Meccanismi di neurodegenerazione in modelli di malattie mitocondriali

Mitocon Bologna, 5/6/2015

Mitochondria and neurons

•High need for Ca++ buffering

•ATP production

•Maintenance of membrane potential

•Release and reuptake of neurotransmitters

Transport to long distance site

Long half-life

Mitochondrial quality control pathways

Dual activity of the m-AAA protease in mitochondria

Protein degradation

Protein activation

Complete proteolysisto peptides

Protein processing

Nolden et al., Cell 2005

The human m-AAA proteases

IMS

matrix

Hetero-oligomericm-AAA protease

Homo-oligomericm-AAA protease

AFG3L2 AFG3L2 paraplegin

MTS TM AAA PDTM

Hereditary spastic paraplegia and mutations in SPG7/paraplegin

•Autosomal recessive

•Pure forms: weakness and spasticity of the lower limbs

•Complex forms: cerebellar signs, nystagmus, muscular wasting, optic pallor, peripheral neuropathy

AFG3L2 is mutated in spinocerebellar ataxia SCA28 and in a rare form of spastic-ataxia

Loss of balancelimb and gait ataxiaDysarthria

Di Bella et al., Nature Genetics, 2010

Y616C

Heterozygous mutations: SCA28 Homozygous mutation:SPAX5

Early onset spastic gait

Generalized tonic-clonic and myoclonic seizures

Progressive motor degeneration, with dysarthria and dysphagia and complete loss of ambulation.

Ataxia

Pierson et al. PLOS genetics, 2011

What is the pathogenic mechanism underlying neurological diseases linked to mutations in subunits

of the m-AAA protease ?

Mouse models of m-AAA protease subunits

Spg 7 -/-

Ferreirinha et al. J. Clin. Invest., 2004

•Late onset motor phenotype; normal vitality and fertility

•4,5 months: Ultrastructural mitochondria abnormalities in synaptic mitochondria

•7 months: retrograde axon degeneration in long spinal tracts and peripheral nerves

Afg3l2 -/-

Maltecca et al. J. Neurosci., 2008

•Severe phenotype, die at P16.

•Impairment of axonal development with delayed myelination and poor radial growth

•Abnormal mitochondria in neuronal cell bodies

LoxP LoxP

frt

ex1 ex2 ex3 ex6ex5ex4 ex7

LoxP

ex1 ex2 ex3 ex6 ex7

deleted

floxed

L7-Cre

CAMKII-Cre

Afg3l2 deficiency in adult neurons

Deletion of Afg3l2 from Purkinje cells causes neuronal degeneration associated with

neuroinflammation

Early-onset COX deficiency in Purkinje neurons

WT Afg3l2 PC-KO

Combined COX (brown)-SDH (blue) staining

Almajan et al. JCI, 2013

Reduced mitochondrial protein synthesis in Afg3l2-deleted brain mitochondria

Almajan et al. JCI, 2013

BN-PAGE

CII (70kDa)

MRPL32

Brain Liver Heart

wt ko wt kowt ko

Reduced levels of mature MRPL32 in Afg3l2-deficient mice

Almajan et al. JCI, 2013

Impaired mito-ribosome assembly in AFG3L2-deleted mitochondria

Almajan et al. JCI, 2013

What about the mitochondrial network?

X

Mito-YFPSTOP

XL7 Cre

Afg3l2 flox/flox

Abnormalities of the mitochondrial network occur very early and before any sign of degeneration

Control RNAi Afg3l2 RNAi

Downregulation of Afg3l2 in primary cortical neurons

Arun Kondadi

Downregulation of Afg3l2 in primary neurons leads to impaired anterograde transport of

mitochondria

Kondadi et al. EMBO J, 2014

Transport of dysfunctional mitochondria in axons is inhibited

Which is the signal? Is fragmentation causing the transport defect?

A stress pathway affected mitochondrial dynamics

Fusion

Content mixing

Maintenance ofmitochondrial integrity

Fission

Mitochondrial fragmentation

Mitophagy Apoptosis

+

Destabilization ofL-OPA1

Formation ofS-OPA1

Cell survival Cell survival Cell death

OMA1

Loss of Δψm

ATPLack of AFG3L2

Mitochondrial defects in Afg3l2-depleted neurons are independent of OMA1 activation

Kondadi et al. EMBO J, 2014

Deletion of Afg3l2 in adult brain causes neurodegeneration and tau hyperphosphorylation

Sara Montagner

fl/fl fl/fl: CaMKIIa-Cre fl/fl fl/fl: CaMKIIa-Cre

Nissl staining p-tau: AT8

Deletion of Afg3l2 in adult brain causes tau hyperphosphorylation and activation of PKA and ERK

kinases

Kondadi et al. EMBO J, 2014

Tau downregulation partially rescues the mitochondrial transport defects of Afg3l2-depleted neurons

Kondadi et al. EMBO J, 2014

Can modulation of tau affect mitochondrial transport in neurons?

NAC treatment rescues mitochondrial transport defects and decrease phospho-tau level in

primary neurons

Kondadi et al. EMBO J, 2014

AFG3L2 deficiency

ROS

Stress-induced mitochondrial fragmentation mediated by OMA1

respiratory deficiency

Anterograde mitochondrial transport

phospho-tau

ATP levels

Neurodegeneration

?

Impaired mitochondrial protein synthesisReduced assembly of respiratory chain subunits

Eva AlmajanArun Kumar Kondadi

Shuaiyu WangSara MontagnerPaola Martinelli

Esther Barth

Thomas LangerRicarda RichterAnne Korwitz

Nikolai KladtAstrid SchaussCECAD

Nils-Göran Larsson,MPI for ageing research

Acknowledgements

Spg7 and Afg3l2 are differently expressed in the brain

The global dosage of m-AAA proteases is important to prevent neurodegeneration in

the mouse

Developmental axon defects

Late-onset axonal degeneration

Spg7 -/-

Spg7 -/- Afg3l2 -/-

Afg3l2 -/-

Afg3l2 -/+

Late-onset cerebellar dysfunction

Spg7 -/- Afg3l2 -/-

Early-onset neurodegeneration

Embryonic lethal

Ferreirinha et al. J. Clin. Invest., 2004

Maltecca et al. J. Neurosci., 2008

Maltecca et al. J. Neurosci., 2009

Martinelli et al. HMG., 2009

tau mediated neurodegeneration and mitochondria

Yoshiyama Y et al. J Neurol Neurosurg Psychiatry doi:10.1136/jnnp-2012-303144

Microtubules appear disrupted in Afg3l2 KO brain

WT KO