12. 6Dept. Medical Genetics, University of British Columbia, Vancouver, Canada. 13. 14. 15. Abstract. 16. LRRK2 mutations produce end-stage Parkinson's ...
1 2 3
Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice
4
Mattia Volta2*, Dayne A. Beccano-Kelly3*, Sarah A. Paschall4*, Stefano Cataldi4+, Sarah MacIsaac4+,
5
Naila Kuhlmann4, Chelsie A. Kadgien4, Igor Tatarnikov4, Jesse Fox4, Jaskaran Khinda4, Emma Mitchell4,
6
Sabrina Bergeron4, Heather Melrose5, Matthew J. Farrer6$ & Austen J. Milnerwood1$
7
+ $
8
1
Dept. Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Canada
9
2
Institute for Biomedicine, EURAC, Bolzano, Italy
10
3
Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, U.K.
11
4
Graduate Program in Neurosciences, University of British Columbia, Vancouver, Canada
12
5
Mayo Clinic, Jacksonville, Florida, U.S.A.
13
6
Dept. Medical Genetics, University of British Columbia, Vancouver, Canada
* equal contributions
14 15 16
Abstract
17
LRRK2 mutations produce end-stage Parkinson’s disease (PD) with reduced nigrostriatal
18
dopamine. Conversely, asymptomatic carriers have increased dopamine turnover and altered
19
brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and
20
mitochondrial abnormalities occur in aged mutant knock-in (GKI) mice. Conversely, cultured
21
GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic
22
glutamate and dopamine function across ages. Young GKI exhibit more vertical exploration,
23
elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These
24
phenomena decline with age, but are stable in littermates. In young GKI, dopamine transients are
25
slower, independent of DAT, increasing dopamine extracellular lifetime. Slowing of dopamine
26
transients is observed with age in littermates, suggesting premature ageing of dopamine synapses
27
in GKI. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes,
28
making them amenable to investigation of early pathophysiological, and later parkinsonian-like,
29
alterations. This model will prove valuable in efforts to develop neuroprotection for PD.
30 31 32
1
33
Introduction
34
Parkinson’s disease (PD) is clinically diagnosed when patients present with characteristic
35
progressive motor symptoms, although post-mortem detection of Lewy pathology and nigral cell
36
loss are currently required for confirmation. A recent study suggests nigral cell death may be as
37
low as 0-10% 1-3 years from diagnosis, whereas dopamine functional markers such as tyrosine
38
hydroxylase (TH) and dopamine transporter (DAT) are profoundly reduced at the earliest points
39
assessed 1. The rapid and near complete loss of dopamine functional markers at (or within a few
40
years of) diagnosis argues that ongoing clinical deterioration over several years is due to loss of
41
compensatory mechanisms and / or dysfunction of non-dopaminergic neurons.
42
Although motor symptoms respond well to current therapy (e.g., dopamine replacement
43
by L-DOPA or deep brain stimulation; DBS), PD is a multisystem disorder with a host of L-
44
DOPA unresponsive features. All patients suffer a range of non-motor symptoms, many of which
45
appear to precede motor onset by years or decades 2–8. Cognitive dysfunction is seen in ~40% of
46
newly diagnosed PD cases 9–11 in the form of deficits in attention, executive function, verbal
47
fluency and visuospatial processing, rather than memory per se (although memory is often also
48
impaired) 6,12. This dysexecutive / subcortical syndrome is thought to be due to impaired cortico-
49
striatal basal ganglia processing for action selection 2,13,14. There are no effective symptomatic
50
treatments for many of these non-motor issues, nor are there currently any disease-modifying
51
(neuroprotective) interventions.
52
While the etiology for most Parkinson patients remains unknown, asides age, gene
53
mutations contribute most risk 15. Pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2)
54
account for ~1% of all PD, of which LRRK2 G2019S is most frequent; identified in ~30% of
55
cases in some ethnicities 16,17. Affected LRRK2 individuals develop late-onset, L-DOPA-
56
responsive motor parkinsonism that is clinically and often pathologically indistinguishable from
57
idiopathic PD 18,19. Dopamine PET imaging of affected LRRK2 mutation carriers reveals
58
progressive neurochemical alterations similar to those of sporadic PD, namely impaired
59
presynaptic dopamine function 20,21. Further study in LRRK2 families reveals surprising
60
alterations in clinically asymptomatic, otherwise healthy, mutation carriers, including: i) early
61
increases in dopamine turnover by PET 22, ii) changes in cortical connectivity by resting state
62
MRI and neurochemical changes 20,23,24, and iii) alterations in cognitive tests of executive
63
function 25. Advances in our understanding of PD argue cell death and overt motor dysfunction
2
64
are late occurrences, preceded by dysfunction of dopaminergic and non-dopaminergic systems.
65
In this light, in model systems modelling PD etiology, the underlying pathophysiology and
66
phenotypes should also be expected to be initially subtle, progressive, and include dysfunction of
67
multiple neuronal systems, prior to cell loss.
68
We engineered the LRRK2 G2019S substitution into the endogenous mouse gene
69
(G2019S knock-in mice; GKI) which in vivo produced reductions in basal and pharmacologically
70
evoked nigrostriatal dopamine release in mice aged >12 months by microdialysis 26. This
71
Parkinson’s-like deficit was not observed at 6 months, and occurred despite a normal
72
compliment of nigral neurons and nigrostriatal dopamine markers (TH). Contrastingly, in
73
cortical neurons cultured from the same GKI mice, we observed increases in glutamatergic and
74
GABAergic synaptic transmission at 21 days in vitro 27.
75
To investigate this disparity, we probed dopamine and glutamate release, neuronal
76
morphology, synaptic proteins and behavior in young and aged GKI mice. Young animals
77
exhibit increased exploratory rearing behavior and increases in striatal glutamate and dopamine
78
transmission. As GKI mice age, they exhibit less exploratory rearing and reductions in both
79
glutamate and dopamine transmission. However, extracellular lifetime of single dopamine
80
release events is enhanced in young GKI animals, and maintained in aged animals, at which
81
point wild-type littermate values have increased to parity. Several measures demonstrate the
82
LRRK2 G2019S mutation produces alterations in young adult mice, most of which decline with
83
age, prior to ages where hypodopaminergia, mitochondrial and tau pathology are observed. We
84
provide further evidence that GKI mice provide a valuable model in which to probe early
85
pathophysiological effects of mutant LRRK2, and later classically PD-like deficits. We conclude
86
that understanding the early pathophysiological changes in etiological models may offer the best
87
hope for development of neuroprotection in PD and related diseases.
88 89
Materials & Methods
90
G2019S knock-in mice and behavioral testing
91
C57Bl/6J wild-type (WT) and Lrrk2 G2019S knock-in heterozygous (GKI) mice 26,27 were
92
maintained according to Canadian Council on Animal Care regulations. To avoid confounds of
93
oestrus cycle upon behavior and neural connectivity, only male animals were used in this work.
94
Mice undergoing surgery were weighed at age of use, and all other mice in the colony were
3
95
weighed at a single time point to produce an age vs. weight plot. Separate cohorts of adult
96
animals were tested (once only) at 1-6 and 12-18 months of age. After familiarization to handling
97
over three days with the operator, mice underwent the following behavioral paradigms and
98
videos were analyzed post-hoc using ANY-maze (Stoelting) behavioral tracking software, as
99
previously 28,29. Open field (OF) test: mice explored an arena (48cm x 48cm) for 15 min.
100
Cylinder test: mice were placed in a 1l beaker and video recorded for 5min. The number of
101
rearings and forelimb wall contacts were scored manually off-line. All testing and analysis was
102
performed experimenter blind.
103 104
Electrophysiology
105
Whole-cell patch clamp recording was conducted in striatal projection neurons (SPNs) in 300μm
106
thick coronal slices from 1-18 month-old male mice, as in 30,31. To help preserve cell viability,
107
slices from >12 month old animals were pre-incubated in recovery solution containing (in mM:
108
93 NMDG, 93 HCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 Glucose, 5 Sodium
109
Ascorbate, 2 Thiourea, 3 Sodium Pyruvate, 10 MgSO4.7H2O, 0.5 CaCl2.2H2O, pH 7.3-7.4,
110
300-310 mOsm) for 15min at 34˚C prior to transfer to a holding chamber. Slices were held and
111
perfused at RT with artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5
112
KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 10 glucose, pH 7.3–7.4, 300–310 mOsm).
113
Cells were visualized by IR-DIC on an Olympus BX51 microscope (20x + 4x magnifier) and
114
SPNs visually identified by somatic size (8–20 μm), morphology and location within the
115
dorsolateral striatum, 50–150 μm beneath the slice surface. Data were acquired by Multiclamp
116
700B amplifier digitized at 10 kHz, filtered at 2 kHz and analyzed in Clampfit10 (Molecular
117
Devices). Pipette resistance (Rp) was 5–8 MΩ when filled with (in mM): 130 Cs
118
methanesulfonate, 5 CsCl, 4 NaCl, 1 MgCl2, 5 EGTA, 10 HEPES, 5 QX-314, 0.5 GTP, 10 Na2-
119
phosphocreatine, and 5 MgATP, 0.1 spermine, pH 7.2, 290 mOsm. Tolerance for series
120
resistance (Rs) was 22 months 26).
240
Longo et al., (2014) found homozygous GKI mice exhibited hyperactivity at all ages
241
tested 35; although such a result might be impacted by lower body weight in mutants. In
242
agreement, we previously observed an increase (~10%) in open field activity in a small cohort of
243
homozygous GKI mice at 6 (but not 12) months in the founding colony 26; whereas hyperactivity
244
was not observed at any age in heterozygous mice 26. Here we tested large cohorts in the open
245
field exploration task and, in agreement with our previous report, found no significant effects
246
(Figure 1.B.i, WT n=66 & 34, GKI n=72 & 44, for 12 months, respectively; 2-way
247
ANOVA: F(1,212)=0.09, 0.002, 0.007 and p=0.8, 0.9 & 0.9 for age, genotype and interaction,
248
respectively). We similarly found no evidence for thigmotaxis at either age point, suggesting a
8
249
lack of anxiety- or anxiolytic-like phenotypes, which may have altered open field exploration
250
(center path ratios were not significantly different).
251
Contrastingly, in the cylinder exploration test conducted in a smaller environment that
252
may stimulate mice or relieve some stress of open field testing, we found significant age and
253
age-genotype interaction effects (Figure 1.B.ii, WT n=50 & 33, GKI n=59 & 49, for 12
254
months, respectively; 2-way ANOVA: F(1,187)=23.6, 7.3, and p=0.0001, 0.008 for age and
255
genotype-age interaction, respectively). Post-test analysis demonstrated a significant ~25%
256
increase in rearing events in GKI mice aged 35% increase in miniature excitatory postsynaptic current (mEPSC) frequency at 3 weeks
271
in vitro 27. To determine whether similar alterations to cortical/thalamic glutamate release occur
272
in GKI mouse brain, we conducted whole-cell patch-clamp recording of dorsolateral striatal
273
medium-sized spiny projection neurons (MSNs or SPNs; referred to herein as SPNs) in slices
274
acutely prepared from young and aged GKI and WT littermate mice (Figure 2). Intrinsic
275
membrane properties were as predicted for SPNs 30,33,38, and although there were statistically
276
significant age effects upon membrane capacitance (Cm), membrane resistance (Rm) and decay
277
time constants (Tau m), there were no genotype or genotype-age interaction effects (see Figure
278
2.Supplement 1; 2-way ANOVA values included). Thus, intrinsic membrane properties of SPNs
279
are not altered by the presence of G2019S LRRK2.
9
280
Analysis of spontaneous EPSCs (sEPSCs, Figure 2.A) revealed a significant main age
281
effect upon event amplitude (Figure 2.A.i. 2-way ANOVA; F(1,165)=3.92, p=0.049, WT n=40(17)
282
& 31(12), GKI n=53(20) & 44(15), for 1-3 and >12 months, respectively), but no genotype or
283
genotype-age interaction effects (F(1,165)=2.1, 1.8, p=0.15 & 0.18, respectively). Contrastingly,
284
analysis of sEPSC frequencies revealed significant main genotype and age effects (Figure 2.A.ii.
285
2-way ANOVA; F(1,165)=6.3, 17.3, p=0.013, 0.0001, respectively), and a significant interaction
286
between genotype and age (F(1,165)=7.8, p=0.006). Post-tests demonstrated a significant ~47%
287
increase in sEPSC frequency in GKI SPNs aged 1-3m, relative to WT littermates (p=0.0004).
288
This phenomenon was significantly reduced with age in GKI mice (p=0.0001), but not WT
289
animals (p=0.6). To more discretely interrogate the age-dependency of this large increase in
290
sEPSC event frequency, comparisons were made between GKI and WT data sets in approximate
291
1, 3, 12 and 18 month groupings (Figure 1. Supplement 2, RM-ANOVA values included); inter-
292
event interval cumulative probabilities demonstrated that frequency is higher in GKI SPNs at 1
293
and 3, but not 12 or 18 months of age.
294
A recent report by Matikainen-Ankney et al., (2016) also found a similar increase in SPN
295
event frequency in slices from similar GKI mice (aged 12 months, no such genotype interaction was
414
observed (Figure 3.B.i). The data demonstrate that early hypersensitivity of dopamine release to
415
chronic D2 activation in GKI slices is not present in slices from older animals.
416
Striatal dopamine transmission is tightly regulated by dopamine transporter (DAT)-
417
mediated reuptake; DAT levels, location and activity dictate the sphere of influence and,
418
dominating over diffusion, are the major determinant of released dopamine’s extracellular
419
lifetime in the striatum53–55. There was a significant main effect of genotype (Figure 3.B.ii. No
420
Drug; 2-way ANOVA; F(1,81)=9.73, p=0.003) upon single response decay times (Tau); decays
421
were significantly slower in slices from GKI mice aged 3m than WT controls (post-test p=0.01),
422
but similar in older slices. Slower DA transients might be expected to result from reduced DAT
423
levels or activity, but we found no genotype, age or interaction effects upon DAT (or TH)
424
staining (Figure 3. Supplement 1.A. ANOVA values included). To further probe DAT we also
425
assessed protein levels biochemically and found a significant main effect of genotype, due to
426
increased DAT protein (relative to GAPDH) in GKI brains (Figure 3. Supplement 1.B. ANOVA
427
values included).
428
Although increased DAT protein suggests slower transients are not due to a paucity of
429
DAT, it is possible that slowing of DA transients in GKI is due to a functional impairment in
430
DAT clearance after release. Therefore, in a separate set of experiments we evoked release in a
431
high concentration of the DAT blocker GBR12909 (1uM, IC50=6.63nM; Figure 3.B.ii). In the
432
absence of DAT re-uptake, as expected, WT decays were approximately twice as long (No Drug
433
442.1±38.9ms, +GBR12909 788.2±ms); however, there remained significant main genotype and
434
age effects (Figure 3.B.ii. +GBR12909; 2-way ANOVA; F(1,97)=8.55 & 15.35, p=0.004 &
14
435
0.0002, respectively). Consistent with results in the presence of DAT reuptake, when DAT was
436
blocked decays were significantly longer in slices from GKI mice than WT controls aged 3m
437
(post-test p=0.014). Decays in WT animals slowed with age (post-test p=0.002) to an extent
438
similar to both young old GKI decay times.
439
The data demonstrate augmented dopamine transmission in young GKI mice revealed by
440
repeated stimuli and extracellular lifetime of individual pulses, which is not due to a deficit in
441
DAT-dependent clearance. Moreover, an age-dependent increase in stimulated striatal dopamine
442
signalling (increased extracellular lifetime) appears to occur in GKI mice at an earlier time point.
443
As stimulated release is not impaired in older GKI slices, we conclude that the latent reduction in
444
extracellular dopamine levels observed at 12 (but not 6) months by microdialysis 26 is a result of
445
changes to the endogenous regulation of nigrostriatal dopamine release, rather than impaired
446
vesicular release per se. Interestingly, in agreement with our data here, another recent report in
447
similar knock-in mice by Longo and colleagues 56 found that DAT levels and activity are
448
increased in GKI brains from animals aged 12 (but not 3) months. In this light, synaptic DA
449
release seen here may appear similar to WT in aged GKI slices (or even reduced by
450
microdialysis 26) due to increased clearance through DAT, masking a persisting increase in DA
451
release.
452 453
Summary & Conclusions
454
Familial and idiopathic PD are now accepted as complex motor and non-motor syndromes
455
resulting from dysfunction to multiple neurotransmitter systems and cell types throughout the
456
peripheral and central nervous system 15,57. Although some treatments are effective for motor
457
dysfunction, none slow or prevent the neurodegenerative process. Many non-motor symptoms
458
antedate motor manifestations (and clinical diagnosis) by years to decades; demonstrating
459
protracted pathological processes. In this light, investigations of PD etiology in model systems,
460
especially short-lived rodents, should expect initially subtle, potentially progressive, dysfunction
461
of multiple neuronal systems, prior to (or without) overt motor dysfunction and cell loss 15,57.
462
GKI mice harbor a single point mutation, which confers the greatest genetic risk for PD
463
in humans. We find that spontaneous exploration is significantly elevated in certain tasks in
464
young mice, in broad agreement with work by other labs in similar animals 35. This exploratory
465
hyperactivity is seemingly context dependent, observed in cylinder exploration but not evident in
15
466
the open field; as such we predict this reflects changes in motivation, rather than motor function
467
per se. We expect ongoing investigations of these animals in more complex environments and
468
tasks will uncover other phenotypes. We previously observed that presynaptic glutamatergic
469
release is elevated in cortical cultures from GKI mice 27, suggesting altered synapse development
470
and / or mature function. Here we find that similar increases are observed in glutamate release
471
onto striatal neurons in brain slices from young GKI animals, without changes in synapse
472
number. This increase is not preserved as animals age, with GKI mice exhibiting a pronounced
473
down-regulation of spontaneous glutamate release. A recent independent study of similar GKI
474
animals drew similar conclusions 39, and the weight of evidence demonstrates early alterations to
475
synaptic function produced by endogenous G2019S mutations.
476
Our previous study revealed reduced extracellular dopamine levels in the striata of aged
477
(12 months), but not young (6 month) GKI mice 26. Our analyses of dopamine transmission here
478
demonstrates that reductions in vivo are unlikely to be due to impaired release or DAT reuptake,
479
and are likely a consequence of altered regulation of dopamine release and/or nigral burst firing
480
patterns in vivo. In young GKI mouse slices, dopamine release was elevated upon repeated
481
stimulation, and the extracellular lifetime of dopamine was consistently augmented. This
482
suggests that, similar to glutamate synapses, dopamine transmission is elevated in young animals
483
by the G2019S mutation, an elevation which is lost with ageing. Recent brain imaging studies
484
demonstrate that clinically manifest LRRK2 mutation carriers develop deterioration of the
485
dopamine and serotonin systems, similarly to sporadic PD patients 22,58; however, non-manifest
486
mutation carriers exhibit increased striatal dopamine turnover 22 and early increases in serotonin
487
transporter binding 58. We conclude that the GKI mouse is a valuable model in which to probe
488
the etiology and early pathophysiology of LRRK2, and potentially sporadic, parkinsonism.
489
Interventions against such pathophysiological processes in these models may provide the
490
functional neuroprotection, so desperately lacking for Parkinson’s disease and related disorders.
16
491
492 493 494 495 496 497 498 499 500 501 502 503 504 505
Figures
Figure 1. Increased exploratory behavior in young GKI mice declines with age. A) Animal body weight over time. There was no main effect of genotype upon mouse weight, but there was a significant interaction between weight and age (see text and Figure 1 supplement 1). Weights were not significantly at any age up to 500 days (i; animal n shown in ii); however, in the oldest age-group there here was a statistically significant increase in GKI mean weight (post-test *p=0.018). B) Spontaneous exploratory behavior in young and old mice. Analysis of open field exploration showed no genotype (p=0.9) or interaction (p=0.9) effects on total distance travelled (i). In the cylinder test for vertical exploration (ii) there was a significant interaction between genotype and age upon rearing events (p=0.008). At 12m old mice (ii). Upon repeated stimulation there were significant (post-test *p=0.05, **p=0.01), elevations in dopamine peak release in GKI mice, relative to WT controls at 3 months (ii). In WT and GKI slices from >12 month mice, peak dopamine release was more variable, there a was no modulation induced by repeated stimulation, as in younger slices, nor were there genotype differences. There was a significant age-dependent increase in D2 autoreceptor mediated paired-pulse depression (iii; 3m n=20(5) & 13(5), >12m n=18(6) & 30(11) for WT and GKI, respectively, post-test *** p=0.001), reflected by reduced paired-pule ratios (PPR), but there was no main effect of genotype (p=0.7) or genotype-age interaction (p=0.99). B) The D2 agonist quinpirole (10uM) equally suppressed peak evoked dopamine release in WT and GKI slices from 3m mice after a 10-minute wash-in; however, continued exposure
20
548 549 550 551 552 553 554 555 556 557 558 559 560
revealed a trend toward a main effect of genotype (p=0.07) on repeated release, and a significant genotype-time interaction (p=0.039, post-test *p=0.049 at 18min post application). This effect was not observed in >12 month slices (i). Evoked dopamine transient decay time revealed a significant main genotype effect (ii. No drug, 3m n=20(5) & 13(5), >12m n=19(6) & 33(11) for WT and GKI, respectively p=0.0025) due to significantly longer decay times (slower transients) in 3m GKI slices (post-test *p=0.027). In >12m slices, WT decay times had increased to a value similar to 3 month GKI (ii. No drug). The presence of the presynaptic dopamine transporter (DAT) blocker GBR (10uM, ii. +GBR12909) increased all transient decay times, as expected. There were significant main effects of age and genotype (3m n =17(6) & 23(8), >12m n=31(9) & 30(11), p=0.0002 & 0.0043 for WT and GKI, respectively) upon transient lifetime; in the absence of dopamine re-uptake, 3 GKI transients were still significantly slower than WT controls (post-test *p=0.014), and significant age-dependent increases in dopamine decay times were observed in WT slices (post-test *p=0.0024).
21
561 562 563 564 565 566 567 568 569
Figure 1. Supplement 1. Weights of WT, GKI and GKI homozygous animals are similar until advanced ages. Animal body weight over time. There was no main effect of genotype upon mouse weight, but there was a significant interaction between weight and age (2-way ANOVA, genotype p=0.17, interaction p=0.049). Weights were not significantly at any age up to 500 days; however, in the oldest age-group there was a statistically significant increase in GKI (Holm-Sidak post-test *p=0.018) and GKI Homo (post-test ##p=0.009) mean weight, relative to WT animals.
22
570 571 572 573 574 575 576 577
Figure 2. Supplement 1. There are no genotype-dependent alterations to SPN intrinsic membrane properties. Whole-cell recording of medium-sized spiny striatal projection neurons (SPNs) in membrane test mode and analysis of intrinsic membrane properties. While significant age-dependent changes in membrane capacitance (Cm), resistance (Rm) and decay time (Taum) were observed, there were no genotype or interaction effects. 2-way ANOVA detailed below.
23
578 579 580 581 582 583 584 585 586 587 588 589
Figure 2. Supplement 2. GKI SPN sEPSC frequency is similarly increased in 1 and 3 month slices, and similar to WT in 12 and 18 month slices. Whole-cell recording in dorsolateral striatal SPNs and analysis of sEPSCs recorded at -70mV. Higher sEPSC frequencies (as reflected by decreased inter-event intervals) underlie significant main genotype effects to similar extents in slices from 1 and 3 month animals, but there were no significant effects of genotype in 12 or 18 month slices. Details of 2-way RM-ANOVA and Holm-Sidak post-tests are shown below.
24
590 591 592 593 594 595 596 597 598 599 600 601 602
Figure 2. Supplement 3. Spontaneous EPSCs recorded in dorsolateral striatum in the coronal slice preparation are TTX insensitive. A.i) Example traces of spontaneous EPSCs (sEPSCs) in whole-cell voltage-clamp (WCVC) recordings in striatal projection neurons (SPN) of the dorsolateral striatum, prior to, and in the presence of, tetrodotoxin (TTX; 1uM) rendering any remaining responses miniature EPSCs (mEPSCs). ii) There was no significant effect of TTX upon mean sEPSC amplitude (paired t-test p=0.82) or frequency (paired t-test p=0.29) within each cell. Expressed as a percentage of the initial sEPSC mean value, neither mEPSC amplitudes nor frequencies were significantly altered by TTX application (onesample t-test against 100, p=0.7 & 0.2 respectively). The weak trend in frequency was towards increases in GKI, rather than the decreases predicted if action potential-dependent events were contributing to mean sEPSC frequency.
25
603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618
Figure 2. Supplement 4. There are no differences in the number of postsynaptic specializations, or presynaptic excitatory nerve terminal markers in the striatum of young or aged GKI mice. A) Representative bright-field images of Golgi-impregnated medium-sized spiny projection neurons (SPNs) of the dorsolateral striatum from WT and GKI mice aged 1-3m (i), with inserts showing zoom of areas marked in main image by dashed lines. There were significant increases in the density of postsynaptic protrusions as animals aged between 1-3 and >12 months, but there were no significant differences between WT and GKI dendritic protrusions at either age (WT n=57(9) & 18(3), GKI n=56(9) & 16(3), for 1-3 & >12, respectively). B) Representative confocal micrograph of VGluT1 and VGluT2 immunofluorescence staining in 2 month-old WT dorsolateral striatum, demonstrating the expected pattern of a higher density of VGluT1 than VGluT2 glutamatergic terminals and very little overlap between VGluT1 and VGluT2 (i). There were no significant effects of age or genotype upon VGluT1 or VGluT2 cluster densities (ii; WT n=8(4) & 9(4), GKI n=8(4) & 7(4), for 2m & >12m, respectively). Details of 2-way ANOVA and Holm-Sidak post-tests are shown below.
26
619 620 621 622 623 624 625 626 627 628 629
630 631 632 633 634 635 636 637
Figure 2. Supplement 5. Paired-pulse facilitation profiles in the striatum of aged GKI mice are similar & GKI mutant PPRs are insensitive to dopamine agonism and antagonism. Whole-cell patch clamp recording and eEPSC paired-pulse experiments. i) There were no significant differences in glutamatergic paired-pulse ratio at >12 months, similarly to the 1-3m age point in the absence of quinpirole (Fig.2.C). At 1m of age, when WT and GKI PPRs differ significantly in the presence of quinpirole (Fig.2.C), GKI PPRs were insensitive to both dopamine agonism (ii. 10uM quinpirole) and antagonism (iii. 10uM remoxipride). Details of 2-way ANOVA and Holm-Sidak post-tests are shown below.
Figure 2. Supplement 6. Spontaneous EPSCs recorded in GKI dorsolateral striatum SNPs are remoxipride insensitive. Spontaneous EPSCs (sEPSCs) were recorded prior to, and in the presence of, remoxipride (Remox; 10uM). A) There was no significant effect of remoxipride upon mean sEPSC amplitude (paired t-test p=0.86) or frequency (paired t-test p=0.80) within each cell. Expressed as a percentage of the initial sEPSC mean value, neither amplitude nor frequency was significantly altered by remoxipride application (one-sample t-test against 100, p=0.84 & 0.74, respectively).
27
638 639 640 641 642 643 644 645 646
Figure 2. Supplement 7. There were no differences in GKI sEPSC frequency between D1- and D2dopamine receptor expressing SPNs, and no consistent effect of quinpirole upon eEPSC peak or PPR in either cell type. Whole-cell patch clamp recording in D1 vs. D2-type SPNs in GKI slices. A) There were no differences in sEPSC mean event frequency in either cell type at young (1-3m) or old (>12m) age points. B) The D2 agonist quinpirole had no consistent modulatory effect upon D1 or D2 eEPSC peak amplitudes, or Paired-pulse ratios expressed as post-application values over pre-application (C). 2-way ANOVA and unpaired Student’s t-test values are shown.
28
647 648 649 650 651 652 653 654 655 656 657 658 659
Figure 3. Supplement 1. Early alterations in GKI dopamine release are not associated with reductions in nigrostriatal dopamine markers and persist despite genotype-dependent increases in DAT protein levels by western blot. A) Staining for presynaptic dopamine markers DAT and tyrosine hydroxylase (TH; i) showed highly specific enrichment in the striatum; however, there were no age or genotype differences (ii). Although variable, the data suggest there is no loss of either terminal marker in GKI striatum. B) Striatal DAT levels were also assessed by western blot (i); although variable results in young GKI suggest no loss of DAT. ii) A significant main genotype effect (due to increased DAT levels in aged GKI) revealed increases in striatal DAT levels in the presence of endogenous G2019S. 2-way values are shown.
29
660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Kordower, J. H. et al. Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease. Brain 136, 2419–2431 (2013). Goldman, J. G. & Postuma, R. Premotor and nonmotor features of Parkinson’s disease. Curr Opin Neurol 27, 434–441 (2014). Chaudhuri, K. R. & Schapira, A. H. Non-motor symptoms of Parkinson’s disease: dopaminergic pathophysiology and treatment. Lancet Neurol. 8, 464–474 (2009). Jenner, P. et al. Parkinson’s disease--the debate on the clinical phenomenology, aetiology, pathology and pathogenesis. J Park. Dis 3, 1–11 (2013). Weintraub, D., Comella, C. L. & Horn, S. Parkinson’s disease--Part 2: Treatment of motor symptoms. Am. J. Manag. Care 14, S49-58 (2008). Weintraub, D., Comella, C. L. & Horn, S. Parkinson’s disease--Part 3: Neuropsychiatric symptoms. Am. J. Manag. Care 14, S59-69 (2008). Weintraub, D., Comella, C. L. & Horn, S. Parkinson’s disease--Part 1: Pathophysiology, symptoms, burden, diagnosis, and assessment. Am. J. Manag. Care 14, S40-8 (2008). Tolosa, E., Compta, Y. & Gaig, C. The premotor phase of Parkinson’s disease. Park. Relat Disord 13 Suppl, S2-7 (2007). Broeders, M. et al. Cognitive change in newly-diagnosed patients with Parkinson’s disease: a 5year follow-up study. J Int Neuropsychol Soc 19, 695–708 (2013). Pedersen, K. F., Larsen, J. P., Tysnes, O. B. & Alves, G. Prognosis of mild cognitive impairment in early Parkinson disease: the Norwegian ParkWest study. JAMA Neurol 70, 580–586 (2013). Litvan, I. et al. MDS Task Force on mild cognitive impairment in Parkinson’s disease: critical review of PD-MCI. Mov Disord 26, 1814–1824 (2011). Goldman, J. G., Aggarwal, N. T. & Schroeder, C. D. Mild cognitive impairment: an update in Parkinson’s disease and lessons learned from Alzheimer’s disease. Neurodegener Dis Manag 5, 425–443 (2015). Calabresi, P., Picconi, B., Tozzi, A., Ghiglieri, V. & Di Filippo, M. Direct and indirect pathways of basal ganglia: a critical reappraisal. Nat Neurosci 17, 1022–1030 (2014). Gerfen, C. R. & Surmeier, D. J. Modulation of striatal projection systems by dopamine. Annu Rev Neurosci 34, 441–466 (2011). Volta, M., Milnerwood, A. J. & Farrer, M. J. Insights from late-onset familial parkinsonism on the pathogenesis of idiopathic Parkinson’s disease. Lancet Neurol. 14, (2015). Ozelius, L. J. et al. LRRK2 G2019S as a cause of Parkinson’s disease in Ashkenazi Jews. N. Engl. J. Med. 354, 424–425 (2006). Hulihan, M. M. et al. LRRK2 Gly2019Ser penetrance in Arab-Berber patients from Tunisia: a case-control genetic study. Lancet Neurol 7, 591–594 (2008). Haugland, R. P. in Handbook of fluorescent probes and research products (ed. Gregory, J.) 554– 572 (Molecular probes Inc., 2002). Gaig, C. et al. Nonmotor symptoms in LRRK2 G2019S associated Parkinson’s disease. PLoS One 9, e108982 (2014). Adams, J. R. et al. PET in LRRK2 mutations: comparison to sporadic Parkinson’s disease and evidence for presymptomatic compensation. Brain 128, 2777–2785 (2005). Nandhagopal, R. et al. Progression of dopaminergic dysfunction in a LRRK2 kindred: a multitracer PET study. Neurology 71, 1790–1795 (2008). Sossi, V. et al. Dopamine turnover increases in asymptomatic LRRK2 mutations carriers. Mov Disord 25, 2717–2723 (2010). Helmich, R. C. et al. Reorganization of corticostriatal circuits in healthy G2019S LRRK2 carriers. Neurology 84, 399–406 (2015). Vilas, D. et al. Clinical and imaging markers in premotor LRRK2 G2019S mutation carriers. Park. Relat Disord 21, 1170–1176 (2015).
30
711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761
25. 26. 27. 28.
29.
30.
31.
32.
33. 34. 35.
36. 37.
38.
39.
40. 41. 42.
43. 44.
Thaler, A. et al. Neural correlates of executive functions in healthy G2019S LRRK2 mutation carriers. Cortex 49, 2501–2511 (2013). Yue, M. et al. Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice. Neurobiol. Dis. 78, (2015). Beccano-Kelly, D. A. et al. Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice. Front. Cell. Neurosci. 8, (2014). Beccano-Kelly, D. A. et al. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory. Hum Mol Genet (2014). doi:10.1093/hmg/ddu543 Volta, M. et al. Chronic and acute LRRK2 silencing has no long-term behavioral effects, whereas wild-type and mutant LRRK2 overexpression induce motor and cognitive deficits and altered regulation of dopamine release. Park. Relat. Disord. 21, (2015). Beccano-Kelly, D. A. et al. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory. Hum. Mol. Genet. 24, (2015). Milnerwood, A. J. et al. Early Increase in Extrasynaptic NMDA Receptor Signaling and Expression Contributes to Phenotype Onset in Huntington’s Disease Mice (DOI:10.1016/j.neuron.2010.01.008). Neuron 65, (2010). Ade, K. K., Wan, Y., Chen, M., Gloss, B. & Calakos, N. An Improved BAC Transgenic Fluorescent Reporter Line for Sensitive and Specific Identification of Striatonigral Medium Spiny Neurons. Front. Syst. Neurosci. 5, 32 (2011). Milnerwood, A. J. et al. Memory and synaptic deficits in Hip14/DHHC17 knockout mice. Proc. Natl. Acad. Sci. U. S. A. 110, (2013). Petkau, T. L. et al. Synaptic dysfunction in progranulin-deficient mice. Neurobiol. Dis. 45, 711– 722 (2012). Longo, F., Russo, I., Shimshek, D. R., Greggio, E. & Morari, M. Genetic and pharmacological evidence that G2019S LRRK2 confers a hyperkinetic phenotype, resistant to motor decline associated with aging. Neurobiol Dis 71, 62–73 (2014). Trinh, J. et al. A comparative study of Parkinson’s disease and leucine-rich repeat kinase 2 p.G2019S parkinsonism. Neurobiol. Aging 35, 1125–1131 (2014). Kachergus, J. et al. Identification of a novel LRRK2 mutation linked to autosomal dominant parkinsonism: evidence of a common founder across European populations. Am J Hum Genet 76, 672–680 (2005). Milnerwood, A. J. et al. Early Increase in Extrasynaptic NMDA Receptor Signaling and Expression Contributes to Phenotype Onset in Huntington’s Disease Mice. Neuron 65, 178–190 (2010). Matikainen-Ankney, B. A. et al. Altered Development of Synapse Structure and Function in Striatum Caused by Parkinson’s Disease-Linked LRRK2-G2019S Mutation. J Neurosci 36, 7128– 7141 (2016). Cepeda, C. et al. Differential electrophysiological properties of dopamine D1 and D2 receptorcontaining striatal medium-sized spiny neurons. Eur J Neurosci 27, 671–682 (2008). Sara, Y., Virmani, T., Deak, F., Liu, X. & Kavalali, E. T. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45, 563–573 (2005). Ramirez, D. M. O., Khvotchev, M., Trauterman, B. & Kavalali, E. T. Vti1a Identifies a Vesicle Pool that Preferentially Recycles at Rest and Maintains Spontaneous Neurotransmission. Neuron 73, 121–134 (2012). Crawford, D. C. & Kavalali, E. T. Molecular underpinnings of synaptic vesicle pool heterogeneity. Traffic 16, 338–364 (2015). Milnerwood, A. J. & Raymond, L. A. Corticostriatal synaptic function in mouse models of Huntington’s disease: early effects of huntingtin repeat length and protein load. J Physiol 585, 817–831 (2007).
31
762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792
45. 46. 47.
48. 49.
50. 51. 52. 53. 54. 55. 56. 57. 58.
Parisiadou, L. et al. LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity. Nat Neurosci 17, 367–376 (2014). Mei, Y. et al. Adult restoration of Shank3 expression rescues selective autistic-like phenotypes. Nature (2016). doi:10.1038/nature16971 Akopian, G. & Walsh, J. P. Reliable long-lasting depression interacts with variable short-term facilitation to determine corticostriatal paired-pulse plasticity in young rats. J Physiol 580, 225– 240 (2007). Ding, J., Peterson, J. D. & Surmeier, D. J. Corticostriatal and thalamostriatal synapses have distinctive properties. J Neurosci 28, 6483–6492 (2008). Sciamanna, G., Ponterio, G., Mandolesi, G., Bonsi, P. & Pisani, A. Optogenetic stimulation reveals distinct modulatory properties of thalamostriatal vs corticostriatal glutamatergic inputs to fast-spiking interneurons. Sci. Rep. (2015). doi:10.1038/srep16742 Bamford, N. S. et al. Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals. Neuron 42, 653–663 (2004). Kreitzer, A. C. & Malenka, R. C. Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson’s disease models. Nature 445, 643–647 (2007). Phillips, P. E., Hancock, P. J. & Stamford, J. A. Time window of autoreceptor-mediated inhibition of limbic and striatal dopamine release. Synapse 44, 15–22 (2002). Rice, M. E., Patel, J. C. & Cragg, S. J. Dopamine release in the basal ganglia. Neuroscience 198, 112–137 (2011). Jones, S. R. et al. Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc Natl Acad Sci U S A 95, 4029–4034 (1998). Gainetdinov, R. R. Dopamine transporter mutant mice in experimental neuropharmacology. Naunyn Schmiedebergs Arch Pharmacol 377, 301–313 (2008). Longo, F. et al. Age-dependent dopamine transporter dysfunction and Serine129 phospho-αsynuclein overload in G2019S LRRK2 mice. Acta Neuropathol. Commun. 5, 22 (2017). Kalia, L. V. & Lang, A. E. Parkinson’s disease. Lancet 386, 896–912 (2015). Wile, D. J. et al. Serotonin and dopamine transporter PET changes in the premotor phase of LRRK2 parkinsonism: cross-sectional studies. Lancet Neurol. 16, 351–359 (2017).
32
