Dopaminergic, serotonergic, and noradrenergic deficits in Parkinson disease

OBJECTIVE: People with Parkinson disease (PD) frequently develop dementia, which is associated with neocortical deposition of alpha-synuclein (α-syn) in Lewy bodies and Lewy neurites. In addition, neuronal loss and deposition of aggregated α-syn also occur in multiple subcortical nuclei that project to neocortical, limbic, and basal ganglia regions. Therefore, we quantified regional deficits in innervation from these PD-affected subcortical nuclei, by measuring the neurotransmitters and neurotransmitter transporter proteins originating from projections of dopaminergic neurons in substantia nigra pars compacta, serotonergic neurons in dorsal raphé nuclei, noradrenergic neurons in locus coeruleus, and cholinergic neurons in nucleus basalis of Meynert. METHODS: High-performance liquid chromatography and novel enzyme-linked immunosorbent assays were performed to quantify dopaminergic, serotonergic, noradrenergic, and cholinergic innervation in postmortem brain tissue. Eight brain regions from 15 PD participants (with dementia and Braak stage 6 α-syn deposition) and six age-matched controls were tested. RESULTS: PD participants compared to controls had widespread reductions of dopamine transporter in caudate, amygdala, hippocampus, inferior parietal lobule (IPL), precuneus, and visual association cortex (VAC) that exceeded loss of dopamine, which was only significantly reduced in caudate and amygdala. In contrast, PD participants had comparable deficits of both serotonin and serotonin transporter in caudate, middle frontal gyrus, IPL, and VAC. PD participants also had significantly reduced norepinephrine levels for all eight brain regions tested. Vesicular acetylcholine transporter levels were only quantifiable in caudate and hippocampus and did not differ between PD and control groups. INTERPRETATION: These results demonstrate widespread deficits in dopaminergic, serotonergic, and noradrenergic innervation of neocortical, limbic, and basal ganglia regions in advanced PD with dementia

Quantifying regional α -synuclein, amyloid β, and tau accumulation in Lewy body dementia

OBJECTIVE: Parkinson disease (PD) is defined by the accumulation of misfolded α-synuclein (α-syn) in Lewy bodies and Lewy neurites. It affects multiple cortical and subcortical neuronal populations. The majority of people with PD develop dementia, which is associated with Lewy bodies in neocortex and referred to as Lewy body dementia (LBD). Other neuropathologic changes, including amyloid β (Aβ) and tau accumulation, occur in some LBD cases. We sought to quantify α-syn, Aβ, and tau accumulation in neocortical, limbic, and basal ganglia regions. METHODS: We isolated insoluble protein from fresh frozen postmortem brain tissue samples for eight brains regions from 15 LBD, seven Alzheimer disease (AD), and six control cases. We measured insoluble α-syn, Aβ, and tau with recently developed sandwich ELISAs. RESULTS: We detected a wide range of insoluble α-syn accumulation in LBD cases. The majority had substantial α-syn accumulation in most regions, and dementia severity correlated with neocortical α-syn. However, three cases had low neocortical levels that were indistinguishable from controls. Eight LBD cases had substantial Aβ accumulation, although the mean Aβ level in LBD was lower than in AD. The presence of Aβ was associated with greater α-syn accumulation. Tau accumulation accompanied Aβ in only one LBD case. INTERPRETATION: LBD is associated with insoluble α-syn accumulation in neocortical regions, but the relatively low neocortical levels in some cases suggest that other changes contribute to impaired function, such as loss of neocortical innervation from subcortical regions. The correlation between Aβ and α-syn accumulation suggests a pathophysiologic relationship between these two processes

Calpain Cleavage of Brain Glutamic Acid Decarboxylase 65 Is Pathological and Impairs GABA Neurotransmission

Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2–3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission

Effect of high K⁺ stimulation of rat brain synaptosomes on cleavage of fGAD65.

<p><b>A.</b> Representative immuno-blot image showing progression in cleavage of fGAD65 to form tGAD65 when rat brain synaptosomes were stimulated with increasing K⁺ concentration (lanes 2–4). A non-stimulated synaptosomal fraction (lane 1) served as a control. The samples were analyzed by immuno-blotting using monoclonal GAD65-specific antibody; GAD6. The 58 KDa cleaved product tGAD65 was found to accumulate proportional to the increase in KCl concentration. <b>B.</b> Densitometric analyses of the conversion of fGAD65 to tGAD65 in rat brain synaptosomes treated with increasing K⁺ concentration expressed as % of control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g001" target="_blank">Fig. 1B</a>, Ctrl). Data are presented as mean ± SEM, n = 4 independent experiments. <i>*P</i><0.05 and <i>^#P</i><0.05 are statistically significant differences between the levels of fGAD65 and tGAD65 of 50 mM KCl treatment group and the control respectively. <i>**P</i><0.001 and <i>^##P</i><0.001 are statistically significant differences in the levels of fGAD65 and tGAD65 between 100 mM KCl treatment group and the control respectively.</p

Effect of calpain treatment of GABAergic SVs on newly synthesized GABA uptake.

<p>Uptake of newly synthesized GABA was conducted in the presence (•) or absence (▪) of calpain enzyme. [³H] Glu was used as a substrate for the synthesis of newly synthesized [³H] GABA. Values are mean of ± SEM (n = 3). The specific uptake was obtained by subtracting the non-specific uptake from the total uptake and was expressed as pmol per mg of protein.</p

Cleavage of SV-associated fGAD65 as a function of KCl concentration:

<p><b>A.</b> Representative immuno-blot demonstrating the isolation of enriched SVs, relatively free from any contaminating cytosolic fractions, other than that of Golgi and ER fractions, as indicated by immuno-blotting against synaptosophysin (SV marker), calnexin (ER marker) and 58K Protein (Golgi marker). <b>B.</b> Representative immuno-blot demonstrating cleavage of SV-associated fGAD65 directly on the SVs, on account of a dose-dependent KCl stimulation. <b>C.</b> Quantitative densitometric comparison, expressed as relative percentage with respect to SVs, of the levels of expression of fGAD65 and synaptophysin in SVs vs Cyt (cytosolic) fractions. Values are mean of ± SEM (n = 3). *<i>P</i><0.05 and <i>**P</i><0.001 are statistically significant differences in the levels of expression between fGAD65 and synaptophysin in the SV fractions when compared tocyt fractions. <b>D.</b> Densitometric analyses of the changes in the expressions of fGAD65 and tGAD65 in the SV fractions. Untreated SVs served as a control. Data points are mean of ± SEM (n = 3). <i>*P</i><0.05 and <i>^#P</i><0.05 are statistically significant differences between 50 mM KCl treatment group and the control group respectively. **P<0.005 and ##P<0.005 are statistically significant differences between 100 mM KCl group and the control group.</p

Time dependent differential expression of GAD65 and GAD67 mRNA upon exposure to 100 mM KCl in primary rat neuronal cell cultures.

<p>A. Representative gel image of GAD65 and GAD67 mRNA separated on a 1.5% agarose gel. Lane 1: Control; Lanes 2–5: 10 min, 1 hr, 4 hr and 8 hr exposure to 100 mM KCl respectively. GAD65 and GAD67 mRNA were differentially regulated under the same conditions of exposure to 100 mM KCl for different time points as indicated in lanes 2–5. B. Quantitative analyses of GAD65 and GAD67 mRNA expression shown as % of control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g004" target="_blank">Fig. 4B</a>, Ctrl). Values are mean of ± SEM (n = 4); <i>^##P</i><0.05 statistically significant differences between GAD67 mRNA expression of 10 min exposure group and the control. <i>*P</i><0.05 statistically significant differences between GAD65 mRNA expression of 1 hr and 4 hr exposure groups with the control respectively. <i>**P</i><0.005 is the statistically significant difference between GAD65 mRNA expression between 8 hr exposure group and the control.</p

Identification of truncated forms of GAD and corresponding elevation of glutamate levels in a MCAO model.

<p><b>A.</b> Representative immuno-blot demonstrating the presence of truncated forms of GAD65 and GAD67 and corresponding elevation of calpastatin. N, Normal; I, Ischemic. <b>B.</b> Densitometric analysis of the immuno-blot images. Data points are mean of ± SEM (n = 3). **<i>P</i><0.005 and *<i>P</i><0.05 are statistically significant differences when compared to their respective controls. <b>C.</b> Corresponding changes in the levels of glutamate before and during ischemia. Values are mean of ± SEM (n = 4). *<i>P</i><0.005 is the statistically significant differences between during occlusion and before occlusion.</p

Effect of high K⁺ stimulation of rat brain synaptosomes on total GAD65 activity.

<p><b>A.</b> Total GAD65 activity in the rat brain synaptosomes was measured under conditions promoting cleavage of fGAD65. Synaptosomes were stimulated with increasing concentrations of K⁺ as indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2A</a>; 10 mM KCl group, 50 mM KCl group and 100 mM KCl group). Unstimulated synaptosomes served as the control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2A</a>; Ctrl group). Total GAD65 activity comprising the activity of both fGAD65 and tGAD65 after stimulation was measured using a combinational approach of immunoprecipitation and radiometric GAD activity assays. First, soon after stimulation, total GAD65 was pulled down by immunoprecipitation using monoclonal GAD65 antibody; GAD6. Next, after normalizing the synaptosomal lysates between groups, the pulled down complex was analyzed using the classical radiometric method for measuring GAD activity. The values are mean ± SEM (n = 4); <i>*P</i><0.05 and <i>**P</i><0.005 are statistically significant differences between 50 mM KCl and 100 mM KCl treatment groups with the control group respectively. <b>B.</b> Effect of calpain inhibitor on total GAD65 activity was evaluated by pre-incubating the synaptosomes with 1 µM of calpain inhibitor peptide as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2B</a>. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2A</a>, the synaptosomes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2B</a>, were subjected to 50 mM KCl treatment with or without the calpain inhibitor, followed by synaptosomal lysis, normalization, immunoprecipitation and then the total GAD65 activity was measured by radiometric GAD activity assays. Data are represented as ± mean SEM (n = 3); <i>*P</i><0.05 are the statistically significant differences between K⁺ (50 mM) and the control groups. <i>^#P</i><0.05 are the statistically significant differences between K⁺ (50 mM) and the K⁺+Calp Inhi (50 mM KCl and 1 µM calpain inhibitor) groups.</p

Sequence of primers used in the RT-PCR reaction.

<p>Abbreviations used: G3PDH: Glyceraldehyde-3-phosphate dehydrogenase.</p