α - Synuclein in Lewy bodies and Lewy neurites : a histopathological signature of PD

1. Alpha-synuclein: Pathology, mitochondrial dysfunction and neuroinflammation in Parkinson’s disease

2. Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantianigra pars compacta (SNc) and accumulation of insoluble cytoplasmic protein inclusions referred to as Lewy bodies and Lewyneurites. The precise mechanism underlying the pathogenesis of PD is not yet understood. Accumulating evidence suggests that soluble α-synucleinaggregates, known as oligomers, play a significant role in the PD neurodegenerative process by impairing many subcellular functions

4. In this lecture, we summarize the current data describing what is known about α-synuclein physiology and how changes in α-synuclein biology can contribute to PD. We further discuss a role for mitochondrial dysfunction and neuroinflammation in PD and how α-synuclein is involved in many aspects of neuronal function including mitochondrial homeostasis. Understanding how the different structural forms of α-synuclein influence mitochondrial function (and vice versa), may ultimately help us better design PD therapeutics.

5. α-Synuclein in Lewy bodies and Lewyneurites: a histopathologicalsignature of PD Lewy bodies and Lewyneurites are abnormal inclusions that accu- mulate within neurons in PD. Lewy body and Lewyneurite pathology have been described in several other neurodegenerative diseases including Lewy body dementia (LBD) and multiple system atrophy (MSA). The major component of the Lewy bodies and Lewyneurites is insoluble α-synuclein fibrils

6. SNCA mutations in PD The vast majority of PD cases are sporadic with an average age at onset of 60 years of age. Approximately 5–10% of patients are diagnosed with a monogenic form of the disease, which typically has an earlier age at onset and is accompanied by more extensive neuropathology. Current advancements in understanding sporadic PD have been mainly driven by studies focused on elucidating the functional roles of genes identified as monogenic forms of PD. Mutations in SNCA, LRRK2, and VPS35 genes are highly penetrant and cause autosomal dominant forms of PD. The SNCA gene encodes the human α-synuclein protein. The presence of either a point mutation (e.g. A53T; A30P and E46K) in the SNCA gene or a whole locus multiplication will result in an autosomal dominant form of PD.

7. α-Synuclein is an abundant 140-residue neuronal protein, that, under physiological conditions, is found mainly in neuronal presynaptic terminals, close to synaptic vesicles. It is a member of a conserved family of proteins that also includes β-synuclein and γ-synuclein, and was originally described as the precursor protein for the non-amyloid component of Alzheimer's disease senile plaques. The protein is intrinsically unfolded, which means that in the purified form at neutral pH it lacks an ordered secondary or tertiary structure. Upon binding to membranes or synthetic vesicles containing acidic phospholipids, however, it assumes an α-helical structure WHAT IS THE PHYSIOLOGICAL ROLE OF α-Synuclein?

8. Although its exact functions are unknown, α-synuclein is assumed to help in the regulation of synaptic-vesicle release and to provide a stabilising effect on complexes of SNARE family proteins

9. Proposed model of native α-Syn function. Native α-Syn binds at the same time to the synaptic vesicle membrane and synaptobrevin-2. Synaptic vesicle fusion with the presynaptic plasma membrane is mediated by the three neuronal SNARE proteins synaptobrevin-2 (on the synaptic vesicle membrane), SNAP-25 and syntaxin-1 (on the presynaptic plasma membrane), which form the synaptic SNARE-complex. Native α-Syn clusters synaptic vesicles depending on binding to synaptobrevin-2 and the vesicles themselves, which may result in a local increase of synaptic vesicles at the presynaptic plasma membrane, and a subsequent increase in SNARE-complex formation.

10. Structure of α-synuclein There are conflicting reports on the native state of α-synuclein α- Synuclein is a small 140 amino acid protein and is divided into three distinct regions: a positively charged N-terminal region, a central hydro- phobic region that has a high propensity to aggregate , and a highly acidic C-terminal domain. One hypothesis suggests α-synuclein exists as an intrinsically disordered protein or unstructured monomer.

11. Local structural properties of monomeric α-Syn Under normal physiological conditions, α-Syn is present in monomeric unstructured form as well as presynaptic vesicle-bound form. The initial attempts for studying the structure of α-Syn were made using traditional biophysical and biochemical methods. These included circular dichroism(CD) and Fourier transform infrared (FTIR) spectroscopy for determining the secondary structure; size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS) for determining the overall conformation and compaction of the protein. All together these studies showed that suggesting that inside cells α-Syn may have an unstructured yet with compact structure

12. Alternatively, α-synuclein may exist as a tetramer and the use of denaturing detergents and cell lysis approaches may destabilize the tetramer resulting in a monomer. Missense mutations in α-synucleingene can decrease the tetramer:monomer ratio causing a shift favoring a pathogenic state. High levels of the soluble oligomeric form, rather than the insoluble fibril form of α-synucleinis now proposed to be pathogenic species in PD

13. Concentration-dependent α-synucleinoligomerisation and aggregation Recombinant α-synuclein incubated under certain conditions in vitro assumes an oligomeric conformation and is gradually converted to β-sheet-rich, fibrillar structures that resemble the Lewy bodies and neurites found in human neuropathological samples. This process is termed aggregation and is thought to underlie the toxic potential of α-synuclein Which particular species of α-synuclein are toxic has been debated. Some evidence favoursfully fibrillaror the intermediate soluble oligomeric species.

14. A major factor that could drive the aggregation and neurotoxic effects of α-synuclein is the total concentration of the protein. Whether total α-synuclein concentrations are increased in the brains of patients with PD is unclear. Levels of α-synuclein are regulated by a balance of synthesis, degradation, and secretion. The ubiquitin proteasome system and the autophagy-lysosome pathway, (which involves microautophagy, macroautophagy, and chaperone-mediated autophagy) are the two major quality-control systems postmitotic neurons use to maintain intracellular proteostasis. The mechanism of α-synuclein degradation remains unclear. Some reports suggest that monomeric α-synuclein can be degraded by the proteasome.

15. Post-translational modifications and α-synucleinaggregation The α-synuclein protein undergoes extensive post-translational modifications, including phosphorylation, nitration, and DA(Dopamine) modification, that all seem to favor oligomerization. Interestingly, both nitrated and phosphorylated forms of α-synuclein are present in the brains of PD patients, suggesting that these modifications may contribute to PD pathology. In the brains of healthy individuals only a small fraction (~4%) of total α-synuclein is phosphorylated at residue Serine-129 (Ser-129). In contrast, phos- phorylation at Ser-129 is the most prevalent (~90%) PTM form of α-sy- nuclein detected in PD brains containing Lewy bodies

17. Kinases phosphorylating α-synuclein. The domain structure of α-synuclein showing phosphorylation at serine 129 by members of the polo-like kinase (PLK), casein kinase (CK), and G protein coupled receptor kinase (GRK) families. Pathogenic α-synuclein missense mutations are indicated with arrows.

18. α-Syn and Parkinson’s disease Parkinson’s disease (PD) is one of the most common neurological disorders affecting over ten million people globally. The incidence of PD increases steadily with age; the highest prevalence being in the population older than 80 years of age. PD is an age-associated degenerative disease affecting central as well as peripheral nervous systems of the individual. Pathologically, PD is characterized by progressive neurodegeneration in specific regions of the brain, i.e. the dopaminergic neurons in the substantianigra pars compacta (SNc) (the major center in the brain for coordination of body movements)

19. The loss of these neurons results in a gradual decline of the sufferer’s motor skills, cognitive functions, and other processes. The clinical signs and symptoms of PD include tremor, postural instability, slowness of movement, rigidity, behavioral problems, anxiety hallucinations, and sometimes dementia. Degeneration of dopaminergic neurons in the SNcregion and a reduction in the levels of the neurotransmitter dopamine causes most of the clinical symptoms of PD The dopamine replacement therapy via oral administration of levodopa (L-dopa) is the most effective and currently available treatment for the disease symptoms. only symptomatic relief and does not halt or slow down the progression of the disease.

20. Besides the degeneration of dopaminergic neurons, another important pathological feature of PD includes the presence of widespread proteinaceouscytoplasmic inclusions, i.e., Lewy bodies (LBs) and Lewyneurites (LNs) in the neurons surviving in SNcregion. These inclusion bodies mostly contain the filamentous aggregates of phosphorylated and ubiquitinated α-Syn protein. Many studies demonstrate the link between α-Syn aggregation and PD phenotype . However, the actual factors that lead to the formation of these abnormal proteinaceousinclusions and how these are associated with the neurotoxicity occurring in PD are still unknown. Despite several studies done so far, the pathogenic mechanisms underlying PD are still obscure, and hence, the disease is incurable till date.

21. α-Syn mediated pathogenesis in PD To understand the role of α-Syn in mediating PD pathogenesis, studies were performed with α-Synknock out mice. No difference was observed between the architecture of neurons and the synaptic terminals of knockout mice and that of normal mice. Although they displayed certain deficits in the nigrostriatal dopamine pathway, they did not develop any abnormalities or PD characteristics. This study suggested that PD is due to a gain of toxic function by α-Syn rather than due to loss of its normal function.

22. The gain of toxic function by α-Syn is associated with the accumulation of α-Syn, which results in its aggregation The rate of α-Syn synthesis and clearance maintains the level of protein in CNS. α-Syn clearance and degradation take place by direct proteolysis, with the aid of chaperones, autophagy and proteasome-mediated degradation Failure of any of these pathways results in the accumulation of α-Syn in cells. For instance, the collapse of the lysosomal degradation pathway (to remove abnormal forms of α-Syn) promoted the inclusion formation in cells

23. α-Syn aggregation and effect of disease-associated familial mutations Earlier, PD was considered to be an aging-related disorder with anunknownspecific cause or genetic component. However, this notion changed in the late 1990s with the identification of mutations in the SNCA gene associated with early-onset, familial forms of PD. Since then, intensive genetic analysis studies have identified at least 22 different genes containing causal mutations, which are either associated with familial PD or are risk factors for PD. these mutations account only for a < 10 % of all the PD cases; the rest of the cases being sporadic

24. Mutations in the SNCA gene, which encodes for α-Syn is known to be directly associated with the disease pathogenesis. Mutations in SNCA and gene duplication or triplication cause a familial and sporadic form of the disease. Six dominant mutations (A30P, E46K, A53T, A53E, H50Q, G51D) and one recently reported A53V have been discovered till date which are genetically linked to a familial form of PD

25. The aggregation of α- Synfollows a nucleation-dependent polymerization pathway, which is monitored by an amyloid-specific Thioflavin T dye. The aggregation pathway consists of three phases: (I) lag phase,whichis a rate-limiting step and proceeds with the formation of the aggregation-competent nucleus; (II) elongation phase, where the nucleus converts into protofibrils and higher order aggregating species and (III) stationary phase, where majority of the soluble protein is converted into amyloid fibrils, and dynamic equilibrium between fibrils and monomers is reached

26. Effect of familial PD mutations on disease pathogenesis. (A) The correlation scheme between lag time of aggregation and PD onset. x-axis represents the lag time during the aggregation of α-Syn and its mutants based on the in vitro studies. The Y-axis represents the age of the onset of the disease in patients harboring these mutations. (B) The scheme showing primary and secondary nucleation processes of α-Syn during its aggregation. Familial PD mutations can alter these processes in a way that they form different type and amount of oligomers during aggregation pathway, which can dictate the pathophysiology of the disease.

27. Lysosomal effects of α-synuclein Wild-type α-synuclein is degraded by CMA upon binding to the CMA-specific receptor LAMP-2A. CMA=chaperone-mediated autophagy. ER=endoplasmic reticulum. GCase=glucocerebrosidase.

28. In patients carrying the Ala30Pro and Ala53Thr mutations of SNCA, which are linked to Parkinson's disease, or where α-synuclein is modified by dopamine, binding is stronger and α-synuclein is not internalised, which leads to inhibition of degradation of α-synuclein and other CMA substrates. This dysfunction can lead to macroautophagy alterations and accumulation of autophagic vacuoles, which might result in neuronal death.

29. Some studies have shown that increased concentrations of α-synuclein are associated with impaired formation of autophagic vacuoles through interaction with Rab1a, which leads to suppression of macroautophagy.

30. Inhibition of chaperone-mediated autophagy leads to increased aggregation of high-molecular-weight and detergent-insoluble α-synuclein species in neuronal cells, which suggests that this process has a crucial role in the prevention of oligomerisation or aggregation of α-synuclein. In-vivo support for this idea is provided by a study that showed enhanced chaperone-mediated, autophagy-dependent degradation of α-synuclein in mouse substantianigra under conditions of stress, such as that induced by an excess of α-synuclein. Furthermore, expression of HSC70 and LAMP-2A, which have regulatory roles in chaperone-mediated authophagy, might be reduced in the brains of patients with PD compared with that in age-matched control brains. This difference would further support the theory that dysfunctional chaperone-mediated autophagy is implicated in PD pathogenesis.

31. In particular, chaperone mediated autophagy (CMA) has been proposed to affect α-synuclein turnover and metabolism.

32. Substrates with a KFERQ motif are recognized by a complex of chaperones, which then bind to the lysosome-associated membrane protein type 2A (LAMP-2A), acting as the receptor for this pathway CMA activity is directly correlated with levels of LAMP-2A, and importantly LAMP-2A expression is decreased in the SN of PD brains . Reductionin LAMP-2A levels has been reported to result in the accumulation of α-synuclein and nigral cell death

33. Steps and physiological functions of CMA. (A) Proteins degraded by CMA are identified in the cytosol by a chaperone complex that, upon binding to the targeting motif in the substrate protein (1), brings it to the surface of lysosomes (2).

34. Binding of the substrate to the cytosolic tail of the receptor protein LAMP-2A promotes LAMP-2A multimerization to form a translocation complex (3). Upon unfolding, substrate proteins cross the lysosomal membrane (4) ….

35. ….assisted by a luminal chaperone and reach the lysosomal matrix where they undergo complete degradation (5). (B) General and cell-type specific functions of CMA and consequences of CMA failure in different organs and systems.

36. Impairment of CMA by pathogenic proteins contributes to neurodegeneration

37. (A) Mechanisms of CMA failure in Parkinson's disease. Many PD-related proteins bear CMA-targeting motifs (α-synuclein, UCH-LI and LRRK2 shown here) (top). LRRK2 has eight CMA-targeting motifs but only the sequence of the most commonly used is shown. Both wild-type α-synuclein and LRRK2 are degraded by CMA.

38. Mutant forms of these proteins and of UCH-L1 bind abnormally to LAMP-2A, albeit via different mechanisms, leading to blockage of their own degradation as well as degradation of other CMA substrates. Dopamine-modified α-synuclein and abnormally high levels of wild-type LRRK2 also impair CMA. Failure of CMA causes accumulation and aggregation of these toxic proteins that could contribute to Lewy body formation in PD. Alterations of CMA by mutant LRRK2 and UCH-L1 show converging toxic effects on α-synuclein aggregation.

39. (B) Perturbation of CMA by mutant tau in tauopathies. wild-type tau protein is a bona fide CMA substrate carrying two CMA-targeting motifs (top). Pathogenic variants of tau fail to translocate fully into the lysosomal lumen. Such inefficient translocation promotes partial cleavage of tau and formation of tau oligomers at the lysosomal membrane resulting in destabilization of lysosomal membrane and lysosomal leakage. Release of lysosomal tau oligomers into the cytosol may act as a precursor for further tau aggregation.

40. Dysfunctional degradation pathways and α-synuclein aggregation Several proteolytic systems, including the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) participate in the degradation of α-synuclein. The UPS is mainly responsible for the degradation of short-lived soluble proteins, while the autophagy-lysosome pathway degrades long-lived macromolecules, cytosolic components and dysfunctional organelles. Failure of these functionally inter-connected proteolytic systems can be accompanied by an accumulation of aggregated α-synuclein, that ultimately interferes with proper cellular function and contributes to PD pathogenesis

41. Mitochondrial dysfunction in Parkinson’s disease Mitochondrial dysfunction is proposed to be central to the pathogenesis of both sporadic and familial PD. New data indicates that α-synucleincan Interact with mitochondria by binding to the outer mitochondrial membrane and can be imported under certain conditions, as well as interact with the F-type ATPase

42. α –synuclein in mitochondrial pathology α -synuclein is primarily a cytoplasmic protein, but a fraction of it is imported into mitochondria, where α -synuclein associates with complex I in the inner mitochondrial membrane. Studies with knockout mice suggest that α -synuclein controls synaptic vesicle dynamics and may regulate mitochondrial membrane lipid composition and complex I activity. Familial PD mutations, oxidative stress (nitration) and dopamine adducts accelerate the formation of toxic α synucleinproto-fibrils, which may permeabilize the outer mitochondrial membrane, resulting in the loss of the mitochondrial membrane potential ( △ Ψ m), cytochrome c release and apoptosis. Increased mitochondrial levels of α -synuclein in PD have been associated with reduced complex I activity and oxidative stress.

44. Complex I inhibition by α-synuclein Numerous studies have shown that overexpression of α-synuclein impairs mitochondrial complex or complex IV activity.

45. Effects of α-synuclein on mitochondria Accumulation of monomeric or oligomeric α-synuclein occurs in the outer mitochondrial membrane, where it interacts strongly with cardiolipin. Overexpression of α-synuclein induces mitochondrial fragmentation, which leads to increased removal of mitochondria through mitophagy. α-Synuclein can also accumulate within the inner mitochondrial membrane and inhibit the activity of complex I of the respiratory chain. Wild-type forms of Parkinand PINK1 can inhibit α-synuclein-induced mitochondrial fragmentation, but stimulate mitophagy. Mitochondrial fragmentation and inhibition of complex I activity can lower mitochondrial membrane potential, which leads to increased production of ROS and neuronal death. Δψm=membrane potential. ROS=reactive oxygen species.

46. α-Synuclein, LRRK2, tau, and cytoskeletal effects Genome-wide association studies have identified strong associations with PD for SNCA, MAPT (which encodes microtubule-associated protein tau), and LRRK2. The identification of these genes in such analyses does not necessarily mean that their protein products interact synergistically or otherwise to facilitate the pathogenesis of PD. Functional links might, however, exist between the proteins that affect the cytoskeleton.

47. Oligomerisation of α-synuclein could destabilise cytoskeletal units, which in turn might accelerate the formation of α-synuclein oligomers and further cytoskeletal disruption and result in the neuritic degeneration observed in synucleinopathies

48. Effects of α-synuclein on the cytoskeleton α-Synucleinenhances the phosphorylation of the microtubule-stabilising protein, tau, possibly via GSK3β or other kinases. Reduced interaction of tau with the microtubule network alters its stability. Similarly, α-synuclein can directly affect the microtubule network by binding with fully polymerised microtubules or the component monomers, α-tubulin and β-tubulin.

49. Some studies show that accumulation of α-synuclein inhibits microtubule polymerisation, but others show stimulation of polymerisation. Reciprocal interaction between depolymerised microtubules and α-synuclein that triggers oligomerisation of α-synuclein and leads to a pathological state has been proposed. Similarly to α-synuclein, LRRK2 can affect microtubule stability through increased phosphorylation of tau, through GSK3β or Ste20 kinases, or directly by increased phosphorylation of β-tubulin.