Agnieszka Ciesielska

Estrogen and Cytokines Production - The Possible Cause of Gender Differences in Neurological Diseases

Naturally occurring sexual dimorphism has been implicated in the risk, progression and recovery from numerous neurological disorders. These include head injury, multiple sclerosis (MS), stroke, and neurodegenerative diseases (Parkinsons disease (PD), Alzheimers disease (AD) or amyotrophic lateral sclerosis (ALS). Accumulating evidence suggests that observed differences between men and women could result from estrogens wide range of effects within the mammalian central nervous system (CNS), with its neuroprotective effect being one of the most important. It seems possible that neuroprotective activity of estrogen could be partially a result of its anti-inflammatory action. It has been well established that inflammation plays an important role in the etiopathogenesis and manifestation of brain pathological changes. In this regard, an important role has been suggested for pro-inflammatory cytokines produced by activated glial cells, neurons and immune cells that invade brain tissue. Within the CNS, cytokines stimulate inflammatory processes that may impair blood-brain barrier permeability as well as promote apoptosis of neurons, oligodendrocytes and induce myelin damage. Given that estrogen may modulate cytokine expression, coupled with the fact that gender differences of cytokine production are apparent in animal models of PD and MS, suggests an important connection between hormonal-cytokine link in neurodegeneration. Indeed, while MS patients and mice subjected to experimental autoimmune encephalomyelitis (EAE) display gender specific alterations of IFN-gamma and IL-12, variations of TNF and IL-6 were associated with PD. Also in case of more acute neurodegenerative conditions, such as stroke, the effect of IL-6 gene G-174C polymorphism was different in males and females. Given that our understanding of the role of estrogen on cytokine production and accompanying CNS pathological conditions is limited, the present reviews aims to present some of our recent findings in this area and further evaluate the evidence that may be relevant to the design of new hormonal anti-inflammatory treatment strategies for neurodegenerative diseases.

Gender differences in neurological disease: role of estrogens and cytokines.

Increasing evidence suggests that inflammatory response may be a critical component of different brain pathologies. However, the role played by this reaction is not fully understood. The present findings suggest that neuroinflammtory mediators such as cytokines may be involved in a number of key steps in the pathological cascade of events leading to neuronal injury. This hypothesis is strongly supported by experimental and clinical observations indicating that inhibition of the inflammatory reaction correlates with less neuronal damage. Estrogens are thought to play a role in the sex difference observed in many neurological diseases with inflammatory components including stroke, Alzheimers and Parkinsons diseases, multiple sclerosis, or amyotrophic lateral sclerosis. Clinical and experimental studies have established estrogen as a neuroprotective hormone in these diseases. However, the exact mechanisms involved in the neuroprotective effects of estrogens are still unclear. It is possible that the beneficial effects of these hormones may be dependent on their inhibitory activity on the inflammatory reaction associated with the above-mentioned brain pathologies. Here, we review the current clinical and experimental evidence with respect to the inflammation-modulating effects of estrogens as one potential explanatory factor for sexual dimorzphism in the prevalence of numerous neurological diseases.

Cerebral Infusion of AAV9 Vector-encoding Non-self Proteins Can Elicit Cell-mediated Immune Responses

There is considerable interest in the use of adeno-associated virus serotype 9 (AAV9) for neurological gene therapy partly because of its ability to cross the blood-brain barrier to transduce astrocytes and neurons. This raises the possibility that AAV9 might also transduce antigen-presenting cells (APC) in the brain and provoke an adaptive immune response. We tested this hypothesis by infusing AAV9 vectors encoding foreign antigens, namely human aromatic L-amino acid decarboxylase (hAADC) and green fluorescent protein (GFP), into rat brain parenchyma. Over ensuing weeks, both vectors elicited a prominent inflammation in transduced brain regions associated with upregulation of MHC II in glia and associated lymphocytic infiltration. Transduction of either thalamus or striatum with AAV9-hAADC evinced a significant loss of neurons and induction of anti-hAADC antibodies. We conclude that AAV9 transduces APC in the brain and, depending on the immunogenicity of the transgene, can provoke a full immune response that mediates significant brain pathology. We emphasize, however, that these observations do not preclude the use of AAV serotypes that can transduce APC. However, it does potentially complicate preclinical toxicology studies in which non-self proteins are expressed at a level sufficient to trigger cell-mediated and humoral immune responses.

Anterograde Axonal Transport of AAV2-GDNF in Rat Basal Ganglia

We elucidated the effects of parkinsonian degeneration on trafficking of AAV2-GDNF in the nigro-striatum (nigro-ST) of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Vector infused into striatum (ST) was transported to substantia nigra (SN), both pars compacta (SNc), and pars reticulata (SNr). In the lesioned hemisphere, glial cell line-derived neurotrophic factor (GDNF) immunoreactivity was only found in SNr consistent with elimination of SNc dopaminergic (DA) neurons by 6-OHDA. Further analysis showed that striatal delivery of AAV2-GDNF resulted in GDNF expression in globus pallidus (GP), entopeduncular nucleus (EPN), and subthalamic nucleus (STN) in both lesioned and unlesioned hemispheres. Injection of vector into SN, covering both SNc and SNr, resulted in striatal expression of GDNF in the unlesioned hemisphere but not in the lesioned hemisphere. No expression was seen in GP or EPN. We conclude that adeno-associated virus serotype 2 (AAV2) is transported throughout the nigro-ST exclusively by anterograde transport. This transport phenomenon directs GDNF expression throughout the basal ganglia in regions that are adversely affected in Parkinsons disease (PD) in addition to SNc. Delivery of vector to SN, however, does not direct expression of GDNF in ST, EPN, or GP. On this basis, we believe that striatal delivery of AAV2-GDNF is the preferred course of action for trophic rescue of DA function.

Cyclooxygenases mRNA and protein expression in striata in the experimental mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration to mouse

Cyclooxygenases (COX) are associated with complex alteration in many pathologies of the central nervous system (CNS). Increased expression of COX-2 has been shown in injured or degenerated neurons, thus suggesting that COX-2 may contribute to neuronal damage. In this study, we present the expression of COX-1 and COX-2 mRNA and protein in striatum following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to mice. MPTP causes an acute damage of dopaminergic neurons especially in the nigrostriatal dopaminergic system, thus diminishing dopamine (DA) content in striatum and decreasing the number of dopaminergic cells in the pars compacta of the substantia nigra (SN). C57Bl mice have received 60 mg/kg of MPTP introperitoneally. A group of mice received also rofecoxib 10 mg/kg from the 1st day following MPTP administration. Dopamine content in striatum (high-performance liquid chromatography-HPLC), mRNA expression of COX-1 and -2 (reverse transcriptase-polymerase chain reaction technique-RT-PCR), COX-1 and -2 protein content (immunoblotting) have been measured on day 1st, 3rd, 7th, 14th and 21st after the injury. We have found that COX-1 mRNA expression is not changed following MPTP administration, but COX-2 gene and protein expression in striatum increases from the 3rd to the 7th and 14th days, and diminishes on the 21st day. Production of prostaglandins is augmented only briefly after MPTP treatment and did not correlate with increased COX-2 mRNA and COX-2 protein production. Thus, the increase of COX-2 expression does not follow the acute stage of cell death but rather the recovery period after the injury. We also demonstrate that COX-2 activity inhibition by rofecoxib (10 mg/kg), which has been started 1 day after the injury, has not neuroprotective effect. Our study suggests that COX-2 does not contribute to neurons death following MPTP administration and that the inhibition of COX-2 activity is not beneficial to neurons injured by MPTP. However, COX-2 mRNA and protein expressions increase after MPTP injury; the role of these findings remains obscure.

GDNF signaling implemented by GM1 ganglioside; failure in Parkinson's disease and GM1-deficient murine model

GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling has been linked to loss of dopaminergic neurons in Parkinsons disease (PD). This study demonstrates attenuated GDNF signaling in neurons deficient in ganglio-series gangliosides, and restoration of such signaling with LIGA20, a membrane permeable analog of GM1. GM1 is shown to associate in situ with GFRα1 and RET, the protein components of the GDNF receptor, this being necessary for assembly of the tripartite receptor complex. Mice wholly or partially deficient in GM1 due to disruption of the B4galnt1 gene developed PD symptoms based on behavioral and neuropathological criteria which were largely ameliorated by gene therapy with AAV2-GDNF and also with LIGA20 treatment. The nigral neurons of PD subjects that were severely deficient in GM1 showed subnormal levels of tyrosine phosphorylated RET. Also in PD brain, GM1 levels in the occipital cortex, a region of limited PD pathology, were significantly below age-matched controls, suggesting the possibility of systemic GM1 deficiency as a risk factor in PD. This would accord with our finding that mice with partial GM1 deficiency represent a faithful recapitulation of the human disease. Together with the previously demonstrated age-related decline of GM1 in human brain, this points to gradual development of subthreshold levels of GM1 in the brain of PD subjects below that required for effective GDNF signaling. This hypothesis offers a dramatically different explanation for the etiology of sporadic PD as a manifestation of acquired resistance to GDNF.

Age- and sex-differences in the nitric oxide synthase expression and dopamine concentration in the murine model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Parkinsons disease (PD) is an age- and sex-related neurodegenerative disorder of unknown aetiology. The involvement of nitric oxide synthase (NOS) in the etiopathogenesis of PD is quite well documented. We decided to examine changes in dopamine (DA) levels as well as iNOS, nNOS, eNOS mRNA and protein expression in the striatum of C57BL male and female (2- and 12-month old) mice in the course of PD-related neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The significantly decreased level of DA was previously observed in male than in female, irrespective of age. In young mice the recovery of DA was significantly greater in female compared to male mice. On the contrary, both in male and female old animals the low concentration of DA was extended up to 21 days post MPTP injection. The increases in iNOS protein expression post MPTP intoxication occurred more rapidly in male (young and old) than in female mice. The pattern of changes in iNOS protein expression was also different in young versus aged mice. nNOS protein expression increased earlier in young male than young female mice. No changes were observed in eNOS expression. In conclusion, our results support the hypothesis of the involvement of iNOS and nNOS, but not eNOS in neurodegenerative processes. Our findings suggest that age- and sex-differences in DA concentration and iNOS expression as well as sex-differences of nNOS expression after intoxication may depend on the increased susceptibility of males as well as older animals to toxic effect of MPTP and aggravated process of recovery in old brains.

The influence of AAV2-mediated gene transfer of human IL-10 on neurodegeneration and immune response in a murine model of Parkinson's disease

BACKGROUND The aim of this study was to examine the effect of AAV2-hIL-10 (vector containing cDNA for human interleukin 10) on dopaminergic system activity (measured as DA levels and TH mRNA expression in mouse striata), and other monoamine and amino acid neurotransmitters concentration as well as development of inflammatory processes (measured as TGF-β, IFN-γ and GFAP mRNA expression) in a murine MPTP neurotoxicant model of Parkinsons disease. METHODS Male C57BL/6 mice 12 months-old were used in this study. AAV2-hIL-10 vector was bilaterally administered into striatum at 14, 21 or 28 days prior to MPTP intoxication. Animals were sacrificed at 7 days following MPTP injection. The expression of hIL-10 (human interleukin 10) was examined by ELISA. Striatal monoamine and amino acid neurotransmitters were measured by HPLC method. TH, TGF-β, IFN-γ and GFAP mRNA expression was examined by RT-PCR method. RESULTS MPTP treatment dramatically reduced DA levels and decreased TH mRNA expression in mouse striata, effects that were significantly impeded by AAV2-hIL-10 administration prior to MPTP intoxication. AAV2-hIL-10 infusion increased IFN-γ, TGF-β and GFAP mRNA expression. CONCLUSIONS Our data suggest that the transfer of AAV2-hIL-10 into the striatum may play a neuroprotective role in the mouse MPTP model of PD and these effects are mediated by the anti-inflammatory action of IL-10.

Influence of Age and Gender on Cytokine Expression in a Murine Model of Parkinson’s Disease

Objective: The neuroinflammatory reaction has been linked with Parkinson’s disease. One of the hypotheses to explain the significance of age and gender (male predominance) effects on neurodegeneration in Parkinson’s disease may result from a link between these risk factors and the inflammatory processes. Here, we investigated the expression of inflammatory mediators in relation to 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine (MPTP)-induced neurodegenerative processes in nigrostriatal pathway in young and aged male and female mice.Methods and Results: We simultaneously assessed striatal tyrosine hydroxylase (TH) protein concentrations (Western blotting) and cytokine (TNFα, IFNγ, IL-1β, IL-6 and TGFβ1) mRNA levels (RT-PCR) in young and aged (2- and 12-month-old) C57BL/6 male and female mice after 6 h, 1, 3, 7, 14, 21 days after MPTP intoxication.Western blotting analysis showed that at the early time points, males showed a greater reduction in striatal TH versus females. Additionally, in contrast to the aged mice, in young males and females the TH concentration gradually increased between the 7th and the 21st day after intoxication. The increases in TNFα, IL-1β and IFNγ after intoxication were faster in both young and aged males than females. In males (both ages), we observed an increase in TGFβ1 at the early time points. In contrast, in females (both ages) TGFβ1 was elevated at later time points. MPTP caused an increase in IL-6 in males and females, but this increase was significantly higher in females. Conclusions: A gender and age skewing of the cytokine gene expression in the striatum after intoxication may be related to the greater susceptibility in males as well as older animals to the detrimental effects of MPTP.

Decreased inflammation and augmented expression of trophic factors correlate with MOG-induced neuroprotection of the injured nigrostriatal system in the murine MPTP model of Parkinson's disease.

The response of the immune system during injury of the central nervous system may play a role in protecting neurons. We have previously reported that immunization with MOG 35-55 prior to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury of the dopaminergic system promotes less dopamine depletion and less dopaminergic damage of neurons in mice. In this study, we evaluate the influence of MOG immunization on the inflammatory reaction that occurs at the place of injury. C57Bl male mice, 2 and 12 months old, received i.p. injections of MPTP (40 mg/kg) and some groups animals also received an additional injection with myelin oligodendrocyte glycoprotein (MOG) 35-55 in CFA 6 days before MPTP administration. MPTP caused a common inflammatory reaction characterized by microglial activation, infiltration of T cells into the substantia nigra and striatum and increased expression of mRNA encoding pro-inflammatory cytokines (IL-1 beta, TNFalpha, INF gamma) and trophic factors (TGFbeta, GDNF). MOG immunization prior to MPTP administration significantly diminished the microglial reaction and reduced the levels of infiltrating CD8+ lymphocytes. The number of CD4+ T cells remained at the same level as in the MPTP group. Expression of pro-inflammatory cytokines was diminished. The mRNA expression of GDNF was significantly higher in the MOG pretreated mice relative to the MPTP group, both in the 2 month old and 12 month old groups. Since MOG immunization prior to MPTP intoxication appears to prevent nigrostriatal injury, the observed decrease of inflammation and increase of GDNF mRNA expression in the injured areas might represent one of the mechanisms of observed neuroprotection.