PubMed: 22611162

Inhibition of tau aggregation in a novel Caenorhabditis elegans model of tauopathy mitigates proteotoxicity.
Human molecular genetics
Baumeister R | Biernat J | Fatouros C | Koushika SP | Mandelkow E | Mandelkow EM | Pir GJ | Schmidt E

Evidence 51af0efa73

Overall, Tau, both in the soluble and in the insoluble fractions, was phosphorylated to a higher degree in the pro-aggregant strain compared with the other two strains.

Evidence a8ee67c9f0

We supplemented the growth medium of syn- chronized L1 larvae (pro-aggregant strain) with 25 m M MB and measured their locomotion speed as day 1 young adults. This treatment led to 15% amelioration of locomotion (Sup- plementary Material, Fig. S6A).

Evidence 3965789e6f

At the biochemical level, MB treatment altered Tau solubility, shifting the equilibrium towards more soluble Tau and reduced detergent-insoluble Tau by  35% in the pro-aggregant strain (Fig. 8A, quantitation in 8B), con- sistent with its anti-aggregation properties (31).

Evidence afafc7dbfd

For this purpose, we focused on the well-characterized DK280 mutation (10–13), which specifically leads to aggregation-mediated toxicity.

Evidence 06a2453b26

Aberrant phosphorylation and aggregation of Tau have been linked to axonal transport problems, synaptic malfunction and degeneration (6).

Evidence 38bc578cc2

Resistance to aldicarb can arise from either a pre- or a post-synaptic perturbation, whereas resistance to levamisole typically indicates a post-synaptic defect (50). Animals of the pro-aggregant strain displayed a mild resist- ance to aldicarb, producing a paralysis profile intermediate between the sensitive wild-type N2 and the resistant rab-3(js49) strain, which we used as controls (Fig. 5A).

Evidence 9d8edb20d9

This indicated that SNB-1 failed to properly accumulate at the presynaptic termini of young adult pro-aggregant worms.

Evidence d3a3e3d6f0

We crossed WyEx2709 into the pro-aggregant strain (resulting in strain BR6011) and discov- ered that this regular mitochondrial distribution was distorted.

Evidence 98adac4485

The velocity of mitochondrial transport in the pro-aggregant strain was lower than in wild-type (mean + SD ¼ 171 + 111 versus 256 + 117 nm/s), whereas the anti-aggregant strain (194 + 131 nm/ s) did not substantially differ from wild- type (Fig. 7D).

Evidence 8969f6b8b5

We then applied, in the 96-well liquid culture format, the two most promising hit compounds obtained in a mammalian cell model of Tau toxicity (57), namely the phenylthiazolyl- hydrazide derivatives Bsc3094 and bb14, and observed a similar amelioration effect in locomotion (Supplementary Ma- terial, Fig. S6B).

Evidence 9c15c8b269

We next analysed the most prominent Tau aggregation inhibitor compound from a recent- ly published in vitro screen (compound #16 in reference 33), which belongs to the ATPZ class of Tau inhibitors (5-amino-3-(4-chlorophenyl)-N-cyclopropyl-4-oxo-3,4-dihyd- rothieno[3,4- D ]pyridazine-1-carboxamide, referred to as cmp16 for simplicity, structure shown in Fig. 9A). This com- pound prevents Tau fibril formation in vitro, and is able to cross the mammalian blood–brain barrier, an attribute that makes it favourable for clinical applications (33).

Evidence 512a95e88b

At 100 m M , we observed improved locomotion of treated animals. These animals moved approximately 1.6 times faster than DMSO-treated controls (Fig. 9A).

Evidence 108110a815

Treatment with cmp16 diminished the progressive accumulation of neurite gaps in the motor neurons of the pro-aggregant animals compared with the DMSO-treated controls (from 3.2 + 1 gaps at day 5 of the DMSO-treated strains to 2.4 + 1 gaps of the cmp16-treated strains, P , 0.05) (Fig. 9B). Lower accumulation of structural damage in neurons can be interpreted as a sign of reduced neu- rodegeneration (22,58).

Evidence 4d5dfe0690

BSc3094 resulted in 40% decrease in the detergent-insoluble Tau (FA fraction) in pro-aggregant animals,

Evidence 113063f9b8

In one set of C. elegans strains, we expressed the pathological FDTP-17 mutant DK280, which enhances aggregation, whereas the other set harbours, in addition to DK280, the proline substitutions I277P and I308P (PP), which act as b-sheet breakers and prevent aggregation (15).

Evidence c6e261cbb8

The motility of the pro-aggregant worms was considerably enhanced upon apply- ing RNAi against F3DK280 (speed ¼ 70.7 + 17 mm/s for RNAi-treated versus 40.3 + 17 for control), whereas the anti- aggregant worms showed no difference upon treatment (Sup- plementary Material, Fig. S1C).

Evidence 73ecca8f02

In contrast, we observed severe developmental defects in the pro-aggregant strain (BR5707) that manifest as increased numbers of persist- ent gaps in both the ventral and dorsal neural cords (mean + SD ¼ 2.7 + 1.4 gaps at the L3 stage).

Evidence b95a7ff359

In contrast, the pro-aggregant strain showed frequent occurrence of gaps (27.8 + 4.8% of day 1 adult animals), similar to those observed in the GABAergic neurons (Fig. 3E and Supplementary Material, Fig. S3).

Evidence cdd4e637d5

From these data, we conclude that the continued expression of FL Tau V337M and F3DK280 is toxic for the neurons and as a conse- quence, the development of the nervous system is perturbed.

Evidence 0a28e0e357

In add- ition, only FL Tau V337M was phosphorylated at the KXGS motif (Fig. 2A, mid panel, 12E8), S396 and S404 (PHF-1 epitope) (Fig. 2A, lower panel, PHF-1).


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