Protein Quality Control Pathways at the Crossroad of Synucleinopathies

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Protein Quality Control Pathways at the Crossroad of

Synucleinopathies

Eduardo de Mattos, Anne Wentink, Carmen Nussbaum-Krammer, Christian

Hansen, Steven Bergink, Ronald Melki, Harm Kampinga

To cite this version:

Eduardo de Mattos, Anne Wentink, Carmen Nussbaum-Krammer, Christian Hansen, Steven Bergink,

et al.. Protein Quality Control Pathways at the Crossroad of Synucleinopathies. Journal of Parkinson’s

disease, Amsterdam : b : IOS Press, In press, 14 pp. �10.3233/JPD-191790�. �cea-02458907�

Uncorrected Author Proof

IOS Press

Review

1

Protein Quality Control Pathways

at the Crossroad of Synucleinopathies

2

3

Eduardo P. De Mattos

a

_{, Anne Wentink}

b

_{, Carmen Nussbaum-Krammer}

b

_{, Christian Hansen}

c

_,

Steven Bergink

a

, Ronald Melki

d

and Harm H. Kampinga

a,∗

4 5

a

_{Department of Biomedical Sciences of Cells & Systems, University Medical Center Groningen, University of}

Groningen, Groningen, Netherlands

6 7

b

_{Center for Molecular Biology of Heidelberg University (ZMBH), and German Cancer Research Center (DKFZ),}

DKFZ-ZMBH Alliance, Heidelberg, Germany

8 9

c

_{Molecular Neurobiology, Department of Experimental Medical Science, Lund, Sweden}

10

d

_{Institute Francois Jacob (MIRCen), CEA and Laboratory of Neurodegenerative Diseases, CNRS,}

Fontenay-Aux-Roses Cedex, France

11 12

Accepted 2 January 2020

Abstract. The pathophysiology of Parkinson’s disease, dementia with Lewy bodies, multiple system atrophy, and many others

converge at alpha-synuclein (␣-Syn) aggregation. Although it is still not entirely clear what precise biophysical processes act

as triggers, cumulative evidence points towards a crucial role for protein quality control (PQC) systems in modulating

␣-Syn

aggregation and toxicity. These encompass distinct cellular strategies that tightly balance protein production, stability, and

degradation, ultimately regulating

␣-Syn levels. Here, we review the main aspects of ␣-Syn biology, focusing on the cellular

PQC components that are at the heart of recognizing and disposing toxic, aggregate-prone

␣-Syn assemblies: molecular

chaperones and the ubiquitin-proteasome system and autophagy-lysosome pathway, respectively. A deeper understanding of

these basic protein homeostasis mechanisms might contribute to the development of new therapeutic strategies envisioning

the prevention and/or enhanced degradation of

␣-Syn aggregates.

13 14 15 16 17 18 19 20 21

Keywords: Alpha-synuclein, synucleinopathies, protein homeostasis, protein aggregation, molecular chaperones,

ubiquitin-proteasome system, autophagy

22 23

INTRODUCTION

24

Alpha-synuclein (␣-Syn) was first identified in

25

human brain extracts more than 25 years ago [1, 2],

26

and since then many physiological roles have been

27

ascribed to this small protein. Although

␣-Syn has no

28

defined tridimensional structure in aqueous solution

29

[3] and is soluble under most physiological

condi-30

tions [4], it can adopt beta-strand rich conformations

31

∗_{Correspondence to: Harm H. Kampinga, Ant. Deusinglaan 1,}

Building 3215, 5th Floor, FB30, 9713 AV Groningen, Nether-lands. Tel.: +31 50 3616143; Fax: +31 50 3616111; E-mail: h.h.kampinga@umcg.nl.

favoring the formation of amyloid fibrils in several

32

neurodegenerative diseases, collectively known as

33

synucleinopathies [5–7]. For instance,

␣-Syn aggre-

34

gates are found in distinctive neuronal structures

35

known as Lewy bodies (LBs) and Lewy neurites

36

(LNs) in idiopathic and familial forms of Parkinson’s

37

disease (PD) and dementia with Lewy bodies [8],

38

and in glial cytoplasmatic inclusions in multiple sys-

39

tem atrophy [9–12]. However, instead of being able

40

to adopt only one type of structure, recent studies

41

revealed that aggregated

␣-Syn possess distinct con-

42

formations (polymorphs) with unique cytotoxicity

43

profiles [13–17]. This suggests that different synu-

44

Uncorrected Author Proof

cleinopathies arise from distinct

␣-Syn polymorphs,

45

as indeed proposed by experiments in animal models

46

[18, 19].

47

Although the initial events leading to

␣-Syn

aggre-48

gation and toxicity in vivo are still poorly understood,

49

several lines of evidence suggest that cellular

pro-50

tein quality control (PQC) pathways play a central

51

role in these processes. Among these are the

molecu-52

lar chaperones and the two main protein degradation

53

pathways, namely the ubiquitin-proteasome system

54

(UPS) and autophagy-lysosome pathway (ALP) [20].

55

Here, we review basic molecular and cellular

princi-56

ples of

␣-Syn aggregation and their connection with

57

PQC components, with a special emphasis on the

58

suppression of

␣-Syn aggregation and/or toxicity by

59

molecular chaperones.

60

ALPHA-SYNUCLEIN STRUCTURE AND

61

FUNCTION

62

The N-terminal domain of

␣-Syn contains

sev-63

eral motifs with amphipathic properties allowing

64

for interactions with membranes (binding to lipid

65

vesicles) and that can serve in protein-protein

inter-66

actions [21] (Fig. 1). The central portion (residues

67

61 to 95) contains the non-amyloid-beta

compo-68

nent of Alzheimer’s disease amyloid (NAC) motif

69

[1], which is both sufficient and required for

amy-70

loid formation [6, 22–24]. The C-terminal region

71

has an important role in shielding the NAC motif

72

from aggregation [6, 24]. Deletion of only the last

73

10 amino acids is already sufficient to accelerate

␣-74

Syn aggregation in vitro, and this effect is stronger

75

upon larger C-terminal truncations up to amino

76

acids 102–120 [24–26].

␣-Syn is subject to several

77

post-translational modifications (PTMs), including

78

N-terminal acetylation, ubiquitylation,

SUMOyla-79

tion, nitration, and phosphorylation [27–32], with

80

diverse consequences for its function and

propen-81

sity to aggregate (detailed below). Several roles

82

have been ascribed to

␣-Syn, including facilitating

83

the assembly of N-ethylmaleimide-sensitive factor

84

attachment protein receptor (SNARE)-complexes at

85

presynaptic neuron terminals that mediate release of

86

neurotransmitters [33, 34], and induction of mem-

87

brane curvatures [35], among many others [36].

88

SNCA MUTATIONS REVEAL UNIQUE

89

FEATURES OF

␣-SYN TOXICITY AND

90

AGGREGATION

91

Two types of mutations in the SNCA gene have

92

been linked to autosomal dominant forms of PD,

93

highlighting distinct mechanisms by which

␣-Syn

94

aggregation can be triggered: (i) increased gene

95

dosage and (ii) point mutations enhancing

␣-Syn

96

aggregation propensity. The latter, including A30P

97

[37], E46K [38], H50Q [39, 40], G51D [41], and

98

A53T [42], A53V [43], and A53E [44] (see Fig. 1),

99

have been discovered by genetic screens in fami-

100

lies with hereditary PD and directly influence

␣-Syn

101

aggregation to different extents and via discrete

102

pathways [45]. Mutants such as

␣-Syn

A53T

largely

103

enhance

␣-Syn aggregation into fibrils [45, 46], most

104

likely by changing the conformational landscape

105

that

␣-Syn populates towards aggregation-prone con-

106

formers, without disrupting vesicular interactions

107

[21]. In contrast, the A30P mutation does not

108

markedly modulate

␣-Syn aggregation compared

109

to overexpression of

␣-Syn

WT

in cellular models

110

[46–48]. Instead, it abolishes

␣-Syn interaction with

111

lipid vesicles both in vitro [21, 49] and in vivo [50],

112

which may lead to a buildup of cytosolic

␣-Syn lev-

113

els, and eventually contributes to

␣-Syn aggregation.

114

This implies that

␣-Syn aggregation is also extremely

115

dependent on its concentration and can even be trig-

116

gered by the wild type protein [50, 51]. In fact,

117

familial PD cases caused by duplications or triplica-

118

tions of the SNCA locus have been identified [52–56],

119

Fig. 1. Domain structure of the human alpha-synuclein (␣-Syn) protein. ␣-Syn comprises three basic domains: an N-terminal amphipathic region, a central non-␤-amyloid component (NAC) domain, and a C-terminal acidic domain. Seven membrane-interacting amino acid motifs are also present in the first half of the protein. The region preceding the NAC domain concentrates all pathogenic␣-Syn mutations identified so far. Numbers on the upper part of the structure refer to amino acid positions.

Uncorrected Author Proof

with increased gene dosage correlating with earlier

120

age at onset of disease [57].

121

MODELLING

␣-SYN AGGREGATION:

122

SEEDED VERSUS NON-SEEDED

123

CONDITIONS

124

As for any aggregation-prone protein,

␣-Syn

125

molecules adopt conformations that allow the

126

establishment of non-native interactions between

127

molecules and their coalescence into

thermody-128

namically unstable assemblies [58]. It is upon

129

conformational transition to more regular and

com-130

plementary interfaces [59] that stable seeds are

131

generated, capable of acting as conformational

tem-132

plate of the amyloid state [58]. In contrast, the highly

133

stable preformed

␣-Syn aggregates commonly used

134

in studies grow by incorporation of

␣-Syn molecules

135

to their ends, as the binding of additional molecules

136

to fibrillar ends generates an incorporation site for

137

another molecule [58]. The spontaneous aggregation

138

of

␣-Syn into amyloid fibrils thus is a multi-step

139

process during which various intermediates are

gen-140

erated that provide copious opportunities for PQC

141

interference.

142

The exogenous provision of preformed fibrils

143

(seeded aggregation) bypasses the initial requirement

144

for seed formation and allows the rapid incorporation

145

of

␣-Syn monomers to their ends [58], presenting

146

a more limited number of conformational states at

147

which PQC components can interfere. The

molecu-148

lar mechanisms of chaperone modulation of

␣-Syn

149

aggregation in spontaneous versus seeded

aggrega-150

tion are thus likely to differ significantly. Indeed,

151

some chaperones interfere with unseeded

aggrega-152

tion (e.g., DNAJB6 [60]), whilst others selectively act

153

on the elongation of preformed seeds (e.g., HSPB5)

154

[61]).

155

The distinction between unseeded and seeded

156

␣-Syn aggregation is thus extremely important to

157

our understanding of the

␣-Syn aggregation process

158

and PQC effects thereon. Cellular studies aimed at

159

investigating PQC components in

␣-Syn aggregation

160

are most often unable to clearly determine whether

161

unseeded, seeded or both processes are targeted and

162

to what extent.

163

Non-seeded

α-Syn aggregation

164

It has been surprisingly difficult to consistently

165

model spontaneous, non-seeded

␣-Syn aggregation

166

in cellular and organismal models. In fact, recent

167

nuclear magnetic resonance data showed that

␣-Syn

168

at physiological concentrations remains largely in a

169

monomeric, highly dynamic state in cells [4]. Since

170

the crowded cellular environment is expected to facil-

171

itate

␣-Syn aggregation, these data strongly suggest

172

the existence of agents (such as molecular chaperones

173

and protein degradation machineries) that efficiently

174

counteract

␣-Syn aggregation under normal circum-

175

stances.

176

To date, most studies investigating de novo,

177

non-seeded,

␣-Syn aggregation have relied on over-

178

expression of either wildt-type (WT) or mutant

179

variants of

␣-Syn. In one of the earliest models,

180

␣-Syn inclusion formation was detected in human

181

neuroglioma H4 cells and mouse primary corti-

182

cal neurons only upon overexpression of

␣-Syn

183

constructs (

␣-Syn

WT

,

␣-Syn

A30P

, or

␣-Syn

A53T

) har-

184

boring distinct C-terminal tags of variable sizes,

185

which affected proteasomal clearance [47]. Since

186

untagged

␣-Syn variants remained soluble, the tag

187

potentiated aggregation probably through the expo-

188

sure of the NAC region. Others have employed the

189

co-expression of

␣-Syn with distinct aggregation-

190

prone proteins that co-localize with

␣-Syn in LBs to

191

trigger inclusion formation, such as synphilin-1 [45,

192

62–65] and tubulin polymerization-promoting pro-

193

tein (TPPP/p25␣) [66], but it is not entirely clear

194

whether these are indeed active drivers of

␣-Syn

195

aggregation. Some studies have also used bimolecu-

196

lar fluorescence complementation assays to assess de

197

novo

␣-Syn aggregation [45, 67, 68]. In these cases,

198

fluorescence is reconstituted and detected upon co-

199

expression of two

␣-Syn constructs fused to either

200

the N-or C-terminus halves of a fluorescent protein

201

(for example, the split Venus-system). However, it

202

is still neither clear whether such assemblies are of

203

fibrillar nature, as the interaction of little as two

␣-

204

Syn molecules is already sufficient to reconstitute

205

fluorescence, nor to what extent the reconstitution

206

of the functional fluorescent protein drives assem-

207

bly. Nevertheless, some degree of

␣-Syn fibrillation

208

was detected upon overexpression of distinct split

209

Venus-␣-Syn in flies [68]. In any case, true detergent-

210

insoluble

␣-Syn aggregates are either usually not

211

observed in unseeded

␣-Syn models, or they com-

212

prise only a small fraction of the total

␣-Syn pool,

213

highlighting the urgent need for better models to doc-

214

ument

␣-Syn aggregation.

215

Seeded

α-Syn aggregation

216

Seeded aggregation experiments have been instru-

217

Uncorrected Author Proof

of

␣-Syn pathology (see for instance [26, 69–71]).

219

Indeed, most of the

␣-Syn literature relies on

exper-220

iments in which an exogenous source of

␣-Syn

221

amyloid fibrils is administered to cells or animals

222

in order to trigger aggregation of the endogenous

␣-223

Syn (i.e., the

␣-Syn pool generated by cells de novo,

224

even if it consists of an artificial transgene). In these

225

cases, exogenous

␣-Syn fibrils come from either in

226

vitro reactions using recombinant

␣-Syn [69] or from

227

fibrils isolated from animal models or human

post-228

mortem tissue [19, 26, 72]. As stated above, structural

229

differences of

␣-Syn fibrils may lead to different

230

synucleinopathies [15, 73]. However, it should be

231

noted that there is currently no evidence

demonstrat-232

ing that human pathology starts upon exposure to

233

exogenous

␣-Syn seeds [36], suggesting that factors

234

such as cellular stress may trigger the formation of

235

the first

␣-Syn seeds.

236

␣-SYN AGGREGATION AND TOXICITY

237

IN THE CONTEXT OF PQC SYSTEMS

238

Molecular chaperones

239

Suppression of

α-Syn aggregation by Hsp70

240

machines

241

Molecular chaperones are at the heart of several

242

PQC pathways and have been extensively

impli-243

cated as protective agents against protein aggregation

244

and neurodegeneration [74]. Here, we will

primar-245

ily focus on the action of Hsp70 machines on

␣-Syn

246

aggregation and toxicity. The human genome encodes

247

for multiple Hsp70 isoforms and these Hsp70s act

248

with the help of many co-factors a system that we

249

refer to as the Hsp70 machines.

250

Purified Hsp70s (e.g., HSPA1A or HSPA8) alone

251

can almost completely block

␣-Syn fibrillation at

252

substoichiometric ratios, generating small aggregates

253

composed of both proteins [25, 75–77]. Interestingly,

254

addition of recombinant Hsp70-interacting protein

255

(Hip) to reactions containing Hsp70 and monomeric

256

␣-Syn completely blocked Hsp70 co-aggregation

257

and led to sustained inhibition of

␣-Syn

aggrega-258

tion in an ATP-dependent manner [78], highlighting

259

the importance of additional co-factors for

maxi-260

mal suppression of

␣-Syn aggregation by Hsp70

261

machines (see below). Purified Hsp70s (HSPA1A

262

or HSPA8) have been shown to bind a range of

263

␣-Syn assemblies, including monomers [77],

pre-264

fibrillar [76, 78], and fibrillar species [75, 79,

265

80].

␣-Syn amino acid stretches that are bound

266

by Hsp70s span residues 10–45 and 97–102 [77].

267

Besides the suppression of

␣-Syn nucleation, Hsp70s

268

also bind to

␣-Syn seeds [75] and prevent fibril

269

elongation [79, 80]. These latter findings are con-

270

sistent with a holdase function of Hsp70s against

271

␣-Syn fibril elongation, possibly shielding fibrillar

272

ends from further incorporation of

␣-Syn molecules

273

[79, 80].

274

In cells, co-expression of Hsp70 decreased the

275

amount of high molecular weight

␣-Syn species [64,

276

65], probably by stabilization of

␣-Syn in assembly-

277

incompetent states [81]. This could account for

278

decreased cytotoxicity of

␣-Syn upon overexpression

279

of Hsp70 [65, 82]. Indeed, Hsp70 overexpression in

280

primary neurons markedly decreased the size, but not

281

the amount, of secreted

␣-Syn species [82]. Since

282

Hsp70 was also detected in the culture medium, it

283

was proposed to bind monomeric or low molecu-

284

lar weight pre-fibrillar

␣-Syn assemblies and prevent

285

further aggregation into mature fibrils [82].

286

At the organismal level, mice overexpressing both

287

␣-Syn and the rat HspA1 showed a 2-fold reduc-

288

tion in 1% Triton-X100-insoluble

␣-Syn-containing

289

aggregates, compared to animals expressing

␣-Syn

290

only [65]. In the fruit fly Drosophila melanogaster,

291

selective expression of

␣-Syn

WT

,

␣-Syn

A30P

, or

␣-

292

Syn

A53T

in dopaminergic neurons for 20 days led to

293

a 50% cell loss, but this could be fully rescued by

294

targeted co-expression of the human Hsp70 isoform

295

HSPA1L [83]. Interestingly, despite its cytoprotective

296

effects, HSPA1L did not inhibit

␣-Syn inclusion for-

297

mation, but rather co-localized with

␣-Syn in LB-like

298

structures, suggesting that Hsp70 binding reduced

299

toxic interactions of

␣-Syn with other biomolecules.

300

Such phenomenon is conserved from flies to humans,

301

with evidence for the accumulation of not only

302

Hsp70, but also its cochaperones Hsp40/DNAJs and

303

Hsp110/NEFs, into LBs and LNs from patients with

304

PD, dementia with Lewy bodies, and other synucle-

305

inopathies [63, 83]. Indeed, the titration of Hsp70s

306

out of solution by misfolded

␣-Syn has been hypoth-

307

esized to contribute to disease onset due to lowering

308

of the functional pool of Hsp70 available for protein

309

quality control pathways [78, 84].

310

In vitro, the suppression of

␣-Syn aggregation by

311

either Hsp70 (HSPA1A) or Hsc70 (HSPA8) does not

312

require ATP/ADP cycling [25, 75, 78, 80], nor co-

313

chaperones, such as DNAJB1 [78]. In fact, DNAJB1,

314

which stimulates Hsp70 cycling, even counteracts

315

such sequestering activity [76]. However, these fac-

316

tors are essential for the proper function of Hsp70

317

in quality control pathways in vivo [85]. Indeed,

318

Uncorrected Author Proof

Hsp70 co-chaperones also successfully prevents

␣-320

Syn aggregation and/or toxicity in cell and mouse

321

models of PD. Of special relevance in this context

322

is the large family of Hsp40/DNAJ proteins, which

323

are regarded as the main determinants of specificity

324

of Hsp70 machines, since different DNAJs bind to

325

distinct client proteins and deliver those to Hsp70

326

[85, 86]. Thus, DNAJs could be exploited to

maxi-327

mize the activity of Hsp70 machines towards specific

328

substrates. For example, besides inhibiting

␣-Syn

329

aggregation in vitro [76], DNAJB1 almost completely

330

abolished

␣-Syn inclusion formation in cells

overex-331

pressing

␣-Syn [63]. This has also been shown for

332

DNAJB6 and its close homologue DNAJB8 [51],

333

both of which also strongly suppress the

aggrega-334

tion of other amyloidogenic polypeptides, including

335

expanded polyglutamine-containing proteins [60, 87]

336

and the amyloid-beta protein [88]. Interestingly,

␣-337

Syn aggregation was not suppressed by a DNAJB6

338

mutant that does not interact with Hsp70 [51],

339

strengthening the notion that cooperation between

340

distinct components of Hsp70 machines is essential

341

for optimal function. Despite these examples, little is

342

still known on the contribution of different DNAJs

343

and/or NEFs to the Hsp70-dependent suppression of

344

␣-Syn aggregation in vivo. Similar to recently

devel-345

oped in vitro screens for inhibiting tau aggregation

346

[89], or enhancing

␣-Syn disaggregation (see below)

347

[90], further comparative studies using distinct

com-348

positions of Hsp70 machines are urgently required to

349

better understand and manipulate Hsp70 machines in

350

synucleinopathies.

351

Disaggregation of

α-Syn fibrils by Hsp70

352

machines

353

The diversity and complexity of Hsp70 machines

354

is also highlighted by studies investigating the

poten-355

tial of these systems to disaggregate pre-existing

356

␣-Syn amyloids. For instance, although Hsp70 alone

357

does not modify or disaggregate mature

␣-Syn fibrils

358

at relevant time-scales in vitro [75, 91], a specific

359

Hsp70/HSPA-Hsp40/DNAJ-Hsp110/NEF

combina-360

tion showed powerful, ATP-dependent disaggregase

361

activity against

␣-Syn amyloids [90]. Indeed, optimal

362

fragmentation and depolymerization of

␣-Syn fibrils

363

was detected upon combining Hsc70/HSPA8 with

364

Hdj1/DNAJB1 and the NEF Apg2/HSPA4, but not

365

upon addition of other Hsp70 machine members, such

366

as Hsp70/HSPA1A, DNAJA1, DNAJA2, or BAG1.

367

Moreover, a precise stoichiometry between these

368

components was crucial for productive

disaggrega-369

tion [90, 91], further illustrating the tight balance

370

between specificity and levels of chaperones/co-

371

chaperones for the activity of Hsp70 machines. It is

372

still not known whether Hsp70-mediated disaggrega-

373

tion of

␣-Syn also occurs in vivo, but it is tempting

374

to speculate that the breakup of fibrils into smaller,

375

more soluble assemblies facilitates their process-

376

ing by downstream PQC components, as discussed

377

below. However, it is equally possible that disaggre-

378

gation could be detrimental and facilitate

␣-Syn seed

379

propagation. Further studies are necessary to clarify

380

these issues.

381

Clearance of

α-Syn assemblies via protein

382

degradation machineries

383

The two major cellular protein degradation

384

machineries comprise the UPS and ALP, with the

385

latter encompassing both autophagosome-dependent

386

and independent pathways [92]. There is an intricate

387

and tightly regulated crosstalk between proteasomal

388

and lysosomal pathways engaged in the processing

389

of

␣-Syn, as several studies reported preferential

390

degradation of

␣-Syn via the UPS or ALP [31,

391

93–98]. Moreover,

␣-Syn (WT or distinct mutants)

392

overexpression can impair the activity of both the

393

UPS [99, 100] and distinct components of the ALP

394

[66, 101–104], which would act in a progressive

395

pathogenic feedback loop to accelerate aggregation

396

and toxicity. Whether UPS or ALP lead to the degra-

397

dation of

␣-Syn assemblies is still actively debated.

398

Recent findings suggest, however, that the UPS has

399

a more prominent role in degrading smaller

␣-Syn

400

assemblies at least when protein quality systems are

401

highly active, as is generally the case in young,

402

healthy organisms [99]. Autophagic activity seems

403

to be more required for larger

␣-Syn assemblies and

404

upon increased

␣-Syn burden, due to either muta-

405

tions that lead to

␣-Syn accumulation or decreased

406

activity of other PQC components, as observed with

407

aging [99].

␣-Syn has also been shown to be recog-

408

nized and degraded by other cellular (extracellular)

409

proteases not directly linked to the UPS and ALP

410

pathways [105, 106]. However, the extent to which

411

such enzymes are required for proper

␣-Syn turnover

412

and/or inhibition of propagation is still poorly under-

413

stood.

414

PTMs also play a role in

␣-Syn processing and

415

act as important sorting hubs to distinct protein

416

degradation machineries. For instance, the covalent

417

binding of ubiquitin to

␣-Syn, via either mono-

418

(monoUb) or polyubiquitylation (polyUb) in dis-

419

Uncorrected Author Proof

Fig. 2. Targeting and processing of alpha-synuclein (␣-Syn) by protein quality control (PQC) pathways. Left: in normal conditions, in which the cellular PQC capacity is in balance with the␣-Syn burden, soluble as wells as pre-fibrillar ␣-Syn assemblies (after disassembly) have been shown to be targeted to and degraded by several PQC components. The initial survey of␣-Syn species might be performed by molecular chaperones (1), which can facilitate the sorting of␣-Syn to distinct degradative routes, such as the ubiquitin-proteasome system (UPS; 2), a ubiquitin-independent proteasomal degradation pathway (3), chaperone-mediated autophagy (CMA; 4), macroautophagy (5), secretion via endosomes (6) [162], and proteolytic digestion by intracellular (7) or extracellular proteases. Right: in aged organisms or pathological conditions, the␣-Syn burden surpasses the cellular PQC capacity, leading to ␣-Syn accumulation and subsequent aggregation. Fibrillar␣-Syn assemblies can trap several biomolecules, including molecular chaperones (8), which contributes to chaperone depletion and decreases PQC capacity. Similarly,␣-Syn aggregation has been linked to impairment of different steps of macroautophagy (9), CMA (10), and proteasomal degradation (11). In some experimental setups, increased␣-Syn levels can also lead to increased autophagic flux and destruction of organelles, such as mitochondria (12).␣-Syn species can also be secreted to the extracellular space and taken up by neighboring cells (13), where they seed the aggregation of soluble␣-Syn species (14). ␣-Syn aggregation additionally impairs the intracellular trafficking of other proteins, such as the lysosomal enzyme glucocerebrosidase (GCase; 15). Decreased lysosomal GCase activity, due to either mislocalization of wildtype (wt) GCase or mutant GCase variants (16), leads to accumulation of GCase substrates (such as glycosylceramide; 17), which might potentiate␣-Syn aggregation. See main text for further mechanistic details and references. ER: endoplasmic reticulum; Hsc70: heat shock cognate 71 kDa protein; LAMP2a: lysosome-associated membrane protein 2 isoform a; poly-Ub: poly-ubiquitin.

fate of

␣-Syn. For instance, the co-chaperone CHIP

421

(carboxyl terminus of Hsp70-interacting protein),

422

a ubiquitous E3 Ub-ligase and crucial downstream

423

effector of Hsp70 machineries [85], was shown to

424

promote

␣-Syn degradation via both the UPS and

425

ALP [107]. Also, while monoubiquitylation by the

426

E3 ubiquitin-ligase SIAH targeted

␣-Syn to the UPS,

427

removal of the ubiquitin moiety by the deubiquitylase

428

USP9X favored

␣-Syn degradation via

macroau-429

tophagy [108]. Yet another ubiquitin-ligase (Nedd4)

430

facilitated the binding of K63-linked polyUb chains

431

to

␣-Syn and promoted its lysosomal degradation

432

via the ESCRT pathway [109]. Depending on its

433

assembly state, other PTMs such as SUMOylation,

434

phosphorylation, nitration, O-GlcNAcylation,

oxida-435

tion, and dopamine-modification can also modulate

436

␣-Syn processing via downstream degradation path-

437

ways [30, 31, 110–114]. In this context, the main

438

findings associated to the partition of

␣-Syn between

439

the UPS and ALP are discussed below and illustrated

440

in Fig. 2.

441

Ubiquitin-proteasome system

442

In mammalian cells, the central player of the UPS

443

is the 26S proteasome, a large, ATP-dependent multi-

444

protein complex devoted to the selective destruction

445

of target proteins [115]. Evidence for the degrada-

446

tion of

␣-Syn via proteasomes comes from both

447

in vitro [116] and cellular studies [117–119], with

448

not only monomeric, but maybe also pre-fibrillar

␣-

449

Syn species (after dissociation) being targeted to this

450

Uncorrected Author Proof

␣-Syn and catalyze the addition of either mono- or

452

polyUb chains with either cytoprotective or toxic

con-453

sequences depending on the specific experimental

454

setup, presumably due to differential impact on

cellu-455

lar

␣-Syn half-life [109, 117, 120, 121]. Unmodified

456

␣-Syn can also be degraded by proteasomes via an

457

Ub-independent pathway [118], particularly relevant

458

for phosphorylated

␣-Syn at serine 129 (␣-Syn

pS129

)

459

[119]. A mutant mimicking

␣-Syn

pS129

(␣-Syn

S129E

)

460

was shown to be a poor autophagic substrate [111],

461

re-emphasizing the complementary importance of the

462

different degradation pathways. Several lines of

evi-463

dence also suggest that an increased

␣-Syn burden

464

inhibits proteasomal activity, which in turn might

465

lead to a further increase in

␣-Syn levels, thus

estab-466

lishing a pathogenic feedback loop favoring

␣-Syn

467

aggregation [100, 104, 122, 123].

468

Autophagy-lysosome pathway

469

The numerous reports on

␣-Syn degradation via

470

the ALP highlight the importance of

lysosomal-471

dependent regulation of

␣-Syn levels [124]. Not

472

surprisingly, a plethora of therapeutic strategies

473

targeting the ALP have been explored to tackle

␣-474

Syn aggregation and toxicity (reviewed in [125]).

475

The ALP comprises catabolic processes that

con-476

verge at the lysosome, being usually divided

477

in three distinct types: macroautophagy,

microau-478

tophagy, and chaperone-mediated autophagy (CMA)

479

[126]. Macroautophagy relies on the engulfment

480

of substrates within autophagosomes, which are

481

double-layered membrane vesicles that sequester

482

intracellular components and target them to

lyso-483

somes for degradation [127]. Most long-lived

484

proteins, protein aggregates and even whole damaged

485

organelles are degraded via macroautophagy [92,

486

128]. The importance of macroautophagy for

nor-487

mal cellular function is exemplified by experiments

488

in which loss of macroautophagy in neurons led to

489

accumulation of ubiquitylated proteins and

inclu-490

sion bodies, and neurodegeneration [129]. Moreover,

491

mutations in different autophagy-related genes, such

492

as ATG5, lead to genetic diseases with neurologic

493

phenotypes in humans [130].

494

Data supporting a role for macroautophagy in

495

the degradation of monomeric and pre-fibrillar

␣-496

Syn assemblies come mainly from studies detecting

497

␣-Syn buildup upon exposure of cell lines

over-498

expressing either WT or mutant

␣-Syn variants

499

to the inhibitor of autophagosome formation

3-500

methyladenine [93, 95, 97, 131]. In vivo,

overexpres-501

sion of beclin-1, which is involved in autophagosome

502

formation via the phosphatidylinositol 3-phosphate

503

kinase complex, rescued neurological deficits of

␣-

504

Syn transgenic mice [131]. Yet, beclin-1 is involved

505

in other endosomal pathways, not directly linked to

506

macroautophagy [132], which may contribute to the

507

reduction of

␣-Syn levels and improved performance

508

of animals overexpressing

␣-Syn [131]. Impairment

509

of lysosomes, toward which all ALP components

510

converge, with bafilomycin A1 also resulted in

␣-Syn

511

buildup, further supporting a role for the ALP in

␣-

512

Syn degradation [96, 125, 133, 134]. Nonetheless,

513

whether macroautophagy is capable of degrading

514

aggregated, insoluble

␣-Syn assemblies, such as

515

those present in LBs, is still debated. For instance, in

516

a cell model of endogenous

␣-Syn aggregation upon

517

exposure to exogenous

␣-Syn pre-formed fibrils, ␣-

518

Syn inclusion resisted lysosomal degradation [134].

519

In addition, increasing macroautophagy flux upon

520

␣-Syn overexpression was also shown to have detri-

521

mental effects, ranging from increased degradation of

522

mitochondria (mitophagy) in both cellular [135], and

523

animal models of PD [136] to enhanced secretion of

524

␣-Syn assemblies to the extracellular space [66], that

525

may contribute to the spreading of pathogenic

␣-Syn

526

aggregates. On the other hand, Gao and colleagues

527

(2019) have recently demonstrated enhanced degra-

528

dation of internalized exogenous

␣-Syn pre-formed

529

fibrils in neuronal cell lines upon treatment with dif-

530

ferent autophagy inducers, suggesting that lysosomes

531

might be capable of clearing seeded fibrillar

␣-Syn

532

[137].

533

Different from macroautophagy, CMA encom-

534

passes the selective targeting of substrates to lyso-

535

somes via Hsc70 (HSPA8) and its co-chaperones, to

536

specifically recognize cargo proteins with a KFERQ-

537

like pentapetide motif, and lysosomal-associated

538

membrane protein 2a (LAMP2a)-mediated substrate

539

translocation across lysosomal membranes [138,

540

139]. Several lines of evidence support the involve-

541

ment of CMA in the processing of

␣-Syn [125].

542

In an in vitro lysosomal reconstitution assay,

␣-

543

Syn

WT

was selectively targeted to lysosomes by

544

LAMP2a, and mutations within a KFERQ-like motif

545

in

␣-Syn C-terminus abolished this activity [94].

546

In cultured cells overexpressing

␣-Syn, macroau-

547

tophagy inhibition led to higher

␣-Syn clearance

548

via CMA [95, 140], while

␣-Syn protein levels

549

were increased upon specific knockdown of LAMP2a

550

[141], or HSPA8 [141, 142]. Compared to healthy

551

controls, lower LAMP2a protein levels were detected

552

in brains from early-stage PD patients, accompanied

553

Uncorrected Author Proof

strates, such as myocyte-specific enhancer factor 2D

555

(MEF2D) and nuclear factor of kappa light

polypep-556

tide gene enhancer in B-cells inhibitor alpha (I

κB␣)

557

[143]. The importance of CMA in processing

␣-Syn

558

monomers and dimers, but not pre-fibrillar

assem-559

blies [111], is somewhat diminished by the finding

560

that

␣-Syn steady-state levels were unchanged in

561

Lamp2 knockout mice [144]. This however may be

562

due to developmental adaptations in other PQC

com-563

ponents, such as the UPS, as outlined above, thus

564

masking the influence of CMA. Indeed, in vivo

down-565

regulation of Lamp2a in rats resulted in accumulation

566

of ubiquitin-positive

␣-Syn inclusions in the

substan-567

tia nigra followed by loss of dopaminergic neurons

568

[145]. Additional evidence for CMA involvement

569

in

␣-Syn degradation comes from observations that

570

distinct PTMs, including oxidation, nitration, and

571

modification by oxidized dopamine, impair

␣-Syn

572

degradation via CMA, resulting in its buildup [111].

573

Importantly, similar to the rare

␣-Syn

A30P

and

␣-574

Syn

A53T

mutations [94], dopamine-modified

␣-Syn

575

(present in sporadic PD cases) also interferes with

576

the processing of other CMA substrates [111],

577

further contributing to the imbalance of protein

578

homeostasis.

579

Upon convergence of distinct ALP routes at

lyso-580

somes, soluble

␣-Syn assemblies can be degraded

581

by acidic proteases, such as cathepsin D [146–148].

582

Another lysosomal enzyme that has attracted much

583

attention in synucleinopathies is

glucocerebrosi-584

dase (GCase). While homozygous mutations in the

585

GCase-encoding gene GBA1 cause Gaucher’s disease

586

[149], heterozygous mutations are a well-established

587

risk factor for developing PD [150]. Indeed,

␣-Syn

588

buildup is observed in several models of GCase

defi-589

ciency. This occurs upon pharmacological inhibition

590

of GCase activity in cultured cells [151, 152] and

591

also in GBA1 mutant backgrounds, both in mouse

592

models overexpressing

␣-Syn [153–155] and in

593

PD patient iPS-derived dopaminergic neurons [156,

594

157].

␣-Syn buildup impairs GCase trafficking and

595

targeting to lysosomes [158, 159]. Conversely, rescue

596

of GCase activity in mice overexpressing

␣-Syn

A53T

597

reduced

␣-Syn levels and toxicity [155],

establish-598

ing a pathogenic feedback loop that promotes loss

599

of GCase function, and

␣-Syn accumulation,

aggre-600

gation and, potentially, cell-to-cell transmission of

601

␣-Syn seeds [160, 161]. Altogether, these results

602

suggest that the upregulation of autophagy without

603

a simultaneous improvement of lysosomal capacity

604

might not be a true therapeutic strategy in

synucle-605

inopathies.

CONCLUDING REMARKS

606

The topics discussed here paint a complex picture

607

of cellular strategies engaged in the tight regulation

608

of

␣-Syn protein levels, which ultimately determine

609

its aggregation propensity and associated toxicity.

610

Even though there are still some fundamental gaps

611

in our understanding of

␣-Syn biology, it has become

612

increasingly clear that the activity of dedicated PQC

613

components, such as molecular chaperones, the UPS,

614

and ALP is a crucial line of defense against

␣-

615

Syn-mediated pathology. Failure of these systems

616

(e.g., due to cellular stress, genetic predisposition,

617

or aging) will influence

␣-Syn levels and solubility,

618

eventually leading to disease. However, it is tempt-

619

ing to envision that novel therapeutic strategies to

620

prevent, slow down and/or halt progression of synu-

621

cleinopathies will emerge based on our understanding

622

of protein homeostasis in general and in particular in

623

components that prevent initiation of

␣-Syn protein

624

aggregation or help clearing them before they affect

625

neuronal health and synaptic integrity.

626

ACKNOWLEDGMENTS

627

This is an EU Joint Program – Neurodegenera-

628

tive Disease Research (JPND) project. The project

629

is supported through the following funding organi-

630

zations under the aegis of JPND – www.jpnd.eu.

631

France, National Research Agency (ANR); Ger-

632

many, Federal Ministry of Education and Research

633

(BMBF); Netherlands, Netherlands Organization for

634

Scientific Research (ZonMw); Sweden, Swedish

635

Research Council (VR). We would also like to

636

acknowledge Prof. Bernd Bukau for his support and

637

the Deutsche Forschungsgemeinschaft (SFB1036),

638

AmPro program of the Helmholtz Society and the

639

Landesstiftung Baden-W¨urttemberg.

640

CONFLICT OF INTEREST

641

The authors have no conflict of interest to report.

642

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