Emanuele Buratti. Use of TDP-43 for biomarker and disease-modelling analysis

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1 Emanuele Buratti Use of TDP-43 for biomarker and disease-modelling analysis

2 - Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, and is characterized by the progressive loss of upper and lower motor neurons from the spinal cord, brain stem, and motor cortex, leading to muscle weakness and eventual respiratory failure. - Approximately 5 10% of ALS cases are familial with the remaining 90% being sporadic, indicating that both genetic and environmental factors contribute to risk. - Despite this diverse etiology of disease, 97 % of patients display a common phenotype in disease affected tissues, namely the deposition of the TAR-DNA binding protein (TDP-43).

3 What happens in disease?: Buratti and Baralle., 2009 Adv.Gen, 66:1

4 TDP-43 has been already used for many biomarker analyses: Buratti and Baralle., 2012 TiBS

5 Although promising, as there is a lot of overlap between patients and controls: CSF Circulating Lymphomonocytes (CLS) Noto Y et al. (2011) Amyotroph Lateral Scler 12, De Marco G. (2011) Acta Neuropathol. Method: sandwich ELISA system Method: Western blot quantification using imaging system

6 What does TDP-43 do?:

7 TDP-43 interacts with many cellular factors, including hnrnp proteins: D Ambrogio et al., 2009 Buratti, Budini, and Baralle, 2014

8 Differential splicing of exon 17b in the Sort1 gene following TDP-43 depletion: H17b- human h17b+ TDP-43 (UG)4 (UG) (UG) A T T G T C T C H Human Sort1 exon 17b Toxic! TDP-43 (UG)4 (UG) (UG) G C C T C T C T M Mouse Sort1 exon 17b m17b- mouse m17b + Prudencio M. et al, 2012

9 Notwithstanding an extremely high similarity, there is differential binding of several hnrnp proteins to Human and mouse exon 17b sequences:

10 With just TDP-43 as an exception, removing any of these other hnrnps does not increase inclusion of human exon 17b:

11 The effect, however, can be clearly seen when some of these hnrnps are removed together with TDP-43: especially hnrnp L and hnrnp A1/A2: When TDP-43 is removed, hnrnp A1/A2, hnrnp L, and PTB/nPTB seem to help exon 17b inclusion

12 What happens in patients?, in FTLD-TDP the expression of these proteins can vary a lot both between and among healthy controls and diseased patients (work in collaboration with Leo Petrucelli and Mercedes Prudencio at the Mayo Clinic, Jacksonville):

13 Developing a loss of function model in flies: Flies apparently develop normally but cannot get out of the pupal cage without external help and show a reduced life span. Feiguin et al. 2009

14 Most importantly, recovery of lifespan and paralytic phenotype can be achieved using both the human and Drosophila TDP-43. Feiguin et al. 2009

15 One of the reasons why this can occur is because both human and Drosophila TDP-43s share similar protein binding profiles and hence functional properties. Romano et al. 2014

16 merge Anti-ELAV Antifutsch Hrp38 and TBPH genetically interact to increase locomotor defects and reduced life span. W TBPH - TBPH - hrp38 - hrp38 Romano et al. 2014

17 In addition to hnrnp A1/A2, several other hnrnps are conserved between human and Drosophila (11 genes and 23 sirnas are available):

18 Experimental strategy is twofold (two loss-of-function and one gain-of-function models): 1) testing the effects of each sirna on a haploinsufficient or hypomorphic TBPH background: Haploinsufficient TBPH backgroung In this assay, flies are scored in terms of climbing ability Hypomorphic TBPH background 2) testing the effects of each sirna on a gain-of-function TBPH model in the eye (GMR-GAL4): In this assay, flies are scored in terms of eye pigmentation and necrosis level

19 Removal of hnrnp D/Sqd gives a paralytic phenotype in hypomorphic background and enhances eye phenotype: Haplo Hypo

20 Removal of DAZAP1/Hrb27c gives a lethal phenotype both in the haploinsufficient and hypomorphic backgrounds but suppresses very effectively the eye phenotype:

21 Summary:

22 In human SH-SY-5Y cells, these hnrnps do not influence the cellular expression of TDP-43 or viceversa: These results suggest that the enhancer or suppressor properties of these hnrnps is not just due to the regulation of each other expression but must pass through the regulation of common events still to be identified.

23 Conclusions: - In this year, we have demonstrated that hnrnp proteins in addition to TDP-43 can influence the splicing profile of an important event for the pathology in the Sort1 gene. - Moreover, the levels of these proteins can vary heavily both within the control and disease patients, suggesting that they may act as additional biomarkers. - In parallel, using Drosophila as a model system we have screened the effects of hnrnp proteins on TDP-43 proteinopathy, both at the loss- or gain-of-function level - The effects of these hnrnp depletion will now be tested in human cells using RNAseq technology together with TDP-43 depletion. - The aim will be to determine which genes may be particularly affected by the most effective combination of these proteins with TDP-43 in terms of RNA splicing, stability, transport, or expression. - We will then use these genes to understand which key events may controbute the most to ALS pathology and represent priority targets for therapeutic intervention.

24 Ringraziamenti: