Biology subjects

Woodruff, T. M.

Publications and source records attributed to Woodruff, T. M..

4 recordsLinked to original sources

bioRxiv2020

The 'C3aR antagonist' SB290157 is a partial C5aR2 agonist

Innate immune complement activation generates the C3 and C5 protein cleavage products C3a and C5a, defined classically as anaphylatoxins. C3a activates C3a receptors (C3aR), while C5a activates two receptors (C5aR1 and C5aR2) to exert their immunomodulatory activities. The non-peptide compound, SB290157, was originally reported in 2001 as the first C3aR antagonist. In 2005, the first report on non-selective nature of SB290157 was published, where the compound exerted clear agonistic, not antagonistic, activity in variety of cells. Other studies also documented non-selective activities of this drug in vivo. These findings severely hamper data interpretation regarding C3aR when using this compound. Unfortunately, given the dearth of C3aR inhibitors, SB290157 still remains widely used to explore C3aR biology (>70 publications to date). Given these issues, in the present study we aimed to further explore SB290157s pharmacological selectivity by screening the drug against three human anaphylatoxin receptors, C3aR, C5aR1 and C5aR2, using transfected cells. We first confirmed that SB290157 acts as a potent agonist at human C3aR. We also identified that SB290157 exerts partial agonist activity at C5aR2 by mediating {beta}-arrestin recruitment at higher compound doses. Notably, SB290157s activity at C5aR2 was more potent and efficacious than the current lead C5aR2 agonist P32. Notwithstanding this, SB290157 showed inhibitory effect on C3a-mediated signalling in primary human macrophages. Our results therefore provide even more caution against using SB290157 as a research tool to explore C3aR function. Given the reported immunomodulatory and anti-inflammatory activities of C3aR and C5aR2 agonism, any function observed with SB290157 could be due to these off target activities.

pharmacology and toxicology↗

bioRxiv2020

Glucose clearance and uptake is increased in the SOD1G93A mouse model of amyotrophic lateral sclerosis in an insulin-independent mechanism

Metabolic disturbances are associated with the progression of the neurodegenerative disorder, amyotrophic lateral sclerosis (ALS), however the molecular events that drive energy imbalances in ALS are not completely understood. In this study we aimed to elucidate deficits in energy homeostasis in the SOD1G93A mouse model of ALS. We identified that SOD1G93A mice at mid-symptomatic disease stage have increased oxygen consumption and faster exogenous glucose uptake, despite presenting with normal insulin tolerance. Fasting glucose homeostasis was also disturbed, along with increased liver glycogen stores, despite elevated circulating glucagon, suggesting that glucagon signalling is impaired. Metabolic gene expression profiling of livers indicated that glucose cannot be utilised efficiently in SOD1G93A mice. Overall, we demonstrate that glucose homeostasis and uptake are altered in SOD1G93A mice, which is linked to an increase in insulin-independent glucose uptake and a disturbance in glucagon sensitivity, suggesting glucagon secretion and signalling could be potential therapeutic targets for ALS.

physiology↗

bioRxiv2020

Absence of receptor for advanced glycation end product (RAGE) reduces inflammation and extends survival in the hSOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signaling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1G93A mice lacking RAGE, and compared them to SOD1G93A transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signaling exerted a protective effect on SOD1G93A pathology, slowing disease progression and significantly extending survival by [~]3 weeks, and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS, and highlights RAGE as a potential immune therapeutic target for ALS.

neuroscience↗

bioRxiv2020

A validated quantitative method for the assessment of neuroprotective barrier impairment in neurodegenerative disease models

The blood brain barrier (BBB) and blood spinal cord barrier (BSCB) are highly specialised structures that limit molecule entry from the blood and maintain homeostasis within the central nervous system (CNS). BBB and BSCB breakdown are associated with multiple neurodegenerative diseases. Given the key role of neuroprotective barrier impairment in neurodegeneration, it is important to identify an effective quantitative method to assess barrier integrity in animal models. In the present study, we developed and validated a quantitative method for assessing BBB and BSCB integrity using sodium fluorescein, a compound that outperformed other fluorescent dyes. We demonstrated using this method that multiple CNS regions progressively increase in permeability in models of Huntingtons disease and amyotrophic lateral sclerosis, while biphasic disruption occurred in a mouse model of Alzheimers disease with disease progression. Collectively, we report a quantitative fluorometric marker with validated reproducible experimental methods, that allows the effective assessment of BBB and BSCB integrity in animal models. This method could be useful to further the understanding of the contribution of these neuroprotective barriers to neurodegeneration processes.

neuroscience↗