Investigation of the in vivo role of parkin by manipulation of parkin expression in rodent models of Parkinson's disease

Parkinson s disease (PD) is the most common neurodegenerative movement disorder. It is neuropathologically characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and the presence of intracellular alfa-synuclein (alfa-SYN) containing inclusions called Lewy bodies. Although the etiology of PD is still incompletely understood, the discovery of genes linked to familial PD has tremendously improved our knowledge on the molecular mechanisms involved in the pathogenesis of familial as well as sporadic PD, since both forms share clinical and neuropathological features. Mutations in the parkin gene cause early-onset autosomal recessive PD. The parkin protein functions as an E3-ubiquitin ligase and is involved in a wide range of cellular functions including the ubiquitin-proteasome system, mitochondrial quality control and activation of signal transduction cascades. Overexpression of parkin in cell culture and in vivo has been reported to have a neuroprotective effect against a variety of genetic and toxic insults. Furthermore, there is also evidence that inactivation of parkin by oxidative, nitrosative and dopamine stress may play a role in sporadic PD. These data suggest that parkin has a therapeutic potential for the treatment of not only familial but also sporadic PD. However, the mechanism-of-action underlying the neuroprotective capacity of parkin and more in general the in vivo function of parkin and how parkin mutations lead to the development of PD has not yet been completely elucidated. First, we aimed to further validate the therapeutic effect of parkin in an A53T alfa-SYN rAAV2/7 genetic rat model of PD that was recently developed in our lab. Point mutations and multiplications of alfa-SYN cause a rare familial autosomal dominant form of PD and a fibrillar form of alfa-SYN is the main protein component of Lewy bodies, which makes alfa-SYN a key protein in PD pathogenesis. The neuroprotective capacity of parkin has previously been shown by our research group using lentiviral vectors in a PD model based on the neurotoxin 6-OHDA. Since recombinant adeno-associated viral (rAAV) vectors have a better tropism and specificity for dopaminergic neurons in vivo than lentiviral vectors, we expected that overexpression of parkin with rAAV vectors would improve the neuroprotective activity of parkin. However, despite several optimizations of the experimental conditions, we did not observe a neuroprotective capacity of parkin in this alfa-SYN rat model. Clinical parkin mutations are most probably loss-of-function mutations. Therefore, in a second part of this thesis, we aimed to explore the effect of parkin deficiency on the survival of dopaminergic neurons in rodents, in an attempt to increase our understanding of the processes leading to dopaminergic cell death in parkin-associated PD patients. Parkin knockout mice fail to recapitulate dopaminergic degeneration, which might be explained by compensatory adaptations. Therefore, in a first strategy, we hypothesized that acute downregulation of parkin in the dopaminergic neurons of adult brain, might overcome these compensatory issues and induce a more pronounced phenotype. We achieved acute, locoregional knockdown of parkin in the SN of adult rats by injection of a rAAV vector coding for a functional micro-RNA based short hairpin sequence directed against rat parkin. However, this did not result in motor deficits or a clear dopaminergic cell death. In a second strategy to mimic the situation in humans, we overexpressed the T240R clinical parkin mutant in the SN of adult rodents. Cell culture studies and Drosophila studies have put forward a dominant negative effect for the T240R-parkin mutant. We aimed to translate those findings to the rodent brain and overexpressed T240R-parkin and human wild type parkin as a control in the dopaminergic neurons of adult rats using rAAV vectors. Surprisingly, we found that overexpression of not only T240R-parkin but also human wild type parkin induced a progressive and dose-dependent dopaminergic cell death in rats. This degeneration was specific for parkin, since similar overexpression of eGFP did not lead to nigral degeneration. These results warrant caution to the development of therapeutic strategies for PD based on overexpression of parkin or enhancing parkin activity, since this might be deleterious at long-term for dopaminergic neurons. In a third part of this thesis we investigated how loss of parkin function affects alfa-SYN induced neurotoxicity. Therefore, we used two complementary strategies: nigral rAAV2/7-mediated overexpression of human wild type alfa-SYN in parkin knockout mice and acute, locoregional downregulation of parkin in an A53T alfa-SYN rAAV2/7 rat model of PD. We found that the absence of parkin did not increase the vulnerability of the dopaminergic neurons to cell death induced by wild type alfa-SYN. However, the number of cells positive for alfa-SYN phosphorylated at serine residue 129, which is considered to be a pathological form of alfa-SYN, was significantly higher in the parkin knockout mice compared to the wild type mice. On the contrary, local downregulation of parkin significantly increased the sensitivity to A53T alfa-SYN mediated dopaminergic cell death, without affecting the phosphorylation of alfa-SYN. Those data support a functional relationship between parkin deficiency and alfa-SYN neurotoxicity.Altogether, the results of this thesis indicate that equilibrated and well-controlled levels of parkin are crucial for dopaminergic cell survival.status: publishe

Formerly undetected giant cell arteritis revealed at hysterectomy.

status: Published onlin

Diffusely Metastasized Adenocarcinoma Arising in a Mucinous Carcinoid of the Ovary: A Case Report

Mucinous (goblet cell) carcinoids are a rare type of ovarian carcinoid tumors. Only a limited number of primary mucinous carcinoids of the ovary have been reported in the literature. We describe the case of a 55-year-old woman with a diffusely metastasized adenocarcinoma arising in a primary ovarian mucinous carcinoid. The differential diagnosis with a metastatic goblet cell carcinoid from the appendix or elsewhere can be very challenging. In our case, especially the immunohistochemical profile of the tumor with diffuse positivity for cytokeratin 7 and PAX8, and no expression of cytokeratin 20 and CDX2, directed us toward a primary ovarian origin. Expression of PAX8 in ovarian mucinous carcinoid has never been reported before.status: accepte

Gastrointestinal stromal tumour (GIST) arising in a colonic duplication cyst: case report

status: publishe

Long-term overexpression of human wild-type and T240R mutant parkin in rat substantia nigra induces progressive dopaminergic neurodegeneration

Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson disease (PD). The pathogenic mechanisms of how parkin mutations lead to the development of PD are not fully understood. Studies of cell cultures and of Drosophila have suggested a dominant negative effect for the clinical parkin mutant T240R. Conversely, the neuroprotective capacity of parkin has been widely reported; this suggests that the parkin protein may have a potential therapeutic role in PD. Here, we aimed to develop a novel genetic rodent model of PD by overexpression of T240R-parkin and human wild-type parkin as a control in the dopaminergic neurons of adult rats using adeno-associated viral vectors (rAAV2/8). Surprisingly, we found that overexpression not only of T240R-parkin but also of human wild-type parkin induced progressive and dose-dependent dopaminergic cell death in rats, starting from 8 weeks after injection. This degeneration was specific for parkin because similar overexpressionof enhanced green fluorescent protein did not lead to nigral degeneration. Our results warrant caution to the development of therapeutic strategies for PD based on overexpression of parkin or enhancing parkin activity because this might be deleterious for dopaminergic neurons in the long-term.status: publishe

Intravascular Leiomyomatosis as a Rare Cause of Nonthrombotic Pulmonary Embolism

Intravascular leiomyomatosis (IVL) is a very rare condition. It is characterized by the proliferation of benign smooth muscle cells within vascular structures without invasion of these tissues. Symptoms depend on the site of origin and the extent of invasion. Rarely, this neoplasm is located in the inferior vena cava or in the pulmonary vasculature potentially causing symptoms of dyspnea, chest pain, or syncope. We report the case of a 53-year-old woman who was referred to our hospital with extensive pulmonary embolism comprising of a subtotal occlusion of the right pulmonary artery with extension into the left pulmonary artery. Due to persistent dyspnea (New York Heart Association class II) despite anticoagulation, after a six-week period, imaging was repeated and showed stable findings. As she was not responding to adequate anticoagulant therapy, intima sarcoma of the pulmonary artery was suspected, and a pulmonary endarterectomy (PEA) was performed. A smooth, white, intravascular mass was easily and completely removed. Analysis demonstrated a lesion consisting of cells without atypia, showing expression of alpha-smooth muscle actin (alpha SMA) and desmin with partial expression of estrogen receptor (ER) and progesterone receptor (PR), leading to the diagnosis of intravascular leiomyomatosis. The patient fully recovered. Complete surgical removal of the intravascular tumor is recommended to relieve symptoms and prevent possible complications. Clinicians have to be aware that in unresolved pulmonary embolism, nonthrombotic and rare causes, like an intima sarcoma or intravascular leiomyomatosis, should be considered.status: publishe

Intravascular Leiomyomatosis as a Rare Cause of Nonthrombotic Pulmonary Embolism

Intravascular leiomyomatosis (IVL) is a very rare condition. It is characterized by the proliferation of benign smooth muscle cells within vascular structures without invasion of these tissues. Symptoms depend on the site of origin and the extent of invasion. Rarely, this neoplasm is located in the inferior vena cava or in the pulmonary vasculature potentially causing symptoms of dyspnea, chest pain, or syncope. We report the case of a 53-year-old woman who was referred to our hospital with extensive pulmonary embolism comprising of a subtotal occlusion of the right pulmonary artery with extension into the left pulmonary artery. Due to persistent dyspnea (New York Heart Association class II) despite anticoagulation, after a six-week period, imaging was repeated and showed stable findings. As she was not responding to adequate anticoagulant therapy, intima sarcoma of the pulmonary artery was suspected, and a pulmonary endarterectomy (PEA) was performed. A smooth, white, intravascular mass was easily and completely removed. Analysis demonstrated a lesion consisting of cells without atypia, showing expression of alpha-smooth muscle actin (alpha SMA) and desmin with partial expression of estrogen receptor (ER) and progesterone receptor (PR), leading to the diagnosis of intravascular leiomyomatosis. The patient fully recovered. Complete surgical removal of the intravascular tumor is recommended to relieve symptoms and prevent possible complications. Clinicians have to be aware that in unresolved pulmonary embolism, nonthrombotic and rare causes, like an intima sarcoma or intravascular leiomyomatosis, should be considered

Serous tubal intraepithelial carcinoma-like and pagetoid tubal metastasis of an ovarian large cell neuroendocrine carcinoma : peculiar metastatic growth patterns of a rare tumor

A large cell neuroendocrine carcinoma of the ovary is presented because of tubal metastases with peculiar growth patterns: pagetoid and serous tubal intraepithelial carcinoma-like. The possibility that a tubal intraepithelial carcinoma could represent a metastasis should be considered by pathologists