Marina Romero-Ramos

Neuroimmunological processes in Parkinson's disease and their relation to α-synuclein: microglia as the referee between neuronal processes and peripheral immunity.

The role of neuroinflammation and the adaptive immune system in PD (Parkinsons disease) has been the subject of intense investigation in recent years, both in animal models of parkinsonism and in post-mortem PD brains. However, how these processes relate to and modulate α-syn (α-synuclein) pathology and microglia activation is still poorly understood. Specifically, how the peripheral immune system interacts, regulates and/or is induced by neuroinflammatory processes taking place during PD is still undetermined. We present herein a comprehensive review of the features and impact that neuroinflamation has on neurodegeneration in different animal models of nigral cell death, how this neuroinflammation relates to microglia activation and the way microglia respond to α-syn in vivo. We also discuss a possible role for the peripheral immune system in animal models of parkinsonism, how these findings relate to the state of microglia activation observed in these animal models and how these findings compare with what has been observed in humans with PD. Together, the available data points to the need for development of dual therapeutic strategies that modulate microglia activation to change not only the way microglia interact with the peripheral immune system, but also to modulate the manner in which microglia respond to encounters with α-syn. Lastly, we discuss the immune-modulatory strategies currently under investigation in animal models of parkinsonism and the degree to which one might expect their outcomes to translate faithfully to a clinical setting.

Distribution of Na/K‐ATPase alpha 3 isoform, a sodium‐potassium P‐type pump associated with rapid‐onset of dystonia parkinsonism (RDP) in the adult mouse brain

The Na+/K+‐ATPase1 alpha subunit 3 (ATP1α3) is one of many essential components that maintain the sodium and potassium gradients across the plasma membrane in animal cells. Mutations in the ATP1A3 gene cause rapid‐onset of dystonia parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. To achieve a better understanding of the pathophysiology of the disease, we used immunohistochemical approaches to describe the regional and cellular distribution of ATP1α3 in the adult mouse brain. Our results show that localization of ATP1α3 is restricted to neurons, and it is expressed mostly in projections (fibers and punctuates), but cell body expression is also observed. We found high expression of ATP1α3 in GABAergic neurons in all nuclei of the basal ganglia (striatum, globus pallidus, subthalamic nucleus, and substantia nigra), which is a key circuitry in the fine movement control. Several thalamic nuclei structures harboring connections to and from the cortex expressed high levels of the ATP1α3 isoform. Other structures with high expression of ATP1α3 included cerebellum, red nucleus, and several areas of the pons (reticulotegmental nucleus of pons). We also found high expression of ATP1α3 in projections and cell bodies in hippocampus; most of these ATP1α3‐positive cell bodies showed colocalization to GABAergic neurons. ATP1α3 expression was not significant in the dopaminergic cells of substantia nigra. In conclusion, and based on our data, ATP1α3 is widely expressed in neuronal populations but mainly in GABAergic neurons in areas and nuclei related to movement control, in agreement with RDP symptoms. J. Comp. Neurol. 519:376‐404, 2011.

The relation between α-synuclein and microglia in Parkinson’s disease: Recent developments

Recent research suggests a complex role for microglia not only in Parkinsons disease but in other disorders involving alpha-synuclein aggregation, such as multiple system atrophy. In these neurodegenerative processes, the activation of microglia is a common pathological finding, which disturbs the homeostasis of the neuronal environment otherwise maintained, among others, by microglia. The term activation comprises any deviation from what otherwise is considered normal microglia status, including cellular abundance, morphology or protein expression. The microglial response during disease will sustain survival or otherwise promote cell degeneration. The novel concepts of alpha-synuclein being released and uptaken by neighboring cells, and their importance in disease progression, positions microglia as the main cell that can clear and handle alpha-synuclein efficiently. Microglias behavior will therefore be a determinant on the diseases progression. For this reason we believe that the better understanding of microglias response to alpha-synuclein pathological accumulation across brain areas and disease stages is essential to develop novel therapeutic tools for Parkinsons disease and other alpha-synucleinopathies. In this review we will revise the most recent findings and developments with regard to alpha-synuclein and microglia in Parkinsons disease.

α-Synuclein expression is modulated at the translational level by iron.

Several studies have suggested an interaction between &agr;-synuclein protein and iron in Parkinson’s disease. The presence of iron together with &agr;-synuclein in Lewy bodies, the increase of iron in the substantia nigra and the correlation between polymorphism of the several genes implicated in iron metabolism and Parkinson’s disease, support a role for iron in the neurodegeneration. Analysis of post mortem brains revealed increased amount of insoluble &agr;-synuclein protein despite unchanged/reduced levels of &agr;-synuclein mRNA in Parkinson’s disease. Interestingly, on the basis of the presence of a putative iron responsive element in the 5′-UTR, it has been suggested that there is a possible iron-dependent translational control of human &agr;-synuclein mRNA. Considering the similarity between the sequences present in human &agr;-synuclein mRNA and the ferritin iron responsive element, we postulated that iron deficiency would decrease the translation of &agr;-synuclein mRNA. Here we used HEK293 cells treated with iron chelator deferoxamine or ferric ammonium citrate to verify the possible iron-dependent translational control of human &agr;-synuclein biosynthesis. We show that the amount of polysome-associated endogenous human &agr;-synuclein mRNA decreases in presence of deferoxamine. Our data demonstrate that human &agr;-synuclein expression is regulated by iron mainly at the translational level. This result not only supports a role for iron in the translational control of &agr;-synuclein expression, but also suggests that iron chelation may be a valid approach to control &agr;-synuclein levels in the brain.

Long-term polarization of microglia upon α-synuclein overexpression in nonhuman primates.

We have previously shown that persistent α-synuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) α-synuclein. However, A53T α-synuclein overexpression induced neurodegeneration that resulted in nigral dopaminergic cell death. Here, we analyze the microglia population in the midbrain of these animals by stereological quantification of Iba1+ cells. Our data here show that monkeys overexpressing A53T α-synuclein showed a long-term increase in microglia presenting macrophagic morphology. However, wt α-synuclein overexpression, despite the absence of dopaminergic cell death, resulted in a permanent robust increase of the microglia population characterized by a range of distinct morphological types that persisted after 1 year. These results confirm that the microglial response differs depending on the type of α-synuclein (wt/A53T) and/or whether α-synuclein expression results in cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, the microglial response is modulated by events related to α-synuclein expression in substantia nigra and persists in the long term. The data presented here is in agreement with that previously observed in a recombinant adeno-associated virus (rAAV) α-synuclein rat model, thereby validating both the findings and the model, and highlighting the translational potential of the rodent model to higher species closer to humans.

Viral Vector Mediated Overexpression of Human alpha-Synuclein in the Nigrostriatal Dopaminergic Neurons: A New Model for Parkinson's Disease.

Parkinsons disease is predominantly a dopamine deficiency syndrome, which is produced in the brain by the loss of cells located in a small area in the ventral midbrain called the substantia nigra. Complete unilateral dopamine lesions, based on the administration of toxic substances (ie, 6-hydroxy-dopamine in rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice and primates) have been extremely useful in testing strategies of replacement. For example, the functional and biochemical impact of the transplanted ventral mesencephalic dopaminergic progenitors has been characterized to a large extent, using the complete lesion model in rats. Over the last decade, however, studies addressing the ability of neurotrophic factors to protect injured dopamine cells prompted researchers to make available partial and progressive lesion models to allow a window of opportunity to interfere the disease progression. Recent findings relating alpha-synuclein with Parkinsons disease pathology have opened new possibilities to develop alternative models based on the overexpression of this protein using recombinant adeno-associated viral vectors, which is valuable not only for helping to better understand its involvement in the disease process, but also to more closely resemble the neurodegeneration found in Parkinsons disease.

Chronic intranasal deferoxamine ameliorates motor defects and pathology in the α-synuclein rAAV Parkinson's model

Parkinsons disease is characterized by neuronal death in the substantia nigra and the presence of intracellular inclusions of α-synuclein in the Lewy bodies. Several lines of data support a role for iron in Parkinsons disease: iron is present in Lewy bodies, iron accumulates in the dopaminergic neurons in the substantia nigra, and Parkinsons disease is correlated with polymorphisms of several genes implicated in iron metabolism. Furthermore, iron can compromise the solubility of α-synuclein through direct interaction and can induce neurotoxicity in vitro. Here, we investigate the possible neuroprotective effect of the iron chelator deferoxamine in vivo to elucidate whether iron chelation can provide meaningful therapy for Parkinsons disease. Hence, we used a Parkinsons disease animal model based on unilateral injection of a recombinant adeno-associated viral vector encoding α-synuclein in the rat midbrain. Rats were treated with a novel deferoxamine delivery approach: 6 mg of the compound was administered intranasally three times a week for 3 or 7 weeks. The behavior of the animals and histopathological changes in the brain were analyzed. Our data show that although intranasal administration of deferoxamine in rats did not protect them from dopaminergic cell death, it did decrease the number of the pathological α-synuclein formations at the terminal level. In addition, this treatment resulted in changes in the immune response and an overall partial improvement in motor behavior. Taken together, our data show that in vivo iron chelation can modulate α-synuclein-induced pathology in the central nervous system. Our data suggest that chronic administration of intranasal deferoxamine may be a valid approach to limiting the mishandling of α-synuclein in the central nervous system observed in Parkinsons disease and slowing disease progression.

Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease.

Alpha-synuclein phosphorylated at serine 129 (S129) is highly elevated in Parkinsons disease patients where it mainly accumulates in the Lewy bodies. Several groups have studied the role of phosphorylation at the S129 in α-synuclein in a rat model for Parkinsons disease using recombinant adeno-associated viral (rAAV) vectors. The results obtained are inconsistent and accordingly the role of S129 phosphorylation in α-synuclein toxicity remains unclear. This prompted us to re-examine the neuropathological and behavioral effects of the S129 modified α-synuclein species in vivo. For this purpose, we used two mutated forms of human α-synuclein in which the S129 was replaced either with an alanine (S129A), to block phosphorylation, or with an aspartate (S129D), to mimic phosphorylation, and compared them with the wild type α-synuclein. This approach was similar in design to previous studies, however our investigation of dopaminergic degeneration also included performing a detailed study of the α-synuclein induced pathology in the striatum and the analysis of motor deficits. Our results showed that overexpressing S129D or wild type α-synuclein resulted in an accelerated dopaminergic fiber loss as compared with S129A α-synuclein. Furthermore, the motor deficit seen in the group treated with the mutant S129D α-synuclein appeared earlier than the other two forms of α-synuclein. Conversely, S129A α-synuclein showed significantly larger pathological α-synuclein-positive inclusions, and slower dopaminergic fiber loss, when compared to the other two forms of α-synuclein, suggesting a neuroprotective effect of the mutation. When examined at long-term, all three α-synuclein forms resulted in pathological accumulations of α-synuclein in striatal fibers and dopaminergic cell death in the substantia nigra. Our data show that changes in the S129 residue of α-synuclein influence the rate of pathology and neurodegeneration, with an overall deleterious effect of exchanging S129 to a residue mimicking its phosphorylated state.

Vaccination strategies for Parkinson disease: Induction of a swift attack or raising tolerance?

Parkinson disease is the second most common neurodegenerative disease in the world, but there is currently no available cure for it. Current treatments only alleviate some of the symptoms for a few years, but they become ineffective in the long run and do not stop the disease. Therefore it is of outmost importance to develop therapeutic strategies that can prevent, stop, or cure Parkinson disease. A very promising target for these therapies is the peripheral immune system due to its probable involvement in the disease and its potential as a tool to modulate neuroinflammation. But for such strategies to be successful, we need to understand the particular state of the peripheral immune system during Parkinson disease in order to avoid its weaknesses. In this review we examine the available data regarding how dopamine regulates the peripheral immune system and how this regulation is affected in Parkinson disease; the specific cytokine profiles observed during disease progression and the alterations documented to date in patients’ peripheral blood mononuclear cells. We also review the different strategies used in Parkinson disease animal models to modulate the adaptive immune response to salvage dopaminergic neurons from cell death. After analyzing the evidence, we hypothesize the need to prime the immune system to restore natural tolerance against α-synuclein in Parkinson disease, including at the same time B and T cells, so that T cells can reprogram microglia activation to a beneficial pattern and B cell/IgG can help neurons cope with the pathological forms of α-synuclein.

Anti-Inflammatory Modulation of Microglia via CD163-Targeted Glucocorticoids Protects Dopaminergic Neurons in the 6-OHDA Parkinson's Disease Model.

Increasing evidence supports a decisive role for inflammation in the neurodegenerative process of Parkinsons disease (PD). The immune response in PD seems to involve, not only microglia, but also other immune cells infiltrated into the brain. Indeed, we observed here the infiltration of macrophages, specifically CD163+ macrophages, into the area of neurodegeneration in the 6-hydroxydopamine (6-OHDA) PD model. Therefore, we investigated the therapeutic potential of the infiltrated CD163+ macrophages to modulate local microglia in the brain to achieve neuroprotection. To do so, we designed liposomes targeted for the CD163 receptor to deliver dexamethasone (Dexa) into the CD163+ macrophages in the 6-OHDA PD model. Our data show that a fraction of the CD163-targeted liposomes were carried into the brain after peripheral intravenous injection. The 6-OHDA-lesioned rats that received repeated intravenous CD163-targeted liposomes with Dexa for 3 weeks exhibited better motor performance than the control groups and had minimal glucocorticoid-driven side effects. Furthermore, these animals showed better survival of dopaminergic neurons in substantia nigra and an increased number of microglia expressing major histocompatibility complex II. Therefore, rats receiving CD163-targeted liposomes with Dexa were partially protected against 6-OHDA-induced dopaminergic neurodegeneration, which correlated with a distinctive microglia response. Altogether, our data support the use of macrophages for the modulation of brain neurodegeneration and specifically highlight the potential of CD163-targeted liposomes as a therapeutic tool in PD. SIGNIFICANCE STATEMENT The immune response now evident in the progression of Parkinsons disease comprises both local microglia and other immune cells. We provide evidence that CD163+ macrophages can be a target to modulate brain immune response to achieve neuroprotection in the 6-hydroxydopamine model. To do so, we targeted the CD163+ population, which to a low but significant extent infiltrated in the neurodegenerating area of the brain. Specially designed liposomes targeted for the CD163 receptor were loaded with glucocorticoids and injected peripherally to modify the infiltrated CD163 cells toward an anti-inflammatory profile. This modification of the CD163 population resulted in a distinctive microglial response that correlated with decreased dopaminergic cell death and better motor performance.