MTM S.r.l. Milano

Renton, Chi and Trainor, 2014

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Modified from Renton, Chi and Trainor, 2014

Mainly a defect in RNA metabolism/transport and cell homeostasis

LOF mutations in translation factors and their regulators often result in nervous system defects, e.g. synaptic malfunction

There is a striking link between RNA binding proteins (RBPs) and ALS. For example, mutations in the RBPs, hnRNPA1 and A2B1, TDP-43, and FUS are strongly associated with this disease.

The self-assembly ability of these proteins is enhanced by ALS-causing mutations.

Geschwind and Levitt, 2007; Gkogkas et al., 2013; Kim et al., 2013; Sreedharan et al., 2008; Kwiatkowski et al., 2009; Vance et al.,2009; Kim et al., 2013

Advantages of localized protein synthesis

Segregation of proteins w/o changes in sequence / structure

Quicker response to external stimuli

Energetic advantage of transporting mRNA (true? 1 mRNA, many nascent peptides)

UTR can contain alternatively spliced mRNA localization signals

Neurons could benefit the most from localized protein synthesis (highly polarized cells)

Evidence for axonal protein synthesis:

Coding RNAs, non-coding RNAs and tRNAs are found in the axon

Periaxoplasmic ribosomal plaques are found in the

axon During development, growing axons respond to

stimuli when detached from cell body (not after cycloheximide treatment)

The need for axonal protein synthesis

Axonal damage precedes the demise of the neuronal soma in ALS

Recently discovered causative mutations of ALS strike genes that are ubiquitously expressed but neurons possessing meter-long axons are particularly affected

Ratio of an average pyramidal neuron's cell body to axonal volume

Heiman et al., 2008

Goal: immunoprecipitation of ribosomes and enrichment for translated RNAs loaded onto them ! RNA purification ! RNAseq ! translatome profiling

Small subunit

Large subunit

RPL GFP

Use tagged RP as a transgene ! mouse line

Generation of genetically engineered conditional mouse lines expressing the chimeric RP in genetically defined neural cell lineages

Final goals: Generation of a platform for high-throughput

translatome profiling in ALS models Identification of early disease biomarkers new pathogenetic mechanisms new therapeutic targets.

RPL22 conditional K/in mouse, a.k.a. RiboTag (Sanz et al., 2009) RPL22 at the interface between two 60S subunits, the tag may

cause hindrance non-fluorescent tag close homolog in mammalian genomes, called RPL22-like

RPL10a BAC transgenics (Doyle et al., 2008) RPL10a ROSA26 conditional k/in (Zhou et al., 2013)

RPL10A is exposed to the cytosol in ER ribosomes, but it is buried in cytoplasmic polyribosomes

competition with the endogenous protein

" Eukaryotic ribosome MW:

4.2 MD

" More than 80 proteins

" Which one should we tag?

" Avoid small subunit - Tagging an RPS could hinder polyribosome

formation

" Avoid ribosome-ER interface

" Avoid important sites for ribosome physiology (mRNA entry tunnel, peptide exit tunnel, interaction of eIF and eEF)

" Some chimeric proteins show a strong nucleolar and perinuclear signal 48h after transfection ! good

" Others show a nuclear signal and faint cytoplasmic signal ! bad

HEK293 cells 48h

" Sucrose gradient enables separation of ribosomal subunits and polysomes from cytosolic fraction

" WB shows that both

RPLa-eGFP and RPLb-eGFP behave similarly to endogenous RPL26

NSC34

RPLc-eGFP

RPLd-eGFP

RPLe-eGFP

RPLa-eGFP

RPLf-eGFP

RPLb-eGFP

RPLg-eGFP

RPL26

Azidohomoalanine incorporation assay (metabolic labeling). AHA is a non-toxic methionine analog recognized by a monoclonal Ab.

c d e a f b g h

NSC34

Paola Podini, Angelo Quattrini

RPLa-eGFP

RPLb-eGFP

Hippocampal neurons

Cytosolic eGFP

RPLa-eGFP

Puro RPLa-eGFP DAPI

A

Puro RPLa-eGFP

Primary cortical neurons, 8 DIV

Puromycylation

Cortical neurons, 8 DIV

30 secs

Approximate speed: 0.2-1 m/sec

30 sec

Cortical neurons, 8 DIV

CD1 X

C57BL/6

Delivery of lentiviral

RPLa-eGFP into E12.5

lateral ventricles

Analysis at E17.5 or P5

Luca Muzio

Is RPLa-eGFP expression compatible with normal brain development?

E17.5

RPLa-eGFP In cultured primary neurons:

No toxicity Appropriate subcellular localization Co-localizes with endogenous RPLs

Translational activity: A percentage of RPLa-eGFP positive dots in neurites are

translationally active Live imaging:

Granules containing RPLa-eGFP are transported along neurites at a speed compatible with fast active transport

In vivo: No evidence of toxicity Normal cortical development

Summary Summary

Confocal STED

Pixel size 15nm

STED STED

ImageJ particle analysis

Generation of Tg mice for TRAP in ALS models

31

NSC 34

DAPI DAPI RPLa-eGFP

A

No Cre !

+ Cre !

32

Endogenous locus

5' homology arm 3' homology arm

Knock-in construct

5' homology arm 3' homology arm

polyA polyA

33

5' homology arm

polyA polyA

3' homology arm

Neo allele

Conditional allele

Knock-in allele

5' homology arm 3' homology arm

5' homology arm 3' homology arm

polyA

polyA

polyA

ALS model Cre; RPLa-eGFP

RNAseq

TRAP

Translatome profiling by TRAP in vivo

in murine models of ALS (and other NDDs) vs wt controls in specific neuronal types in neuronal cell body vs the axon (e.g. corticospinal, sciatic) in other CNS and PNS cell lineages (glia, microglia, stem cells) in basal conditions and upon stimulation (metabolic,

pharmacological, electrical, optogenetic)

Characterization of dysregulated cellular pathways Identification / validation of candidate biomarkers and

candidate therapeutic drug targets