Emergent glutamate & dopamine dysfunction in VPS35(D620N) knock-in mice and rapid reversal by LRRK2 inhibition

Emergent glutamate & dopamine dysfunction in VPS35(D620N) knock-in mice and rapid reversal by LRRK2 inhibition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Emergent glutamate & dopamine dysfunction in VPS35(D620N) knock-in mice and rapid reversal by LRRK2 inhibition Anusha Kamesh, Chelsie Kadgien, Naila Kuhlmann, Sean Coady, Emily Hurley, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5167163/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 May, 2025 Read the published version in npj Parkinson's Disease → Version 1 posted 10 You are reading this latest preprint version Abstract The D620N variant in Vacuolar Protein Sorting 35 (VPS35) causes autosomal-dominant, late-onset Parkinson’s disease. VPS35 is a core subunit of the retromer complex that canonically recycles transmembrane cargo from sorting endosomes. Although retromer cargoes include many synaptic proteins, VPS35’s neuronal functions are poorly understood. To investigate the consequences of the Parkinson’s mutation, striatal neurotransmission was assessed in 1-, 3- & 6-month-old VPS35 D620N knock-in (VKI) mice. Spontaneous and optogenetically-evoked corticostriatal glutamate transmission was increased in VKI striatal spiny projection neurons by 6 months, when total striatal glutamate release, quantified by iGluSnFR imaging, showed similarities to wild-type. dLight imaging revealed robust increases in VKI striatal dopamine release by 6 months, which were reversed with acute ex vivo leucine-rich repeat kinase 2 (LRRK2) inhibition. We conclude that increased glutamate and dopamine transmission in VKI mice progressively emerges in young-adulthood, and that dopamine dysfunction is likely the result of sustained, rapidly-reversible, LRRK2 hyperactivity. Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Neuroscience/Diseases of the nervous system Biological sciences/Neuroscience/Synaptic transmission Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction A simplified definition of Parkinson’s disease (PD) is that of a movement disorder, resulting from the death of dopamine neurons in the substantia nigra pars compacta (SNpc). Within ~ 4 years of clinical presentation, there is an almost complete loss of nigrostriatal dopamine axon markers. Whereas, the loss of nigral neurons is less severe (30–60%) and remains fairly stable thereafter 1 . The resilience of a population of nigral neurons 1 , and the restorative effect of dopamine replacement therapy (e.g., L-DOPA) against motor symptoms 2 offer hope that at least some function can be restored in remaining neurons after disease onset. Unfortunately, no available PD treatment has been shown to slow or prevent disease progression. Furthermore, several disturbances that precede motor dysfunction by many years, e.g., rapid eye movement (REM) sleep behaviour disorder, anosmia, constipation, mood changes, and cognitive decline; are unresponsive to L-DOPA 3 – 5 . Many neuronal populations, in addition to nigral dopamine neurons, degenerate in PD; notably in the pedunculopontine nucleus and locus coeruleus containing cholinergic and noradrenergic neurons, as well as glutamatergic nuclei in the cortex and thalamus 6 – 8 . Thus, the evidence implicates multiple neurotransmitter systems in both early symptom manifestation and disease progression. A point of convergence of multiple neurotransmitter systems in PD pathology are the spiny projection neurons (SPNs) of the dorsolateral striatum 9 – 11 . SPNs integrate extensive cortical and thalamic glutamate input with modulation by nigrostriatal dopamine to direct behavioural action selection, via downstream basal ganglia nuclei 12 . Too much striatal glutamate and dopamine transmission, however, may be toxic 13 – 19 and could act as the initial pathophysiological stress that causes retrograde degeneration and selective loss of SNpc dopaminergic inputs 8 , 20 . PD is thought to emerge from the combination of genetic predisposition and environmental stress, especially age 21 . To investigate how these factors precipitate a Parkinsonian state, we developed VKI mice expressing the VPS35 D620N variant linked to clinically-typical PD 22 – 26 . Heterozygote VKI mice (which model the autosomal dominant presentation in humans) develop tau pathology and nigral loss at > 16 months 27 , 28 . Thus, VKI mice model human PD pathology at advanced ages and are appropriate for the study of early pathophysiological mechanisms. We previously reported increased glutamate transmission in VKI cortical cultures and increased dopamine release in brain slices of young VKI mice 22 , 23 . These changes correlated with hyperphosphorylation of leucine-rich repeat kinase 2 (LRRK2) substrates, the kinase believed to be hyperactive in LRRK2-familial and idiopathic PD 21 – 23 , 29 . In cultures, despite reversal of LRRK2 substrate hyperphosphorylation, glutamate alterations were resistant to LRRK2 inhibition 23 . Contrastingly, 1-week in vivo LRRK2 inhibition rescued decreased dopamine transporter (DAT) protein levels, and normalized dopamine release in VKI brain slices 24 . Here we sought to define the emergence of glutamate and dopamine synaptic dysfunction as young VKI mice mature, and test whether acute LRRK2 kinase inhibition modifies dopamine release. We found that glutamate and dopamine transmission become elevated by 6 months of age. Elevated dopamine release was rapidly reversed by acute (≥ 1.5h) LRRK2 kinase inhibition of slices, without evidence of changes to DAT function. We conclude that striatal glutamate and dopamine transmission is progressively increased in VKI mice throughout young adulthood, and that augmented dopamine release is a function of LRRK2 kinase hyperactivity. The data support the argument that LRRK2 inhibition is likely beneficial against synaptic dysfunction in PD and provide a model framework in which to test the neuroprotective potential of LRRK2 inhibitors. Methods Animals VPS35 D620N knock-in (VKI) were generated as previously described 22 and maintained on a C57Bl6/J wild-type (WT) background. Mice were housed and bred in accordance with the Canadian Council on Animal Care regulations (Animal Use Protocol 2017-7888B). All procedures were approved by and governed in accordance with the Neuro Centre of Neurological Disease Models (Animal Use Protocol 2017-7888B) and Memorial University Animal Care Committee (Animal Use Protocol 18-01-MP). 1-, 3-, and 6-month-old male heterozygous VKI and WT mice were used for all measures of spontaneous glutamate transmission. 3- and 6- month male heterozygous VKI and WT mice were used for all experiments requiring stereotaxic injections as these surgeries were performed >4 weeks in advance of experiments. Genotyping Genotyping of all mice was conducted on tail samples at weaning and on post-mortem ear tissue. Samples were digested in 100µL 10% Chelex (Bio-Rad 142–1253; 20 minutes 95°C, 2x vortex) and centrifuged (2 minutes 12,000 RPM) before DNA-containing supernatant (2µL) was added to PCR master mix (18µL, Qiagen 201203: taq polymerase, DNAse and RNAse-free water, 10X buffer, 10mM dNTPs, and custom DNA oligo primers [ThermoFisher: Forward-TGGTAGTCACATTGCCTCTG, Reverse-ATGAACCAACCATCAATAGGAACAC]). DNA was amplified by PCR (program cycle available upon request) and combined with fluorescent DNA intercalating dye (ZmTech LB-001G) prior to iontophoresis. 10µL of PCR product was run in 4% agarose gel and visualized (BioRad UV gel imager) for the presence of 1 or 2 bands to determine WT vs VKI genotype, respectively. Surgery Channelrhodopsin-2 (ChR2), intensity-based glutamate-sensing fluorescent reporters (iGluSnFR), and D1 dopamine receptor-based fluorescent reporters (dLight1.3b) were delivered by stereotaxic injection of AAV constructs as detailed below. 4-6 weeks prior to slice preparation, mice were subcutaneously injected with carprofen (2-4 mg/mL, 20mg/kg, 0.9% NaCl), left to rest for > 15 minutes, then anaesthetized with isoflurane (5% induction, 1-2% maintenance) and secured in a stereotaxic head frame (Kopf Instruments). The local analgesic, Marcaine, was injected subcutaneously below the site of incision and hair was removed chemically (Nair) or mechanically (Wahl clippers). An incision was made and skull, leveled using Bregma and Lambda as points of reference. A 0.5mm craniotomy was then created (micro-burr dentistry drill) over the site of injection. A 10µL syringe (Nanofil), attached to a microinjector (Harvard Apparatus Pump11 Elite) was lowered, with coordinates zeroed to the brain surface. Following AAV injection (details below), a 5-minute settling period was allowed before removing the needle. The scalp was then rehydrated with Marcaine, sutured (4-0 silk; Ethicon 683G) and reinforced (Vetbond 3M 1469SB), prior to subcutaneous 0.9% NaCl injection to replace fluids (0.2-0.5mL/10mg). Mice were monitored for pain and discomfort after regaining consciousness and returned to home cage in ~1 hour. Post-operative monitoring (3 days) included daily subcutaneous carprofen injection. Construct-specific injection details For ChR2 optogenetic stimulation, AAV9-CAG-ChR2-mCherry (Neurophotonics Centre, Université Laval, lot #834 = 4x10 12 GC/mL) was injected unilaterally (650nL, 1nL/sec) into primary motor cortex (1.5 mm anterior, 1.0 mm lateral, 0.8 mm ventral to Bregma). For optogenetic recording of glutamate release, AAV1.hSyn.iGluSnFr.WPRE.SV40 (Addgene 98929-AAV1, 2.8x10 13 GC/mL) was injected bilaterally (1uL, 2nL/sec) into the dorsolateral striatum (coordinates: 2.0 mm anterior, 1.0 lateral, 3.2 ventral to Bregma). For optogenetic recording of dopamine release, AAV5-CAG-dLight1.3b (Addgene 125560-AAV5, 1.5x10 13 GC/mL) was injected bilaterally (300nL, 1nL/sec) into the dorsolateral striatum. Preparation of acute brain slices 300µm acute coronal brain slices were prepared from restrained mice which were swiftly decapitated. Brains were quickly transferred to ice-cold recovery solution (1 minute; in mM: 93 NMDG, 93 HCl, 2.5 KCl, 1.2 NaH 2 PO 4 , 30 NaHCO 3 , 20 HEPES, 25 glucose, 5 sodium ascorbate, 3 sodium pyruvate, 10 MgSO 4 ·7H 2 O, 0.5 CaCl 2 ·2H 2 O, pH 7.3-7.4, 290-310 mOsm; carbogen-infused). Following this, extra-striatal rostral and caudal brain regions were removed by coronal sectioning, and the remaining block was mounted onto a vibrating-blade microtome platform with sodium acrylate (Leica Microsystems VT 1200S). After hemisection at the midline, coronal sections were cut, and slices were then transferred to warm recovery solution (35°C, 15 minutes). A final transfer of slices into holding chambers containing room-temperature artificial cerebrospinal fluid (aCSF; in mM: 125 NaCl, 2.5 KCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2 MgCl 2 , 2 aCl 2 , 10 glucose, pH 7.2-7.4, 300-310 mOsm, 22-25°C, carbogen-infused) was followed by > 45 minutes settling period before recording in aCSF began. Whole-cell patch-clamp electrophysiology Whole-cell patch-clamp electrophysiology was used to obtain recordings of spontaneous and ChR2-evoked glutamate transmission in SPNs of the dorsolateral striatum (caudate/putamen), as previously 30 . Slices were transferred to a recording chamber perfused with aCSF (22-25°C) containing 100µM picrotoxin (Tocris 1128) at a flow rate of ~1.5mL/min. Slices were visualized on an Olympus BX51 microscope (40x magnification, 2x digital zoom, IR-DIC and Q-Imaging Electro camera). SPNs were identified within the dorsolateral striatum based on somatic size (8-20µM) and distinct morphology, 50-150µM below the surface of the slice. Borosilicate glass capillary tubes (Harvard Apparatus 640805) were pulled using a Sutter P-1000 micropipette puller to form 1µm tip recording pipettes (filled resistance 4-8 MOhms; in mM: 130 Cs methanesulfonate, 5 CsCl, 4 NaCl, 1 MgCl 2 , 5 EGTA, 10 HEPES, 5 QX-314, 0.5 GTP, 10 Na 2 phosphocreatine, 5 MgATP, and 0.1 spermine, pH 7.2, 290mOsm). A motorized micromanipulator (Sutter Instrument MP-285) was used to patch onto identified SPNs, with signals obtained by MultiClamp 700B amplifier in voltage-clamp configuration, filtered at 2kHz and digitized at 10kHz (Molecular Devices Axon Digidata 1440A). Membrane properties were determined with the membrane-test function while holding at -70mV. Synaptic recordings were initiated after a 2-minute settling period, with access resistance tolerance set to 27MW and recordings discarded if D > 10% over the elapsed period. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded over a minimum 2-minute period at -70mV (gain 20) and analyzed in Clampfit10 (Molecular Devices; 5pA peak event threshold, confirmed manually). Non-unitary events were retained for inter-event interval analysis, but only unitary events were used for amplitude and decay constants. Cumulative distributions were used to evaluate amplitude and inter-event intervals in each recorded SPN. Unitary events in each recording were averaged and decay tau measured by 1-term exponential fit. ChR2-evoked post-synaptic currents (ChR2-PSCs) were stimulated by wide-field illumination with blue light (473nm, XCite Series 120Q) transmitted through the 40x immersion objective generating 2.7 mW. 5ms pulses were controlled by a Lambda SC Smart Shutter Controller (Sutter Instruments) administered in trains of 4 pulses with 100ms inter-pulse intervals. Trains were repeated every 30 seconds, 5-10 times and averaged for analysis. Recordings of ChR2-PSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were taken at -70mV (gain 2) averaged across 5 repetitions. AMPAR and N-methyl-D-aspartate receptor (NMDAR) currents were generated by single 5ms pulse stimulations repeated 5x every 30 seconds at -70mV then +40mV to assess AMPAR+NMDAR-mediated ChR2-PSCs, respectively. Peak NMDAR current was estimated at +40mV, 40ms post AMPA peak, and AMPAR currents were then isolated at +40mV by bath application of 10µM D-APV (NMDAR blocker, Tocris 0106). AMPA rectification indices were calculated as a ratio of isolated AMPAR ChR2-PSC peak at +40mV : -70mV. iGluSnFR and dLight imaging Imaging of iGluSnFR and dLight recordings were obtained using a 2X objective focused on dorsolateral STR. Slices were wide-field illuminated with blue light (473nm, CoolLED pE-340fura), with the shutter, EM-CCD camera (Andor iXon Ultra 897), and stimulus isolator (WPI A365) triggered by Clampex & Digidata 1550B). The stimulus isolator was connected to a monopolar tungsten stimulating electrode (A-M Systems 574000) lowered into dorsolateral striatum 50-100µM beneath the surface of the tissue. Recordings were captured using Andor Solis software using 4x4 binning at 205Hz. For these experiments, spontaneous iGluSnFR & dLight transients were excluded from analysis of responses to electrical stimulation. For iGluSnFR recordings, a stimulation train was delivered at fixed stimulation intensity (150uA train of 10 x 0.2ms pulses with 100ms inter-pulse intervals). No-stimulation (for background & bleach subtraction) and stimulation trials were alternated at 1-minute intervals, repeated every 2 minutes, until 5 no-stimulation trials were recorded. An 11 th pulse was delivered with each repetition of the stimulation trial, with increasing inter-pulse intervals between 500-5000ms. The same protocol was used to obtain pulse train dLight recordings after 2-pulse dLight stimulation with 4s inter-pulse intervals, delivered at 50-400uA, every 2 minutes to generate stimulus response curves. Responses were then assessed to determine the stimulus intensity corresponding to 50-70% maximum response (FIJI software) for subsequent pulse train dLight experiments. iGluSnFR and dLight videos were converted to the change in fluorescence intensity over time (DF/F; FIJI software) with peak and decay analyzed in Clampfit (decays measured by the 1-term exponential function of each trace). Absolute and normalized peak and decay of pulse train responses were derived from the average of 4 responses. Acute LRRK2 kinase inhibition Slices from 6-month-old mice were pre-incubated ≥1.5hours and then bath-perfused during dLight recording with LRRK2 kinase inhibitor MLi-2 (500 nM, Tocris 5756, 45% Captisol® in PBS) or vehicle-control (45% Captisol® in PBS 377.6µL/L aCSF). Captisol® vehicle was also present in all 3-month-old dLight recordings to be able to compare dLight recordings across age. Statistical Analysis All data presentation and statistics were conducted in GraphPad Prism10. Data were tested against ROUT outlier analysis with a liberal maximum false discovery rate Q=1%. Rarely, identified outliers were suppressed from the dataset prior to parametric or non-parametric distributions testing (D’Agostino and Pearson). Unpaired t-test/1-way ANOVA (if parametric) or Mann-Whitney U-test/Kruskal-Wallis testing (if non-parametric) were used to analyse data. Post-hoc analysis was performed if ANOVA p <0.05 by Tukey’s (parametric) or Dunn’s (non-parametric) multiple comparisons tests as appropriate. All 2-way ANOVA comparisons used Šídák post-hoc analyses. Asterisks represent p p > 0.05. Data are presented as n=observations from (n) animals (e.g., WT = 6(3) is 6 observations from 3 WT animals). Results VKI SPNs show progressive increase in spontaneous activity by 6 months We compared spontaneous excitatory postsynaptic currents (sEPSCs) by whole-cell patch-clamp as before 30 in VKI and WT SPNs from 1-, 3-, and 6-month-old mice (Figure 1 A). Although similar at 1 month, we found that VKI sEPSCs begin to diverge from WT at 3 months with larger amplitudes. VKIs exhibit robustly elevated amplitude and frequency (ie. reduced inter-event interval) by 6 months (Figure 1B-D). The decay of sEPSCs was faster in VKI SPNs at 3 months but normalized to WT levels at 6 months. An age-dependent decrease in WT sEPSC amplitude and frequency was absent in VKI SPNs (Table S2). We also observed early reductions in membrane tau of 1-month-old VKI SPNs, that normalized to WT levels by 3 months (Figure S1 A,B,C iii). VKI SPNs also trend towards increased membrane capacitance at 3- and 6-months, but not at 1 month (Figure S1 A,B,C i). Membrane resistance was reduced by 6 months in VKI SPNs (Figure S1 A, B, C, ii). As with sEPSC properties, membrane capacitance and resistance measures were altered with age within WT SPNs, but not in VKI neurons (Table S2). Together, age-dependent changes to passive membrane properties, and a progressive (relative) increase in the amplitude, frequency, and decay of spontaneous glutamate transmission is observed in VKI SPNs. Corticostriatal glutamate transmission is progressively increased in VKI mice by 6 months To determine whether increased frequency and amplitude of sEPSCs are due to changes in corticostriatal inputs, optogenetic stimulation of glutamate release onto patched SPNs was conducted at 3 and 6 months (Figure 2 A i). Light stimulation of local cortical terminals generated ChR2-induced post-synaptic currents (ChR2-PSCs). Glutamatergic currents through AMPA and NMDA receptors were isolated by membrane gating properties (ie. holding potential) and by pharmacology with the NMDAR blocker, D-AP5 (Figure 2 A ii). At 3 months, we found no change to VKI AMPAR- or NMDAR-mediated current amplitude. The ratio of NMDA:AMPA current and AMPAR-rectification indices were likewise similar (Figure 2 B). In contrast, we found AMPAR and NMDAR currents were both (equally) elevated in 6-month-old VKIs (Figure 2 C i-ii). There was no change in NMDA:AMPA ratio, or AMPA rectification index at this age (Figure 2 C iii-iv). To assess presynaptic probability of release, we quantified paired-pulse ratios (PPRs) at 100ms intervals (Figure 2 D). There was no significant difference to PPRs in VKI neurons at 3 months, but a significant reduction in PPR was observed in 6-month-old VKI SPNs, indicative of elevated probability of presynaptic release (Pr). Taken together, the data show corticostriatal glutamate transmission becomes elevated in VKI SPNs, relative to WT littermates, and that this correlates with an equivalent increase in both AMPAR and NMDAR transmission. Total striatal glutamate release appears reduced in 3-month-old VKIs and normalizes to WT levels by 6 months. In addition to changes in presynaptic release, glutamate currents recorded in SPNs are modulated by regulation of postsynaptic responsiveness, thalamic glutamate, midbrain dopamine, and GABAergic interneuron activity 31–38 . To directly assess presynaptic release, extracellular glutamate transients were quantified with the intensity-based glutamate-sensing reporter, iGluSnFR1 (Figure 3 A) in response to fixed intensity pulse-trains (10x10Hz) delivered by local striatal electrical stimulation. The amplitude of iGluSnFR responses was markedly reduced in 3-month-old VKI striata, and there was a significant interaction between the normalized peak and pulse number (Figure 3 B i-ii and Table S1). The recovery capacity of iGluSnFR responses following the pulse train was similar at 3 months, as was the decay of the pulse train response (Figure 3 B iii-iv). At 6 months, iGluSnFR responses were similar to WT responses across all measures (Figure 3 C). Interestingly, a significant age-dependent reduction in the amplitude of iGluSnFR transients was only observed for WT and not VKI responses (Table S2). No age dependent changes were observed in other parameters except for the decay of iGluSnFR responses in VKI striata between 3 and 6 months, which appear significantly increased with age. In summary, total striatal glutamate release is reduced in VKI mice at 3 months, but similar to WT at 6 months, suggesting that total presynaptic glutamate release is relatively increased in the VKI striatum with age. Increased striatal dopamine release appears in 6-month-old VKI mice We measured extracellular dopamine transients in the dorsolateral striatum using the dLight fluorescence reporter, evoked by electrical stimulation in slices from 3- and 6-month-old mice (Figure 4-6). We did not observe any changes to dLight amplitudes, or responses to pulse-train stimulation in 3-month-old VKI striata (Figure 4 A); however, recovery capacity following pulse train stimulation was significantly reduced in VKIs. At 6 months, dLight response peaks were significantly increased in VKI brain slices, without any change in decay tau, or PPR at 4s intervals (Figure 5 and Table S1). Pulse train response peaks in VKIs were accordingly increased, without changes in pulse-train response pattern (Figure 6). However, recovery following train stimulation was reduced at smaller intervals and increased at larger intervals. WT responses show amplitude reductions between 3 and 6 months, which are absent in VKI responses (Table S2). There is also a significant change to the pattern of normalized responses to pulse train stimulation with age in VKI striata, but not WT. Decay of responses were not significantly different in either WTs or VKIs when comparing 3- to 6-month-old mice. Therefore, the data indicate dopamine release is progressively increased in VKIs, relative to WT, by 6 months old. Increased dopamine release in VKI striata is reversed with acute LRRK2 kinase inhibition We investigated whether the robustly elevated dopamine release in 6-month-old VKI striata could be targeted with acute LRRK2 kinase inhibition. Slices prepared for dLight recordings were incubated either in vehicle Captisol® or in 500nM of LRRK2 kinase inhibitor, MLi-2, for > 1.5 hours (Figure 5 A). Elevated VKI dLight responses were reversed by LRRK2 inhibition, without altering decay constants, or 4s interval PPRs (Figures 4-6, and Table S1). Although increased amplitudes were similarly reversed in pulse-train experiments, there were no significant effect of MLi-2 on the pattern of responses to stimulation trains. Further, MLi-2 had no effect on WT dLight responses. In conclusion, acute LRRK2 kinase inhibition with MLi-2 reversed the elevated dopamine release in VKI striata at 6 months, without altering dopamine release in WT. Discussion Previously, we found elevated glutamate transmission in 3- to 4-week-old VKI mouse cortical cultures 23 and elevated striatal dopamine transmission in slices from VKI mice aged 3 months 22 , 24 . Here, we find corticostriatal glutamatergic transmission onto SPNs is normal at 1 month, but progressively increases to > 50% over WT by 6 months. Intriguingly, total striatal glutamate release measured by iGluSnFR was reduced at 3 months and increased to match WT levels by 6 months. In contrast, striatal dopamine release measured by dLight was markedly elevated in VKI striata by 6 months and reversed by acute LRRK2 kinase inhibition. SPNs of the dorsolateral striatum are the initial point of convergence for control of behavioural action selection, habit formation, and movement initiation 9 , 12 , 39 . These functions are all altered in PD, and pathology is clearest first in nigrostriatal axons 1 . Our data suggest SPNs in VKI mice develop changes to passive membrane properties throughout young-adulthood, which could reflect functional and/or anatomical differences. Membrane capacitance is proportional to membrane area, whereas membrane resistance inversely correlates to process diameter (and usually correlates with capacitance). These properties were bidirectionally altered in 6-month-old VKI SPNs, indicative of a larger soma, neurites, and/or processes, and increased leak channel conductance 40 , 41 . We observed a subtle increase in amplitude and reduced decay time of spontaneous glutamate events in 3-month-old VKI SPNs, but no changes to optogenetically-evoked corticostriatal AMPAR or NMDAR currents. This suggests any modestly increased response to quantal release in a proportion of synapses is not sufficient to be detected in large currents generated by activity-dependent release. In light of relatively unchanged corticostriatal transmission in SPNs, it was surprising to see that iGluSnFR measures of total glutamate release were markedly reduced in VKI mice at 3 months. This could be explained by an increased number of active synapses, with lower individual release probabilities, or increased postsynaptic responsiveness. Since we previously quantified no change to glutamate synapse number in VKI mice at 3 months 22 , it may be that increased postsynaptic responsiveness exists as an attempt to compensate for one another (or vice versa ), thereby keeping transmission onto SPNs within a homeostatic range. Alternatively, glutamate release onto SPNs may be unaltered, but reduced at other non-SPN synapses (e.g., excitatory input to cholinergic interneurons 32 , 34 ), inviting future interrogation by cell-type. By 6 months, clearer increases in SPN spontaneous event amplitude were accompanied by increased frequency (ie. decreased inter-event intervals). At this age, VKI SPN corticostriatal AMPAR- and NMDAR-current amplitudes were also increased ~ 50% over WT. More frequent spontaneous activity and higher PPRs, which we also observe in corticostriatal evoked responses, are usually interpreted as increased probability of presynaptic release. Larger AMPAR and NMDAR currents could also be a result of higher postsynaptic receptor numbers. We and others have reported increased GluA1-containing AMPAR receptor expression with VPS35 D620N and LRRK2 G2019S knock-in 23 , 42 , but increases to both NMDAR and AMPAR expression, here, would have to be equal to explain the lack of difference in NMDAR:AMPAR current ratios here. Thus, the parsimonious explanation is that of increased probability of release, as we reported in vitro 23 . Here, direct measures of total striatal presynaptic release with iGluSnFR found no difference between VKI and WT striata, suggesting that corticostriatal transmission onto SPNs must be selectively increased in VKIs. Alternatively, local electrical stimulation may activate the negative tuning of corticostriatal synapses through presynaptic D2 receptors 43 , which would reduce total striatal glutamate release by iGluSnFR imaging, but not affect the direct activation of corticostriatal release with ChR2. Together, the data reveal an emergent increase in cortical glutamate transmission onto VKI SPNs by 6 months, perhaps to compensate for a reduction in total glutamate release observed at 3 months (or vice versa ). While not required for diagnosis, changes to glutamate transmission are observed in PD, and likely impact non-motor and motor features of the disease 5 , 20 , 44 . Elevated glutamate transmission is seen early in PD-relevant mice carrying knock-in LRRK2 G2019S mutations, and those overexpressing synuclein 19 , 30 , 45 – 47 . Therefore, the progressive dysregulation of glutamate in young adult VKI mice is consistent with other models of PD, and may represent a direct contribution to (or compensation against) pathophysiological progression to degeneration. Previously, we found dopamine release was elevated in VKI mice at 3 months by fast-scan cyclic voltammetry (FSCV) 22 , 24 . This was accompanied by slower decay constants, indicative of reduced DAT activity, and a reduction of DAT protein expression 22 , 24 . Strikingly, increased dopamine release in 3-month-old VKI slices was rescued by chronic 1-week in vivo administration of LRRK2 kinase inhibitor, MLi-2 24 . LRRK2 kinase inhibition also normalized the reduced DAT levels, which we concluded was causal to the rescue 24 . Here, we aimed to determine whether the rescue can be observed with shorter-term LRRK2 inhibition (≥ 1.5h), indicating that dysregulation of dopamine release machinery is LRRK2 kinase-dependent, and not due to systemic effects of LRRK2 kinase inhibition. As FSCV offers low temporal (10Hz) and spatial resolution (limited by the carbon fiber electrode surface area), we employed dLight1.3b to assay dopamine release across the entire dorsolateral striatum at 20x higher temporal resolution. Considering our published data, we were surprised that dLight recordings showed no difference between VKI and WT dopamine release at 3 months, other than a reduction in the speed of recovery from train stimulation. In contrast, 6-month-old VKI mice displayed a robust elevation in the amplitude of dLight responses, while decay constants were unaltered. The capacity to re-release dopamine after train stimulation was biphasically altered at this age, with shorter time intervals showing a reduced recovery capacity, and longer time intervals showing an increased recovery capacity. Together, this suggests dopamine release is robustly elevated in VKI by 6 months old, and that dopamine axons repolarise, repackage, and re-release dopamine more readily at intervals > 1s. Decay constants, and short-term depression (which would be observed as depletion of PPRs during high-frequency trains) are both indicators of DAT function 48 – 51 . As we saw no difference in these measures, we posit that increased dopamine release is independent of DAT function, as in LRRK2 G2019S knock-in mice 30 . Differences between our published 3-month VKI voltammetry 22 , 24 and dLight data here maybe explained by biological and/or technical differences. Three data sets on mice from the same founding colony now show elevated dopamine release, albeit at slightly different ages 22 , 24 . While institutional housing conditions may modify phenotype progression, technical differences between slice treatment might also explain the discrepancy. Here, unlike for previous voltammetry data, slices were bathed in NMDG recovery solution to increase slice viability. Recordings were performed in the vehicle for MLi2, Captisol®, a biopolymer that has the potential to increase neurotransmitter release 52 – 57 , potentially masking differences between genotypes at 3 months. Disparities in the recording modality might also change the interpretation of results at 3 months. Direct comparison of FSCV and dLight shows both techniques report the expected increase in dopamine release duration and decay with nomifensine or cocaine (blocking DAT); however, peak changes are only observed with FSCV 48 . FSCV relies on the area of a carbon fiber electrode onto which dopamine can diffuse, which will increase under conditions of DAT inhibition, and translate to an increase in the peak of FSCV transients 48 . If the spread of extracellular dopamine is increased in VKI at 3 months this would translate to peak changes in voltammetry, but not dLight. Further investigation of the spread dLight fluorescence may reveal whether diffusion is altered at ages preceding clear increases in dLight peaks in VKI mice. Despite these caveats, repeated assessment of dLight across ages in VKI mice reveals increased dopamine release in young-adulthood, which is sensitive to acute LRRK2 kinase inhibition. Elevated dopamine release in 6-month VKI was returned to WT levels by ≥ 1.5h slice treatment with LRRK2 inhibitor MLi-2, relative to vehicle-treated slices from the same brain. This occurred without changes to parameters dictated by DAT or D2 receptors (decay rate / pulse train PPR, and 4s PPR 49 , 58 ). Therefore, we conclude LRRK2 kinase hyperactivity and inhibition can alter dopamine release independent of canonical DAT and D2 receptor activity. Both DAT and D2 receptors appear to be VPS35 cargoes, as is the dopamine vesicular monoamine transporter (VMAT) 23 , 59 , 60 . It may be that aberrant LRRK2 kinase activity increases vesicular packing of dopamine via VMAT, as VMAT2 levels are elevated in VKI striatal tissue 22 , 24 . This aligns with our observation that dopamine release is elevated during pulse-trains without significantly altering release probability (P2/P1 PPR, 100ms intervals). LRRK2 variants convey the highest genetic risk for PD, with several pathogenic variants linked to clinically-typical familial parkinsonism 21 . Brain scans of LRRK2 PD patients show impaired presynaptic dopamine function 61 , but asymptomatic LRRK2 mutation carriers exhibit higher dopamine turnover, indicating increased release 62 . Dopamine release measured by FSCV is also elevated in 3-month-old LRRK2 G2019S knock-in mice 30 , consistent with our results in VKI mice 22 , 24 . Early increases in dopamine release decline in LRRK2 knock-in mice 30 , and a similar pattern is observed in α-synuclein overexpressing PD mice 63 . Together, evidence from VKI, LRRK2 and synuclein mouse models are consistent with early elevations in dopamine release in humans 62 . LRRK2 and VPS35 canonically function in endolysosomal regulation and trafficking, and synaptic vesicles (SVs) are highly specialised, neural-specific, endosomes 64 . As with other membranes, the SV cycle is orchestrated by Rabs, several of which are phosphorylated by LRRK2. This includes the canonically synaptic-vesicle associated Rab3&5 65,66 and classically-endolysosomal Rab10&12, that have recently been shown to be as enriched as Rab3&5 on SVs 67 . Both VPS35 and LRRK2 can alter synaptic vesicle docking and size 68 , although how remains speculative. Conceptually, LRRK2 phosphorylation of Rab3 might be expected to increase SV availability by facilitating biogenesis, trafficking, and priming of vesicles at the active zone. Rab3 tethering to Rab-Interacting Molecule (RIM) 69 regulates vesicle fusion, without which DA release is blocked 70 . Other than Rab3, the role of Rabs at SVs is somewhat unclear, and effects of LRRK2-Rab phosphorylation almost entirely unknown. Finally, while Rabs have recently taken centre stage, several SV proteins were previously implicated as LRRK2 substrates; including, auxillin, endophilinA, dynamin, & synapsin 47 , and we show here that LRRK2 kinase inhibition can have rapid effects on dopamine release. Further investigation of VPS35 mutant, and LRRK2 inhibitor effects, on dopamine and glutamate vesicle cycles seems justified. Given the ubiquitous expression of VPS35 and LRRK2, it likely that onset of PD-like dysfunction and degeneration results from the accumulation of low-level cellular insults over years in mice, or decades in humans. Sustained elevations in striatal neurotransmission, or even compensatory action towards it, likely increases cellular stress. This in turn, could lead to the accumulation of excitotoxic damage which may not be effectively cleared in the presence of the VPS35 (or LRRK2) mutation 71 . Increased energy demands and calcium buffering required by increased synaptic activity may also contribute to mitochondrial dysfunction observed in many PD scenarios, including VPS35 patient-derived neurons 72 . These may eventually lead to axon degeneration in more vulnerable neuronal populations, such as nigrostriatal dopamine neurons. In summary, we present evidence of progressively elevated neurotransmission in young-adult mice with VPS35 D620N mutations, which we propose will become neurotoxic if sustained. However, acute inhibition of LRRK2 kinase rapidly reverses the increased dopamine release, offering hope as a viable neuroprotective strategy. Targeting early neuronal hyperactivity may be the optimal therapeutic window for disease-modifying PD treatments, especially for those known to carry PD-causal gene variants. 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Supplementary Files Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 03 May, 2025 Read the published version in npj Parkinson's Disease → Version 1 posted Editorial decision: Revision requested 14 Nov, 2024 Reviews received at journal 13 Nov, 2024 Reviews received at journal 28 Oct, 2024 Reviewers agreed at journal 19 Oct, 2024 Reviewers agreed at journal 18 Oct, 2024 Reviewers agreed at journal 15 Oct, 2024 Reviewers invited by journal 15 Oct, 2024 Editor assigned by journal 15 Oct, 2024 Submission checks completed at journal 14 Oct, 2024 First submitted to journal 27 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5167163","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":378291826,"identity":"6d914235-41e6-4770-949b-d1ebf56251e8","order_by":0,"name":"Anusha Kamesh","email":"","orcid":"","institution":"McGill University","correspondingAuthor":false,"prefix":"","firstName":"Anusha","middleName":"","lastName":"Kamesh","suffix":""},{"id":378291827,"identity":"99af7dd4-45fb-4a6c-9bfc-6af31a46c9ab","order_by":1,"name":"Chelsie Kadgien","email":"","orcid":"","institution":"McGill University","correspondingAuthor":false,"prefix":"","firstName":"Chelsie","middleName":"","lastName":"Kadgien","suffix":""},{"id":378291828,"identity":"f7fe8801-63fd-4252-a75d-4e77832d2b63","order_by":2,"name":"Naila Kuhlmann","email":"","orcid":"","institution":"McGill University","correspondingAuthor":false,"prefix":"","firstName":"Naila","middleName":"","lastName":"Kuhlmann","suffix":""},{"id":378291830,"identity":"84086160-acb0-4309-94cd-a814d85d26ed","order_by":3,"name":"Sean Coady","email":"","orcid":"","institution":"McGill University","correspondingAuthor":false,"prefix":"","firstName":"Sean","middleName":"","lastName":"Coady","suffix":""},{"id":378291832,"identity":"0f770f6e-a47c-4f3f-b4b8-729722f75214","order_by":4,"name":"Emily Hurley","email":"","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Hurley","suffix":""},{"id":378291834,"identity":"1f05ed4d-5564-4d06-882b-508678e296df","order_by":5,"name":"Matthew Parsons","email":"","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Parsons","suffix":""},{"id":378291836,"identity":"92572c84-f57f-4950-9355-24bed7ecc53b","order_by":6,"name":"Austen Milnerwood","email":"data:image/png;base64,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","orcid":"","institution":"McGill University","correspondingAuthor":true,"prefix":"","firstName":"Austen","middleName":"","lastName":"Milnerwood","suffix":""},{"id":378291837,"identity":"a56d491d-4a69-4b1b-8198-de49605cbda8","order_by":7,"name":"Jessica Barron","email":"","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Barron","suffix":""}],"badges":[],"createdAt":"2024-09-27 19:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5167163/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5167163/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41531-025-00948-7","type":"published","date":"2025-05-03T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79548222,"identity":"4f4a4e08-5e98-4048-a213-db0af1c6ea11","added_by":"auto","created_at":"2025-03-31 06:17:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2313867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpontaneous glutamate transmission increase in dorsolateral striatal VKI SPNs emerges by 6 months.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e \u003cstrong\u003ei) \u003c/strong\u003eSchematic depicting whole-cell patch-clamp recording of spiny projection neurons (SPNs) in the dorsolateral striatum (STR) from acute coronal brain sections also containing cortex (CTX). \u003cstrong\u003eii)\u003c/strong\u003e Representative trace of voltage-clamp recording of spontaneous excitatory post-synaptic current (sEPSC) from dorsolateral striatal SPNs. Representations of amplitude, inter-event interval, and decay tau measurements annotated in red. \u003cstrong\u003eB) \u003c/strong\u003eAt 1 month, \u003cstrong\u003ei)\u003c/strong\u003ecumulative probability distributions of sEPSC amplitude are not significantly different between VKI and WT SPNs (2-way ANOVA Interaction \u003cem\u003ep\u003c/em\u003e\u0026gt;0.99, genotype \u003cem\u003ep\u003c/em\u003e=0.22). \u003cstrong\u003eii)\u003c/strong\u003eCumulative probability distributions of inter-event intervals are not significantly different between VKI and WT SPNs (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.99, genotype \u003cem\u003ep\u003c/em\u003e=0.93). \u003cstrong\u003eiii) \u003c/strong\u003esEPSC decay tau is not significantly different between VKI and WT SPNs (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.43). \u003cstrong\u003eC \u003c/strong\u003eAt 3 months,\u003cstrong\u003e i) \u003c/strong\u003ecumulative probability distributions show VKI SPNs have more, larger amplitude sEPSC than WT SPNs (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.02, genotype \u003cem\u003ep\u003c/em\u003e=0.21). \u003cstrong\u003eii.\u003c/strong\u003e sEPSC inter-event intervals are not significantly different between genotypes (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.10, genotype \u003cem\u003ep\u003c/em\u003e=0.95). \u003cstrong\u003eiii. \u003c/strong\u003eAverage sEPSC decay tau is significantly faster in VKI vs WT neurons (Mann-Whitney U-test \u003cem\u003ep\u003c/em\u003e=0.01). \u003cstrong\u003eD \u003c/strong\u003eAt 6 months, \u003cstrong\u003ei) \u003c/strong\u003ecumulative distributions show a significantly higher number of larger-amplitude sEPSCs in VKI neurons (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, genotype \u003cem\u003ep\u003c/em\u003e=0.02). \u003cstrong\u003eii. \u003c/strong\u003eCumulative distributions of sEPSC inter-event intervals indicate VKI neurons have shorter inter-event intervals than WT neurons (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, genotype \u003cem\u003ep\u003c/em\u003e=0.06). \u003cstrong\u003eiii. \u003c/strong\u003eAverage sEPSC decay taus are not significantly different in VKI vs WT neurons (Mann-Whitney U-test \u003cem\u003ep\u003c/em\u003e=0.65). Asterisks denote pairwise comparisons; * = 0.05\u0026gt;\u003cem\u003ep\u003c/em\u003e\u0026gt;0.01, ** = 0.01\u0026gt;\u003cem\u003ep\u003c/em\u003e\u0026gt;0.001, *** = 0.001\u0026gt;\u003cem\u003ep\u003c/em\u003e\u0026gt;0.0001, **** = \u003cem\u003ep\u003c/em\u003e\u0026gt;0.0001. 2-way ANOVA comparisons listed if \u003cem\u003ep\u003c/em\u003e\u0026lt;0.10.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/4807b3113125b65b5dca66bf.png"},{"id":79548219,"identity":"69d6de8c-b3f5-4fcd-a0dc-0a92f5812720","added_by":"auto","created_at":"2025-03-31 06:17:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1856772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvoked corticostriatal glutamate transmission increases in VKI SPNs by 6 months.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA i) \u003c/strong\u003eSchematic depicting ChR2 viral injection into motor cortex 4-6 weeks prior to preparation of acute coronal slices containing CTX and STR. Whole-cell patch-clamp recordings were obtained from dorsolateral striatal SPNs +/- NMDAR inhibitor, APV. \u003cstrong\u003eii) \u003c/strong\u003eRepresentative ChR2-PSC traces in response to single pulses of light. SPNs that were held at membrane potentials of -70mV or +40mV (in black), and +40mV in the presence of AP5 (in cyan). \u003cstrong\u003eB \u003c/strong\u003eAt 3 months, \u003cstrong\u003ei) \u003c/strong\u003ethe peak amplitude of AMPAR-mediated currents at -70mV are not significantly different between WT and VKI SPNs (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.46). \u003cstrong\u003eii) \u003c/strong\u003eAmplitude of NMDAR-mediated currents are not significantly different between genotypes at +40mV (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.87). \u003cstrong\u003eiii) \u003c/strong\u003eRatios of +40mV NMDAR current to -70mV AMPAR current are not significantly different between WT and VKI SPNs (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.60). \u003cstrong\u003eiv) \u003c/strong\u003eRectification Index of AMPA current is not significantly different between WT and VKI SPNs (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.45). \u003cstrong\u003eC \u003c/strong\u003eAt 6 months, \u003cstrong\u003ei) \u003c/strong\u003eVKI SPNs show larger currents than WT SPNs, in peak amplitude of both AMPAR-mediated\u003cstrong\u003e \u003c/strong\u003ecurrent (Mann-Whitney U-test \u003cem\u003ep\u003c/em\u003e=0.01) and \u003cstrong\u003eii) \u003c/strong\u003eNMDAR-mediated current (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.0005). \u003cstrong\u003eiii) \u003c/strong\u003eRatio of NMDA:AMPA currents are not significantly different between genotypes (Unpaired t-test \u003cem\u003ep=\u003c/em\u003e0.90). \u003cstrong\u003eiv) \u003c/strong\u003eAMPA Rectification Index is not significantly different between WT and VKI SPNs (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.67). \u003cstrong\u003eD i) \u003c/strong\u003eRepresentative ChR2-PSCs with 4 pulses of ChR2 stimulation (in red), 100ms inter-pulse interval. \u003cstrong\u003eii) \u003c/strong\u003eAt 3 months, the paired-pulse ratio (PPR) of 4 responses to ChR2 stimulation, normalized to the first response, is not significantly different between VKI and WT SPNs (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.34, genotype \u003cem\u003ep\u003c/em\u003e=0.22). \u003cstrong\u003eiii) \u003c/strong\u003eAt 6 months, the paired-pulse ratio is significantly reduced in VKI vs WT SPNs (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.001, genotype \u003cem\u003ep\u003c/em\u003e=0.04).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/0d5ba57bc44920f3f8ab8e02.png"},{"id":79548223,"identity":"21e20aea-0ce5-44e4-986a-6d74b1346b6c","added_by":"auto","created_at":"2025-03-31 06:17:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2351066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal striatal glutamate release in VKIs normalize to WT levels by 6 months.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA i) \u003c/strong\u003eGraphic depicting viral injection of iGluSnFR in dorsolateral striatum 4-6 weeks prior to acute slice preparation. \u003cstrong\u003eii)\u003c/strong\u003e Area of iGluSnFR expression in coronal slices containing CTX and STR depicted in green along with stimulus electrode (Stim) placed in dorsolateral STR. \u003cstrong\u003eiii) \u003c/strong\u003eVisualization of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) change in fluorescence relative to baseline (ΔF/F) in dorsolateral STR in response to local electrical stimulation (Stim). Low to high ΔF/F as a ratio of 0 to 1 represented as a gradient from purple to white, respectively. Boundaries of the corpus callosum (CC) separate CTX and STR in a coronal brain slice. Dashed circle represents region of interest (ROI) of 20 pixels used for iGluSnFR analysis. \u003cstrong\u003eii) \u003c/strong\u003eRepresentative iGluSnFR response corresponding to trains of 10x10Hz pulses of electrical stimulation followed by 11\u003csup\u003eth\u003c/sup\u003e pulse at increasing interval with each of 4 repetitions. Black trace represents 1\u003csup\u003est\u003c/sup\u003e of 4 stimulations with subsequent stimulations overlayed in maroon. \u003cstrong\u003eB \u003c/strong\u003eAt 3 months, \u003cstrong\u003ei)\u003c/strong\u003e VKI vs WT iGluSnFR response size (ΔF/F) is significantly smaller in VKI brain slices (2-way ANOVA\u003cstrong\u003e \u003c/strong\u003einteraction \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, genotype \u003cem\u003ep\u003c/em\u003e=0.007). \u003cstrong\u003eii) \u003c/strong\u003eWhen normalized to 1\u003csup\u003est\u003c/sup\u003e pulse (Norm. P1), VKI iGluSnFR responses are significantly different from WT (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.006, genotype \u003cem\u003ep\u003c/em\u003e\u0026gt;0.99). \u003cstrong\u003eiii) \u003c/strong\u003eVKI and WT recovery after 10x10Hz stimulation, normalized to 1\u003csup\u003est\u003c/sup\u003e pulse, are similar (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.84, genotype \u003cem\u003ep\u003c/em\u003e=0.50). \u003cstrong\u003eiv)\u003c/strong\u003e Decay of the last response in pulse train (P10) is similar between VKI and WT iGluSnFR responses (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.17). \u003cstrong\u003eC \u003c/strong\u003eAt 6 months, \u003cstrong\u003ei)\u003c/strong\u003e VKI vs WT iGluSnFR response size is similar in VKI and WT brain slices (2-way ANOVA\u003cstrong\u003e \u003c/strong\u003einteraction \u003cem\u003ep\u003c/em\u003e=0.90, genotype \u003cem\u003ep\u003c/em\u003e=0.40). \u003cstrong\u003eii) \u003c/strong\u003eNormalized VKI iGluSnFR responses are not significantly different from WT (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.77, genotype \u003cem\u003ep\u003c/em\u003e=0.87). \u003cstrong\u003eiii) \u003c/strong\u003eVKI recovery capacity after 10x10Hz stimulation is similar to WT capacity (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.63, genotype \u003cem\u003ep\u003c/em\u003e=0.12). \u003cstrong\u003eiv)\u003c/strong\u003e Decay of the last response to 10x10Hz stimulation is not different between VKI and WT iGluSnFR responses (Mann-Whitney U-test \u003cem\u003ep\u003c/em\u003e=0.33).\u003c/p\u003e","description":"","filename":"Figure31.png","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/df0ce7926d4729445c9128bc.png"},{"id":79548926,"identity":"8dbbd141-279e-41f6-89a4-2cd2917cd3a2","added_by":"auto","created_at":"2025-03-31 06:25:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1869214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edLight responses to electrical stimulation are not altered in VKI brain slices at 3 months.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA i) \u003c/strong\u003eGraphic depicting stereotaxic injection of AAVs encoding for dLight in dorsolateral STR 4-6 weeks prior to acute slice preparation. Area of dLight expression in coronal slices containing CTX and STR depicted in blue with Stim placed in dorsolateral STR. \u0026nbsp;\u003cstrong\u003eii) \u003c/strong\u003eVisualization of dLight change in fluorescence from baseline (ΔF/F) in in response to local electrical stimulation (ΔF/F values of 0 to 1 represented as a gradient from purple to white, respectively). Boundaries of the CC separate CTX and STR in a coronal brain slice. Dashed circle represents ROI of 40 pixels used for dLight analysis. \u003cstrong\u003eiii) \u003c/strong\u003eRepresentative dLight response in dorsolateral striatum corresponding to 2 pulses delivered with 4s inter-pulse interval.\u003cstrong\u003e \u003c/strong\u003eAt 3 months \u003cstrong\u003eiv) \u003c/strong\u003eIncreasing stimulus intensity did not alter size of dLight responses in VKI vs WT STR (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.69, genotype \u003cem\u003ep\u003c/em\u003e=0.83). \u003cstrong\u003ev)\u003c/strong\u003e Decay of the 1\u003csup\u003est\u003c/sup\u003e response peak (P1) is similar between VKI and WT brain slices (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.57). \u003cstrong\u003evi)\u003c/strong\u003e PPR at 4s (P2 Peak/P1 Peak) is similar between VKI and WT brain slices (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.21). \u003cstrong\u003eB i) \u003c/strong\u003eRepresentative dLight response in dorsolateral STR corresponding to 4x stimulation with 10x10Hz pulse train and subsequent recovery stimulation.\u003cstrong\u003e \u003c/strong\u003eAt 3 months \u003cstrong\u003eii)\u003c/strong\u003e dLight responses were not significantly different in size between VKI and WT brain slices (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.99, genotype \u003cem\u003ep\u003c/em\u003e=0.85). \u003cstrong\u003eiii) \u003c/strong\u003edLight responses relative to 1\u003csup\u003est\u003c/sup\u003e pulse (P1) were not different between VKIs and WTs (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.69, genotype \u003cem\u003ep\u003c/em\u003e=0.45). \u003cstrong\u003eiv)\u003c/strong\u003e Recovery capacity following 10x10Hz stimulation are lower in VKI brain slices (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.81, genotype \u003cem\u003ep\u003c/em\u003e=0.06). \u003cstrong\u003ev) \u003c/strong\u003eDecay of the pulse train response is not significantly different in VKI vs WT brain slices (Unpaired t-test \u003cem\u003ep\u003c/em\u003e=0.28).\u003c/p\u003e","description":"","filename":"Figure41.png","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/77cec6c45b561d27e302cc2c.png"},{"id":79548217,"identity":"4540deb5-1a30-42f1-8b2a-9663c226ebd5","added_by":"auto","created_at":"2025-03-31 06:17:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1123513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDopamine release in VKI brain slices at 6 months is elevated and can be reduced with acute MLi-2 treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA i) \u003c/strong\u003eGraphic depicting stereotaxic injection of AAVs encoding for dLight into the dorsolateral striatum 4-6 weeks prior to acute slice preparation. Coronal slices containing CTX and STR were subsequently incubated ≥1.5h with either vehicle Captisol® or 500nM MLi-2 prior to recording. dLight expression is depicted in blue with Stim in dorsolateral STR. \u003cstrong\u003eii)\u003c/strong\u003e Representative 2-pulse stimulation with 4s inter-pulse interval with accompanying dLight response.\u0026nbsp; \u003cstrong\u003eB \u003c/strong\u003edLight responses were analyzed together (WT veh vs VKI veh vs WT MLi-2 vs VKI MLi-2) and graphed separately. \u003cstrong\u003ei)\u003c/strong\u003e VKI responses are increased across increasing stimulus intensities compared to WT responses (2-way ANOVA interaction p=0.0003, genotype p=0.0001, Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test @50µA \u003cem\u003ep\u003c/em\u003e=ns, 100-400 µA \u003cem\u003ep\u003c/em\u003e \u0026gt;0.05). \u003cstrong\u003eii) \u003c/strong\u003eMLi-2 treatment does not alter WT dLight responses (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test \u003cem\u003ep\u003c/em\u003e=ns).\u003cstrong\u003e iii) \u003c/strong\u003eMLi-2 treatment reduces elevated dLight responses in VKI brain slices (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test @50 µA \u003cem\u003ep\u003c/em\u003e=0.07, @100-400 µA \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003cstrong\u003e iv) \u003c/strong\u003eDecay of 1\u003csup\u003est\u003c/sup\u003e response is not different between VKI and WT brain slices and is not sensitive to MLi-2 treatment (Kruskal-Wallis \u003cem\u003ep\u003c/em\u003e=0.66). \u003cstrong\u003ev) \u003c/strong\u003ePPR at 4s interval is not different between VKI and WT brain slices and is not sensitive to MLi-2 treatment (Kruskal-Wallis \u003cem\u003ep\u003c/em\u003e=0.69).\u003c/p\u003e","description":"","filename":"Figure51.png","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/ae741c1479d2ed225048a8ef.png"},{"id":79548924,"identity":"24ddede8-38cb-4949-be6d-eb502fc65119","added_by":"auto","created_at":"2025-03-31 06:25:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1756410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStriatal dopamine release with train stimulation is larger in VKI brain slices and sensitive to acute MLi-2 treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA i) \u003c/strong\u003eGraphic depicting stereotaxic injection of AAVs encoding for dLight into the dorsolateral STR 4-6 weeks prior to acute slice preparation. Coronal slices containing CTX and STR were subsequently incubated \u0026gt;1.5h with either vehicle Captisol® or 500nM MLi-2 prior to recording. dLight expression is depicted in blue with Stim in dorsolateral STR. \u003cstrong\u003eii)\u003c/strong\u003e Representative response to pulse train stimulation (10x10Hz with repeated 4x with increasing 11\u003csup\u003eth\u003c/sup\u003e recovery pulse interval). \u003cstrong\u003eB-D \u003c/strong\u003edLight response parameters are shown separated by genotype in vehicle treatment and within\u003cstrong\u003e \u003c/strong\u003egenotypes by treatment condition. Statistics were performed using 2-way ANOVA on WT veh vs VKI veh vs WT MLi-2 vs VKI MLi-2 with relevant post-hoc comparisons. \u003cstrong\u003eB i) \u003c/strong\u003edLight response size is significantly higher in VKI vs WT vehicle-treated brain slices (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, genotype \u003cem\u003ep\u003c/em\u003e=0.003, Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test @P1-P4 \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, P5 \u003cem\u003ep\u003c/em\u003e=0.06, P6-P10 \u003cem\u003ep\u003c/em\u003e=ns). \u0026nbsp;\u003cstrong\u003eii) \u003c/strong\u003edLight responses, when normalized to the first peak, are similar between VKI and WT vehicle-treated brain slices (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, genotype \u003cem\u003ep\u003c/em\u003e=0.2393, Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test @P1-P10 \u003cem\u003ep\u003c/em\u003e=ns). \u003cstrong\u003eiii) \u003c/strong\u003eRecovery capacity following high frequency stimulation is significantly altered in VKI vs WT vehicle-treated brain slices (2-way ANOVA interaction \u003cem\u003ep\u003c/em\u003e=0.02, genotype \u003cem\u003ep\u003c/em\u003e=0.19, Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test @500ms \u003cem\u003ep\u003c/em\u003e=0.04, 1000-5000ms \u003cem\u003ep\u003c/em\u003e=ns). \u003cstrong\u003eC i) \u003c/strong\u003edLight response peaks in WT brain slices are not altered with acute MLi-2 treatment (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test \u003cem\u003ep\u003c/em\u003e=ns). \u003cstrong\u003eii) \u003c/strong\u003edLight responses normalized to the first peak are not altered with MLi-2 treament (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test \u003cem\u003ep\u003c/em\u003e=ns). \u003cstrong\u003eiii) \u003c/strong\u003eRecovery after train stimulation is not altered with MLi-2 in WT brain slices (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test \u003cem\u003ep\u003c/em\u003e=ns). \u003cstrong\u003eD i) \u003c/strong\u003edLight response peaks in VKI brain slices are significantly reduced with MLi-2 treatment (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test @P1-P10 \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). \u003cstrong\u003eii) \u003c/strong\u003eNormalized dLight responses\u003cstrong\u003e \u003c/strong\u003ein VKI brain slices are not altered with MLi-2 treatment (Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test \u003cem\u003ep\u003c/em\u003e=ns). \u003cstrong\u003eiii) \u003c/strong\u003eRecovery after train stimulation is not altered with MLi-2 treatment in VKI brain slices\u003cstrong\u003e \u003c/strong\u003e(Šídák's\u0026nbsp;multiple comparisons\u0026nbsp;test \u003cem\u003ep\u003c/em\u003e=ns).\u003cstrong\u003e E) \u003c/strong\u003eDecay of pulse train response is not significantly different between VKI vs WT brain slices with vehicle or MLi-2 treatment (1-way ANOVA \u003cem\u003ep\u003c/em\u003e=0.21).\u0026nbsp;\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"Figure62.png","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/c899c81546cf80c6ea6ffb5e.png"},{"id":81988024,"identity":"51badb90-f5f6-4ab6-bcf6-e61a965f5713","added_by":"auto","created_at":"2025-05-05 16:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13390694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/d3fbc0d9-6243-460e-a0dd-9292a12052ca.pdf"},{"id":79548220,"identity":"0ff2252b-bd2c-40c7-8874-0ec825b5f3a6","added_by":"auto","created_at":"2025-03-31 06:17:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":392051,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5167163/v1/23aaf0c882dfc0f5e69133f3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emergent glutamate \u0026 dopamine dysfunction in VPS35(D620N) knock-in mice and rapid reversal by LRRK2 inhibition","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA simplified definition of Parkinson\u0026rsquo;s disease (PD) is that of a movement disorder, resulting from the death of dopamine neurons in the substantia nigra pars compacta (SNpc). Within ~\u0026thinsp;4 years of clinical presentation, there is an almost complete loss of nigrostriatal dopamine axon markers. Whereas, the loss of nigral neurons is less severe (30\u0026ndash;60%) and remains fairly stable thereafter\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The resilience of a population of nigral neurons\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and the restorative effect of dopamine replacement therapy (e.g., L-DOPA) against motor symptoms\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e offer hope that at least some function can be restored in remaining neurons after disease onset.\u003c/p\u003e \u003cp\u003eUnfortunately, no available PD treatment has been shown to slow or prevent disease progression. Furthermore, several disturbances that precede motor dysfunction by many years, e.g., rapid eye movement (REM) sleep behaviour disorder, anosmia, constipation, mood changes, and cognitive decline; are unresponsive to L-DOPA\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Many neuronal populations, in addition to nigral dopamine neurons, degenerate in PD; notably in the pedunculopontine nucleus and locus coeruleus containing cholinergic and noradrenergic neurons, as well as glutamatergic nuclei in the cortex and thalamus\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Thus, the evidence implicates multiple neurotransmitter systems in both early symptom manifestation and disease progression.\u003c/p\u003e \u003cp\u003eA point of convergence of multiple neurotransmitter systems in PD pathology are the spiny projection neurons (SPNs) of the dorsolateral striatum\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. SPNs integrate extensive cortical and thalamic glutamate input with modulation by nigrostriatal dopamine to direct behavioural action selection, via downstream basal ganglia nuclei\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Too much striatal glutamate and dopamine transmission, however, may be toxic\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and could act as the initial pathophysiological stress that causes retrograde degeneration and selective loss of SNpc dopaminergic inputs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePD is thought to emerge from the combination of genetic predisposition and environmental stress, especially age\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To investigate how these factors precipitate a Parkinsonian state, we developed VKI mice expressing the VPS35 D620N variant linked to clinically-typical PD\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Heterozygote VKI mice (which model the autosomal dominant presentation in humans) develop tau pathology and nigral loss at \u0026gt;\u0026thinsp;16 months\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Thus, VKI mice model human PD pathology at advanced ages and are appropriate for the study of early pathophysiological mechanisms.\u003c/p\u003e \u003cp\u003eWe previously reported increased glutamate transmission in VKI cortical cultures and increased dopamine release in brain slices of young VKI mice\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These changes correlated with hyperphosphorylation of leucine-rich repeat kinase 2 (LRRK2) substrates, the kinase believed to be hyperactive in LRRK2-familial and idiopathic PD\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In cultures, despite reversal of LRRK2 substrate hyperphosphorylation, glutamate alterations were resistant to LRRK2 inhibition\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Contrastingly, 1-week \u003cem\u003ein vivo\u003c/em\u003e LRRK2 inhibition rescued decreased dopamine transporter (DAT) protein levels, and normalized dopamine release in VKI brain slices\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere we sought to define the emergence of glutamate and dopamine synaptic dysfunction as young VKI mice mature, and test whether acute LRRK2 kinase inhibition modifies dopamine release. We found that glutamate and dopamine transmission become elevated by 6 months of age. Elevated dopamine release was rapidly reversed by acute (\u0026ge;\u0026thinsp;1.5h) LRRK2 kinase inhibition of slices, without evidence of changes to DAT function. We conclude that striatal glutamate and dopamine transmission is progressively increased in VKI mice throughout young adulthood, and that augmented dopamine release is a function of LRRK2 kinase hyperactivity. The data support the argument that LRRK2 inhibition is likely beneficial against synaptic dysfunction in PD and provide a model framework in which to test the neuroprotective potential of LRRK2 inhibitors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVPS35 D620N knock-in (VKI) were generated as previously described\u003csup\u003e22\u003c/sup\u003e and maintained on a C57Bl6/J wild-type (WT) background. Mice were housed and bred in accordance with the Canadian Council on Animal Care regulations (Animal Use Protocol 2017-7888B). All procedures were approved by and governed in accordance with the Neuro Centre of Neurological Disease Models (Animal Use Protocol 2017-7888B) and Memorial University Animal Care Committee (Animal Use Protocol 18-01-MP). 1-, 3-, and 6-month-old male heterozygous VKI and WT mice were used for all measures of spontaneous glutamate transmission. 3- and 6- month male heterozygous VKI and WT mice were used for all experiments requiring stereotaxic injections as these surgeries were performed \u0026gt;4 weeks in advance of experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenotyping of all mice was conducted on tail samples at weaning and on post-mortem ear tissue. Samples were digested in 100\u0026micro;L 10% Chelex (Bio-Rad 142\u0026ndash;1253; 20 minutes 95\u0026deg;C, 2x vortex) and centrifuged (2 minutes 12,000 RPM) before DNA-containing supernatant (2\u0026micro;L) was added to PCR master mix (18\u0026micro;L, Qiagen 201203: taq polymerase, DNAse and RNAse-free water, 10X buffer, 10mM dNTPs, and custom DNA oligo primers [ThermoFisher: Forward-TGGTAGTCACATTGCCTCTG, Reverse-ATGAACCAACCATCAATAGGAACAC]). DNA was amplified by PCR (program cycle available upon request) and combined with fluorescent DNA intercalating dye (ZmTech LB-001G) prior to iontophoresis. 10\u0026micro;L of PCR product was run in 4% agarose gel and visualized (BioRad UV gel imager) for the presence of 1 or 2 bands to determine WT vs VKI genotype, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChannelrhodopsin-2 (ChR2), intensity-based glutamate-sensing fluorescent reporters (iGluSnFR), and D1 dopamine receptor-based fluorescent reporters (dLight1.3b) were delivered by stereotaxic injection of AAV constructs as detailed below. 4-6 weeks prior to slice preparation, mice were subcutaneously injected with carprofen (2-4 mg/mL, 20mg/kg, 0.9% NaCl), left to rest for \u003cu\u003e\u0026gt;\u003c/u\u003e15 minutes, then anaesthetized with isoflurane (5% induction, 1-2% maintenance) and secured in a stereotaxic head frame (Kopf Instruments). The local analgesic, Marcaine, was injected subcutaneously below the site of incision and hair was removed chemically (Nair) or mechanically (Wahl clippers). An incision was made and skull, leveled using Bregma and Lambda as points of reference. A 0.5mm craniotomy was then created (micro-burr dentistry drill) over the site of injection. A 10\u0026micro;L syringe (Nanofil), attached to a microinjector (Harvard Apparatus Pump11 Elite) was lowered, with coordinates zeroed to the brain surface. Following AAV injection (details below), a 5-minute settling period was allowed before removing the needle. The scalp was then rehydrated with Marcaine, sutured (4-0 silk; Ethicon 683G) and reinforced (Vetbond 3M 1469SB), prior to subcutaneous 0.9% NaCl injection to replace fluids (0.2-0.5mL/10mg). Mice were monitored for pain and discomfort after regaining consciousness and returned to home cage in ~1 hour. Post-operative monitoring (3 days) included daily subcutaneous carprofen injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruct-specific injection details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor ChR2 optogenetic stimulation, AAV9-CAG-ChR2-mCherry (Neurophotonics Centre, Universit\u0026eacute; Laval, lot #834 = 4x10\u003csup\u003e12\u003c/sup\u003e GC/mL) was injected unilaterally (650nL, 1nL/sec) into primary motor cortex (1.5 mm anterior, 1.0 mm lateral, 0.8 mm ventral to Bregma). For optogenetic recording of glutamate release, AAV1.hSyn.iGluSnFr.WPRE.SV40 (Addgene 98929-AAV1, 2.8x10\u003csup\u003e13\u0026nbsp;\u003c/sup\u003eGC/mL) was injected bilaterally (1uL, 2nL/sec) into the dorsolateral striatum (coordinates: 2.0 mm anterior, 1.0 lateral, 3.2 ventral to Bregma). For optogenetic recording of dopamine release, AAV5-CAG-dLight1.3b (Addgene 125560-AAV5, 1.5x10\u003csup\u003e13\u003c/sup\u003e GC/mL) was injected bilaterally (300nL, 1nL/sec) into the dorsolateral striatum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of acute brain slices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e300\u0026micro;m acute coronal brain slices were prepared from restrained mice which were swiftly decapitated. Brains were quickly transferred to ice-cold recovery solution (1 minute; in mM: 93 NMDG, 93 HCl, 2.5 KCl, 1.2 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 30 NaHCO\u003csub\u003e3\u003c/sub\u003e, 20 HEPES, 25 glucose, 5 sodium ascorbate, 3 sodium pyruvate, 10 MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.5 CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, pH 7.3-7.4, 290-310 mOsm; carbogen-infused). Following this, extra-striatal rostral and caudal brain regions were removed by coronal sectioning, and the remaining block was mounted onto a vibrating-blade microtome platform with sodium acrylate (Leica Microsystems VT 1200S). After hemisection at the midline, coronal sections were cut, and slices were then transferred to warm recovery solution (35\u0026deg;C, 15 minutes). A final transfer of slices into holding chambers containing room-temperature artificial cerebrospinal fluid (aCSF; in mM: 125 NaCl, 2.5 KCl, 25 NaHCO\u003csub\u003e3\u003c/sub\u003e, 1.25 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2 MgCl\u003csub\u003e2\u003c/sub\u003e, 2 aCl\u003csub\u003e2\u003c/sub\u003e, 10 glucose, pH 7.2-7.4, 300-310 mOsm, 22-25\u0026deg;C, carbogen-infused) was followed by \u003cu\u003e\u0026gt;\u003c/u\u003e45 minutes settling period before recording in aCSF began.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-cell patch-clamp electrophysiology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-cell patch-clamp electrophysiology was used to obtain recordings of spontaneous and ChR2-evoked glutamate transmission in SPNs of the dorsolateral striatum (caudate/putamen), as previously\u003csup\u003e30\u003c/sup\u003e. Slices were transferred to a recording chamber perfused with aCSF (22-25\u0026deg;C) containing 100\u0026micro;M picrotoxin (Tocris 1128) at a flow rate of ~1.5mL/min. Slices were visualized on an Olympus BX51 microscope (40x magnification, 2x digital zoom, IR-DIC and Q-Imaging Electro camera). SPNs were identified within the dorsolateral striatum based on somatic size (8-20\u0026micro;M) and distinct morphology, 50-150\u0026micro;M below the surface of the slice. Borosilicate glass capillary tubes (Harvard Apparatus 640805) were pulled using a Sutter P-1000 micropipette puller to form 1\u0026micro;m tip recording pipettes (filled resistance 4-8 MOhms; in mM: 130 Cs methanesulfonate, 5 CsCl, 4 NaCl, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 5 EGTA, 10 HEPES, 5 QX-314, 0.5 GTP, 10 Na\u003csub\u003e2\u003c/sub\u003e phosphocreatine, 5 MgATP, and 0.1 spermine, pH 7.2, 290mOsm). A motorized micromanipulator (Sutter Instrument MP-285) was used to patch onto identified SPNs, with signals obtained by MultiClamp 700B amplifier in voltage-clamp configuration, filtered at 2kHz and digitized at 10kHz (Molecular Devices Axon Digidata 1440A). Membrane properties were determined with the membrane-test function while holding at -70mV. Synaptic recordings were initiated after a 2-minute settling period, with access resistance tolerance set to 27MW\u0026nbsp;and recordings discarded if\u0026nbsp;D\u003cu\u003e\u0026gt;\u003c/u\u003e10% over the elapsed period. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded over a minimum 2-minute period at -70mV (gain 20) and analyzed in Clampfit10 (Molecular Devices; 5pA peak event threshold, confirmed manually). Non-unitary events were retained for inter-event interval analysis, but only unitary events were used for amplitude and decay constants. Cumulative distributions were used to evaluate amplitude and inter-event intervals in each recorded SPN. Unitary events in each recording were averaged and decay tau measured by 1-term exponential fit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChR2-evoked post-synaptic currents (ChR2-PSCs) were stimulated by wide-field illumination with blue light (473nm, XCite Series 120Q) transmitted through the 40x immersion objective generating 2.7 mW. 5ms pulses were controlled by a Lambda SC Smart Shutter Controller (Sutter Instruments) administered in trains of 4 pulses with 100ms inter-pulse intervals. Trains were repeated every 30 seconds, 5-10 times and averaged for analysis. Recordings of ChR2-PSCs mediated by \u0026alpha;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were taken at -70mV (gain 2) averaged across 5 repetitions. AMPAR and N-methyl-D-aspartate receptor (NMDAR) currents were generated by single 5ms pulse stimulations repeated 5x every 30 seconds at -70mV then +40mV to assess AMPAR+NMDAR-mediated ChR2-PSCs, respectively. Peak NMDAR current was estimated at +40mV, 40ms post AMPA peak, and AMPAR currents were then isolated at +40mV by bath application of 10\u0026micro;M D-APV (NMDAR blocker, Tocris 0106). AMPA rectification indices were calculated as a ratio of isolated AMPAR ChR2-PSC peak at +40mV : -70mV. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiGluSnFR and dLight imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImaging of iGluSnFR and dLight recordings were obtained using a 2X objective focused on dorsolateral STR. Slices were wide-field illuminated with blue light (473nm, CoolLED pE-340fura), with the shutter, EM-CCD camera (Andor iXon Ultra 897), and stimulus isolator (WPI A365) triggered by Clampex \u0026amp; Digidata 1550B). The stimulus isolator was connected to a monopolar tungsten stimulating electrode (A-M Systems 574000) lowered into dorsolateral striatum 50-100\u0026micro;M beneath the surface of the tissue. Recordings were captured using Andor Solis software using 4x4 binning at 205Hz. For these experiments, spontaneous iGluSnFR \u0026amp; dLight transients were excluded from analysis of responses to electrical stimulation. For iGluSnFR recordings, a stimulation train was delivered at fixed stimulation intensity (150uA train of 10 x 0.2ms pulses with 100ms inter-pulse intervals). No-stimulation (for background \u0026amp; bleach subtraction) and stimulation trials were alternated at 1-minute intervals, repeated every 2 minutes, until 5 no-stimulation trials were recorded. An 11\u003csup\u003eth\u003c/sup\u003e pulse was delivered with each repetition of the stimulation trial, with increasing inter-pulse intervals between 500-5000ms. The same protocol was used to obtain pulse train dLight recordings after 2-pulse dLight stimulation with 4s inter-pulse intervals, delivered at 50-400uA, every 2 minutes to generate stimulus response curves. Responses were then assessed to determine the stimulus intensity corresponding to 50-70% maximum response (FIJI software) for subsequent pulse train dLight experiments. \u0026nbsp;iGluSnFR and dLight videos were converted to the change in fluorescence intensity over time (DF/F; FIJI software) with peak and decay analyzed in Clampfit (decays measured by the 1-term exponential function of each trace). Absolute and normalized peak and decay of pulse train responses were derived from the average of 4 responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcute LRRK2 kinase inhibition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSlices from 6-month-old mice were pre-incubated \u0026ge;1.5hours and then bath-perfused during dLight recording with LRRK2 kinase inhibitor MLi-2 (500 nM, Tocris 5756, 45% Captisol\u0026reg; in PBS) or vehicle-control (45% Captisol\u0026reg; in PBS 377.6\u0026micro;L/L aCSF). Captisol\u0026reg; vehicle was also present in all 3-month-old dLight recordings to be able to compare dLight recordings across age.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presentation and statistics were conducted in GraphPad Prism10. Data were tested against ROUT outlier analysis with a liberal maximum false discovery rate Q=1%. Rarely, identified outliers were suppressed from the dataset prior to parametric or non-parametric distributions testing (D\u0026rsquo;Agostino and Pearson). Unpaired t-test/1-way ANOVA \u0026nbsp;(if parametric) or Mann-Whitney U-test/Kruskal-Wallis testing (if non-parametric) were used to analyse data. \u003cem\u003ePost-hoc\u0026nbsp;\u003c/em\u003eanalysis was performed if ANOVA \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 by Tukey\u0026rsquo;s (parametric) or Dunn\u0026rsquo;s (non-parametric) multiple comparisons tests as appropriate. All 2-way ANOVA comparisons used \u0026Scaron;\u0026iacute;d\u0026aacute;k post-hoc analyses. Asterisks represent \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 from pair-wise comparisons. All statistical analyses are described in figure legends, and \u003cem\u003epost-hoc\u003c/em\u003e analyses are reported in Tables S1 \u0026amp; S2. Trends are defined as 0.10 \u0026gt; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05. \u0026nbsp;Data are presented as n=observations from (n) animals (e.g., WT = 6(3) is 6 observations from 3 WT animals).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eVKI SPNs show progressive increase in spontaneous activity by 6 months\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe compared spontaneous excitatory postsynaptic currents (sEPSCs) by whole-cell patch-clamp as before\u003csup\u003e30\u003c/sup\u003e in VKI and WT SPNs from 1-, 3-, and 6-month-old mice (Figure 1 A). Although similar at 1 month, we found that VKI sEPSCs begin to diverge from WT at 3 months with larger amplitudes. VKIs exhibit robustly elevated amplitude and frequency (ie. reduced inter-event interval) by 6 months (Figure 1B-D). The decay of sEPSCs was faster in VKI SPNs at 3 months but normalized to WT levels at 6 months. An age-dependent decrease in WT sEPSC amplitude and frequency was absent in VKI SPNs (Table S2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also observed early reductions in membrane tau of 1-month-old VKI SPNs, that normalized to WT levels by 3 months (Figure S1 A,B,C iii). VKI SPNs also trend towards increased membrane capacitance at 3- and 6-months, but not at 1 month (Figure S1 A,B,C i). Membrane resistance was reduced by 6 months in VKI SPNs (Figure S1 A, B, C, ii). As with sEPSC properties, membrane capacitance and resistance measures were altered with age within WT SPNs, but not in VKI neurons (Table S2). Together, age-dependent changes to passive membrane properties, and a progressive (relative) increase in the amplitude, frequency, and decay of spontaneous glutamate transmission is observed in VKI SPNs. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorticostriatal glutamate transmission is progressively increased in VKI mice by 6 months\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether increased frequency and amplitude of sEPSCs are due to changes in corticostriatal inputs, optogenetic stimulation of glutamate release onto patched SPNs was conducted at 3 and 6 months (Figure 2 A i). Light stimulation of local cortical terminals generated ChR2-induced post-synaptic currents (ChR2-PSCs). Glutamatergic currents through AMPA and NMDA receptors were isolated by membrane gating properties (ie. holding potential) and by pharmacology with the NMDAR blocker,\u0026nbsp;D-AP5 (Figure 2 A ii). At 3 months, we found no change to VKI AMPAR- or NMDAR-mediated current amplitude. The ratio of NMDA:AMPA current and AMPAR-rectification indices were likewise similar (Figure 2 B). In contrast, we found AMPAR and NMDAR currents were both (equally) elevated in 6-month-old VKIs (Figure 2 C i-ii). There was no change in NMDA:AMPA ratio, or AMPA rectification index at this age (Figure 2 C iii-iv).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess presynaptic probability of release, we quantified paired-pulse ratios (PPRs) at 100ms intervals (Figure 2 D). There was no significant difference to PPRs in VKI neurons at 3 months, but a significant reduction in PPR was observed in 6-month-old VKI SPNs, indicative of elevated probability of presynaptic release (Pr). Taken together, the data show corticostriatal glutamate transmission becomes elevated in VKI SPNs, relative to WT littermates, and that this correlates with an equivalent increase in both AMPAR and NMDAR transmission. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal striatal glutamate release appears reduced in 3-month-old VKIs and normalizes to WT levels by 6 months.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to changes in presynaptic release, glutamate currents recorded in SPNs are modulated by regulation of postsynaptic responsiveness, thalamic glutamate, midbrain dopamine, and GABAergic interneuron activity\u003csup\u003e31\u0026ndash;38\u003c/sup\u003e. To directly assess presynaptic release, extracellular glutamate transients were quantified with the intensity-based glutamate-sensing reporter, iGluSnFR1 (Figure 3 A) in response to fixed intensity pulse-trains (10x10Hz) delivered by local striatal electrical stimulation. The amplitude of iGluSnFR responses was markedly reduced in 3-month-old VKI striata, and there was a significant interaction between the normalized peak and pulse number (Figure 3 B i-ii and Table S1). The recovery capacity of iGluSnFR responses following the pulse train was similar at 3 months, as was the decay of the pulse train response (Figure 3 B iii-iv). At 6 months, iGluSnFR responses were similar to WT responses across all measures (Figure 3 C). Interestingly, a significant age-dependent reduction in the amplitude of iGluSnFR transients was only observed for WT and not VKI responses (Table S2). No age dependent changes were observed in other parameters except for the decay of iGluSnFR responses in VKI striata between 3 and 6 months, which appear significantly increased with age. In summary, total striatal glutamate release is reduced in VKI mice at 3 months, but similar to WT at 6 months, suggesting that total presynaptic glutamate release is relatively increased in the VKI striatum with age. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncreased striatal dopamine release appears in 6-month-old VKI mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe measured extracellular dopamine transients in the dorsolateral striatum using the dLight fluorescence reporter, evoked by electrical stimulation in slices from 3- and 6-month-old mice (Figure 4-6).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWe did not observe any changes to dLight amplitudes, or responses to pulse-train stimulation in 3-month-old VKI striata (Figure 4 A); however, recovery capacity following pulse train stimulation was significantly reduced in VKIs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 6 months, dLight response peaks were significantly increased in VKI brain slices, without any change in decay tau, or PPR at 4s intervals (Figure 5 and Table S1). Pulse train response peaks in VKIs were accordingly increased, without changes in pulse-train response pattern (Figure 6). However, recovery following train stimulation was reduced at smaller intervals and increased at larger intervals. WT responses show amplitude reductions between 3 and 6 months, which are absent in VKI responses (Table S2). There is also a significant change to the pattern of normalized responses to pulse train stimulation with age in VKI striata, but not WT. Decay of responses were not significantly different in either WTs or VKIs when comparing 3- to 6-month-old mice. Therefore, the data indicate dopamine release is progressively increased in VKIs, relative to WT, by 6 months old.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncreased dopamine release in VKI striata is reversed with acute LRRK2 kinase inhibition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether the robustly elevated dopamine release in 6-month-old VKI striata could be targeted with acute LRRK2 kinase inhibition. Slices prepared for dLight recordings were incubated either in vehicle Captisol\u0026reg; or in 500nM of LRRK2 kinase inhibitor, MLi-2, for \u003cu\u003e\u0026gt;\u003c/u\u003e1.5 hours (Figure 5 A). Elevated VKI dLight responses were reversed by LRRK2 inhibition, without altering decay constants, or 4s interval PPRs (Figures 4-6, and Table S1). Although increased amplitudes were similarly reversed in pulse-train experiments, there were no significant effect of MLi-2 on the pattern of responses to stimulation trains. Further, MLi-2 had no effect on WT dLight responses. In conclusion, acute LRRK2 kinase inhibition with MLi-2 reversed the elevated dopamine release in VKI striata at 6 months, without altering dopamine release in WT.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreviously, we found elevated glutamate transmission in 3- to 4-week-old VKI mouse cortical cultures\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and elevated striatal dopamine transmission in slices from VKI mice aged 3 months\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Here, we find corticostriatal glutamatergic transmission onto SPNs is normal at 1 month, but progressively increases to \u0026gt;\u0026thinsp;50% over WT by 6 months. Intriguingly, total striatal glutamate release measured by iGluSnFR was reduced at 3 months and increased to match WT levels by 6 months. In contrast, striatal dopamine release measured by dLight was markedly elevated in VKI striata by 6 months and reversed by acute LRRK2 kinase inhibition.\u003c/p\u003e \u003cp\u003eSPNs of the dorsolateral striatum are the initial point of convergence for control of behavioural action selection, habit formation, and movement initiation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These functions are all altered in PD, and pathology is clearest first in nigrostriatal axons\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Our data suggest SPNs in VKI mice develop changes to passive membrane properties throughout young-adulthood, which could reflect functional and/or anatomical differences. Membrane capacitance is proportional to membrane area, whereas membrane resistance inversely correlates to process diameter (and usually correlates with capacitance). These properties were bidirectionally altered in 6-month-old VKI SPNs, indicative of a larger soma, neurites, and/or processes, and increased leak channel conductance\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe observed a subtle increase in amplitude and reduced decay time of spontaneous glutamate events in 3-month-old VKI SPNs, but no changes to optogenetically-evoked corticostriatal AMPAR or NMDAR currents. This suggests any modestly increased response to quantal release in a proportion of synapses is not sufficient to be detected in large currents generated by activity-dependent release. In light of relatively unchanged corticostriatal transmission in SPNs, it was surprising to see that iGluSnFR measures of total glutamate release were markedly reduced in VKI mice at 3 months. This could be explained by an increased number of active synapses, with lower individual release probabilities, or increased postsynaptic responsiveness. Since we previously quantified no change to glutamate synapse number in VKI mice at 3 months\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, it may be that increased postsynaptic responsiveness exists as an attempt to compensate for one another (or \u003cem\u003evice versa\u003c/em\u003e), thereby keeping transmission onto SPNs within a homeostatic range. Alternatively, glutamate release onto SPNs may be unaltered, but reduced at other non-SPN synapses (e.g., excitatory input to cholinergic interneurons\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e), inviting future interrogation by cell-type.\u003c/p\u003e \u003cp\u003eBy 6 months, clearer increases in SPN spontaneous event amplitude were accompanied by increased frequency (ie. decreased inter-event intervals). At this age, VKI SPN corticostriatal AMPAR- and NMDAR-current amplitudes were also increased\u0026thinsp;~\u0026thinsp;50% over WT. More frequent spontaneous activity and higher PPRs, which we also observe in corticostriatal evoked responses, are usually interpreted as increased probability of presynaptic release. Larger AMPAR and NMDAR currents could also be a result of higher postsynaptic receptor numbers. We and others have reported increased GluA1-containing AMPAR receptor expression with VPS35 D620N and LRRK2 G2019S knock-in\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, but increases to both NMDAR and AMPAR expression, here, would have to be equal to explain the lack of difference in NMDAR:AMPAR current ratios here. Thus, the parsimonious explanation is that of increased probability of release, as we reported \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Here, direct measures of total striatal presynaptic release with iGluSnFR found no difference between VKI and WT striata, suggesting that corticostriatal transmission onto SPNs must be selectively increased in VKIs. Alternatively, local electrical stimulation may activate the negative tuning of corticostriatal synapses through presynaptic D2 receptors\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, which would reduce total striatal glutamate release by iGluSnFR imaging, but not affect the direct activation of corticostriatal release with ChR2. Together, the data reveal an emergent increase in cortical glutamate transmission onto VKI SPNs by 6 months, perhaps to compensate for a reduction in total glutamate release observed at 3 months (or \u003cem\u003evice versa\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eWhile not required for diagnosis, changes to glutamate transmission are observed in PD, and likely impact non-motor and motor features of the disease\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Elevated glutamate transmission is seen early in PD-relevant mice carrying knock-in LRRK2 G2019S mutations, and those overexpressing synuclein\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Therefore, the progressive dysregulation of glutamate in young adult VKI mice is consistent with other models of PD, and may represent a direct contribution to (or compensation against) pathophysiological progression to degeneration.\u003c/p\u003e \u003cp\u003ePreviously, we found dopamine release was elevated in VKI mice at 3 months by fast-scan cyclic voltammetry (FSCV)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This was accompanied by slower decay constants, indicative of reduced DAT activity, and a reduction of DAT protein expression\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Strikingly, increased dopamine release in 3-month-old VKI slices was rescued by chronic 1-week \u003cem\u003ein vivo\u003c/em\u003e administration of LRRK2 kinase inhibitor, MLi-2\u003csup\u003e24\u003c/sup\u003e. LRRK2 kinase inhibition also normalized the reduced DAT levels, which we concluded was causal to the rescue\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Here, we aimed to determine whether the rescue can be observed with shorter-term LRRK2 inhibition (\u0026ge;\u0026thinsp;1.5h), indicating that dysregulation of dopamine release machinery is LRRK2 kinase-dependent, and not due to systemic effects of LRRK2 kinase inhibition. As FSCV offers low temporal (10Hz) and spatial resolution (limited by the carbon fiber electrode surface area), we employed dLight1.3b to assay dopamine release across the entire dorsolateral striatum at 20x higher temporal resolution.\u003c/p\u003e \u003cp\u003eConsidering our published data, we were surprised that dLight recordings showed no difference between VKI and WT dopamine release at 3 months, other than a reduction in the speed of recovery from train stimulation. In contrast, 6-month-old VKI mice displayed a robust elevation in the amplitude of dLight responses, while decay constants were unaltered. The capacity to re-release dopamine after train stimulation was biphasically altered at this age, with shorter time intervals showing a reduced recovery capacity, and longer time intervals showing an increased recovery capacity. Together, this suggests dopamine release is robustly elevated in VKI by 6 months old, and that dopamine axons repolarise, repackage, and re-release dopamine more readily at intervals\u0026thinsp;\u0026gt;\u0026thinsp;1s. Decay constants, and short-term depression (which would be observed as depletion of PPRs during high-frequency trains) are both indicators of DAT function\u003csup\u003e\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. As we saw no difference in these measures, we posit that increased dopamine release is independent of DAT function, as in LRRK2 G2019S knock-in mice\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDifferences between our published 3-month VKI voltammetry\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and dLight data here maybe explained by biological and/or technical differences. Three data sets on mice from the same founding colony now show elevated dopamine release, albeit at slightly different ages\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. While institutional housing conditions may modify phenotype progression, technical differences between slice treatment might also explain the discrepancy. Here, unlike for previous voltammetry data, slices were bathed in NMDG recovery solution to increase slice viability. Recordings were performed in the vehicle for MLi2, Captisol\u0026reg;, a biopolymer that has the potential to increase neurotransmitter release\u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54 CR55 CR56\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, potentially masking differences between genotypes at 3 months. Disparities in the recording modality might also change the interpretation of results at 3 months. Direct comparison of FSCV and dLight shows both techniques report the expected increase in dopamine release duration and decay with nomifensine or cocaine (blocking DAT); however, peak changes are only observed with FSCV\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. FSCV relies on the area of a carbon fiber electrode onto which dopamine can diffuse, which will increase under conditions of DAT inhibition, and translate to an increase in the peak of FSCV transients\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. If the spread of extracellular dopamine is increased in VKI at 3 months this would translate to peak changes in voltammetry, but not dLight. Further investigation of the spread dLight fluorescence may reveal whether diffusion is altered at ages preceding clear increases in dLight peaks in VKI mice. Despite these caveats, repeated assessment of dLight across ages in VKI mice reveals increased dopamine release in young-adulthood, which is sensitive to acute LRRK2 kinase inhibition.\u003c/p\u003e \u003cp\u003eElevated dopamine release in 6-month VKI was returned to WT levels by \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;1.5h slice treatment with LRRK2 inhibitor MLi-2, relative to vehicle-treated slices from the same brain. This occurred without changes to parameters dictated by DAT or D2 receptors (decay rate / pulse train PPR, and 4s PPR\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e). Therefore, we conclude LRRK2 kinase hyperactivity and inhibition can alter dopamine release independent of canonical DAT and D2 receptor activity. Both DAT and D2 receptors appear to be VPS35 cargoes, as is the dopamine vesicular monoamine transporter (VMAT)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. It may be that aberrant LRRK2 kinase activity increases vesicular packing of dopamine via VMAT, as VMAT2 levels are elevated in VKI striatal tissue\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This aligns with our observation that dopamine release is elevated during pulse-trains without significantly altering release probability (P2/P1 PPR, 100ms intervals).\u003c/p\u003e \u003cp\u003eLRRK2 variants convey the highest genetic risk for PD, with several pathogenic variants linked to clinically-typical familial parkinsonism\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Brain scans of LRRK2 PD patients show impaired presynaptic dopamine function\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, but asymptomatic LRRK2 mutation carriers exhibit higher dopamine turnover, indicating increased release\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Dopamine release measured by FSCV is also elevated in 3-month-old LRRK2 G2019S knock-in mice\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, consistent with our results in VKI mice\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Early increases in dopamine release decline in LRRK2 knock-in mice\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and a similar pattern is observed in α-synuclein overexpressing PD mice\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Together, evidence from VKI, LRRK2 and synuclein mouse models are consistent with early elevations in dopamine release in humans\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLRRK2 and VPS35 canonically function in endolysosomal regulation and trafficking, and synaptic vesicles (SVs) are highly specialised, neural-specific, endosomes\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. As with other membranes, the SV cycle is orchestrated by Rabs, several of which are phosphorylated by LRRK2. This includes the canonically synaptic-vesicle associated Rab3\u0026amp;5\u003csup\u003e65,66\u003c/sup\u003e and classically-endolysosomal Rab10\u0026amp;12, that have recently been shown to be as enriched as Rab3\u0026amp;5 on SVs\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Both VPS35 and LRRK2 can alter synaptic vesicle docking and size\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, although how remains speculative. Conceptually, LRRK2 phosphorylation of Rab3 might be expected to increase SV availability by facilitating biogenesis, trafficking, and priming of vesicles at the active zone. Rab3 tethering to Rab-Interacting Molecule (RIM)\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e regulates vesicle fusion, without which DA release is blocked\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Other than Rab3, the role of Rabs at SVs is somewhat unclear, and effects of LRRK2-Rab phosphorylation almost entirely unknown. Finally, while Rabs have recently taken centre stage, several SV proteins were previously implicated as LRRK2 substrates; including, auxillin, endophilinA, dynamin, \u0026amp; synapsin\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, and we show here that LRRK2 kinase inhibition can have rapid effects on dopamine release. Further investigation of VPS35 mutant, and LRRK2 inhibitor effects, on dopamine and glutamate vesicle cycles seems justified.\u003c/p\u003e \u003cp\u003eGiven the ubiquitous expression of VPS35 and LRRK2, it likely that onset of PD-like dysfunction and degeneration results from the accumulation of low-level cellular insults over years in mice, or decades in humans. Sustained elevations in striatal neurotransmission, or even compensatory action towards it, likely increases cellular stress. This in turn, could lead to the accumulation of excitotoxic damage which may not be effectively cleared in the presence of the VPS35 (or LRRK2) mutation\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Increased energy demands and calcium buffering required by increased synaptic activity may also contribute to mitochondrial dysfunction observed in many PD scenarios, including VPS35 patient-derived neurons\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. These may eventually lead to axon degeneration in more vulnerable neuronal populations, such as nigrostriatal dopamine neurons.\u003c/p\u003e \u003cp\u003eIn summary, we present evidence of progressively elevated neurotransmission in young-adult mice with VPS35 D620N mutations, which we propose will become neurotoxic if sustained. However, acute inhibition of LRRK2 kinase rapidly reverses the increased dopamine release, offering hope as a viable neuroprotective strategy. Targeting early neuronal hyperactivity may be the optimal therapeutic window for disease-modifying PD treatments, especially for those known to carry PD-causal gene variants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.K. performed most animal surgeries and experiments, contributed to the experimental design and curation, performed data analysis, and co-wrote the manuscript with A.J.M.C.A.K. contributed intellectually to the interpretation of findings and training of A.K.N.K. performed initial animal surgeries and contributed to the training of A.K.S.C. collected a subset of iGluSnFR recordings E.H. prepared slices and collected recordings for a subset of iGluSnFR experimentsJ.C.B. performed animal surgery for a subset of iGluSnFR experimentsM.P.P. contributed to the experimental design and analysis of iGluSnFR experiments and to the training of A.K.A.J.M. contributed experimental analytical design and curation, training of A.K., and co-wrote the manuscript with A.K.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKordower, J. 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Neurosci.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 10613\u0026ndash;10628 (2015).\u003c/li\u003e\n\u003cli\u003eHanss, Z. \u003cem\u003eet al.\u003c/em\u003e Mitochondrial and Clearance Impairment in p.D620N VPS35 Patient-Derived Neurons. \u003cem\u003eMovement Disorders\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 704\u0026ndash;715 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5167163/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5167163/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe D620N variant in Vacuolar Protein Sorting 35 (VPS35) causes autosomal-dominant, late-onset Parkinson\u0026rsquo;s disease. VPS35 is a core subunit of the retromer complex that canonically recycles transmembrane cargo from sorting endosomes. Although retromer cargoes include many synaptic proteins, VPS35\u0026rsquo;s neuronal functions are poorly understood. To investigate the consequences of the Parkinson\u0026rsquo;s mutation, striatal neurotransmission was assessed in 1-, 3- \u0026amp; 6-month-old VPS35 D620N knock-in (VKI) mice. Spontaneous and optogenetically-evoked corticostriatal glutamate transmission was increased in VKI striatal spiny projection neurons by 6 months, when total striatal glutamate release, quantified by iGluSnFR imaging, showed similarities to wild-type. dLight imaging revealed robust increases in VKI striatal dopamine release by 6 months, which were reversed with acute \u003cem\u003eex vivo\u003c/em\u003e leucine-rich repeat kinase 2 (LRRK2) inhibition. We conclude that increased glutamate and dopamine transmission in VKI mice progressively emerges in young-adulthood, and that dopamine dysfunction is likely the result of sustained, rapidly-reversible, LRRK2 hyperactivity.\u003c/p\u003e","manuscriptTitle":"Emergent glutamate \u0026amp; dopamine dysfunction in VPS35(D620N) knock-in mice and rapid reversal by LRRK2 inhibition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 06:17:12","doi":"10.21203/rs.3.rs-5167163/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-14T19:45:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-13T18:10:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-28T16:12:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169134550833705810467964107891209475002","date":"2024-10-19T21:18:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254769739671395319869834554083996261726","date":"2024-10-18T19:42:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313353761981891414753858992240868110638","date":"2024-10-15T23:04:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-15T22:58:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-15T16:53:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-14T23:25:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2024-09-27T19:09:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53e30414-611c-4d31-ab31-8df4d98bbb09","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40284946,"name":"Biological sciences/Neuroscience/Cellular neuroscience"},{"id":40284947,"name":"Biological sciences/Neuroscience/Diseases of the nervous system"},{"id":40284948,"name":"Biological sciences/Neuroscience/Synaptic transmission"}],"tags":[],"updatedAt":"2025-05-05T16:04:19+00:00","versionOfRecord":{"articleIdentity":"rs-5167163","link":"https://doi.org/10.1038/s41531-025-00948-7","journal":{"identity":"npj-parkinsons-disease","isVorOnly":false,"title":"npj Parkinson's Disease"},"publishedOn":"2025-05-03 15:57:11","publishedOnDateReadable":"May 3rd, 2025"},"versionCreatedAt":"2025-03-31 06:17:12","video":"","vorDoi":"10.1038/s41531-025-00948-7","vorDoiUrl":"https://doi.org/10.1038/s41531-025-00948-7","workflowStages":[]},"version":"v1","identity":"rs-5167163","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5167163","identity":"rs-5167163","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}