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The Role of Galectin-3 in Amyotrophic Lateral Sclerosis Disease Progression Open Access

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Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig's disease, is a neurodegenerative disease that primarily affects the upper and lower motor neurons and results in paralysis, and death from respiratory complications. At any given time, there are approximately 25,000 patients living with ALS in the USA. The median age of onset is 55 and the lifetime ALS risk is estimated to be between 1/600 and 1/1000 (McGuire et al. et al., 1996). There is no effective treatment for the disease, and the underlying mechanisms that result in motor neuron degeneration are not well understood. Four major pathological hallmarks of ALS have been defined in animal models of motor neuron degeneration and these include neuroinflammation, glutamate induced excitotoxicity, aberrant protein aggregation, and oxidative stress. Importantly, the mutant superoxide dismutase 1 (mSOD1) mouse model of neurodegeneration replicates important features of neuroinflammation and oxidative injury that are observed in both sporadic and familial forms of the disease. The goal of this dissertation was to increase our understanding of the molecular pathogenesis of ALS, using the mSOD1 mouse model at early, pre-symptomatic stages of the disease process. Our method was to use mRNA microarray time series data of spinal cord in mSOD1 and normal control mice, and then identify biological pathways that are involved in the earliest stages of mSOD1 motor neuron degeneration. Candidate pathways were then studied using both in vitro, and in vivo models, including human ALS spinal cords to define potential relevance to ALS disease progression. We analyzed temporal gene expression patterns in the spinal cord from mSOD1 mice from an asymptomatic stage of disease to end-stage of the disease, as well as age-matched controls (36 microarrays). Three proteins, galectins -1, -3, and -9, from a larger galectin family of proteins that bind ß-galactoside residues on glycoproteins were, found to be upregulated in the mSOD1 model at early stages, with galectin-3 in particular showing significant early changes that both anticipated and then tracked with disease progression. Galectin -3 and -9 but not galectin-1 was found to be increased in post-mortem autopsy tissue from patients with the sporadic form of ALS. Galectin-3 was the only galectin found to be upregulated at an asymptomatic stage (by 10-fold relative to non-disease controls) of disease progression and predominantly localized to microglia in the mSOD1 mouse. The role of galectin-3 is not well defined in the CNS but both neuroprotective roles and neurotoxic roles have been attributed to its presence in the CNS. Following this preliminary data, the overriding hypothesis of this proposal is that galectin-3 plays both neuroprotective and neurotoxic roles in the progression of ALS, and that these roles may be stage-specific and drive or dictate symptoms. To identify the role of galectin-3 in ALS disease progression, we bred diseased SOD1G93A mice to galectin-3 knockout mice (Gal-3-/-) to yield SOD1G93A / Gal-3-/- double transgenic mice. Relative to SOD1G93A / Gal-3-/- mice, SOD1G93A / Gal-3+/+ mice performed better in functional neurological tests and had significantly increased survival (slower progression through disease). In addition, SOD1G93A / Gal-3+/+ mice exhibited a decreased state of neuroinflammation as evidenced by lower expression of ectodermal dysplasia 1 (ED1) (a microglial activation marker) and TNF-α. SOD1G93A / Gal-3+/+ mice also exhibited a decrease in neurodegeneration which coincided with a decrease in oxidative stress as measured by levels of protein carbonyl modifications. Excitotoxicity is known to be a major pathological feature of ALS, and recent data suggest a strong interplay between both excitotoxicity and inflammation in the disease. We next explored the neuroprotective functions of galectin-3, within the context of excitotoxic insult by glutamate, and the neuroimmunomodulatory effects on glutamate uptake, which have been shown to limit and exacerbate excitotoxic damage. We found that galectin-3 protects against excitotoxicity, and that such neuroprotection is associated with altered glutamate uptake, potentially through the regulation of specific glutamate transporters glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST), whose expression at the cell surface of astrocytes is directly altered by galectin-3. We also determined that galectin-3 was neuroprotective against excitotoxic insults, even in isolated motor neurons that have no glial interaction. We proposed that this protective effect is likely mediated, at least in part, through the galectin-3 carbohydrate-binding domain. To identify galectin-3 binding partners in the mSOD1G93A model of motor neuron degeneration, we combined immunoprecipitation together with subcellular proteomics, to identify proteins that interact with galectin-3. We identified 31 proteins that were uniquely expressed in SOD1G93A / Gal-3+/+ mice and 21 proteins that were uniquely expressed in non-diseased controls. Of the 52 proteins, we chose to explore the expression profile of 3 proteins using immunoblot analysis. These proteins included Stra6, nucleolin, and Nlrp6, and were chosen for validation based on their potential relevance to ALS disease progression. We found that only Nlrp6, which was decreased in the diseased transgenic models compared to wild-type controls, was differentially expressed in a non-galectin-3 mediated manner. Nlrp6 is a component of the inflammasome, a complex involved in acute and chronic inflammation. Because inflammation is increased in the mSOD1 mouse, the observation that Nlrp6 is downregulated in disease is unexpected, and it may mean that inflammasome mediated inflammation in not an important factor in ALS. Our data suggests that galectin-3 may subserve a neuroprotective role in ALS, in part, through modulation of neuroinflammation, excitotoxicity and oxidative injury and in part through direct action at motor neurons. In addition, we have established a list of galectin-3 binding partners that can be further investigated for a potential role in ALS progression. These studies promote evidence that galectin-3 is a therapeutic target in ALS and strategies to modulate its expression are like to be beneficial to ALS. Further, since galectin-3 is already highly expressed in ALS, it may be useful to design galectin-3 mimetics that allow for more efficacious therapeutic results.

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