membranous structures) (see also Additional file 13A for day-1 old animals). b Bar diagram of anti-aggregant Tau construct TauAT+PP with A152T mutation plus two additional proline substitutions in the hexapeptide motifs (htau40A152TI 277 PI 308 P). c 30 day-1 adult animals from non-tg, Ex[Tauwt], Ex[TauAT] and its anti-aggregant variant Ex[TauAT+PP] after lysis in 1 x sample buffer, then subjected to 10 % PAGE and subsequent western blot analysis using K9JA-pan-tau antibody. Ex[Tauwt], Ex[TauAT] and its anti-aggregant variant Ex[TauAT+PP] carrying the respective Tau transgenes as extrachromosomal arrays (Ex) show comparable levels of Tau expression. Tubulin serves as loading control. d Micrographs of the worms. The anti-aggregant variant of mutant TauAT (TauAT+PP) is equally toxic and produces a similar paralytic phenotype as the single mutant TauAT. Note the absence of tracks and coiled body in Ex[TauAT+PP] similar to Ex[TauAT]. e Mean thrashing assay of day-1 old adult animals carrying Tauwt, TauAT or its anti-aggregant variant (TauAT+PP) transgenes as extrachromosomal arrays. Ex[TauAT+PP] shows less thrashes than the non-tg (~5 % of non-tg) or Ex[Tauwt] worms (~9 % of Tauwt worms). But there is no difference between the Ex[TauAT] and its anti-aggregant variant Ex[TauAT+PP], indicating that the toxicity does not depend on amyloidogenic aggregation. Non-tg strain serves as control. Error bars denote SEM, n ≥ 30. ***P < 0.001, ns., not significant. One-way ANOVA with Tukey’s test applied for multiple comparisons. f Immunostaining of day-1 old Tauwt-lo, Tauwt-hi and TauAT-lo with conformation-specific antibody MC1. Tau in TauAT-lo worms adopts a pathological state as seen by dense staining in the nerve ring and ventral cord. White arrows show stained neuronal processes either in the nerve ring or ventral cord region. Tauwt (-lo and -hi) show only mild staining occasionally. g Worm extracts prepared from mixed stage adults in buffer C, resolved by native PAGE and immunoblotted with K9JA show Tau enriched in soluble high molecular weight complexes in TauAT worm extracts. In addition to two bands common to both Tauwt-lo and TauAT-lo lysates (~170 KDa and >250 KDa marked by asterisks), a smear in the range of 72-95 KDa corresponding to lower oligomeric species and a higher band (> > 250 KDa, black arrowhead) can be seen in the TauAT-lo lysate. h Mean number of bends per 30 s of TauAT-lo worms treated either with DMSO (solvent control) or with 50 or 100 μM concentrations each of known aggregation inhibitors of Tau (Rhodanine compound bb14, PTH compound BSc3094 in DMSO). The lack of rescue indicates that the toxicity of TauAT is based on some mechanism distinct from aggregation (for comparison see [17]). Error bars denote SEM. One-way ANOVA with Tukey’s test applied for multiple comparisons (ns., not significant)
Fig. 9
Fig. 9
N-terminal tau fragments (wild-type or with A152T mutation) are not toxic to C. elegans neurons. a Bar diagram depicts the N-terminal fragment of tau (amino acids Met1-Leu243) derived from full-length Tau. The pan-neuronal snb-1 promoter drives the expression of wild-type N-t- or mutant N-t-fragment (Tauwt-Nt and TauAT-Nt respectively) in C. elegans neurons. b Blot showing the protein expression levels in worms carrying N-terminal fragments or full-length tau transgenes as extrachromosomal arrays. 30 synchronized day-1 old adult worms from each transgene (Ex[Tauwt-Nt], Ex[TauAT-Nt], Ex[Tauwt] and Ex[TauAT]) were lysed and subjected to western blot analysis using N-terminal Tau specific DA9 antibody (epitope at aa 100-130). Non-tg worms serve as control and tubulin as loading control. c Age-related comparison of rate of body thrashing in liquid for days 1, 5 and 7 from the respective transgenic animals. In contrast to full-length tau (Tauwt or TauAT), expression of N-terminal Tau fragments in C. elegans neurons (Tauwt-Nt or TauAT-Nt) cause only slight reductions (~10-13 %) in thrashing at day 1 and day 5. The data points represent the mean (±SEM) thrashing rate and time point, n ≥ 30. Two-way ANOVA followed by Bonferroni correction was used for multiple comparisons. d Immunostaining of animals expressing either wild-type- or mutant-N-t tau fragments at different time points with N-terminal tau-specific antibody SA4473. Top panels depict nerve ring ganglion while the bottom panels show nerve cords. N-terminal Tau fragments show localization restricted to cell bodies and nuclei (white arrows), as nerve processes are largely invisible, in contrast to the situation with full-length Tau (see Fig. 1c)

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