The BDNF Val66Met polymorphism (rs6265) enhances dopamine neuron graft efficacy and side-effect liability in rs6265 knock-in rats - PubMed (original) (raw)

The BDNF Val66Met polymorphism (rs6265) enhances dopamine neuron graft efficacy and side-effect liability in rs6265 knock-in rats

Natosha M Mercado et al. Neurobiol Dis. 2021 Jan.

Abstract

Prevalent in approximately 20% of the worldwide human population, the rs6265 (also called 'Val66Met') single nucleotide polymorphism (SNP) in the gene for brain-derived neurotrophic factor (BDNF) is a common genetic variant that can alter therapeutic responses in individuals with Parkinson's disease (PD). Possession of the variant Met allele results in decreased activity-dependent release of BDNF. Given the resurgent worldwide interest in neural transplantation for PD and the biological relevance of BDNF, the current studies examined the effects of the rs6265 SNP on therapeutic efficacy and side-effect development following primary dopamine (DA) neuron transplantation. Considering the significant reduction in BDNF release associated with rs6265, we hypothesized that rs6265-mediated dysfunctional BDNF signaling contributes to the limited clinical benefit observed in a subpopulation of PD patients despite robust survival of grafted DA neurons, and further, that this mutation contributes to the development of aberrant graft-induced dyskinesias (GID). To this end, we generated a CRISPR knock-in rat model of the rs6265 BDNF SNP to examine for the first time the influence of a common genetic polymorphism on graft survival, functional efficacy, and side-effect liability, comparing these parameters between wild-type (Val/Val) rats and those homozygous for the variant Met allele (Met/Met). Counter to our hypothesis, the current research indicates that Met/Met rats show enhanced graft-associated therapeutic efficacy and a paradoxical enhancement of graft-derived neurite outgrowth compared to wild-type rats. However, consistent with our hypothesis, we demonstrate that the rs6265 genotype in the host rat is strongly linked to development of GID, and that this behavioral phenotype is significantly correlated with neurochemical signatures of atypical glutamatergic neurotransmission by grafted DA neurons.

Keywords: BDNF; Neural grafting; Parkinson’s disease; Val66Met; Val68Met; rs6265.

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Conflict of interest statement

Declaration of Competing Interest

None.

Figures

Fig. 1.

Experimental design. a Experimental timeline of surgical procedures, behavioral evaluation, and drug treatment. b Experimental groups and final group size “_N_” upon completion of the study. c Schematic depicting the design of experiments. Ventral mesencephalic tissue was dissected from wild-type embryonic day 14 Sprague-Dawley rat pups, dissociated, then transplanted into wild-type Val68Val and homozygous Met68Met rats. Abbreviations: 6-OHDA = 6-hydroxydopamine, Amph rotation = amphetamine rotation behavioral assessment, LD = levodopa, LID = levodopa-induced dyskinesia, GID = graft-induced dyskinesia, LD GID = levodopa-mediated GID assessment, Amph GID = amphetamine-mediated GID assessment, sac = sacrifice, WT = wild-type, BDNF = brain-derived neurotrophic factor, SNP = single nucleotide polymorphism, DA = dopamine

Fig. 2.

Behavioral measures of graft functional efficacy. a Total LID score (primary behavioral endpoint) for Val68Val and Met68Met rats throughout levodopa priming (pre-graft period) and for 9 weeks post-engraftment. Dyskinesia severity scores were not significantly different between genotypes in sham grafted rats; thus, sham subjects were combined into one group post-graft. Inset graph depicts total dyskinesia score over time for sham grafted subjects, separated by genotype. Statistics: Non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test at each time point. Week 5:**p = 0.0015 Met68Met-DA vs Sham, †p = 0.0302 Met68Met-DA vs Val68Val-DA; Week 6: *p = 0.0284 Met68Met-DA vs Sham, †p = 0.0475 Met68Met-DA vs Val68Val-DA; Week 8: *p = 0.0198 Met68Met-DA vs Sham; Week 9: ***p = 0.0003 Met68Met-DA vs Sham. Sham groups were not significantly different at any of the post-graft time points (_p_≥ 0.12 for all time points post-graft). b Pre-graft LID severity time course showing individual animal responses during levodopa priming days 1, 12, and 25. Rats were rated at 20, 70, 120, 170, and 220 mins post-levodopa each day. c LID severity time course and total LID scores showing individual animal responses at weeks 3, 6, and 9 post-engraftment. Non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test at each time point. d Amphetamine rotational asymmetry (secondary behavioral endpoint), measured at 7 weeks pre-graft and 7 weeks post-graft. Data are expressed as net ipsilateral rotations (i) and ipsilateral rotations per min over 90 mins (ii–iii). Unpaired _t_-test with Welch’s correction (ii) and one-way ANOVA with Šídák’s post-hoc test (iii). Abbreviations: LID = levodopa-induced dyskinesia, DA = dopamine graft, CCW = counterclockwise

Fig. 3.

Behavioral measures of graft dysfunction. a,b Peak amphetamine-induced (a) and levodopa-induced (b) GID severity score at week 10 post-engraftment. Non-parametric Kruskal-Wallis test with Dunn’s post-hoc comparisons. c,d Time course of amphetamine-mediated (c) and levodopa-mediated (d) GID behavior at week 10 post-engraftment. e Spearman correlation between amphetamine-mediated peak GID behavior and levodopa-mediated peak GID behavior. Abbreviations: GID = graft-induced dyskinesia, Amph = amphetamine, DA = dopamine graft

Fig. 4.

Graft histology in parkinsonian striatum of Met68Met rats and their wild-type (Val68Val) counterparts. a Representative micrographs of THir striatum demonstrating more extensive grafted DA neurite outgrowth in Met68Met host striatum. Scale bars = 100 μm (10x micrographs), 1000 μm (1x micrographs). b Corresponding THir nigral tissue sections showing near-complete depletion of host SNc DA neurons in the lesioned hemisphere. Scale bar = 1000 μm. c Stereologically estimated total number of grafted DA neurons. Mean ± SEM. Unpaired _t_-test. d Stereologically estimated total graft volume. Mean ± SEM. Unpaired _t_-test. e Schematic illustrating fields of view used for analysis of grafted DA neurite outgrowth. Proximal and distal regions are denoted by “1” and “2,” respectively. f Average grafted DA neurite density proximal and distal to the graft border. Mean ± SEM. Two-way repeated measures ANOVA with Šídák’s post-hoc test. g Comparison of distal grafted DA neurite outgrowth separated into regions surrounding the graft. Mean ± SEM. Unpaired _t_-tests and mixed-effects model. Red bar indicates maximum and minimum neurite density means for Met68Met subjects. h Distal grafted DA neurite density compared with endogenous DA innervation of intact contralateral striatum. Mean ± SEM. Two-way repeated measures ANOVA with Šídák’s post-hoc test. Abbreviations: THir = tyrosine hydroxylase immunoreactive, str = striatum, ot = olfactory tubercle, vta = ventral tegmental area, sn = substantia nigra, ctx = cortex, D = dorsal, M = medial, V = ventral, L = lateral, 1 = proximal zone, 2 = distal zone, ns = not significant

Fig. 5.

Impact of Met allele on graft (TH) connectivity with host MSN spines (synaptopodin). a Schematic representation of regions relative to the graft in which confocal images were acquired (left), and representative 3D confocal z-stack of grafted DA neurites in tissue stained for TH and synaptopodin (right). Scale bar = 10 μm. b (i’) Increased magnification of micrograph in panel (a), and (i”) Imaris® 3D reconstruction of DA fiber denoted by (i) in panel (a). Scale bar = 1 μm. c Comparison of TH-synaptopodin contact density normalized to TH surface volume. Mean ± SEM. Two-way mixed-effects model with repeated measures, followed by Šídák’s and Dunnett’s post-hoc tests.Red bar indicates maximum and minimum contact density means in the intact striatum. d Spearman correlation between _proximal_TH-synaptopodin contact density and total LID severity score at 9 wks post-engraftment. e Spearman correlation between _distal_TH-synaptopodin contact density and total LID severity score at 9 wks post-engraftment. Abbreviations: ctx = cortex, str = striatum, 1 = proximal zone, 2 = distal zone

Fig. 6.

Vglut2 mRNA expression in DA neurons grafted into parkinsonian striatum. a Schematic depicting normal mature vs immature DA neuron phenotype, based on data from (El Mestikawy et al., 2011). b,c Vglut2 mRNA expression in THir grafted DA neurons and unidentified grafted TH-negative cells. Cells denoted by (i-iii) in panel (c) are shown with increased magnification in (i’–iii’). Scale bars = 250 μm in panel (b) and 5 μm for insets in this panel; 100 μm in panel (c), or 10 μm for insets in this panel. d Vglut2 mRNA in DA neurons of the naïve adult rat midbrain. DA neurons of the substantia nigra (i) and ventral tegmental area (ii) are shown with increased magnification in (i’) and (ii’), respectively. Vglut2_-negative DA neurons are indicated by solid arrows in (i’) and (ii’), whereas solid arrowheads indicate cells containing Vglut2 mRNA only, and unfilled arrows indicate cells containing colocalized TH-Vglut2. Scale bars = 300 μm for panel (d), and 30 μm for i’ and ii’. e Percentage of DA neurons expressing_Vglut2 mRNA in naïve adult rat midbrain and grafted, parkinsonian striatum. NOTE: Grafted DA neurons are from wild-type donors. Mean ± SEM. Two-way ANOVA with Šídák’s post-hoc test (Naïve Total and Grafted); unpaired _t_-tests (naïve SNc and naïve VTA). Abbreviations: DA = dopamine, VMAT = vesicular monoamine transporter, Glut = glutamate, VGLUT2 = vesicular glutamate transporter 2, TH = tyrosine hydroxylase, SNc = substantia nigra pars compacta, VTA = ventral tegmental area

Fig. 7.

VGLUT2 protein expression in THir DA fibers grafted into parkinsonian striatum. a Computer generated Imaris® 3D reconstruction of confocal z-stack depicting VGLUT2 colocalization within grafted DA neurites. The advanced Imaris® algorithms allow visualization of fine structures (i.e., vesicles) inside cellular elements (i.e., neurites), and allows the cellular element to be visualized at varying levels of transparency (i.e., 50% shown here) to allow visualization of internal elements. Scale bar = 1 μm. b Quantification of VGLUT2 protein located within THir DA fibers, normalized to TH surface volume. TH-VGLUT2 colocalization in the striatum of naïve rats was compared with grafted DA fibers in the parkinsonian striatum and endogenous DA fibers in the intact striatum contralateral to the lesion. Mean ± SEM. Two-way ANOVAs with Tukey’s post-hoc test. c Spearman correlation between TH-VGLUT2 colocalization in grafted DA neurites and total amphetamine-mediated GID score at 10 wks post-engraftment. d Confocal micrograph indicating synaptic apposition (≤ 0.6 μm) between VGLUT2 protein located inside a grafted DA neurite (indicated by open arrow on the right) and PSD95 located adjacent to the DA neurite (indicated by open arrow on the left). The presumed synapse (i) is shown with increased magnification using Imaris® 3D imaging in (i’) and (i”). Scale bars = 2 μm (confocal micrograph), 1 μm (Imaris® 3D reconstructions). % transparency indicates that applied to TH fibers. e Quantification of presumed excitatory VGLUT2-PSD95 synapses made by DA neurites in the grafted striatum and intact contralateral striatum, normalized to TH surface volume. Mean ± SEM. Mixed-effects model with Šídák’s post-hoc test. f Spearman correlation between presumed excitatory VGLUT2-PSD95 synapses made by DA neurites and total amphetamine-mediated GID score at 10 wks post-engraftment. Note that one statistical outlier was removed from the Met68Met group. Abbreviations: VGLUT2 = vesicular glutamate transporter 2, PSD95 = postsynaptic density protein 95, TH = tyrosine hydroxylase, MSN = medium spiny neuron, ns = not significant

Fig. 8.

Excitatory corticostriatal (VGLUT1) synaptic input onto grafted DA neurons. a Schematic depicting “normal” corticostriatal synapse with modulatory DA input (right side of diagram), and “atypical” excitatory glutamatergic synapse onto grafted DA neurons (left side of diagram). b Representative confocal z-stack of tissue stained for VGLUT1, PSD95, and TH proteins. Scale bar = 10 μm. Inset: Increased magnification of a presumed glutamatergic (VGLUT1) synapse onto PSD95 protein located inside a grafted THir DA fiber (≤ 0.6 μm). Inset scale bar = 0.5 μm. c Imaris® 3D reconstruction of the inset image in panel (b). % transparency indicates that applied to TH fibers. d Quantification of presumed corticostriatal (VGLUT1) synapses with PSD95 located inside grafted DA fibers, normalized to TH surface volume. Mean ± SEM. Two-way repeated measures ANOVA with Šídák’s post-hoc test. e Quantification of presumed corticostriatal (VGLUT1) synapses onto grafted DA fibers, regardless of PSD95 presence, normalized to TH surface volume. Mean ± SEM. Two-way repeated measures ANOVA with Šídák’s post-hoc test. Abbreviations: PSD95 = postsynaptic density protein 95, VGLUT1 = vesicular glutamate transporter 1, Glut = glutamate, DA = dopamine, TH = tyrosine hydroxylase

Fig. 9.

Impact of Met allele on presence of serotonin neurons in VM grafts. a Confocal micrographs depicting presence of serotonergic neurons in VM grafts from wild-type donors, in Val68Val and Met68Met host striatum. Inset: Increased magnification of DA neurons (THir) and serotonin neurons (Tph2_-immunoreactive) present in grafted striatum. As shown in these representative images, there was no colocalization of_Tph2 mRNA and TH in VM grafts, and this finding was identical between genotypes. Scale bars = 100 μm in panel (a), 10 μm in inset panels. b Quantification of grafted serotonin neurons relative to the number of grafted DA neurons. Mean ± SEM. Unpaired_t_-test. c Spearman correlation between the proportion of serotonin neurons relative to DA neurons and total amphetamine-mediated GID score at 10 wks post-engraftment. d Spearman correlation between the proportion of serotonin neurons relative to DA neurons and total levodopa-mediated GID score at 10 wks post-engraftment. e Spearman correlation between the proportion of serotonin neurons relative to DA neurons and total LID score at 9 wks post-engraftment. Abbreviations: TH = tyrosine hydroxylase,Tph2 = tryptophan hydroxylase 2

Fig. 10.

Impact of Met allele on Bdnf mRNA expression in grafted DA neurons and Trkb mRNA expression in host striatum. a Schematic illustrating Trkb expression in host dorsolateral striatum and Bdnf expression in grafted DA neurons. b Confocal micrograph of Bdnf mRNA in DA grafted tissue. Cells depicted in (i-iii) are shown at increased magnification in inset (i’–iii’). Scale bars = 100 μm for panel (b), 10 μm for inset images. c Percentage of grafted DA neurons expressing_Bdnf_ mRNA in Val68Val and Met68Met grafted rats. Mean ± SEM. Unpaired _t_-test. NOTE: Bdnf mRNA data are available from only three Val68Val grafted subjects due to limited tissue sections containing grafts. d,f Micrographs of Trkb mRNA expression, presumed to be principally within medium spiny neurons, in grafted and intact dorsolateral striatum of sham grafted (d) and DA grafted (f) rats. Scale bars = 25 μm. e,g Quantification of Trkb mRNA in sham grafted (e) and DA grafted (g) rats depicted in panels (d) and (f), respectively. Mean ± SEM. Two-way repeated measures ANOVAs with Šídák’s post-hoc test. Abbreviations: ctx = cortex, str = striatum, Bdnf = brain-derived neurotrophic factor,Trkb = tyrosine receptor kinase B, DA = dopamine, ns = not significant

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The BDNF Val66Met polymorphism (rs6265) enhances dopamine neuron graft efficacy and side-effect liability in rs6265 knock-in rats - PubMed (original) (raw)