4.1. Upregulation of arrestin and GRK expression in Parkinson's disease with dementia
Our data indicate that a subgroup of patients with PD that had been clinically diagnosed with dementia has higher levels of the two non-visual arrestins and two of GRK subtypes, GRK3, and GRK5, in the striatum when compared with control and PD patients without dementia. Within the striatum, upregulation of both mRNA and protein levels for the arrestins was the most pronounced in CN and VSTR. Increase in the arrestins' and GRKs' protein expression was accompanied by elevation in their mRNA concentrations in multiple striatal regions, often even in those that did not show protein upregulation (). The pattern of arrestin and GRK expression in the striatum of patients with PD but without clinical diagnosis of dementia was surprisingly different. There was absolutely no evidence of elevated expression of any of the proteins in any striatal region examined. We also detected no significant differences between the PD and control groups, although some decrease in GRK3 protein in VSTR and arrestins' mRNA in CN can be inferred from the fact that the values in the PDD group were significantly higher than that in the PD group but not in control. A conspicuous feature of the PDD group was the uniformity of values as compared to both control and PD groups, in which a wide range of values for all arrestin/GRK proteins, particularly for GRK2, were detected. Changes in the expression of other signaling proteins we examined (except DARPP-32) followed a pattern similar to that of arrestins and GRKs: we detected a significant upregulation of ERK1/2 in CN, Akt in CN and VSTR, and GSK3α in VSTR in the PD group with dementia as compared to control and PD groups (). Interestingly, no changes in the expression of any of the signaling proteins were noted in Pu, although the degree of dopaminergic loss was the highest in that striatal region ().
Summary of changes in the expression levels of arrestins, GRKs, and other signaling proteins in the striatal subdivisions in Parkinson's disease and Parkinson's disease with dementia at postmortem1.
What could be the reason for such substantial differences in expression of signaling proteins between these two groups of PD patients? There were no significant differences between the groups in age or sex composition. The postmortem interval was significantly shorter in PDD group (the same as in control). However, the expression of arrestins and GRKs (as well as of other signaling proteins) was not influenced by any of these factors. There was a tendency for the PDD group to have shorter disease duration (time between diagnosis and death), which suggests a faster, more severe disease progression. This is in agreement with published data demonstrating negative impact of dementia in PD on survival [49
]. The question of whether the presence of dementia reflects greater damage to the dopaminergic system has been addressed in our previous study that used a cohort of patients largely overlapping with the one used here [51
]. We found that the loss of DA terminals as measured by the concentrations of DA transporter binding sites, TH immunoreactivity in the basal ganglia, and the number of TH positive neurons in the dorsal, ventral and medial DA cell groups were similar in both subgroups of PD patients. In this study, we confirmed a similar loss of DA transporter and TH in the striatum of both PD groups. According to published reports [21
], there is no difference in vivo
in the rate or extent of dopaminergic degeneration between patients with PD and PD with dementia. Our post mortem results generally support that conclusion. Ito and colleagues [47
] reported that patients with PD and dementia had lower 18-fluoro-DOPA uptake in the ventral striatum and right caudate nucleus as compared to patients with PD only. Interestingly, we also have detected a more pronounced loss of TH in the ventral striatum in PDD group, although the difference was very small. Additionally, in the present study the caudate nucleus and ventral striatum were the regions where most of the detected changes in the arrestin/GRK expression occurred (), in spite of lesser dopaminergic loss in these regions as compared to the putamen. Therefore, specific mesolimbic and caudate dopaminergic dysfunction may be linked to dementia in PD and may also determine the changes in arrestin/GRK concentrations seen in that group.
Some cases of PDD had Alzheimer's disease-like pathology (plaques and tangles) to varying degrees, which could be the reason for their dementia as well as for changes in arrestin/GRK expression we detected. However, other cases had no such pathology, and their values were within the same range. We performed factor analysis on all variables and found that expression levels of signaling molecules studied are represented by one factor, whereas loads of senile plaques and neurofibrillary tangles are represented by a different factor (). Therefore, the load of Alzheimer's pathology is not predictive of the expression levels of arrestins and GRKs. Since the plaque/tangle density and the arrestin/GRK expression are statistically uncorrelated, they seem unlikely to arise from a common cause or to have any causal connections. Consequently, such contrasting patterns of changes in the arrestin/GRK expression in the PD and PD with dementia groups is unlikely to be due to the presence of Alzheimer's pathology.
Therefore, we conclude that deficits in signaling mechanisms specific for PD patients with dementia may be responsible for the pattern of arrestin/GRK expression in these patients. The concentrations of arrestin/GRKs in cultured cells and in vivo
are altered by drugs that directly or indirectly cause persistent stimulation or blockade of GPCRs [7
], which implies the involvement of signaling mechanism in the regulation of the arrestin/GRK expression. The concentration GRK2, a short-lived protein, is regulated by synthesis as well as degradation, both of which are responsive to various signaling pathways (reviewed in [85
]). The activity of the GRK2 promoter is enhanced by stimulation of Gq-coupled GPCRs [91
], whereas Gi-coupled receptors promote degradation of GRK2 in arrestin-dependent manner [84
]. Judging by their higher protein/mRNA ratios, other GRKs seem to be more stable than GRK2 and are likely to be controlled mostly by posttranscriptional mechanisms, but specific information is lacking. Dementia in Alzheimer' disease and PD is associated with similar cholinergic deficits, which are substantially more severe in PD patients with dementia than in cognitively intact PD patients [24
and references therein,40
]. Cholinesterase inhibitors used to treat Alzheimer's disease also seem to be effective against PD-associated dementia [67
], the fact that argues for the central role of cholinergic dysfunctions in PD with dementia. It is possible that the pattern of arrestin/GRK expression in PD with dementia contrasting with that in PD is brought about by cholinergic and other signaling deficits combined with dopaminergic depletion.
Another important consideration is antemortem drug treatment the patients had received. The patients' records indicated that all had been treated with levodopa/carbidopa and many – with DA agonists. Individual doses of levodopa varied greatly within each of the two PD groups. Patients from both groups had also been treated with other drugs, including psychotropic drugs such as antidepressants, benzodiazepines, and antipsychotics. Importantly, there seems to be no substantial difference in the pattern of drug treatment between the two PD groups. Antemortem drug treatment is important, because, as discussed above, drug acting at GPCRs or signal transduction may alter the expression of arrestins and GRKs. Specific information, however, is sparse, because most drug classes have never been tested for their effects on the arrestin/GRK expression. Depression is a common non-motor feature of PD, and PD patients are often treated with antidepressants [64
]. Limiter research performed so far indicate that antidepressants may downregulate GRK2 in the prefrontal cortex of depressed patients at postmortem [29
], and the effects may differ between tricyclic and SSRI (selective serotonin reuptake inhibitor) antidepressants [72
]. Our data demonstrated that L-DOPA reduced the expression of arrestin2 and GRK2 and 6 upregulated in the basal ganglia of MPTP-treated monkeys [13
]. At present, it is unknown whether benzodiazepines or antipsychotics can alter the arrestin/GRK expression. Animal studies are urgently needed to feel this gap.
4.2. Functional significance of changes in arrestin and GRK expression
When dopaminergic neurons die in PD, a number of compensatory events occur in postsynaptic neurons to maintain dopaminergic signaling at a normal level (reviewed in [11
]). If the compensation for DA depletion was aimed at restoring a normal level of G protein-mediated signaling via DA receptors, a down-regulation of arrestins and GRKs in the PD brain should be expected. It is well established that reduction of the arrestin and/or GRK concentration invariably reduces GPCR desensitization and internalization and enhances G protein-mediated signaling [3
]. Therefore, decreased concentrations of arrestin/GRKs in the DA-depleted basal ganglia would impede desensitization of DA receptors and help sustain the G protein-mediated signaling. In this study, we did observe a tendency towards a down-regulation of GRK proteins in the striatum of PD patients. In contrast, in PD patients with dementia, which displayed loss of dopaminergic innervation in the striatum at least as extensive as in the PD group, the opposite happened: the expression of arrestins and GRKs in the striatum was enhanced.
What effect might elevated arresin/GRK levels have on receptor signaling? A wealth of data shows that an increase in arrestin/GRK concentration facilitates desensitization and internalization of various GPCRs and dampens G protein-mediated signaling [45
]. Overexpression of arrestins and GRKs induces excessive degradation of GPCRs in vitro [81
] and in vivo [46
], leading to sustained receptor down-regulation. Earlier we found that D3 DA dopamine receptors were consistently downregulated in the PD group with dementia across the striatal and pallidal subterritories, whereas in non-demented PD cases there was a slight elevation in D3 receptor binding in the rostral basal ganglia [51
]. This result matches the pattern in arrestin/GRK expression observed in this study. Down-regulation of D3 receptors in the PDD group was posttranscriptional as evidenced by lack of changes in the D3 mRNA expression. The elevated arrestin/GRK expression might lead to the D3 receptors down-regulation via enhanced internalization. The DA D3 receptor is resistant to GRK-mediated phosphorylation, and its trafficking is very sensitive to the availability of arrestins and GRKs [55
]. However, D2 DA receptor binding was increased in both PD groups to a similar extent [51
], which appears inconsistent with the contrasting changes in arrestin/GRK expression seen in these groups. PD patients with dementia often respond poorly to L-DOPA, as shown by us [51
] and others [16
]. It is important to note that persistent elevation of arrestin and GRK concentrations, whatever the cause, can lead to reduced signaling via GPCRs, including DA receptors, and, therefore may be a contributing factor to poor therapeutic response to L-DOPA regardless of changes in DA receptor numbers.
Although upregulation of arrestins and GRKs inhibits G protein-mediated signaling, other signaling pathways facilitated by arrestin binding to GPCRs may be enhanced. Arrestins, in addition to their “negative” role in G protein-mediated signaling, have “positive” signaling functions. Arrestins serve as a link between GPCRs and the mitogen-activated protein kinase (MAPK) signaling pathways (reviewed in [37
]). Arrestins work as scaffolds recruiting a number of components of several MAPK cascades to activated GPCRs, thereby mediating activation of the MAPK pathways. In particular, both non-visual arrestins have been shown to activate ERK [66
]. Over-expression of arrestins potentiates arrestin-mediated ERK activation [100
]. Conversely, reduction of the arrestin concentration by siRNAs inhibits ERK activation [3
]. Therefore, the increase in arrestin concentration in the PD with dementia group might facilitate stimulation of the MAPK pathways by GPCRs. We did not directly examine ERK phosphorylation levels in our samples. Proteins are normally rapidly dephosphorylated, and after prolonged post mortem intervals the data would be difficult to interpret. However, an increase in the concentration of total ERK protein in the PD group with dementia may be viewed as evidence of the activation of this pathway, possibly, linked to the upregulation of arrestins.
Recently arrestin3 has been implicated in regulation of Akt pathway [8
]. Persistent activation of D2 DA receptors inhibits Akt. This reduces phosphorylation of its usual substrate glycogen synthase kinase3 (GSK3α,β thereby increasing its activity [8
]. Arrestin3 mediates this action of DA by forming a signaling complex comprising Akt and protein phosphatase2 that dephosphorylates Akt [9
]. Arrestin3 knockout in mice inhibits DA-mediated behavior and abolishes DA-mediated regulation of Akt [9
]. It is still unknown whether only arrestin3 or both non-visual arrestin subtypes regulate DA-dependent Akt signaling. Conceivably, the upregulation of Akt and GSK3α expression seen in the striatum of PD patients with dementia is linked to the elevated concentrations of arrestins. Another interesting aspect is that arrestin2 has recently been shown to act as a messenger between activated GPCRs and the nucleus and directly activates specific promoters such as p27
by facilitating local histone H4 acetylation [52
]. Arrestin2 up-regulation results in enhanced activation of such promoters, and arrestin2 knockdown by siRNA suppresses transcription of these genes [52
]. Therefore, it is possible that elevated arrestin expression in the brain of patients with Parkinson's disease and dementia may modulate activation and/or expression of other signaling molecules either via cytoplasmic signaling mechanisms or via interaction with promoters of specific genes.
Arrestin activity is often contingent upon receptor phosphorylation (reviewed in [35
]). Therefore, changes in the concentration of GRKs modulate arrestin-mediated signaling. Different subclasses of GRKs have differential role in arrestin-dependent cellular functions [54
]. GRK2 and 3 largely mediate “classic” GRK functions, such as receptor phosphorylation, arrestin recruitment, and receptor endocytosis. However, arrestin-mediated ERK activation requires GRK5 or 6. ERK activation is enhanced by overexpression of GRK5 or 6 and inhibited by their knockdown. Manipulations of GRK2/3 concentrations have the opposite effects. So far, this functional antagonism has been demonstrated for two GPCRs, angiotensin II receptor [54
] and V2 vasopressin receptor [92
], and only in cultured cells. It is unclear to what extent these findings are applicable to the in vivo situation in general and to neuronal signaling mechanisms in particular. However, it seems likely that changes in GRK expression observed in PD with dementia cases contribute to specific alterations in arrestin-mediated signaling and, possibly, affect other downstream signaling pathways.
GRKs were traditionally considered selective for GPCRs. However, GRKs phosphorylate other substrates and therefore may have functions not directly related to GPCR regulation. GRK2 and GRK5 bind microtubules and phosphorylate tubulin, possibly promoting microtubule assembly [20
]. Visual arrestin has been shown to bind microtubules [38
], and this ability plays a role in arrestin-mediated visual adaptation [77
]. Non-visual arrestins also bind microtubules, demonstrating even higher affinity [39
]. It is conceivable that coordinated action of arrestins and GRKs at microtubules is important for function of neuronal cytoskeleton, and that it may be altered by enhanced expression of arrestins and GRKs in PD patients with dementia. GRK2 and GRK5 also phosphorylate synucleins, with GRK5 preferring α-synuclein as a substrate [90
]. GRK5-mediated phosphorylation of α-synuclein inhibits its interaction with phospholipids and phospholipase D2 acting as negative control of α-synuclein function in vesicle trafficking [90
]. It is possible that upregulation of GRK5 in the brains of PD patients with dementia modifies the α-synuclein activity and/or metabolism, possibly promoting Lewy body formation. Interestingly, we have observed higher levels of cortical and striatal insoluble α-synuclein in the PDD group as compared to PD and control (Joyce, Borwege and Osredkar, unpublished observation). It is important to note that α-synuclein phosphorylation by GRK5 is controlled by GPCR activity [90
] and, therefore, is likely to be affected by neurotransmitter deficits and drug treatment regiment.
Previously, we have examined the alterations in arrestin/GRK expression in the MPTP-treated Macaque monkeys [13
]. Non-human primates treated with neurotoxin MPTP to induce dopaminergic degeneration are considered the golden standard of animal models of PD. We have found that MPTP lesion significantly upregulated arrestin2 and two GRKs, GRK2 and 6, in most basal ganglia regions. Chronic L-DOPA treatment brings the expression of all proteins back to normal regardless whether dyskinesia develops in response to L-DOPA. Increased expression of arrestins and GRKs in MPTP-treated monkeys was accompanied by enhanced ERK activation and elevated total ERK concentration. In fact, MPTP-treated drug-naive monkeys resemble the PD with dementia group of human patients in terms of changes in arrestin/GRK concentrations, whereas the PD group is similar to L-DOPA-treated MPTP monkeys. Patients in both groups of this cohort had been treated with dopaminergic drugs during their illness. The only difference is that the PD group as a rule retained good therapeutic response to L-DOPA whereas most PDD patients became resistant to the medication. It is tempting to speculate that PD patients with dementia are resistant to L-DOPA on the therapeutic level because they are resistant to the drug on the molecular level. L-DOPA may down-regulate arrestin/GRK expression in the brain of some patients keeping them responsive to medication. At the same time those with persistently elevated arrestin/GRK expression become resistant to the therapeutic drug effects. Dementia seen in these patients may be linked to abnormally enhanced arrestin/GRK expression.
Many limitations inherent to postmortem studies often make it difficult to understand functional significance of postmortem findings. Any human disease exists as a plethora of individual forms that limited postmortem cohorts fail to represent fully. Postmortem specimens are often derived from elderly patients at late stages of the disease and invariably suffering from other disorders. Therefore, it is hardly possible to follow the progression of the disease or identify its unique mechanisms. Patients are treated antemortem with a wide variety of drugs that are likely to interfere with postmortem measurements. Inevitable postmortem delay may induce drastic changes in molecular structure. Specimens in the present study have remarkably short postmortem delay but it is still much longer than in animal experiments. Most importantly, postmortem studies cannot prove causal relationship between the disease symptoms and molecular features, because targeted experimental manipulations are not possible. Therefore, experimental analysis of the functional role of arrestins and GRKs in PD is best accomplished in animal models of PD that are amenable to experimental and genetic manipulations. However, there is no way to model in animals the combination of PD with dementia, which is not uncommon in human patients. The unique heuristic value of postmortem studies lies in the ability to study a real human disease at structural and molecular resolution not achievable by any other method applicable to the human brain.
Substantial changes in the expression levels of arrestins and GRKs in the striatum of patients with PDD described here suggests the involvement of these regulatory proteins in PD and/or dementia pathology and identifies them as novel targets for therapeutic intervention. It is possible that enhanced expression of the components of the GPCR regulation machinery seen in the PD with dementia cohort may be induced by dopaminergic denervation similarly to what happens in the MPTP monkey. In patients in which dopaminergic drugs fail to suppress arrestin/GRK expression, elevated concentration of these proteins may confer resistance to therapeutic effects of dopaminergic drugs and propagate pathology by interfering with the functions of the cytoskeleton and/or that of α-synuclein. Deciphering signaling mechanisms leading to the enhanced expression of arrestins and GRKs as well as downstream signaling events unleashed by high concentrations of arrestins and GRKs is critical for understanding of the mechanisms of the PD-associated dementia and for designing effective treatment strategies.