Astrocytes in Parkinson’s disease
Master Thesis Neuroscience & Cognition
Ischa Bruinsma
October – December 2009
Parkinson’s disease is a common neurodegenerative disorder with a high prevalence in people
over 65. Its main h...
1. Introduction
Though Parkinson’s disease was first described in 1817 its pathology is still not fully
understood. Seve...
of inherited PD, genetic linkage studies have identified 13 PARK loci to date. Molecular
genetics studies have identifie...
MPTP is a by-product of the chemical synthesis of a meperidine analogue with potent heroin-
like effects. It can induce ...
2005). After injection dopaminergic neurons in the SNpc die within 24 hours, show apoptotic
morphology and decreased α-s...
mice models overexpressing α-synuclein using various promoters and mutations. A problem
with choosing a promoter, is tha...
Astrocytes express glial fiblillary acidic protein (GFAP) which is an intermediate filament
involved in controlling the ...
2008). If the dopamine neurons, which spontaneously generate abundant free radicals during
the metabolism of dopamine, ...
2. Astrogliosis
Reactive astrogliosis, whereby astrocytes undergo varying molecular and morphological
changes, is a poo...
and its associated synaptic functions, could be important for the progressive nature of the
pathophysiology associated ...
astrocytic activation parallels the time course of dopaminergic cell death in the SNpc as well
as striatum and GFAP exp...
has also been shown that gluthatione, an important antioxidant enzyme, is downregulated in
the SN of PD patients (Pearc...
could be the trigger for astrogliosis (Sriram et al. 2004). A schematic representation of this
pathway is given in figu...
3. Neurodegenerative & proinflammatory factors in astrocytes
Reactive astrogliosis in response to injury is a mechanism...
space could potentially stimulate astrocytes into an inflammatory state. Monomeric and
aggregated α-syn may be secreted...
major cause of neuronal cell loss in PD. On top of this, increased astrocytic GSH levels lead
to increased GSH release,...
concentration precedes other hallmarks of PD and may be important in the early pathogenesis
of PD.
The reason that main...
the H2O2 they produce because they contain high levels of GSH and gluthatione peroxidase,
which can detoxify H2O2 withi...
microglia gathered in such areas of high ICAM-1 expression, and LFA-1 leukocytes
infiltrated the tissue (Miklossy et al...
4. Neuroprotective effects of astrocytes
Of course, reactive astrogliosis does not happen without a reason. Initially, ...
basal state. NF-κB target genes include many pro-inflammatory proteins. Nurr1 is recruited to
NF-κB on inflammatory gen...
purines (Cicarelli et al. 1999). They release either adenine-based purines, e.g. adenosine and
adenosine triphosphate, ...
4.6 Protective effects of enriched environment
It has been found that enriched environment (EE) has protective effects ...
trophic activity (Nakajima et al. 2001). Reactive astrocytes might mediate increased basic
fibroblast growth factor and...
5. Potential therapies for PD treatment targeting astrocytes
Some of these findings about the role of astrocytes in PD ...
treatment prevented reduction in striatal dopamine and loss of loss of dopaminergic neurons
in the SN. Pre-treatment wi...
6. Discussion
Astrocytes play an important role in neurodegenerative diseases. In situations of neuronal
damage they ca...
Protective effects might be mediated through upregulation of PAR-1 which increases GSH
expression and secretion of neur...
the Nrf2-ARE pathway seems to be a very effective way to prevent neuronal degradation in
the SN and halt PD progression...
List of abbreviations
6-OHDA 6-hydroxydopamine
α-syn α-synuclein
ACM astrocytic culture medium
ARE antioxidant response...
Aderka D, Le JM, Vilcek J. IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in
Fattori E, Lazzaro D, Musiani P, Modesti A, Alonzi T, Ciliberto G. IL-6 expression in neurons of transgenic
mice causes...
Kumar MJ, Nicholls DG, Andersen JK. Oxidative alpha-ketoglutarate dehydrogenase inhibition via subtle
elevations in mon...
Norton WT, Aquino DA, Hozumi I, Chiu FC, Brosnan CF. Quantitative aspects ofreactive gliosis: a review.
Neurochem Res. ...
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Astrocytes in Parkinson's disease

  1. 1. Astrocytes in Parkinson’s disease Master Thesis Neuroscience & Cognition Ischa Bruinsma October – December 2009 Supervisor: Simone van den Berge
  2. 2. 2 Abstract Parkinson’s disease is a common neurodegenerative disorder with a high prevalence in people over 65. Its main hallmark is progressive loss of dopaminergic neurons in the substantia nigra, leading to characteristic motor symptoms such as tremor and bradykinesia. The pathology of this disease is not yet fully understood. Recently it has been suggested that astrocytes might play an important role in disease initiation and progression. In response to neuronal damage astrocytes can enter a state called reactive astrogliosis, which is characterised by hypertrophy and increased expression of glial fibrillary acidic protein. Though initially meant as a protective reaction, this can have both beneficial and detrimental effects on surrounding neurons. In this review the role that astrocytes might play in Parkinson’s disease is discussed. Evidence of astrogliosis in animal models of Parkinson’s disease and post mortem studies of patients is described, implicating the involvement of this process. Subsequently, several factors are discussed that are important during the astrocytic response in Parkinson’s disease, divided in neuroprotective and neurodegenerative effects. Based upon the astrocytic factors that could contribute to disease pathology, some potential therapies for Parkinson’s disease targeting astrocytes are suggested.
  3. 3. 3 Contents Abstract.......................................................................................................................................2 Contents......................................................................................................................................3 1. Introduction ............................................................................................................................4 1.1 Parkinson’s disease...........................................................................................................4 1.2 Animal models of PD .......................................................................................................5 1.3 Astrocytes .........................................................................................................................8 1.4 Astrocytes in the substantia nigra.....................................................................................9 2. Astrogliosis...........................................................................................................................11 2.1 Astrogliosis in animal models of PD..............................................................................12 2.2 Astrogliosis in PD patients .............................................................................................13 2.3 Remote astrocyte activation ...........................................................................................14 2.4 Activation pathway.........................................................................................................14 3. Neurodegenerative & proinflammatory factors in astrocytes ..............................................16 3.1 α-synuclein in astrocytes and Lewy bodies....................................................................16 3.2 ER stress and unfolded proteins .....................................................................................17 3.3 The role of astrocytes in oxidative stress-induced neuronal degradation.......................17 3.4 MAO-B expression in astrocytes and production of reactive oxygen species ...............19 3.5 Immune responses and the role of astrocytes in sustaining inflammation .....................20 3.6 Effect of hormones on astrocyte development and response to injury...........................21 4. Neuroprotective effects of astrocytes ...................................................................................22 4.1 Protective effects of PAR-1 activation...........................................................................22 4.2 Role of Nurr1 in protecting dopaminergic neurons........................................................22 4.3 Anti-inflammatory and neuroprotective effects of hydrogen sulphide ..........................23 4.4 The role of purines in regulation of the astrocytic response ..........................................23 4.5 Astrocyte influence on microglia ...................................................................................24 4.6 Protective effects of enriched environment....................................................................25 4.7 Astrocyte influence on neurogenesis..............................................................................25 4.8 Nrf2-mediated gene transcription inducing a large neuroprotective response...............26 5. Potential therapies for PD treatment targeting astrocytes ....................................................27 6. Discussion.............................................................................................................................29 List of abbreviations .................................................................................................................32 References ................................................................................................................................33
  4. 4. 4 1. Introduction Though Parkinson’s disease was first described in 1817 its pathology is still not fully understood. Several factors are known to contribute to disease initiation and/or progression (Gasser 2009; Weidong Le et al. 2009) but it is not known why certain people develop the disease while others do not. This review focuses on a particular aspect of the disease: the involvement of astrocytes. The importance of astrocytes in normal brain function and their possible involvement in neurodegenerative disorders has only recently been discovered. In response to certain signals, astrocytes can start an active inflammatory response called reactive astrogliosis (Eddleston & Mucke 1993), which is implicated to be important in the pathology of Parkinson’s disease. Here, an overview is given of how astrocytes and reactive astrogliosis might be involved in Parkinson’s disease and different models of this disease. First, an introduction is given explaining some background information on Parkinson’s disease, different animal models for this disease, and normal astrocyte function. Subsequently, the concept of astrogliosis will be explained and some detrimental as well as beneficial factors are presented that might play a role in reactive astrogliosis in Parkinson’s disease. Finally, some potential therapies targeting astrocytes are suggested. 1.1 Parkinson’s disease Parkinson’s disease (PD) is a common neurodegenerative disorder with a prevalence of 1.8 % in people over 65 (Rijk et al. 2000). It is characterised by disabling motor abnormalities such as tremor, muscle rigidity, bradykinesia, postural instability and other accompanying symptoms such as fatigue and speech problems (Purves et al. 2001) . Overall, PD is primarily idiopathic with only a subset of cases (<15% of cases) with a family history of PD. In pedigrees with a patternFigure 1 In Parkinson’s disease neurons are lost in the substantia nigra, which is located in the midbrain. (
  5. 5. 5 of inherited PD, genetic linkage studies have identified 13 PARK loci to date. Molecular genetics studies have identified genes associated with 7 of 13 PARK loci (Harvey et al. 2008). The primary neuropathological hallmark of PD is loss of pigmented dopaminergic neurons in the substantia nigra pars compacta (SNpc), projecting to the striatum. This results in a deficit in striatal dopamine levels, leading to most of the clinical symptoms. In figure 1 the neuronal loss in the SN of a PD patient is shown compared to a healthy individual. Until now, the most common treatment for this disease is administration of levodopa (L-dopa), a dopamine precursor, which increases dopamine levels in the brain. However, especially after chronic administration, this treatment has many side effects and loses its effectiveness (Schapira et al. 2009). Moreover, L-dopa administration does not interfere with disease progression but only alleviates the symptoms. Therefore, it is important to require a deeper understanding of the causes and pathology of PD. Not only to try to prevent the disease but also to develop new therapies that might stop or slowdown disease progression. PD is diagnosed pathologically by the loss of pigmented neurons in the SNpc and is associated with widespread occurrence of Lewy bodies and dystrophic Lewy neurites throughout the central and autonomic nervous system (Gelb et al. 1999). These are abnormal intracellular aggregates of α-synuclein in respectively neurons and axons, that might contribute to disease pathology. It has also been found that oxidative stress plays an important role in disease pathology and might be the main cause of neuronal degradation. In post mortem studies, evidence of increased oxidative stress in the SNpc of PD patients has been observed (Hunot et al. 1996). 1.2 Animal models of PD PD is a multifactorial disease with a complex etiology that results from genetic risk factors, environmental exposures and most likely a combination of both. Rodent models of Parkinsonism aim to reproduce key pathogenic features of the syndrome, including movement disorder induced by the progressive loss of dopaminergic neurons in the substantia nigra, accompanied by the formation of α-synuclein containing Lewy body inclusions. The two most widely used models for PD are the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model and the 6-hydroxydopamine (6-OHDA) lesion model, these and other rodent models are reviewed in Melrose et al. 2006.
  6. 6. 6 MPTP is a by-product of the chemical synthesis of a meperidine analogue with potent heroin- like effects. It can induce a Parkinsonian syndrome in humans almost indistinguishable from PD. MPTP is a potent and irreversible mitochondrial complex I inhibitor whose toxic metabolite MPP+ is selectively transported by the dopamine transporter DAT (Watanabe et al. 2005). The conversion of MPTP in astrocytes and the effect of MPP+ on neurons is shown in figure 2. MPTP causes damage to the dopaminergic pathways identical to that seen in PD with a greater loss of neurons in the SNpc than in the ventral tegmental area (Muthane et al. 1994) and greater loss of nerve terminals in the putamen than in the caudate nucleus (Moratalla et al. 1992). Differences with PD are that MPTP damage has much faster effects and no Lewy bodies are found after MPTP administration in humans, though there is upregulation of α-synuclein expression in rodents and primates (Meredith et al. 2002). Dopaminergic neurons in rats are relatively resistant to MPTP-induced toxicity and in mice susceptibility of the nigrostriatal pathway to neurodegeneration is strain dependant (Muthane et al. 1994). 6-OHDA administration causes nigrostriatal depletion when stereotaxically injected into the SN, median forebrain bundle or striatum. It destroys catecholaminergic neurons through a combination of reactive oxygen species and increased toxic quinones (reviewed by Bove et al. Figure 2 Schematic representation of the conversion of MPTP in astrocytes and the effects of MPP+ on neurons (Vila & Predborski 2003).
  7. 7. 7 2005). After injection dopaminergic neurons in the SNpc die within 24 hours, show apoptotic morphology and decreased α-synuclein mRNA (corresponding with neuronal degeneration), creating a PD like situation. A difference with PD is that Lewy body formation has not been observed in this model. Models based on neurotoxins have enormous value in helping to understand the consequences of nigrostriatal loss and to test symptomatic therapies and interventions. However, there is no clear evidence that the mechanisms of action of these toxins are similar to neuropathological processes occurring in PD. Furthermore, there is no evidence to date that effective neuroprotection against these toxins translates into an effective neuroprotective therapy in humans with PD. Another major limitation of the toxin-based models is that they do not reproduce the pathology and cell loss observed in other brain regions and peripheral tissues of patients, nor the broad range of non-motor symptoms seen in PD. Next to models based on neurotoxicity, there are now also transgenic models available. Genetic mutations identified in familial Parkinsonism have recently provided a new approach to understand the molecular pathways affected. Transgenic models with knock-outs, overexpression or mutations in single genes provide a powerful new set of molecular tools to study etiology. Examples of genes that can be manipulated in PD models are recessively inherited loss-of-function mutations in Parkin, DJ-1 and PTEN-induced putative kinase-1 (PINK1) (for review see Harvey et al. 2008). In the past decade these were found to cause early-onset (< 50 years at onset), L-DOPA-responsive Parkinsonism. The slowly progressive and predominant motor phenotype in these patients suggests a disorder largely restricted to dopaminergic neuronal loss. The majority of patients with Parkin-linked disease demonstrate neuronal loss restricted to the substantia nigra. In contrast, dominantly inherited, gain-of- function mutations in α-synuclein and leucine-rich repeat kinase result in more typical, late- onset, Lewy body Parkinsonism with multi-system involvement (Ross & Farrer 2005). Presently, it is unknown whether genetic causes identified in rare, Mendelian forms of Parkinsonism highlight pathways affected in idiopathic PD. Parkinson’s syndrome most likely results from an intricate combination of gene and gene-environment interactions. The most optimal model for studying PD pathology would be one more closely reflecting sporadic forms of the disease. The only genetic abnormality to date for which good evidence exists in favour of a link with sporadic PD, is overexpression of wild-type α-synuclein via duplication or triplication of the gene (Lee & Trojanowski 2006). Furthermore, patients with sporadic forms of the disease present with abnormal α-synuclein accumulation and aggregates in a subset of central and peripheral neurons (Halliday et al. 2006). There are several transgenic
  8. 8. 8 mice models overexpressing α-synuclein using various promoters and mutations. A problem with choosing a promoter, is that this often restricts expression to a specific area or specific neurons, while models should mimic the broad but regionally selective α-synuclein pathology observed in patients. It has also been found that some modifications, such as double mutation or truncation, are necessary to obtain cell loss and decrease dopamine levels in mice (reviewed by Chesselet 2008). The relevance of these mechanisms of α-synuclein toxicity to sporadic PD remains unclear because patients do not have doubly mutated α-synuclein and the phenotype induced by the truncated protein so far does not mimic that of PD. However, there is a reasonable likelihood that these models share mechanisms that occur in sporadic PD (Chesselet 2008). Mutations accelerate the formation of abnormal forms of the protein that can also be adopted by wild-type α-synuclein, and truncated forms of the protein are found in patient brains (Follmer et al. 2007). Another approach is to overexpress α-synuclein using viral vectors which produces a rapid degeneration of nigrostriatal neurons (Kirik & Bjorklund 2003). This revealed the ability of wild-type α-synuclein to induce nigrostriatal pathology. The well controlled regional and temporal overexpression, and the lack of expression during embryonic and post-natal development, which may better mimic disease conditions and avoid the upregulation of defence mechanisms, are distinct advantages. However, only a subset of neurons is transduced in these models, which again brings up the problem of multiple affected neuronal systems in PD pathology (Chesselet 2004). Of course the shortcomings of available models should not discourage their use and delay progress in the field of therapeutic strategies for PD. Each model has its own advantages and disadvantages and they can be used to test various aspects of the disease. 1.3 Astrocytes Astrocytes are the major cell population within the central nervous system (CNS), they make up 55-60% of total brain cells. Astrocytes are complex highly differentiated cells that are present throughout the entire CNS. They make numerous essential contributions to normal functioning in the healthy CNS. Examples of their many functions are regulation of blood flow, provision of energy metabolites to neurons, participation in synaptic function and plasticity and maintenance of the extracellular balance of ions, fluids and transmitters. Other active functions might be synchronisation of neuronal firing patterns through neurotransmitter release (reviewed by Volterra 2005).
  9. 9. 9 Astrocytes express glial fiblillary acidic protein (GFAP) which is an intermediate filament involved in controlling the shape and movement of astrocytes and is important for astrocyte- neuronal interaction. GFAP-mediated astrocytic processes play a vital role in modulation synaptic efficacy in the CNS. GFAP is also essential for normal white matter architecture and blood-brain barrier integrity (reviewed by Eng et al. 2000). Not too long ago, it was believed that astrocytes were inactive elements just providing a scaffold function in the CNS. Now, it is known that astrocytes express almost the same set of ion channels and receptors as neurons and they can respond to activation and have active modulatory roles in intercellular communication (Seifert et al. 2006; Volterra 2005). It was also found that not all astrocytes are the same with respect to antigen profiles and functional properties. However, not much is known yet about expression and function of these different astrocytes. 1.4 Astrocytes in the substantia nigra It has been known for a long time that not all astrocytes in the brain are the same. Initially, a difference was found between astrocytes in grey and white matter but later it was discovered that there is also regional and even intraregional heterogeneity (Bachoo et al 2004). In the brain, astrocytes have an ordered arrangement with minimal overlap, forming discrete territories in parallel with neuronal and vascular territories (Bushong et al. 2002). Astrocytes in specific brain areas might differ for example in the neurotransmitter receptors they express, their immune response, opiod receptor expression or gap junction coupling (Yeh et al. 2009). Astrocytes can also be divided in one half exhibiting high GFAP expression, low input resistance, a typical irregular cell body with branched processes and low membrane potential and another half with low GFAP expression, larger input resistance and lower glutamate uptake that are not couple through gap junctions (Volterra et al. 2005). Though it is widely known that astrocytes form a heterogeneous population, not much is known about specific properties of astrocytes in certain brain areas such as the substantia nigra, which is important in PD. The reason for the specific loss of dopaminergic neurons in the SN in PD might be based on special properties of the astrocytes in this area. These could be more vulnerable to mutations or susceptible to environmental effects, and could have a different response to injury than astrocytes in other brain areas. It is known, for example, that there are lower numbers of astrocytes in the SN than in other brain areas (Mena & Yebenes
  10. 10. 10 2008). If the dopamine neurons, which spontaneously generate abundant free radicals during the metabolism of dopamine, are less protected by a smaller proportion of guardian cells with high free radical scavenging properties, such as the astrocytes, then this could be an explanation for the increased susceptibility of the nigral dopamine neurons. Astrocytes support the differentiation, survival, pharmacological properties, and resistance to injury of dopamine neurons (Mena & Yebenes 2008). Astrocytes in the striatum have also been found to express relatively high levels of intercellular adhesion molecule-1 (ICAM-1), an inflammatory mediator (Morga et al. 1998), which might make this region more susceptible to inflammation. The SN appears to be particularly vulnerable to inflammatory processes. For example, lipopolysaccharide (LPS), an inducer of immune response, injected into the SN leads to loss of tyrosine hydroxylase(TH)- cells, while there is no cell loss when it is injected into the hippocampus or cortex (Liu et al. 2006). This vulnerability could be due to the high level of oxidative action in dopaminergic neurons or to the possible higher abundance of microglial cells. Another factor making the SN more vulnerable is the high levels of MAO-B in this area in neurons and astrocytes (Damier et al. 1996). MAO-B metabolises dopamine, which is present in particularly high levels in the SN, because it is produced here, and is released by damaged neurons. During conversion of its substrate, MAO-B produces ROS, which increases oxidative stress and can be damaging to neighbouring neurons.
  11. 11. 11 2. Astrogliosis Reactive astrogliosis, whereby astrocytes undergo varying molecular and morphological changes, is a poorly understood hallmark of all central nervous system pathologies. Reactive astrogliosis can be induced by all forms and severities of CNS injury or disease including subtle perturbations. It is not an all-or-nothing response but a finely gradated continuum of progressive changes in gene expression and cellular changes that are subtly regulated by complex intercellular and intracellular signalling. The changes undergone by reactive astrocytes vary with the nature and severity of the injury and are regulated in a context specific manner (Sofroniew 2009). Cellular changes include hypertrophy, and in severe cases, proliferation and scar formation, as can be seen in figure 3 and 4. A very important aspect of astrogliosis is the upregulation of GFAP, which is often used as a marker for reactive astrogliosis (Eddleston & Mucke 1993). In figure 3 an immunohistochemical staining for GFAP is shown in healthy tissue and different severities of astrogliosis, clearly showing morphological changes during reactive astrogliosis. The changes undergone during reactive gliosis have the potential to alter astrocyte activity both through loss and gain of functions (Sofroniew 2009). These changes can have both beneficial and detrimental effects on surrounding neural and non-neural cells. Several aspects of reactive astrogliosis might play a role in PD pathology. Though astrogliosis might be beneficial in many ways, for example in maintaining extracellular glutamate levels and homeostasis in the striatum after dopaminergic neuronal loss, normal astrocytic functions might be compromised during astrogliosis. It has been found for example that the number of glutamate transporters per astrocyte is reduced in a model of chronic PD (Dervan et al. 2004). Changes in astrocyte ability to regulate glutamate, Figure 3 Photomicrographs of immunohistochemical staining of glial fibrillary protein (GFAP) in astrocytes in wild type mice. In healthy tissue and of different gradations of reactive astrogliosis and glial scar formation after tissue insults of different types and different severity. (Sofroniew 2009)
  12. 12. 12 and its associated synaptic functions, could be important for the progressive nature of the pathophysiology associated with Parkinson’s disease. Figure 4 schematic overview of changes undergone by astrocytes during reactive astrogliosis. (Buffo et al. 2009) 2.1 Astrogliosis in animal models of PD In the MPTP model, the start of an astrocytic reaction is observed two days after MPTP application (Kohutnica et al. 1998). This is one day later than the start of the microglial reaction, suggesting that the astrocytic reaction depends on factors released by microglia, such as interleukin-1, which is a known stimulator of astrogliosis (Giulian & Lachman 1985). In the striatum the response is maximal after 5 days and in the SN 14 days after MPTP administration (Kohutnica et al. 1998). Blocking conversion of MPTP into MPP+ with pergyline, a MAO-B inhibitor, inhibits the astrocytic response to MPTP. Although the conversion of MPTP to MPP+ occurs mainly in astrocytes, MPTP alone is not a factor inducing the reaction of these cells. In MPTP induced reactive astrocytes, interleukin-6 (IL-6) expression has also been detected (Kohutnica et al. 1998). It has been shown that IL-6 induces the synthesis of neurotrophic factors, such as nerve growth factor, by astrocytes (Frei et al. 1989), and inhibits the production of neurotoxic agents like tumor necrosis factor α (TNFα)(Aderka et al. 1989). Overexpression of this cytokine leads to an increase in the number of GFAP positive astrocytes (Fattori et al. 1995). Furthermore, in the MPTP model,
  13. 13. 13 astrocytic activation parallels the time course of dopaminergic cell death in the SNpc as well as striatum and GFAP expression remains upregulated, even after most of dopaminergic neurons have died due to MPTP intoxication. These findings implicate that astrocytic reaction occurs after neuronal cell death and might play a role in the propagation of the neurodegenerative process but not in its initiation (Liberatore et al. 1999). In the 6-OHDA model, upregulation of GFAP is seen 24 hours after lesioning, and peaks at 4 days when GFAP levels are almost 4 times as high. This astrocytic response is transient and returns to control levels after 28 days. When 6-OHDA is injected unilaterally a small astrocytic response can be seen in the SN at the controlateral site as well. Furthermore, 6- OHDA injection can induce GFAP changes over long distances in the striatum and even in the cortex (Henning et al. 2008; Sheng et al. 1993). These results suggest that astroglial reaction is triggered directly or indirectly by factors released from damaged neurons. However, it might be that astroglial reaction is triggered only by the injection lesion and not specifically by 6-OHDA damage (Depino et al. 2003). 2.2 Astrogliosis in PD patients Though reactive astrogliosis can readily be found in animal models of PD, autopsy studies of patients clinically and pathologically diagnosed with PD suggest that there might not be any significant astrogliosis in the substantia nigra of PD patients. Some immunohistochemistry experiments on the SN and putamen of PD patients did not find any alterations in the amount of GFAP-immunoreactive astrocytes or in their morphology, compared to control brains. Typical morphological characteristics of astrogliosis, such as hyperthrophy, shortening of cytoplasmic processes and nuclear enlargement, were not found. Moreover, no difference in the expression of metallothioneins was found, which are normally increased as protection to increased oxidative stress (Mirza et al. 2000). However, others did find increased amounts of reactive astrogliosis in PD patients, using GFAP-immunoreactivity (Miklossy et al. 2006). This reactive astrogliosis was accompanied by high inter-cellular adhesion molecule 1(ICAM- 1) expression in the astrocytes and high expression of the counterreceptor Lymphocyte function-associated antigen 1 (LFA-1) in microglia,which might be important for sustaining inflammation (as explained in section 3.5). At post mortem examinations it has also been found that there are increased amounts of NO radicals in brains of PD patients (Hunot et al. 1996). In addition to evidence for increased NO production in PD, impairment of mitochondrial function is also evident (Heales et al. 2004). It
  14. 14. 14 has also been shown that gluthatione, an important antioxidant enzyme, is downregulated in the SN of PD patients (Pearce et al. 1997; Sian et al. 1994) 2.3 Remote astrocyte activation In PD and animal models of PD, astrocytes are not only active at the site of neurodegeneration, the SN, but also in other areas such as the subthalamic nucleus (STN) and globus pallidus (GP)(Henning en al. 2008). It is hypothesised that communication links exist between astrocytes, or between neurons and astrocytes, along neuronal pathways that transmit activating signals in response to neuronal damage, but only if the neuronal pathways are at least partially intact. Analysis of astrocyte activation in two 6-OHDA rat models of PD: partial and complete SNc lesions, by injections of 6-OHDA in the striatum and medial forebrain bundle, respectively, has led to the finding that astrocyte activation after partial lesioning can spread to the GP and STN while complete lesioning results only in astrocyte activation at the lesion site (Henning et al. 2008). Astrocytes can presumably propagate information about neuronal damage or reduced activity through gap-junction linked astrocytic networks. Complete degradation of a neuronal pathway might lead to breakdown of glial communication, for example through loss of gap-junctions, which are no longer necessary for uptake of excess ions or neurotransmitters. 2.4 Activation pathway Though reactive gliosis is a very common phenomenon not much is known about the molecular pathways leading to this reactive state in various neurodegenerative diseases. Analysis of gene expression changes and protein phosphorylation in the MPTP model has identified the JAK-STAT pathway to be involved in astrocyte activation in PD. Administration of MPTP caused rapid phosphorylation by JAK2 and nuclear translocation of STAT3 in striatal astrocytes, prior to the induction of GFAP mRNA and protein (Sriram et al. 2004). Phosphorylated STAT3 can enhance transcription of GFAP possibly via a STAT3 binding site in the GFAP promoter. This indicates that the JAK2/STAT3 pathway is involved in induction of astrogliosis. The JAK2/STAT3 can be activated by several gp130-related cytokines, such as IL-6, leukaemia inhibitory factor (LIF) and oncostatin-M (OSM). Expression of these ligands can be induced by MPTP-mediated neuronal damage, which
  15. 15. 15 could be the trigger for astrogliosis (Sriram et al. 2004). A schematic representation of this pathway is given in figure 5. Figure 5 Schematic diagram showing the involvement of gp130-mediated phosphorylation of JAK2/STAT3 pathway inducing GFAP expression. Upon putative ligand (e.g. IL-6, LIF, OSM) binding, JAK2 and STAT3 are recruited to the gp130-signal transducer,JAK2 phosphorylates the Tyr-705 residue on STAT3. The phosphorylated STAT3 dimerize, translocate to the nucleus and mediates transcriptional activation of astrocytic genes such as GFAP. (Sriram et al. 2004)
  16. 16. 16 3. Neurodegenerative & proinflammatory factors in astrocytes Reactive astrogliosis in response to injury is a mechanism to clean up damage and control its spread. Part of this reaction is the stimulation of inflammatory processes which can help attract other damage controlling cells. However, too much inflammation increases, instead of restricts damage. In some situations, the immune system can start to attack healthy cells instead of just cleaning up damaged ones. Astrocytes can stimulate such neurodegenerative processes and sustain the inflammatory response, sometimes even after the initial trigger is gone. 3.1 α-synuclein in astrocytes and Lewy bodies α-synuclein (α-syn) is a 14 kDa acidic protein concentrated in presynaptic neuronal terminals (Norris et al. 2004). Normal physiological functions are hypothesised to include synaptic vesicle turnover, synaptic plasticity, ubiquitin-proteasome processing and molecular chaperoning. Its mutations are a cause of autosomal dominant PD. Currently there are three known point mutations in the α-syn gene: A30P, E46K and A53T; duplications and triplications also play a role in PD development (Lee & Trojanowski 2006). WT and mutated forms of α-syn can up-regulate ICAM-1 expression and IL-6 secretion in human astrocytes. Mutated forms are more potent for this than WT forms, which might explain why they induce autosomal dominant PD (Klegeris et al. 2006). Aggregated α-syn is a major component of Lewy bodies, which are frequently found in PD (Gelb et al. 1999), also in astrocytes. Astrocytic inclusions in other pathologies, such as progressive supranuclear palsy and corticobasal degeneneration, usually contain tau and not α-syn. Ultrastructurally astrocytic inclusions in PD are composed of a meshwork of randomly arranged, loosely packed α-syn filaments, with diameters of 20-40 nm (Wakabayashi et al. 2000). The distribution of glial cells with inclusions in PD is similar to that of catecholaminergic neurons in the midbrain. The amount of α-syn positive glial inclusions correlates with nigrostriatal neuronal loss (Wakabayashi et al. 2000). Overexpression of α-syn in astrocytes results in cell death (Stefanova et al. 2001) but α-syn aggregation in glial cells takes a long time and degeneration of glial cells is much slower than that of neuronal cells (Wakabayashi et al. 2000). Unaggregated α-syn is an effective stimulator of astrocytes and inflammatory processes. Leakage or excretion of this protein from normal or damaged neurons into the extracellular
  17. 17. 17 space could potentially stimulate astrocytes into an inflammatory state. Monomeric and aggregated α-syn may be secreted via a special endoplasmic/Golgi-independent exocytosis pathway (Lee et al. 2005). Such secretion is enhanced by proteasomal and mitochondrial dysfunction which have been found in PD. α-syn could influence astrocytes through all three major MAPK pathways: ERK1/2, JNK and p38, all of which have been associated with the actions of α-syn. Inhibitors of these MAPK pathways lower IL-6 secretion and ICAM-1 expression in human astrocytes and α-syn significantly increases phosphorylation of ERK1, ERK2, p47 JNK and p38 MAPK (Klegeris et al. 2006). 3.2 ER stress and unfolded proteins It has been recently found that endoplasmic reticulum (ER) stress and aberrant protein degradation might also play an important role in the pathogenesis of neurodegenerative disorders. Homocysteine-induced endoplasmic reticulum protein is a stress-response protein located in the ER membrane of neurons and astrocytes that can help defend against ER stress. In PD this protein is found in neurons and astrocytes in the SN and it is accumulated in Lewy bodies, suggesting a role in their formation. Unfolded proteins in astrocytes could induce the inflammatory response (Slodzinski et al. 2009). 3.3 The role of astrocytes in oxidative stress-induced neuronal degradation When astrocytes are stimulated by inflammatory mediators such as cytokines and lipopolysaccharides, induction of the Ca2+ independent isoform of inducible nitric oxide synthetase (iNOS), leads to generation of nitric oxide (NO) (Bolanos et al. 1994). In situations of neuroinflammation, such as PD, astrocytes might contribute to neuronal cell death by increasing NO production. NO also induces damage to the electron transport chain of brain mitochondria leading to mitochondrial impairment, resulting in more oxidative stress (Bolanaos et al. 1994). NO reacts with O2- to form peroxynitrite and its reactive intermediates which can react indiscriminately with proteins, DNA, and other cell constituents. On the other hand, upon exposure to NO, astrocytes increase their cellular glutathione (GSH) availability, which can react with reactive oxygen species such as NO, reducing NO levels. This can protect against oxidative stress induced neuronal degradation, which might be a
  18. 18. 18 major cause of neuronal cell loss in PD. On top of this, increased astrocytic GSH levels lead to increased GSH release, increasing neuronal protection. NO exposure leads to increased GSH efflux from astrocytes, even after removal of NO. This is not caused by increased permeability of the plasma membrane but is a regulated response (Gegg et al. 2003). Neurons co-cultured with astrocytes approximately double their GSH concentration, protecting them against NO damage (Gegg et al. 2003). Thus, astrocytes can be both protective against oxidative stress by increasing their GSH production and increase oxidative stress via production of NO, damaging neurons. From post mortem brain studies it is known that NO overproduction is found in PD patients. Glial cells marked for high NOS activity and NO radicals have been identified in the SN post mortem examination (Hunot et al. 1996). Furthermore, nitrotyrosine (an index of reactive nitrogen species formation) residues and elevated nitrosylated proteins have been reported, and determination of nitrite in cerebrospinal fluid also implies increased formation of NO within the CNS (Heales et al. 2004). This supports the damaging effects of astrocytes producing NO. Proof of the importance of astrocytic gluthatione for neuronal survival comes from a study using astrocyte-coated dialysis membranes placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium (Chen et al. 2001). Using this technique, it was established that astrocytes can protect neurons against NO induced damage. Furthermore, it was found that gluthatione-depleted astrocytes cannot protect neurons against NO damage and the gluthatione content of astrocytes directly reflects their protective potential. GSH has a special function in protection against NO, but does not influence normal neuronal survival in the absence of NO. Because astrocyte processes form a nearly continuous membrane around neuronal cell bodies and processes, they can reduce the amount of NO reaching neurons by trapping it. GSH can also function as an exchange of reducing equivalents between astrocytes and neurons. Unfortunately, it is known that the protection afforded to neurons by astrocytes is finite, possibly due to a decline in GSH trafficking with chronic iNOS induction (Heales et al. 2004). Depletion of GSH may facilitate production of reactive oxygen species by astrocytes. GSH is important in limiting and repairing the deleterious actions of NO, but GSH levels can be depleted by too high concentrations of NO (Chen et al. 2001). In PD a 40% loss of GSH has been reported in the SN (Sian et al. 1994). This is at odds with elevated GSH as a protection mechanism, but could be explained by the mechanism being transient. This decrease in GSH
  19. 19. 19 concentration precedes other hallmarks of PD and may be important in the early pathogenesis of PD. The reason that mainly neurons are affected in PD could be that neurons seem to be more susceptible to NO exposure. GSH concentration is normally higher in astrocytes than in neurons and in neurons NO exposure leads to a decrease instead of an increase in GSH. There is also a greater inhibition of the mitochondrial respiratory chain in neurons than in astrocytes exposed to NO (Gegg et al. 2003). GSH is synthesised by glutamate-cysteine ligase (GCL), which is the rate limiting enzyme. The increased GSH concentration in astrocytes might be explained by increased GCL activity after NO exposure, while in neurons there is no change in this activity. The high sensitivity of neurons to NO might be caused by low CGL activity and the inability to increase this activity upon NO exposure, which leads to low GSH concentrations and greater susceptibility to oxidative stress. Increased iNOS expression by astrocytes, which produces excessive amounts of NO, could also play a role in inducing increased GFAP expression, a hallmark of reactive astrogliosis. Inflammatory mediators and inducers of NO production induce the expression of GFAP in astrocytes via NO. This induction is inhibited by scavenging NO or inhibiting iNOS, while NO alone is sufficient to stimulate the expression of GFAP in astrocytes, independent of microglia. It has been found that the expression of GFAP in astrocytes can be increased via a NO-GC-cGMP-PKG pathway (Brahmachari et al. 2006), which might be important under neurodegenerative conditions, for example in PD, and could be a pharmacological target for new therapies. 3.4 MAO-B expression in astrocytes and production of reactive oxygen species Monoamineoxidase-B (MAO-B) is found in the brain primarily in non-neuronal cells such as astrocytes and radial glia. The SN contains especially high levels of MAO-B positive astrocytes, which might play an important role in PD pathology (Damier et al. 1996). MAO-B levels are known to increase with age and in association with neurodegenerative diseases (Kumar & Andersen 2004). During oxidation of its substrate, this enzyme reduces oxygen to H2O2, which is a reactive oxygen species (Cohen et al. 1997). It has been postulated that age-related increases in MAO- B activity may contribute to cellular degeneration in the brain due to corresponding increases in the production of this reactive oxygen species. Astrocytes themselves are protected against
  20. 20. 20 the H2O2 they produce because they contain high levels of GSH and gluthatione peroxidase, which can detoxify H2O2 within cells. Surrounding neurons however, are very vulnerable because they contain lower levels of these protective agents, as explained in section 3.3. High amounts of H2O2 produced in astrocytes, expressing high amounts of MAO-B, can diffuse to neurons and contribute to mitochondrial damage and neuronal cell death. MAO-B activity levels have been found to be doubled in the SN in Parkinson’s disease, and to correlate with the percentage of dopaminergic SN cell loss (Damier et al. 1996). In vitro studies have shown that induced increases in astrocytic MAO-B levels result in specific inhibition of mitochondrial complex 1 activity in cultured dopaminergic cells (Kumar et al. 2003). Selective reductions in complex 1 activity have also been associated with PD. It has even been shown that elevations in astrocytic MAO-B result in a relatively selective loss of dopaminergic neurons in the SN (Mallajosyula et al. 2008). This cell loss was accompanied by increased mitochondrial oxidative stress and selective decreases in complex 1 activity along with local microglia activation. These pathological findings correlated with a significant decrease in locomotor activity. Thus, increased MAO-B activity is able to induce several pathological hallmarks of PD (Mallojosyula et al. 2008). This suggests that MAO-B may be a common initiator for these events and provides a novel model for exploring the mechanisms by which these events can occur in the context of the human condition. 3.5 Immune responses and the role of astrocytes in sustaining inflammation Several lines of evidence suggest that inflammatory mediators such as TNFα, NO and interleukin-1, derived from microglia and astrocytes modulate the progression of PD (Teisman & Shulz 2004). Environmental factors, such as infection, may interact with common but less penetrant susceptibility genes to influence the onset of most commonly observed sporadic PD cases. Neuroinflammation in the SN, once initiated, may be self-sustaining, and the SN seems to be particularly vulnerable to inflammation (Klegeris & McGeer 2007). An important contributing protein in inflammation of the SN in PD cases could be ICAM-1. ICAM-1 with its counter receptor LFA-1, is known to play a key role in inflammatory processes. In patients with neuropathologically confirmed PD, high numbers of ICAM-1 positive reactive astrocytes were found (Miklossy et al. 2006). In the SN, these ICAM-1 positive astrocytes were concentrated around residual neurons in areas of heavy neuronal loss. LFA-1 positive
  21. 21. 21 microglia gathered in such areas of high ICAM-1 expression, and LFA-1 leukocytes infiltrated the tissue (Miklossy et al. 2006). ICAM-1 and LFA-1 are known to play a key role in setting the level of inflammation in many other inflammatory diseases, such as rheumatoid arthritis, coronary heart disease, type 1 diabetes and Alzheimers disease (see Miklossy et al. 2006). It is possible that overexpression of ICAM-1 with its ligand LFA-1 in PD patients is responsible for sustaining inflammation in the SN and that this process is responsible for autodestruction of SN dopaminergic neurons (Klegeris et al. 2006). 3.6 Effect of hormones on astrocyte development and response to injury Exposure to hormones is another factor that could influence disease predisposition and severity, possibly through an effect on astrocytes. Differences in hormone exposure might also explain differences in PD prevalence between males and females. Alterations in developmental programming of neuroendocrine and immune system function may critically modulate vulnerability to Parkinson’s disease. It was found that hormonal programming has an important effect on glial response to inflammation and oxidative stress. Studies using glucocorticoid-deficient and estrogen-deprived mice showed that endogenous glucocorticoids and the female hormone estrogen inhibit the aberrant neuroinflammatory cascade, protect astrocytes and microglia from programmed cell death, and stimulate recovery of dopamine neuron functionality, thereby triggering the repair process (Marchetti et al. 2005). In figure 6 a schematic overview can be seen of the effects of estrogen and glucorticoids on activated astrocytes. Figure 6 Schematic overview of the effects of estrogen and glucocorticoids on neurons and astrocytes (Marchetti et al. 2005)
  22. 22. 22 4. Neuroprotective effects of astrocytes Of course, reactive astrogliosis does not happen without a reason. Initially, it is meant to have a protective instead of a neurodegenerative effect. Just like astrocytes support neurons during normal brain function, they continue to offer this support in situations of neuronal damage. Moreover, they can stimulate repair via neurotrophic factors and control the inflammatory response, preventing it from becoming excessive. 4.1 Protective effects of PAR-1 activation A factor that is increased in reactive astrocytes in the SNpc of PD patients is protease activated receptor 1 (PAR-1), a thrombin receptor. Thrombin-mediated activation of human astrocytes results in morphologic changes and increased proliferation characteristic of reactive astrogliosis. Moreover, this results in an increase in glial cell line-derived growth factor and gluthatione peroxidase but not in inflammatory cytokines. Gluthatione peroxidase release from these activated astrocytes has a protective effect on surrounding neurons (Ishida et al. 2006). Increased expression of PAR-1 seems to be a restorative move to protect against neurotoxicity. 4.2 Role of Nurr1 in protecting dopaminergic neurons Nurr 1 belongs to the nuclear receptor (NR)4 family of orphan nuclear receptors and is known to function as a constitutively active transcription factor by binding to target genes as a monomer, homodimer or heterodimer with other receptors (Wang et al. 2003). It has an essential role in development and/or maintenance of dopaminergic neurons. Human mutations resulting in reduced expression of Nurr 1 are associated with late-onset familial PD, indicating that normally Nurr 1 may play a protective role (Le et al. 2003). Indeed it was found that Nurr 1 plays a previously unexpected role in protecting dopaminergic neurons from inflammation-induced neurotoxicity (Saijo et al. 2009). It functions as an inhibitor of inflammatory gene expression in microglia and astrocytes. Astrocytes can act as amplifiers of microglia-derived mediators in the production of neurotoxic factors. Nurr 1 protects the CNS from amplification of inflammatory signalling via microglia-astrocyte communication. Anti-inflammatory activity is mediated by a Nurr1/CoREST transpression pathway that operates in a feedback manner to restore transcription of NF-κB target genes to a
  23. 23. 23 basal state. NF-κB target genes include many pro-inflammatory proteins. Nurr1 is recruited to NF-κB on inflammatory gene promoters via several mediators. It is suggested that the CoREST transpression pathway might be widely used by members of the NR4A family. Reduction of most of the well-established components of the CoREST complex severely compromises the anti-inflammatory activity of Nurr1. Defects in the expression or activity of these proteins could predispose individuals to PD (Saijo et al. 2009). 4.3 Anti-inflammatory and neuroprotective effects of hydrogen sulphide Hydrogen sulphide (H2S) is a physiological product generated by all the tissues in the body and a high production in the brain. It has anti-inflammatory and neuroprotective effects and is also a powerful antioxidant, both directly and indirectly, by keeping other antioxidants such as GSH in a reduced state. It can also react with nitrite ions, neutralising iNOS activity. Both iNOS activity and oxidation products are known to be important in PD pathogenesis, suggesting possible involvement of H2S. In the brain, H2S is synthesised from L-cyteine, via cystathionine-b-synthase (CBS). Astrocytes are the most powerful producers of H2S in the brain, and most strongly express CBS, as was found in post mortem brain tissue (Lee et al. 2009). Inflammatory stimulation causes a reduction in this CBS expression, and thus in H2S anti-inflammatory activity. Endogenous H2S production has a protective effect against release of inflammatory mediators by stimulated astrocytes, this can be stimulated by exposure to SH ions (from H2S in solution). NaSH treatment and endogenous H2S have neuroprotective effects through reduction of toxic materials secreted by glial cells subjected to inflammatory stimuli, and reduce production of inflammatory mediatiors including NO (Lee et al. 2009). Externally supplied SH can also reduce activation of NF-κBwhich is normally induced by inflammatory stimulation of astrocytes, and suggested to be involved in PD (Saijo et al. 2009). 4.4 The role of purines in regulation of the astrocytic response Purines also play a significant role in the pathophysiology of numerous acute and chronic disorders of the central nervous system and might play a role in PD. This hypothesis is supported by the protective effect of adenosine 2A receptor antagonist in PD models and epidemiological studies (Jenner et al. 2009). Astrocytes are the main source of cerebral
  24. 24. 24 purines (Cicarelli et al. 1999). They release either adenine-based purines, e.g. adenosine and adenosine triphosphate, or guanine-based purines, e.g. guanosine and guanosine triphosphate, in physiological conditions and release even more of these purines in pathological conditions. Astrocytes express several receptor subtypes of types for adenine- and guanine-based purines (Cicarelli et al. 2001). Specific enzymes metabolise both adenine- and guanine-based purines after release from astrocytes. This regulates the effects of nucleotides and nucleosides by reducing their interaction with specific membrane binding sites. Adenine-based nucleotides stimulate astrocyte proliferation via an increase in intracellular [Ca2+] and specific effects on proteins. Adenosine also may stimulate astrocyte proliferation, but mostly inhibits astrocyte proliferation, thus controlling excessive reactive astrogliosis. The activation of certain adenosine receptors also stimulates astrocytes to produce trophic factors, which contribute to protect neurons against injuries. Guanosine stimulates the output of adenine-based purines from astrocytes and in addition it directly triggers these cells to proliferate and to produce large amount of neuroprotective factors. These data indicate that adenine- and guanine-based purines released in large amounts from injured or dying cells of CNS may act as signals to initiate brain repair mechanisms widely involving astrocytes (Ciccarelli et al. 2001). 4.5 Astrocyte influence on microglia Although inflammation is an indispensable defense mechanism against pathogens, it often damages surrounding tissues. Therefore, the extent of inflammation should be tightly controlled to maximize the antipathogenic effect, while minimizing tissue damage. Astrocytes might play an important role in this because they can modulate the activity of microglia, which are responsible for the biggest part of the inflammatory response in PD. Reactive oxygen species (ROS) are one of the major signalling molecules capable of modulating microglia activation (Min et al. 2003). Therefore, microglial activation could be regulated by modulating intracellular ROS level. A candidate molecule to regulate intracellular ROS is the antioxidant enzyme heme oxygenase-1 (HO-1), which also has anti- inflammatory effects. Min et al. (2006) demonstrated that astrocyte culture conditioned medium (ACM) enhance HO-1 expression and activity in microglia. Furthermore, treatment with ACM suppressed interferon-γ-induced ROS production, leading to reduced iNOS expression and NO release.
  25. 25. 25 4.6 Protective effects of enriched environment It has been found that enriched environment (EE) has protective effects against neurodegeneration (Anastatasia et al. 2009). EE is defined as a sustained and progressive increase in cognitive and sensorimotor stimuli, with aggregated voluntary physical activity and complex social interactions. EE significantly reduces 6-OHDA degeneration of dopaminergic neurons in the SNpc of adult rats, preserves nigrostriatal projections, and most importantly, improves the dopaminergic function. EE resulted in a marked increase in GFAP expression in the SN after 6-OHDA lesioning (Anastasia et al. 2009). This suggests that an early post-lesion astrocytic reaction may participate in the neuroprotective mechanism. It appears that animals exposed to an EE have an increased ability to respond to the toxic injury in the anterior SNpc, where the most susceptible neuronal population is located. Reactive astrocytes probably participate in endogenous cell repair or neuroprotective mechanisms triggered at very early times following exposure to the toxin, possibly involving release of brain-derived neurotrophic factor, glial cell-line derived neurotrophic factor, and nerve growth factor, among many others. From epidemiological data it is suggested that lifestyle might influence PD etiology and progression. The risk of PD might be influenced, for example, by educational achievement or occupation as well as by exercise (Frigerio et al. 2005; Thacker et al. 2008). The use of enriched environments in experimental situations can model both intellectual stimulation and physical activity. 4.7 Astrocyte influence on neurogenesis It is known that there are progenitor cells in the SN, from which new neurons can be formed (Lie et al. 2002). Stimulating proliferation and differentiation would be very useful to replace the neurons that are lost in PD. Finding the optimal conditions for this would also help in potential stem cell replacement therapies. Astrocytes might help in this process by producing various neurotrophic factors, synthesising extracellular substrates for axonal outgrowth and synaptogenesis, and providing structural support and guiding migration. They can even act as astrocytic progenitors during long-term recovery after brain injury (Liberto et al. 2004). Astrocytes might need to become reactive to induce these properties. It has been found that mesencephalic progenitor cells survive and differentiate better in rat PD models, than in normal rats (Sun et al. 2003), and extracts from dopamine-depleted striatum show a stronger
  26. 26. 26 trophic activity (Nakajima et al. 2001). Reactive astrocytes might mediate increased basic fibroblast growth factor and glial cell-line derived neurotrophic factor levels, or other neurotrophic factors. This can create an environment in which proliferation and differentiation of progenitor cells is stimulated which can potentially be modulated to create new therapies (Chen et al. 2005). 4.8 Nrf2-mediated gene transcription inducing a large neuroprotective response As discussed before, it is known that oxidative stress might be an important cause of neuronal degradation in PD. An endogenous cellular defence mechanism against oxidative stress is the binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE) enhancer sequence. This activates many antioxidant and anti- inflammatory genes as well as growth factors, inducing a large neuroprotective response. Nrf2-ARE activated genes include HO-1, NAD(P)H quinone oxidoreductase-1 and glutathione S-transferases as well as glutathione-synthesizing enzymes glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase modifier subunit (resulting in increased GSH levels). Inflammatory mediators that are downregulated by Nrf2 include iNOS and COX-2. Nrf2 is normally bound to its cytosolic suppressor Keap1, which dissociates in response to oxidative stress, allowing Nrf2 to translocate to the nucleus and induce transcription. Nrf2 is a very general defense mechanism but in the brain it is believed that this response is mainly activated in astrocytes, since over expression of Nrf2 in astrocytes is sufficient to prevent MPTP-induced neuronal cell death (Chen et al. 2009). Induction of this pathway has also been found to reduce astrogliosis (Kanninen et al. 2009). Moreover, Nrf2 translocates to the nucleus after microglial exposure to astrocyte cultured medium, indicating that reactive astrocytes might be able to induce this pathway in other cells (Min et al. 2006).
  27. 27. 27 5. Potential therapies for PD treatment targeting astrocytes Some of these findings about the role of astrocytes in PD can be used to design new PD therapies, targeting astrocytes. The neuroinflammatory hypothesis implies that drugs with an anti-inflammatory mode of action should either arrest, or effectively slow down the neurodegenerative disease progression. The pursuit of novel molecular and cellular targets could be used for future anti-inflammatory drug development. Establishing which of the inflammatory mechanisms are the most powerful in sustaining inflammation, and finding methods to reduce their effects, might be the key to developing truly effective therapy. It has been found that non-aspirin NSAID use is associated with lower risk of PD, though there might be gender differences in this protective effect (Hernan et al. 2006). This clearly indicates that inflammation is involved, and reducing inflammation can have a positive effect. This effect could be due to classical NSAID action on cyclooxygenases (COXs) or their effect on other COX independent targets such as transcription factors NF-κB, NO synthase, and others (Asanuma & Miyazaki 2007). Lee et al. (2009) found that H2S can reduce production of inflammatory mediators by astrocytes and had neuroprotective effects through its antioxidant activity. This indicates there is a considerable therapeutic potential for H2S releasing drugs in the treatment of neurodegenerative disorders characterised by inflammatory processes, such as PD. Damaging effects of inflammation could be reduced by supplementary H2S provided by drugs. H2S releasing NSAIDS such as S-aspirin and S-diclofenac attenuate the neuroinflammation induced by activation of astrocytes. However, consequent actions on blood vessels, where H2S is also synthesised, must be taken into account. Min et al. (2006) showed that astrocytes can regulate microglia activity by inducing HO-1. They also found that mimickers of HO-1 products, such as bilirubin, ferrous iron, and a carbon monoxide-releasing molecule, reduced interferon-γ-induced iNOS expression and/or NO release in microglia. Such components could be used as potential therapies to control the microglial inflammatory response. It has been found for example that overexpression of Nrf2, which induces HO-1, can protect again 6-OHDA (Jakel et al. 2007) as well at MPTP induced damage (Chen et al. 2009) possibly through HO-1 antioxidant and microglia regulatory effects. Another factor that can modulate astrocyte activity,, and could be a potential therapeutic agent, is ONO-2506 (Kato et al. 2003). This substance inhibits the expression of COX-2 or iNOS mRNA, induced in activated astrocytes. In the MPTP-mouse model, ONO-2506
  28. 28. 28 treatment prevented reduction in striatal dopamine and loss of loss of dopaminergic neurons in the SN. Pre-treatment with this drug has no effect, indicating that is does not influence MPTP toxicity but acts on astrocytes which are active later. ONO-2506 acts selectively on astrocytes and modulates their activation or prevents too much activation that may be harmful to neighbouring neurons. Interestingly, in ONO-2506 treated mice there is no reactive astrogliosis peaking at 7 days after MPTP-treatment, as in normal situations, but there is a moderate activation of astrocytes 3 days after MPTP treatment. This suggests that astrocytic activation is facilitated, but to a limited degree, promoting only protective effects (Kato et al. 2003). A very promising therapeutic target is the Nrf2-ARE pathway, which can induce many different protective genes at once. Increased Nrf2-mediated gene transcription can be achieved by overexpression of Nrf2 via injection of lentiviral or other vectors containing this gene, into the SN (Kanninen et al. 2009). This can be done specifically in astrocytes using an astrocyte promoter, or in all cells. Transgene expression then induces specific protection against oxidative stress and reduces inflammation, which might help prevent neuronal degradation in the SN. Another possibility is the oral administration of synthetic triterpenoids which reduce Keap1 binding to Nrf2, increasing Nrf2 translocation to the nucleus, thus also increasing expression of Nrf2-ARE regulated antioxidant and anti-inflammatory genes (Yang et al. 2009). There are many more factors involved in astrogliosis that could be manipulated to control the astroglial reaction, an overview of these is given in Buffo et al. 2009. Of course, manipulation of astrogliosis remains tricky, as astrocyte response is different in different neuropathologies and certain effects might be beneficial as well as detrimental depending on the specific timing.
  29. 29. 29 6. Discussion Astrocytes play an important role in neurodegenerative diseases. In situations of neuronal damage they can become reactive, a process which is called reactive astrogliosis (Eddleston & Mucke 1993). This can be both beneficial, helping with neuronal repair or it can help sustain inflammation, which could lead to more damage. Astrocytes react differently to different types of damage and signals from neighbouring glial or neuronal cells. On top of this, astrocytes form a heterogeneous population and can have different characteristics in different brain areas, influencing their response to damage. From post mortem studies and studies in animal models of PD, it is suggested that astrocytes also play a role in PD (Kohutnica et al. 1998; Sheng et al. 1993). However, not much is known about the specific mechanisms involved. It might be that astrocytes in the SN are particularly vulnerable to certain types of damage or specific mutations, triggering neuronal cell death in this area and inducing PD (Mena & Yebenes 2008; Morga et al. 1998). Or, once astrogliosis is started, astrocytes can sustain and propagate inflammation (Liberatore et al. 1999; Miklossy et al. 2006). On the other hand, the reactive state of astrocytes might help facilitate repair. There are several factors that might have detrimental effects in PD. Mutated α-syn in astrocytes can induce inflammation and aggregated forms of this protein cause neuronal cell death (Klegeris et al. 2006; Stefanova et al. 2001; Wakabayashi et al. 2000). ER stress and abberant protein folding are two more factors implicated in PD that can lead to cell death (Slodzinksi et al. 2009). In their reactive state, astrocytes might also loose functions that are important for normal neuronal survival such as the ability to regulate extracellular glutamate levels (Dervan et al. 2004). Increased MAO-B levels in astrocytes due to age or disease result in increased production of reactive oxygen species, contributing to neuronal cell loss (Damier et al. 1996; Mallajosyula et al. 2008). ROS production can also be induced by inflammatory mediators (Bolanos et al. 1994). Normally this is counteracted by increased GSH expression and efflux but this might be a transient mechanism providing only limited protection (Chen et al. 2001; Heales et al. 2004). Upregulation of ICAM-1 in the SN of PD patients indicates that inflammation in this area might be self sustaining (Miklosssy et al. 2006). It is not known why neurodegenerative effects are sometimes larger than protective effects but this could have something to do with early exposure to hormones, programming astrocytes for a certain response (Marchetti et al. 2005).
  30. 30. 30 Protective effects might be mediated through upregulation of PAR-1 which increases GSH expression and secretion of neurotrophic factors (Ishida et al. 2006). Nurr 1 reduces inflammation via communication with microglia (Saijo et al. 2009). Antioxidants such as H2S can help clean up ROS produced during inflammation (Lee et al. 2009). Adenosine might have protective effects through its stimulation of astrocyte proliferation (Ciccarelli et al. 2001; Jenner et al. 2009). Enriched environment in animal models or possibly lifestyle in patients can also protect against neuronal damage presumably via induction of an early protective astrocytic response (Anastasia et al. 2009). Finally, astrocytes can stimulate neurogenesis via various neurotrophic factors (Chen et al. 2005) and protect against oxidative stress via induction of Nrf2-mediated transcription of antioxidant genes (Chen et al. 2009; Kanninen et al. 2009). Many of the factors increase reactive astrogliosis but this can have negative as well as positive effects. Potential new PD therapies aimed at astrocytes have to find a way to distinguish between these two effects. Inhibiting astrogliosis will suppress neurodegenerative but also many protective effects and necessary homeostasis maintaining functions of astrocytes. Protective effects should be stimulated without simultaneously stimulating degenerative effects, or degenerative effects should be blocked without inhibiting protective effects. This requires specific targeting of receptors and pathways involved in these processes. Some examples are given of potential therapies aimed at astrocyte functioning. However, much better understanding of the role of astrocytes in PD and the molecular mechanisms underlying their effects is needed to find specific therapies that can inhibit the induction or progression of this disease. Especially important would be to find what triggers or initiates the disease so the disease can be recognised more early, and treated from the start, before too many neurons are lost. A promising treatment is also the stimulation of neurogenesis, which might even be able to repair neuronal networks to some extent. A possible way to achieve this would be to manipulate astrocytes in such a way that they favour the survival and differentiation of stem cell transplants and/or natural progenitor cells in the SN (Chen et al. 2005). A very promising treatment is also the induction of Nrf2-mediated gene transcription (Chen et al. 2009; Kanninen et al. 2009; Yang et al. 2009). This activates a large general neuroprotective response through the regulation of antioxidant and anti-inflammatory genes and growth factors. Instead of targeting single molecules and genes this is a relatively easy way to influence many genes at the same time. Activation of Nrf2 gene transcription has already been shown to protect against neuronal cell loss in the MPTP model (Yang et al. 2009). Increasing the neuroprotective and antioxidant potential of astrocytes, via induction of
  31. 31. 31 the Nrf2-ARE pathway seems to be a very effective way to prevent neuronal degradation in the SN and halt PD progression. Astrocytes are a very promising target for the treatment of PD. Conventional therapies which are mostly aimed at neurons, are still not able to stop the progression of the disease or reduce the symptoms for a longer period of time. A problem with therapies targeting neurons is that as long as neuronal death is not prevented, eventually there are not enough neurons left to manipulate. Astrocytes on the other hand only increase their numbers when neuronal cell death increases, and remain in the area. Astrocytes can be manipulated to stop or slow down disease progression and possibly even stimulate repair at later stages of the disease, when many neurons have already died, and other treatments loose effectivity. Therefore, it is very important to continue research into astrocyte function in PD pathology, to get a better understanding of the mechanisms underlying the disease and find possible therapeutic targets.
  32. 32. 32 List of abbreviations 6-OHDA 6-hydroxydopamine α-syn α-synuclein ACM astrocytic culture medium ARE antioxidant response element CBS cystathionine-b-synthase CNS central nervous system COX cyclooxygenase DA dopamine DAT dopamine active transporter EE enriched environment GABA γ-aminobyturic acid GCL glutamate-cysteine ligase GP globus pallidus GSH glutathione HO-1 heme oxygenase 1 IFN-γ interferon γ IL-6 interleukin-6 ICAM-1 intracellular adhesion molecule 1 iNOS inducible nitric oxide synthetase l-dopa levodopa LFA-1 lymphocyte function-associated antigen 1 LPS lipopolysacharide MAO-B monoamine oxidase B MAPK mitogen activated protein kinase MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPP+ 1-methyl-4-phenylpyridinium NF-κB nuclear factor κ B Nrf2 NF-E2-related factor 2 NO nitric oxide NSAID non steroidal anti inflammatory drug NQO1 NADPH quinone oxidoreductase 1 PAR-1 protease activated receptor 1 PD Parkinson’s disease SN(pc) Substantia nigra (pars compacta) STN subthalamic nucleus TH tyrosine hydroxylase TNFα tumor necrosis factor α WT wild type
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