MTM S.r.l.

Milano

Renton, Chi and Trainor, 2014

Mainly a defect in RNA metabolism/transport and cell homeostasis

Modified from Renton, Chi and Trainor, 2014

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)

The need for axonal protein synthesis

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)

Localized protein synthesis in

neurodegenerative disease
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 <1:1000. Same for lower MNs.

Impairment of axonally localized protein synthesis

in MNDs?

Measuring mRNA abundance in diseased cells / tissues

is a main focus of neuropathology.

The abundance of a protein has a stronger correlation

with the translational rate of its mRNA than with total
mRNA abundance

Localized (axonal) translation is often overlooked by

transcriptomic approaches

Thus, it will be critical to profile translating mRNAs

(translatome) in addition to total mRNAs
(transcriptome).

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

GFP

RPL
Large subunit

Small
subunit

Use tagged RP as a transgene ! mouse line

Heiman et al., 2008

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

RPLc-eGFP
RPLd-eGFP

" WB shows that both

RPLa-eGFP and RPLbeGFP behave similarly to
endogenous RPL26

RPLe-eGFP
RPLa-eGFP
RPLf-eGFP
RPLb-eGFP
RPLg-eGFP
RPL26

NSC34

Azidohomoalanine incorporation assay (metabolic labeling).

AHA is a non-toxic methionine analog recognized by a
monoclonal Ab.

RPLa-eGFP

RPLb-eGFP

NSC34
Paola Podini, Angelo Quattrini

Cytosolic eGFP

RPLa-eGFP

Hippocampal neurons

Puromycylation

Puro
RPLa-eGFP
DAPI

Puro
RPLa-eGFP
Primary cortical neurons, 8 DIV

30 secs

Cortical neurons, 8 DIV

30 sec

Cortical neurons, 8 DIV

Approximate speed: 0.2-1 m/sec

Is RPLa-eGFP expression compatible with

normal brain development?

CD1
X
C57BL/6

Delivery of
lentiviral
RPLa-eGFP
into E12.5
lateral
ventricles

Analysis at
E17.5 or P5

Luca Muzio

E17.5

Summary
Summary
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

Pixel size
15nm

Confocal

STED

STED

STED

ImageJ
particle
analysis

Generation of Tg mice for TRAP

in ALS models

DAPI

DAPI
RPLa-eGFP

NSC 34

No Cre !

+ Cre !

31

Endogenous locus

3' homology arm

5' homology arm

Knock-in construct
polyA

polyA

3' homology arm

5' homology arm

32

Neo allele

polyA

polyA

5' homology arm

3' homology arm

Conditional allele
polyA

5' homology arm

Knock-in allele

5' homology arm

polyA

3' homology arm

polyA

3' homology33
arm

ALS model

Cre; RPLa-eGFP

TRAP
RNAseq

 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