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Selective vulnerability in motorSelective vulnerability in motorneuron disordersneuron disorders

Eva Hedlund, PhDEva Hedlund, PhDLudwig Institute for Cancer ResearchLudwig Institute for Cancer ResearchKarolinska Institutet, SwedenKarolinska Institutet, Sweden

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Selective vulnerability in motor neuron disorders

Amyotrophic lateral sclerosis (ALS):

Cortical, spinal and lower cranial nerve motor neuronsdegenerate

Somatic motor neurons, but not autonomic

motor neurons Selective vulnerability among somatic motorneurons:

- Higher cranial nerves damage late or are spared in ALS,SMA and SBMA

The reasons for this selective vulnerability

are unknown

1. arm muscle2. tongue3. cortical (upper) motor neurons4. brainstem (bulbar) motor neurons5. axon bundles6. rib muscles7. spinal motor neurons

8. leg muscle

Spinal bulbar muscular atrophy (SBMA): Spinal and lower cranial nerve motor neurons are lost

Spinal muscular atrophy (SMA):

Spinal motor neurons degenerate

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Cell intrinsic and extrinsic disease mechanisms

Muscle

Motor neuron

Astrocyte

Microglia

Questions: Why are some motor neurons more vulnerable to degeneration than others? Can analysis of the normal intrinsic properties of different motor neuron subpopulations giveclues to mechanisms of relative vulnerability?

?

ToxicSignal?

Activation

signal?

Methodology: Isolation of individual neurons using laser capture microdissection (LCM) and analysis ofgene expression differences using oligomicroarrays (Rat Genome 230 2.0 Array, Affymetrix)

Schwann cells

Interneuron

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Isolation of motor neuron subpopulations with differential vulnerabilityto degeneration in motor neuron disorders

Hedlund et al. 2010 Brain

Laser capture microdissection (LCM) of motor neurons

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Global gene expression analysis of motor neuron subpopulations

Differential Hox gene expression validated the microarray data

Motor neurons in CN12 and cervical spinal cord had more commonality in gene expressionlevels than those in CN3/4 All three motor neuron subpopulations displayed distinct profiles and exhibited genes with

unique expressionHedlund et al. 2010 Brain

Differences in the number ofgenes involved in transcription,RNA -processing, -binding and -splicing and regulation oftranslation

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Protein analysis confirmed differential expression in motor neurons ofCN3/4, CN12 and the cervical spinal cord


Differential expression of multiple proteins withimplications for motor neuron vulnerability andprotection

Can exogenous delivery of CN3/4-restricted genes confer neuroprotection to vulnerablemotor neurons? Developed an in vitro toxicity assay to analyze possible effects on spinal motor neurons.

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The CN3/4-restricted gene IGF-II protected spinal motor neurons fromglutamate-induced toxicity

Astrocyte

AMPA

AMPA

NMDA

Motor neuron

EAAT

Selection criteria: IGF-II support regeneration of motor axons (Caroni and Grandes. 1990 J Cell Biol; Near et al.1992 PNAS)

IGF-I can protect motor neurons in a mouse model of ALS (Kaspar et al. 2003 Science ) We identified a preferential expression of IGF-I and IGF-II within CN3/4 motor neurons

In vitro assay of Glutamate overload-Excitotoxicity

Ca 2+Glu

ROS


L-trans-2,4-pyrrolidine-2,4-dicarboxylic acid (PDC)

+ Glutamate

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Guanine Deaminase, a CN3/4-preferential gene, protected motor neurons


Selection criteria: Guanine deaminase is important for synaptic function and dendritic branching (Firestein et al. 1999Neuron ) (Akum et al. 2004 Nat Neurosci ) (in hippocampal neurons). ALS-associated mutations in TDP-43 attenuates the dendritic function of this protein (Lu etal.2009 Mol Brain ). We identified a restricted expression of guanine deaminase in CN3/4 motor neurons

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Conclusions and future perspectives

Analysis of the intrinsic properties of different motor neurons subpopulations inthe normal animal can give important clues to mechanisms of relative vulnerabilityin motor neuron disorders and may hopefully be used to develop treatments forthese diseases.

Future studies will analyze if the identified molecules can protect neurons after

toxicity is initiated and evaluate the effect in other models of motor neurondegeneration ( in vitro and in vivo ).

Future studies aim to also analyze possible differences in mRNA splicing indifferent motor neuron subpopulations

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Acknowledgements

Harvard Medical School/McLean HospitalOle Isacson (Director)Martin KarlssonTeresia OsbornWesley Ludwig

Supported byALS Association (EH)ALS Research Program Therapeutic Development Award/DOD (OI)The Consolidated Anti-Aging Foundation (OI)National Institute on Aging (TO)

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Alzforum EH 20100914