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Wnt1 and its therapeutic role in Parkson’s Disease

Background Wnt1 gene is a member of the Wnt gene family that encodes for a group of related signaling proteins. It was first found as a proto-oncogene active in mouse mammary tumours and was later found to be present in human with 99% homology. Research in developmental genetics of Drosophila showed that Wnt1 is part of the segment polarity group that dictates Drosophila body patterning in embryogenesis. Wnt gene products are a group of structurally related proteins that are important for brain development in animal embryogenesis. Wnt proteins have been shown to be involved in cellular growth and differentiation in different stages of development in various animal models. They have been associated with the formation of cell polarity, cell shape, and cell division. Wnt signals via a strictly regulated signaling pathway and mutations along this cascade could lead to a range of developmental abnormalities. Wnt proteins have been implicated with numerous degenerative diseases like retinal degeneration and bone density abnormalities. Many types of cancer have also been associated with mutations in the Wnt signaling pathway in both human and mouse, and Wnt is believed to be one example where cancer is closely associated with development.

Cell Proliferation & Apoptosis modulation via Wnt1 signaling cascade The Wnt gene family can affect many different aspects of development through binding to transmembrane Frizzled receptors (Fzd). These aspects of development range from regulation of cell proliferation, apoptosis and cancer growth. When Wnt1 binds to the Frizzled receptor, it induces the release of the protein, β-catenin. The release of β-catenin is through a complex series of activations and deactivations. When the Wnt1 gene product is absent from Frizzled receptor, β-catenin forms a complex with Axin, APC, GSK3 and CK1. CK1 and GSK3 in succession phosphorylate β-catenin, allowing the protein to be degraded. It is degraded as E3 ubiquitin ligase B-Trcp recognizes the phosphorylated protein and targets it for proteosomal degradation. A mutation in any of these cofactors (Axin, APC, etc.) can result in the β-catenin not being degraded. As we will later see, this causes an induction of Wnt1 responsive genes. When there are no mutations in this cascade and Wnt1 is not present, β-catenin would be properly degraded and the genes that are responsive to Wnt1 are not expressed. When Wnt1 is present, it will bind to the Frizzled receptor and form a complex with the receptor and LRP5/6, a transmembrane complex. When Wnt1 binds, Frizzled receptor changes conformation and recruits the cofactor Dvl (Dishevelled) which phosphorylates LRP5/6 and secondarily recruits Axin, one of the cofactors necessary for β-catenin degradation. As a result, Axin is no longer available to create the degradation complex, β-catenin does not become phosphorylated and is thusly not degraded. β-catenin then accumulates in the nucleus where it will become a co-activator for TCF, a cofactor that activates Wnt1-target genes. Therefore, if β-catenin is not present, TCF-TLe/Groucho and histone deactylases (HDAC) serve as Wnt1-target gene repressors, inhibiting the signaling pathway. A loss of function mutation in any of this cascade would cause β-catenin to be overexpressed. The overall effect of Wnt1 expression is the proliferation of cells through the induction of Wnt-target genes. When Wnt1 is absent or a mutation occurs somewhere along the signalling cascade, β-catenin is unable to induce Wnt1-target genes and cell proliferation becomes impaired. Exactly how apoptosis occurs through the absence of Wnt1 signaling is currently unclear but it has been demonstrated that a monoclonal antibody against Wnt1 that blocks its action would induce apoptosis.

Wnt1 and Brain Development The roles of Wnt1 in brain development have been studied predominantly in mice models. Wnt1 has been shown to be very important for the development and proliferation of the mid/hindbrain regions of mouse brain. Wnt1 is associated with the formation and maintenance of the isthmus organizer, the organizing centre of tectum and cerebellum. It is also expressed in the mid/hindbrain organizer (MHO) between midbrain and hindbrain where it was concluded to be important for controlling the proliferation of specific cell populations. In transplantation experiments in mice, Wnt1 has been shown to be responsible for ectopic midbrain and cerebellar tissue. Misexpression of Wnt1 in transgenic mice models showed increased proliferation of neural progenitor cells in dorsal midbrain and caudal midbrain enlargement. Transplantation experiments of Wnt1 into adult caudal midbrain also lead to increased neuron sizes. These experiments indicated that Wnt1 leads to mid/hindbrain-specific proliferation during development. Combined with studies on other development regulatory factors, Wnt1 is believed to be an important mediator of brain patterning and proliferation of specific cells in a strict spatiotemporal manner. Wnt1 functions are closely associated with the activities of many other developmental regulatory factors that either support or inhibit Wnt1 signaling that limits the distribution of Wnt1 in the developing brain. Wnt1 also contribute to the regulation of this network of developmental regulators to facilitate the formation of mid and hindbrain during development. Wnt1 has been shown to be important for the proliferation and differentiation of midbrain dopaminergic precursors in the ventral midbrain. Based on studies in mouse models, Wnt1 affects the development of dopaminergic progenitors in two steps. Wnt1 is involved in the establishment of the dopaminergic progenitor domain by controlling the genetic network in the midbrain early on in development. Wnt1 expression is required for maintaining expression of Otx2, another transcriptional regulator required for dopaminergic neuron development in the midbrain. Wnt1 and Otx2 expressions at the ventral midbrain are also maintained by each other through a positively feedback system and together they support the initial patterning of dopaminergic progenitors by inhibiting the expansion of another regulators, Nkx2-2. Later on in development, Wnt1 also has an important role in the proliferation and differentiation of midbrain dopaminergic progenitors into mature neurons. At this stage, Wnt1 affects regulatory genes of mitosis and accelerates cell cycle progression to enhance cell proliferation. Maintained Wnt1 expression, in addition to Shh signaling from floor plate and fgf8 signaling from midbrain hindbrain boundary, is also required for specifying the cell fate of dopaminergic progenitors and necessary for the complete terminal differentiation of the progenitors into functional, dopamine secreting neurons.

Introduction to Wnt1’s therapeutic role in Parkinson’s Disease Parkinson’s disease is a neurodegenerative disease that is largely idiopathic in nature. A minority of cases of Parkinson’s disease have been associated with mutations in the genes that code for α-synuclein, parkin, leucine-rich repeat kinase 2 or PTEN-induced putative kinase 1. The symptoms, characterized by tremors and rigidity, are caused by the loss of dopamine secreting neurons and accumulation of Lewy bodies in the midbrain. The loss of dopaminergic neurons increases with the advancement of the disease, indicating progressive failure of some unknown underlying mechanism. Many of the available treatments do not address the dopamine loss directly but rather focus on increasing the amount of dopamine in brain by administration of dopamine agonists to compensate for the loss of dopaminergic neurons. There are also other treatments such as gene therapy, neural grafts, and stem cell transplants that attempt to rescue the degeneration of the dopaminergic neurons. The purpose of these therapies is to regenerate healthy neurons that can become a sustainable supply of dopamine and alleviate the symptoms. Stem cell transplantation has been proposed to be the most promising direction. However, it has met limited success due to poor survival rate of transplanted stem cells and de-differentiation of newly generated neurons after a period of time. Wnt1 is known to be involved in the differentiation and proliferation of ventral midbrain dopaminergic neurons from their progenitors during embryonic development and it may be useful for promoting and maintaining the differentiation of dopaminergic stem cells after transplantation. Several mechanisms have been proposed to explain the degeneration of mDA neurons of the mid/hindbrain region. Most researchers now pinpoint the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (“parkinsonian toxin”) model which involves the Wnt1/β-catenin signalling pathway in reactive astrocytes [wiki link] (i.e. cells involved in nervous tissue repair after damage inflicted by injury) .It has been hypothesized that reactive astrocytes lose their plasticity as an organism ages, therefore reducing their regenerative and protective abilities which is also correlated with decreased Wnt1/β-catenin signaling. Further experimentation has shown that the Wnt1/β-catenin is essential for the survival of neurons in DAneuron regions. That in addition with experiments on aged mice that show impairment of the same signaling pathway and inability to recover when exposed to parkinsonian toxin, leads to the conclusion that Wnt1/β-catenin signaling pathway plays a role in the development of Parkinson’s disease.

Experimental Procedure Hypothesis:  Mice models will be used to test the hypothesis that Wnt1 could be used to promote the growth of embryonic neural stem cells after transplantation into dopaminergic neurons in human brains. Methods: Result: If increase in dopamine level in the transplanted area is observed, the hypothesis that drug induced Wnt1 activation promotes differentiation of dopaminergic progenitors is supported in mice. Further studies must be done in primates to accurately test for its effects before human trials.
 * 1) Cultivate mice embryonic stem cells in vitro with growth factors to promote differentiation into dopaminergic progenitors.
 * 2) Genetically modify the enhancer region of Wnt1 in these cells to generate an artificial drug-responsive enhancer that could be conditionally turn on the transcription of Wnt1 through drug administration. The drug should be specific for this enhancer only and cause minimal effects in other parts of the body.
 * 3) Select for genetically modified progenitor cells for their ability to differentiate into functional dopaminergic neurons upon drug activation in vitro and expand them to generate a population of cells.
 * 4) Transplant the stem cells into the striatum of mouse brains.
 * 5) Observe and record the effects of stem cell transplantation by PET scan and behavioural monitoring.
 * 6) When the neurons fail to produce adequate dopamine concentrations and show signs of dedifferentiation, administer the drug intravenously daily. Construct a control group where placebo is given instead of the drug.
 * 7) Observe for change in dopamine production by PET scan and behavioural monitoring and compare with the control group.

Other Pathways Affected As previously described Wnt1 promotes the proliferation of dopaminergic neurons. The loss of dopamine secreting neurons along with the accumulation of Lewy bodies is a characteristic of Parkinson’s disease. Lewy bodies are α-synuclein immunoreactive protein deposits in neurons and glial cells in Parkinson’s disease and causes the cell death of healthy neurons. This means that stem cell transplant is only treating the symptoms of Parkinson’s disease but not as a cure. Ideally if Wnt1 transcription is upregulated in the brain after stem cell transplantation, the amount of dopaminergic neurons will increase. With this the amount of dopamine in the brain will also increase to normal levels and the symptoms of Parkinsons disease will be alleviated. However there are possible side effects and consequences with this method. The Wnt/β-catenin pathway activate genes for cell proliferation. Logically an increase of Wnt1 translation should lead to more activation of the wnt/β-catenin pathway leading to an increase of β-catenin and a net increase in the rate of cell proliferation of affected cells. This could possibly lead to tumors or cancer development in the areas where the treatment is applied. Experiments involving the Wnt signalling pathway have shown that the inhibition of the Wnt signalling pathway is an effective treatment to reduce the rates of tumor cell growth. However, other experimental models have shown that overexpression of β-catenin does not lead to tumorigenesis. This suggests that aberrant regulation of the Wnt pathway – induced by increasing the amount of Wnt1 expressed in the cell – will increase the growth rate of dopaminergic neurons could possibly contribute to tumor growth, but not causing it. Dopamine is essential all throughout the body and many disorders are associated with dopamine imbalances. If dopaminergic neuron production is increased then there is a possibility this will lead to an overproduction of dopamine. Dopamine hyperstimulation is associated with mental disorders such as schizophrenia and depression. Dopamine is also involved in the reward center of the brain. An overexpression of dopamine could lead to addiction like symptoms such as a loss of emotional control and impaired decision making. However, the benefits of this treatment far outweigh the negative potential side effects of this treatment. Rigorous testings in vitro and in vivo should be done during the trials to look for these possible side effects and their solutions.

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