ROSSI MARTINAROSSI MARTINAMADAIO ALESSANDROMADAIO ALESSANDRO

Axonal transport deficits and neurodegenerative diseases

Stéphanie Millecamps & Jean-Pierre JulienCentre de Recherche de l'Institut du Cerveau et de la Moelle Épinière

Université Pierre et Marie CurieNature Review Neuroscience

2013 Mar;14(3):161-76. doi: 10.1038/nrn3380

AXONAL TRANSPORTAxonal transport is a cellular process responsible for movement of mitochondria,

lipids, synaptic vesicles, proteins, and other cell parts (i.e. organelles) to and from a neuron's cell body, through the cytoplasm of its axon (the axoplasm).

Axonal transport occurs throughout the life of a neuron and is essential to its growth and survival:

Provides efficient communication between cell body and axon tip Keeps axons and nerve terminals supplied with proteins, lipids and

mitochondria Partecipates in intracellular neural transmission Clears recycled or misfolded proteins to avoid the build-up of toxic aggregates Allows the neuron to respond effectively to trophic signals or stress insults

MICROTUBULE -BASED AXONAL TRANSPORT Microtubules have a tubular

structure (25 nm in diameter) Microtubules are POLARIZED in

axons: their slower growing minus end (α tubulin) faces the ‑cell body, whereas their faster growing plus end (β tubulin) ‑points towards the axon tips.

They are composed of many α- and β tubulin heterodimers ‑which undergo continuous polymerization and depolymerization (DYNAMIC INSTABILITY)

They are stabilized by microtubule-associated proteins (MAPs) such as tau.

MICROTUBULE -BASED AXONAL TRANSPORT The transport machinery is

based on a complex system of MICROTUBULES

Microtubules in the axon essentially form TRACKS along which various cargoes can be transported by various MOTOR PROTEINS, which require ATP to provide energy. .

The various cargoes that are transported along microtubules in axons move in a SALTATORY FASHION, exhibiting periods of rapid movements, pauses and directional switches.

MICROTUBULE -BASED AXONAL TRANSPORTMOVEMENT IN SALTATORY FASHION

• Filamentous cargoes, such as NEUROFILAMENTS, exhibit long periods of rest (spending on average 73% of the time pausing) and movements mainly in an anterograde direction (that is, towards the cell body) at 0.23 μm per second2,3.

The average transport velocity for neurofilaments depends from a pre-existing neurofilaments structures in vivo. One of the key determinants that curbs the axonal transport of cytoskeleton components is the density of the STATIONARY CYTOSKELETAL NETWORK in the axons, that depends:

1. Axon caliber

2. NFL Availability

3. Stoichiometry of IF proteins.

4. Covalent modifications of NF proteins or phosphorylation change

TRANSPORT IS BIDIRECTIONAL

ANTEROGRADE Fast : membrane-bound organelles and mRNA Slow : cytoplasmic proteins, enzymes and neurofilaments

RETROGRADE Fast : endosomal recycling vesicles, autophagosomes and toxins

METHODS FOR MEASURING AXONAL TRANSPORTMETHODS FOR MEASURING AXONAL TRANSPORT1.1. RADIOACTIVE PULSE-CHASE LABELLING WITH [35S]METHIONINE RADIOACTIVE PULSE-CHASE LABELLING WITH [35S]METHIONINE

• Classic method to analyse the in vivo transport of cytoskeletal proteins along the sciatic nerve or the optic nerve of mice.

• From western blot analysis of the distance covered by radiolabelled proteins at different time intervals, the rate of their transport can be measured.

• This approach has been especially suitable for investigating the in vivo requirement of various intermediate filament proteins for intermediate filament assembly and transport into axons.

METHODS FOR MEASURING AXONAL TRANSPORTMETHODS FOR MEASURING AXONAL TRANSPORT2.2. TIME-LAPSE MICROSCOPY TIME-LAPSE MICROSCOPY

• The monitoring of fluorescent-labelled cargoes can be applied to embryonic The monitoring of fluorescent-labelled cargoes can be applied to embryonic cultured neurons. cultured neurons.

• Study the direction, speed and frequency of pauses of individual cargoes. Study the direction, speed and frequency of pauses of individual cargoes.

• Can be compared in neurons Can be compared in neurons

harbouring different gene mutations.harbouring different gene mutations.

METHODS FOR MEASURING AXONAL TRANSPORTMETHODS FOR MEASURING AXONAL TRANSPORT3.3. In In vitrovitro RETROGRADE TRANSPORT ASSAY RETROGRADE TRANSPORT ASSAY

• It is based on the C-Ter FRAGMENT OF THE TETANUS NEUROTOXIN (TeNT Hc).

• Transport of fluorescently labelled TeNT Hc is monitored by video tracking.

• Can also be applied in living animals.

METHODS FOR MEASURING AXONAL TRANSPORTMETHODS FOR MEASURING AXONAL TRANSPORT4.4. MICROFLUIDIC CULTURE PLATFORM MICROFLUIDIC CULTURE PLATFORM

• Microfluidics is a field of study that uses devices with small channels to control and Microfluidics is a field of study that uses devices with small channels to control and manipulate minute amounts of fluids.manipulate minute amounts of fluids.

• Allows the polarized growth of CNS axons in a fluidically isolated environment without neurotrophins.

• The embedded microgrooves define transport direction, which can be studied by live cell imaging.

• This method is suitable for analysing the trajectories and velocity of individual fluorescent elements. It also allows the investigation of axonal mRNA without detectable somal or dendritic contamination.

• As cumulative evidence suggests that axonal protein synthesis is important in the development, maintenance and regeneration of axons, this method could be valuable in studying the role of mRNA axonal transport and local protein synthesis in neurodegenerative diseases.

TRANSPORT IS BIDIRECTIONAL

ANTEROGRADE → KINESINSKINESINS Fast : membrane-bound organelles and mRNA Slow : cytoplasmic proteins, enzymes and neurofilaments

RETROGRADE Fast : endosomal recycling vesicles, autophagosomes and toxins

MOTOR PROTEINS: KINESIN KINESIN moves from minus end to plus

end

Kinesin 1 is a heterotretramer that is ‑composed of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs).

After the attachment of the motor domain to microtubules and the tail regions to the cargoes, kinesin 1 ‘walks’ ‑along the microtubule

The kinesin front head, which remains bound to microtubules, hydrolyses ATP and that moves the detached rear head towards the microtubule plus end.

The binding of kinesin 1 to microtubules ‑is promoted by tubulin acetylation.

TRANSPORT IS BIDIRECTIONAL

ANTEROGRADE → KINESINS Fast : membrane-bound organelles and mRNA Slow : cytoplasmic proteins, enzymes and neurofilaments

RETROGRADE → DYNACTINDYNACTIN Fast : endosomal recycling vesicles, autophagosomes and toxins

MOTOR PROTEINS: DYNEIN Moves from plus end to minus end.

A multi-subunit complex containing two catalytic heavy chains, two intermediate chains, four light intermediate chains and several light chains.

During cargo transport, one coiled-coil stalk of the heavy chains remains bound to the microtubules while the other one detaches and reattaches to the microtubule, which allows the dynein complex to walk for long distances.

The binding of the cargo to the intermediate and light dynein chains is modulated by DYNACTINDYNACTIN.

MYOSIN Va MYOSIN Va PARTECIPATES AT AXONAL TRANSPORT Involved in the transport of cargoes along

the actin filaments that are enriched in nerve terminals.

Interacting with KHCs directly, with microtubules through its tail domain and with the neurofilament light chain (NFL) through its head motor domain.

Has a crucial role in COUPLING MICROTUBULE- AND ACTIN-BASED TRANSPORT MECHANISMS and could regulate the distribution of cargoes across the microtubule and neurofilament networks.

MOTOR PROTEINS ACTIVITY IS HIGHLY REGULATED PHOSPHORYLATION of the motor proteins or the cargoes.

a) phosphorylation of KLC by glycogen synthase kinase 3β (GSK3β).

release of kinesin from vesicles. LOCAL GRADIENTS of MAPs in axons, such as tau, or by MICROTUBULE POST-

TRANSLATIONAL MODIFICATIONS.

a) low [tau] in the proximal segment of the axon

facilitates kinesin-mediated transport,

b) high [tau] in the axon terminal

facilitates dynein-mediated transport.

c) tubulin acetylation at Lys40 enhance the recruitment of kinesin 1.‑

TRANSPORT DEFECTS IN NEURODENEGERATIVE DISEASEEvidence of axonal transport defects in neurodegenerative diseases emerged from

microscopy studies showing that, in these disorders, some cargoes can accumulate in the perikaryon, the proximal segment of the axon, or the distal part of the axon.

ALZHEIMER DISEASE & OTHER DEMENTIAL PARKINSON DISEASE & PERRY SYNDROME HUNGTINGTON DISEASE UPPER MOTOR NEURON DISEASES CHARCOT-MARIE-TOOTH PERYPHERAL NEUROPHATY LOWER MOTOR NEURON DISEASES AMYOTROPHIC LATERAL SCLEROSIS

ALZHEIMER DISEASE & OTHER DEMETIAL

Mutations in APP and in the presenilin genes are responsible for early-onset, familial forms of Alzheimer’s disease.

Mutations in the gene encoding tau can cause tauopathies, including familial frontotemporal dementia with Parkinsonism.

The pathological forms of APP, tau, PS1 and amyloid β that can be found in Alzheimer’s ‑disease affect fast axonal transport by various mechanisms.

Genetics studies support causal roles for APP, PS1 and tau in dementia.

APP → AMYLOID PRECURSOR PROTEIN PS1 → PRESENILIN 1 Tau → MICROTUBULE-ASSOCIATED PROTEIN

ALZHEIMER DISEASE & OTHER DEMETIAL

Hyperphosphorylation of tau by CDK5–p25 kinase or glycogen synthase kinase 3β (GSK3β) leads to a decrease in the microtubule binding affinity of tau and destabilization of microtubules.

GSK3β activation by tau, mutant presenilin 1 (PS1) or amyloid β (Aβ) impairs cargo ‑attachment to kinesin.

Aβ also damages bidirectional axonal transport of vesicles through the activation of casein kinase 2 (CK2), which prevents cargoes from binding to the motor proteins.

POLYGLUTAMINE DISEASE They are caused by the expansion of a CAG tract in particular genes, leading to the loss of

selected neuronal populations through a dying-back neuropathy and the formation of aggregates that recruit and sequester essential cellular proteins.

Both Huntington’s disease and spinal and bulbar muscular atrophy (SBMA; also known as Kennedy disease), two well-known polyQ diseases, are associated with axonal transport defects.

HUNGTINTON disease is characterized by muscle incoordination, cognitive decline and dementia. It occurs when the number of CAG repeats in the coding region of the gene HUNGTINTIN (HTT) is above 36 → polyQ HTT

The characteristic degeneration of striatal and cortical neurons in mice expressing polyQ HTT is preceded by axonal pathology64, and axonal aggregates of polyQ HTT physically disrupt axonal transport in cultured neurons.

POLYGLUTAMINE DISEASE

In Huntington’s disease, axonal transport defects are caused by disruption to the binding of the motor protein with microtubules or cargoes.

Polyglutamine (polyQ) huntingtin promotes tubulin deacetylation, resulting in binding failure of the motor proteins to the microtubules.

Histone deacetylase 6 (HDAC6) inhibitors can rescue tubulin acetylation and such binding

POLYGLUTAMINE DISEASE

Microtubule binding of kinesin is also prevented by phosphorylation of the kinesin motor domain by polyQ huntingtin-activated c-Jun N-terminal kinase 3 (JNK3).

Huntingtin-associated protein 1 (HAP1) is an adaptor protein that interacts with kinesin light chains and dynactin subunit 1 (DCTN1).

PolyQ huntingtin disrupts HAP1 mediated axonal transport, including that of brain-‑derived neurotrophic factor (BDNF). The loss of neurotrophic support may contribute to neurotoxicity in Huntington’s disease.

HEREDITARY SPASTIC PARAPLEGIA Hereditary spastic paraplegias (HSPs) are clinically heterogeneous inherited upper

motor neuron diseases that are characterized by progressive spasticity and pyramidal weakness, with predominant presentation in the legs.

Numerous HSP-associated genes encode proteins involved in axonal transport and intracellular trafficking, which underlines the vulnerability of long motor neuron axons to alterations in these processes.

Mutations in SPAST (SPG4), the gene encoding SPASTIN, are responsible for 40% of autosomal dominant forms of HSP.

Spastin contains a microtubule-interacting and endosomal-trafficking (MIT) domain, other microtubule-interacting regions and an ATPase with various cellular activities (AAA) domain, and it acts to regulate microtubule dynamics.

• HSP mutations have also been found in ATL1 and zinc finger FYVE domain-containing 27 (ZFYVE27); these genes encode ATLASTIN 1 and PROTRUDIN, respectively, which are two partners of spastin.

HEREDITARY SPASTIC PARAPLEGIA

In HSP mutations can affect the function of the kinesin motor protein KIF5A, which show a reduction in microtubule affinity or exhibit a reduction in transport velocity in vitro.

Microtubule severing by spastin facilitates the axonal transport of microtubules, whereas microtubule bundling impairs such transport.

These findings suggest that mutant spastin interferes with kinesin-mediated transport along microtubules, probably through a novel gain of function, microtubule-independent mechanism that involves activation ‑ ‑of the kinases and phosphatases that regulate molecular motor proteins.

CHARCOT-MARIE-TOOTH DISEASE CMT is the most common hereditary neuropathy and is characterized

by weakness and atrophy of distal muscle and mild sensory loss. The disease is classified into two clinical subtypes:

CMT1: motor nerve conduction velocity is markedly reduced owing to myelin degeneration (myelinopathy)

CMT2: conduction velocity is only slightly subnormal and is caused by axonal degeneration (axonopathy).

Numerous mutations in the gene encoding mitochondrial fusion protein mitofusin 2 (MFN2) have been reported to be associated with CMT2A (one of several subtypes of CMT2).

MFN2 is a mitochondrial outer membrane GTPase that regulates the fusion of mitochondria and the mitochondrial network architecture.

Dominant mutations in the gene encoding heat shock protein β1 (HSPB1) cause CMT2F. HSPB1, like other small heat shock proteins, binds to misfolded proteins to prevent their aggregation.

CHARCOT-MARIE-TOOTH DISEASE

HSBP1 mutant induces microtubule deacetylation, which is rescued by inhibitors of histone deacetylase 6 (HDAC6).

Assembly and transport of neurofilaments are disrupted by mutant HSBP1 and mutant neurofilament light chain (NFL).

A CMT-associated mitofusin 2 (MFN2) mutant variant disrupts the binding of kinesin adaptor protein with mitochondria, producing highly aggregated mitochondria that cluster along the axon.

AMYOTROPHIC LATERAL SCLEROSIS Amyotrophic lateral sclerosis (ALS), the most

common adult-onset motor neuron disease, is characterized by the degeneration of cortical, bulbar and spinal motor neurons.

This degeneration induces progressive muscle wasting, paralysis and spasticity that ultimately lead to respiratory failure.

Electron microscopy studies have revealed that swellings occur in the initial segment of motor axons in patients with ALS and that these swellings contain vesicles, lysosomes, mitochondria and intermediate filaments.

These changes are highly suggestive of axonal transport defects.

Excitotoxicity, a phenomenon associated with ALS, may contribute to transport defects in this disorder.

AMYOTROPHIC LATERAL SCLEROSIS

Mutations affecting dynein complex proteins (such as dynactin subunit 1 (DCTN1)) impair microtubule-based axonal transport.

Glutamate activates c-Jun N-terminal kinase (JNK), which phosphorylates the kinesin motor domain and inhibits kinesin from binding to microtubules.

Glutamate and ALS-linked mutant superoxide dismutase 1 (SOD1) activate p38 and the resulting phosphorylation of kinesin light chain inhibits cargo from binding to the motor protein.

Glutamate and mutant SOD1 activate p38 and CDK5–p25 kinase, which phosphorylate neurofilaments and prevent neurofilament from binding to the motor protein.

CONCLUSIONS

AXONAL TRANSPORT DEFICITS MIGHT ARISE THROUGH VARIOUS MECHANISMS THAT MAY ACT SYNERGISTICALLY.

1.LOSS-of-FUNCTION MUTATION (RARE)

2.ALTERATED KINASE ACTIVITIES

3.DIRECT INTERATIONS BETWEEN MUTANT/MISFOLDED PROTEIN and AXONAL TRANSPORT MACHINERY

4.MITOCHONDRIAL DYSFANCTION ENERGETIC BREAKDOWN

5.ALTERATION IN METABOLISM OF RNA COMPONENT

CONCLUSIONS Many neurodegenerative diseases are characterized by adult-onset, progressive accumulations

of specific proteins in different types of neurons. It is commonly assumed that disturbances in axonal transport are key pathological events that

contribute to neurodegeneration. However, the causal relationship between axonal transport disturbances and degeneration

remains unclear.

The identification of mutations in genes encoding proteins that are known to be involved in axonal transport strongly support the view that defective intracellular transport can directly trigger neurodegeneration.