September 13th BIOS E108 Parkinson’s disease (PD) -Pathology -Diagnosis -  -synuclein

Parkinson’s disease (PD)

Parkinson’s disease First described by James Parkinson in his “Essay on Shaking Palsy” (Paralysis- agitans; 1817). Neurodegenerative disease characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compact (SNpc) and by the accumulation of intracellular fibrillar aggregates, the Lewy Bodies. PD is the second most common neurodegenerative disease, after AD, encountered with aging. 95% of the cases are sporadic, only 5% are genetically inherited. Because of the impact of environmental toxins in PD, developed, industrial countries have an higher incidence of PD cases, compared to undeveloped, non industrialized countries. Differences in the PD diagnosis among countries could also be due to different diagnostic methods, treatments and follow ups. Medium age of onset is after 60 years old (for sporadic PD). About 1% of the population of 60 years old is affected by PD.

Symptoms in Parkinson’s disease -Resting tremor (a tremor of a limb that increases when the limb is at rest) -Bradykinesia (The slowing down and loss of spontaneous and voluntary movement) -Rigidity (increased resistance to the passive movement of a limb) -Postural Imbalance (incapability to keep a steady posture) -Difficulties in the speech A patient “qualifies” for PD when has at least two of these symptoms

Diagnosis of Parkinson’s disease -Clinical diagnosis is very difficult at early stages, and reliable biological markers have not been identified yet. -L-DOPA responsiveness is a reliable indication of PD -Imaging is used, in particular PET scan, Positron Emission Tomography. -Reliable diagnosis only post- mortem. L-DOPA

Parkinson’s diseases: a neurodegenerative disease characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compact (SNpc) and in the nigrostriatal pathway. As a consequence: THE DOPAMINERGIC SIGNALING IS IMPAIRED, THUS THE FUNCTIONS CONTROLLED BY DOPAMINERGIC SIGNALLING ARE LOST, RESULTING IN THE DISEASE, AND IN THE SYMPTOMS THAT CHARACTERIZE IT.

Dauer & Przedborski, 2003, Neuron 39, SNpc degeneration in PD

Interconnections between the Basal Ganglia and the Cortex -Basal Ganglia are located in the mid-brain, and consist of several interconnected subcortical nuclei with major projections to the cerebral cortex, thalamus, and certain brain stem nuclei. -They receive major input from the cerebral cortex, and send their output back to the cerebral cortex. Kandel, Schwartz, Jessell “Priniciples of Neural Science”

Dopaminergic Pathways in normal and PD: the direct and indirect ways Kandel, Schwartz, Jessell “Priniciples of Neural Science”

Dopamine synthesis and signaling

Pathological hallmarks of PD 1- loss of nigrostriatal dopaminergic neurons 2-presence of proteinacious cytoplasmic inclusions, called Lewy Bodies (LBs) 3- the pattern of cell loss parallels the level of expression of the DA transporter (DAT) 4-At the onset of symptoms, DA is depleted ~80% and ~60% of Substantia Nigra Pars Compacta (SNpc) is lost. 5-More damage occurs at the nerve terminals, suggesting that dopaminergic nerve terminals are the primary target for the neurodegeneration.

Lewy Bodies 1-Intracellular lesions not specific of PD, but present also in certain cases of Dementia (Lewy Bodies Dementia) 2-Spherical eosinophilic cytoplasmic protein aggregates, measuring more than 15  M in diameter composed of : a-synuclein, parkin, ubiquitin and neurofilaments 3-Mechanism of toxicity not known: these aggregates could directly damage the cell by disrupting the intraneuronal protein trafficking, or by sequestering soluble proteins crucial for the life of the cell. 4-These inclusions may result from a process aimed at sequestering soluble misfolded proteins in the cytosol.

Deposition of fibrillar proteinacious material in PD Parkinson’s disease: characterized by dopaminergic neuronal loss and by intracellular depositions, the Lewy bodies, comprised of  -synuclein and ubiquitin, as the major components. Other components are proteasome and cytoskeletal proteins and other proteins that interact with  -synuclein. Bossy-Wetzel E, et al., Nat Med Jul;10 Suppl:S2-9. Review.

Risk factors ENVIRONMENTAL/OCCUPATIONAL: direct correlation between these factors and PD 1-Exposure to toxins and pesticides (MPTP, rotenone, paraquat): from eating fruit and vegetables, to drinking well water. Significantly increased risk of developing PD among men who worked more than 10 years on a plantation. These substances inhibit the mitochondrial complex I, thus cause mitochondrial dysfunction. 2-Welding and exposure to heavy metals (such as zinc, amalgam, copper, lead, iron, manganese): they cause oxidative stress and production of ROS species.

Personal habits Tobacco and coffee: inverse correlation to PD Alcohol: non clear inverse correlation to PD Dietary factors Tobacco: Reduced risk of PD among cigarette smokers. Nicotine may be neuroprotective by -stimulating the release of Dopamine -acting as a antioxidant -altering the activity of MAOB Coffee: Significantly decreased risk of PD among coffee drinkers. Caffeine may be neuroprotective by inhibiting adenosine A2 receptor: this may reduce inflammation and sustain the activity of dopamine D2 receptors. In fact, A2A receptor activity partially inhibits locomotor activity by inhibiting dopamine action at D2 receptors.

Alcohol: -Alcohol could sustain dopamine release. However, it isn’t clear whether alcohol consumption increases or reduces the susceptibility to PD. Dietary Factors: -antioxidant: vitamins C and E may help reduce the susceptibility to PD by neutralizing free radicals. High amount of vitamin E have been associated to significantly decreased risks of PD. -fat and fatty acids: High intake of fat could increase the susceptibility to PD by increasing the risk of lipid peroxidation, thus the formation of ROS and free radicals. Among fats, poly-unsaturated ones (omega-3 and omega-6 essential fatty acids, in particular arachidonic acid and linoleic acid) may help reducing the susceptibility to PD. They may reduce inflammation. Iron: Iron is positively correlated to PD. High levels of iron are in the Substantia Nigra pars compacta of PD patients. Iron may induce the formation of free radicals

Other factors Inflammation: It is not clear whether inflammation is a cause or a consequence in the pathogenesis of PD, however, higher intake of non-steroidal anti- inflammatory drugs is inversely correlated to PD. Estrogen: Estrogen may be protective from PD, possibly through antioxidant properties. Intake of estrogen is associated to better performances in the treatment of PD.

Etiology of PD SPORADICGENETIC Environmental toxins: MPTP Paraquat Rotenone Cocaine Endogenous toxins: Reactive Oxygen Species (ROS)  -synuclein Parkin PINK1 LRRK2 UCHL-1 DJ-1

Proteins genetically mutated in inherited PD: role and onset of the disease

Dauer & Przedborski, 2003, Neuron 39, Mechanisms in PD: environmental and genetic factors

Genetic of PD Point mutations or repeats on genes encoding for proteins crucially involved in the pathogenesis of PD. Familial cases. Two cases of autosomal dominant inherited PD:  -synuclein and LRRK2 Five cases of inherited autosomal recessive PD: UCHL-1, parkin, DJ-1, PINK1 They all cause PD symptoms, the same course of the disease, and degeneration of dopaminergic neurons. However, they may have different age of onset. Susceptibility genes (involved in dopamine metabolism, mitochondrial metabolism, detoxification, etc.).

 -synuclein (PARK1/4) 1-Belonging to the family of the synucleins, existing in three forms:  -synuclein,  -synuclein and  -synuclein. Although they all colocalize in the presynaptic terminal, only  -synuclein accumulates in Lewy bodies. 2-  -synuclein gene is expressed on chromosome 4 3-cytosolic protein of 140 aa, approximate molecular weight 14.5 kDa 4-Mostly expressed at a neuronal level, in presynaptic terminals. 5-It is degraded through the ubiquitin-proteasome pathway 6-It exists in 2 different conformations  -helix and  -sheet 7-Its function is still unknown. Probably involved in neurites development and in dopaminergic signaling 8-two mutations account for inherited forms of autosomal dominant PD (A53T; A30P)

 -synuclein A30TA53P A30T A53P In the N-terminal region there are 5-6 imperfect repeats (EKTKEGV). This region acquires  -helical secondary structure upon binding to membrane lipids, whereas the C-terminal region is highly flexible (Eliezer, 2001). NAC=non amyloid component, in this hydrophobic region are aa crucial for  - synuclein aggregation. Kahle et al.,

 - synucleins

Physiological role of  -synuclein 1-  -synuclein is expressed in neuronal tissues, brain and spinal cord, not in muscles and other organs (Maroteaux, 1988). 2-  -synuclein associates to lipid membranes. In nervous system  -synuclein is found in close association to synaptic vesicles, where it may modulate synaptic vesicle function (Kahle 2002), by binding to the lipid component of the vesicles and regulating vesicles and protein trafficking (Jensen, 1998, 1999, 2000) probably through modulation of phospholipase D activity. This binding stabilizes the  -helical conformations of the N-terminal domain of  -synuclein (Davidson 1998, Eliezer, 2001). 3- In dopaminergic neurons,  -synuclein may regulate the pool of DA released in the cytosol.

 -synuclein regulation of presynaptic vesicle cycling

Pathogenic mechanisms of  -synuclein 1- misregulation of the amount of DA released in the cell. 2- docking of  -synuclein with oxidized DA (DA quinone), and formation of protofibrils. 3- formation of protofibrils sequesters  -synuclein available for binding to synaptic vesicles, thus the amount of  -synuclein in the  -helical structure decreases.

 -synucleinopathies

Kahle et al.,  -, but not  -synuclein, forms aggregates in Lewy bodies in PD  -synuclein  -synuclein

 -synuclein mutations (PARK1/4) A53T, A30P and E46K: TOXIC GAIN OF FUNCTION None of these mutations have been found in sporadic cases of PD. These mutants are more prone to misfolding and aggregation compared to wild type  -synuclein. A53T -is the most aggressive form, in vitro it tends to shift into a  -sheet conformation more than the other mutants. -Contursi kindred. Discovered in 1996 in an Italian village. Found also in a Greek kindred. Analysis of DNA mapped a 800kb region that overlapped in all the cases, meaning that all the patients derived by a common ancestor. A30P -discovered in Lower degree of penetrance and later onset than A53T (Penetrance: a term used in genetics that describes the extent to which the properties controlled by a gene, its phenotype, will be expressed). Genomic triplication Triplication of a 2 Mb genomic region containing the  -synuclein gene (Singleton, 2003). This results in overexpression of the gene for  -synuclein, thus in higher susceptibility to PD.

 -synuclein in PD: gain of toxic function both in familial and sporadic PD -All  -synuclein inherited mutations lead to dominant forms of PD -WT  -synuclein binds to DA quinone and forms protofibrils in sporadic PD Is aggregation playing a role in  -synuclein-mediated toxicity? ?

Gain of toxic function Formation of aggregates fibrils or oligomers Loss of physiological function

O2O2 + H 2 O 2 + O 2 - Oxidation of Dopamine and subsequent interaction with  -synuclein or with Cys residues on different substrates: first step to the formation of protofibrils  -synuclein on Tyr, Met or Lys

Common Pathway Model of PD

We know that: -  -synuclein binds lipid membranes, thanks to its N-term region. -  -synuclein exists in 3 different conformations: *bound to lipid membranes *as soluble monomer *as aggregate (oligomers or amyloid fibril) -A53T mutation tends to form aggregates very quickly gain of toxic function? -A30P mutation affects the binding of  -synuclein to lipid membranes loss of physiological function?

Study of  -synuclein aggregates as causative of PD FIBRILSOLIGOMERS

Volles and Lansbury Model of  -synuclein fibrillization

Proposed mechanisms of toxicity of  -synuclein fibrils: As lipid associated structure, they can disrupt membranes integrity, both plasma membranes and intracellular membranes causing: *Disruption/damage of synaptic vesicles, altered release of neurotransmitter. *Disruption/damage of ER and Golgi. This lead to ER stress and altered trafficking of proteins in the cell. *Disruption/damage of mitochondria, causing mitochondrial stress, rupture, thus oxidation. * Disruption/damage of proteasomes. Not clear whether this occurs because of a direct toxic function of  -synuclein on the UPP, or as a consequence of mitochondrial dysfunction.

Identification of Glu residues in  -synuclein that affect fibrils formation A30A53 E35E57  -syn

 -synuclein variants between the  -strands form ring/pore-like aggregates

 -synuclein variants between the  -strands form oligomers

Overexpression of  -syn wt or variant is toxic to dopaminergic neurons of SNPC Tyrosine Hydroxylase staining

Comparison of Dopaminergic neuronal loss: inherited  -syn mutations versus  -syn variants

a-syn variants are toxic for dopaminergic neurons What happens to the neurons treated with the variants?

TH  -syn  -syn variants change the morphology of dopaminergic neurons

 -syn variants foster caspase 3 activation/apoptosis

Hypothesis: Formation of oligomers renders the variants more toxic Are oligomers accumulated in the brain of animals treated with the  -syn variants?

 -syn variants lead to formation of trimers in the insoluble fraction…

…but not in the soluble fraction

WT  -syn Tg …in vivo

Gain of toxic function?

 -syn KO does not affect mitochondrial integrity

Mechanism?

 -syn effects on mitochondrial fragmentation are independent on Drp1

Recombinant oligomeric  -syn directly acts on membranes, clustering and driving the fission of artificial membranes

 -synuclein and autophagy: a pathogenic connection in PD

 -synuclein is detected in autophagic vesicles

 -synuclein is detected in lysosomal vesicles

Mitotic cells Differentiated cells WT and mutant  -synuclein are degraded through autophagy

WT but not mutant  -synuclein is degraded through CMA autophagy

Does autophagy fail in PD? What happens when this occurs? Through what mechanism?

L-Dopa INDUCES preferentially macroautophagy vs CMA: a mechanism occurring specifically in cultured dopaminergic neurons via  -synuclein

Dopamine regulates lysosomal protein degradation modifying  -synuclein

Clusters of oligomeric  -synuclein accumulate in lysosomal fraction depending on dopamine levels

Dopamine-modified  -synuclein tends to form oligomers

DA-modified  -synuclein is detected in the form of aggregates in lysosomes

Conclusions: Dopamine-modified  -synuclein can affect autophagy, inhibiting CMA and favoring macroautophagy. As a consequence,  -synuclein aggregates accumulate in the lysosomes. This process with the time may disrupt the equilibrium that regulate macroautophagy and determine excessive accumulation of  -synuclein aggregates, leading to lysosomal failure and the formation of Lewy bodies. Probably this is a mechanism involved only in sporadic PD.