Developing behavioural testing and MR imaging to evaluate cognitive impairment in models of CNS autoimmunity

This project will investigate cognition and cognitive fatigue in models of CNS autoimmunity. We will use automated touchscreens and fMRI to measure deficits in higher order brain functions. We have two critical milestones in this project: to use touchscreen testing protocols to determine if mouse models demonstrate the same kinds of attention, working memory and cognitive fatigue deficits observed in MS, and to use fMRI to measure higher-order defects in the autoimmune models and identify any changes present that might be associated with cognitive dysfunction and the findings from human studies.https://ir.lib.uwo.ca/brainscanprojectsummaries/1037/thumbnail.jp

Using automated touchscreen tasks for cognitive assessment in a novel model of Parkinson\u27s disease

This research will focus specifically on evaluating cognitive flexibility (the ability to respond and adapt behaviours to changes in the environment), cognitive dysfunction (changes in the intellectual processing and reasoning that impact daily functions, to a greater severity than what might be expected from typical age-related decline) and long-term memory in a PD model using touchscreens.https://ir.lib.uwo.ca/brainscanprojectsummaries/1001/thumbnail.jp

Integrating behavioural, imaging and transcriptional profiling to discover the impact of midlife stress in Alzheimer\u27s disease

We will be integrating this cognitive assessment with imaging of brain structure and function to understand the mechanisms by which a risk factor, in this case modifiable life stress, influences Alzheimer\u27s disease-related decline. The resultant data will be integrated and disseminated using a new open-access neuroinformatics platform developed at Western (MouseBytes.ca), which will become a unique resource for open science investigations and set the standard for sharing of behavioural data across the world.https://ir.lib.uwo.ca/brainscanprojectsummaries/1028/thumbnail.jp

Domains of STIP1 responsible for regulating PrPC-dependent amyloid-β oligomer toxicity.

Soluble oligomers of amyloid-beta peptide (AβO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer\u27s disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AβO binding to PrP(C) and protects neurons from AβO-induced cell death. Here, we investigated the molecular interactions between AβO and STIP1 binding to PrP(C) and their effect on neuronal cell death. We showed that residues located in a short region of PrP (90-110) mediate AβO binding and we narrowed the major interaction in this site to amino acids 91-100. In contrast, multiple binding sites on STIP1 (DP1, TPR1 and TPR2A) contribute to PrP binding. DP1 bound the N-terminal of PrP (residues 23-95), whereas TPR1 and TPR2A showed binding to the C-terminal of PrP (residues 90-231). Importantly, only TPR1 and TPR2A directly inhibit both AβO binding to PrP and cell death. Furthermore, our structural studies reveal that TPR1 and TPR2A bind to PrP through distinct regions. The TPR2A interface was shown to be much more extensive and to partially overlap with the Hsp90 binding site. Our data show the possibility of a PrP, STIP1 and Hsp90 ternary complex, which may influence AβO-mediated cell death

Hyperactivity and attention deficits in mice with decreased levels of stress-inducible phosphoprotein 1 (STIP1)

Stress-inducible phosphoprotein I (STIP1, STI1 or HOP) is a cochaperone intermediating Hsp70/Hsp90 exchange of client proteins, but it can also be secreted to trigger prion protein-mediated neuronal signaling. Some mothers of children with autism spectrum disorders (ASD) present antibodies against certain brain proteins, including antibodies against STIP1. Maternal antibodies can cross the fetus blood-brain barrier during pregnancy, suggesting the possibility that they can interfere with STIP1 levels and, presumably, functions. However, it is currently unknown whether abnormal levels of STIP1 have any impact in ASD-related behavior. Here, we used mice with reduced (50%) or increased STIP1 levels (fivefold) to test for potential ASD-like phenotypes. We found that increased STIP1 regulates the abundance of Hsp70 and Hsp90, whereas reduced STIP1 does not affect Hsp70, Hsp90 or the prion protein. Interestingly, BAC transgenic mice presenting fivefold more STIP1 show no major phenotype when examined in a series of behavioral tasks, including locomotor activity, elevated plus maze, Morris water maze and five-choice serial reaction time task (5-CSRTT). In contrast, mice with reduced STIP1 levels are hyperactive and have attentional deficits on the 5-CSRTT, but exhibit normal performance for the other tasks. We conclude that reduced STIP1 levels can contribute to phenotypes related to ASD. However, future experiments are needed to define whether it is decreased chaperone capacity or impaired prion protein signaling that contributes to these phenotypes

The Transient Receptor Potential Melastatin 2 (TRPM2) Channel Contributes to beta-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment

In Alzheimer\u27s disease, accumulation of soluble oligomers of beta-amyloid peptide is known to be highly toxic, causing disturbances in synaptic activity and neuronal death. Multiple studies relate these effects to increased oxidative stress and aberrant activity of calcium-permeable cation channels leading to calcium imbalance. The transient receptor potential melastatin 2 (TRPM2) channel, a Ca2+-permeable nonselective cation channel activated by oxidative stress, has been implicated in neurodegenerative diseases, and more recently in amyloid-induced toxicity. Here we show that the function of TRPM2 is augmented by treatment of cultured neurons with beta-amyloid oligomers. Aged APP/PS1 Alzheimer\u27s mouse model showed increased levels of endoplasmic reticulum stress markers, protein disulfide isomerase and phosphorylated eukaryotic initiation factor 2 alpha, as well as decreased levels of the presynaptic marker synaptophysin. Elimination of TRPM2 in APP/PS1 mice corrected these abnormal responses without affecting plaque burden. These effects of TRPM2 seem to be selective for beta-amyloid toxicity, as ER stress responses to thapsigargin or tunicamycin in TRPM2(-/-) neurons was identical to that of wild-type neurons. Moreover, reduced microglial activation was observed in TRPM2(-/-)/APP/PS1 hippocampus compared with APP/PS1 mice. In addition, age-dependent spatial memory deficits in APP/PS1 mice were reversed in TRPM2(-/-)/APP/PS1 mice. These results reveal the importance of TRPM2 for beta-amyloid neuronal toxicity, suggesting that TRPM2 activity could be potentially targeted to improve outcomes in Alzheimer\u27s disease

Regulation of Amyloid Oligomer Binding to Neurons and Neurotoxicity by the Prion Protein-mGluR5 Complex

The prion protein (PrPC) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrPC, laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrPC and mGluR5 are co-receptors also for -amyloid oligomers (AOs) and have been shown to modulate toxicity and neuronal death in Alzheimer\u27s disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrPC-mGluR5 or AO-PrPC-mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrPC-mGluR5 has an important role in AO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrPC (Ln-1) binds to PrPC and induces intracellular Ca2+ increase in neurons via the complex PrPC-mGluR5. Ln-1 promotes internalization of PrPC and mGluR5 and transiently decreases AO biding to neurons; however, the peptide does not impact AO toxicity. Given that mGluR5 is critical for toxic signaling by AOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrPC-mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrPC-mGluR5 may modulate signaling intensity by different PrPC ligands

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. Molecular & Cellular Proteomics 12: 10.1074/mcp.M113.031005, 3253-3270, 2013

The Prion Protein Ligand, Stress-Inducible Phosphoprotein 1, Regulates Amyloid-beta Oligomer Toxicity

In Alzheimer\u27s disease (AD), soluble amyloid-beta oligomers (A beta Os) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrPC). However, it is unknown whether other ligands of PrPC can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrPC in the vicinity of the A beta O binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in A beta O toxicity. We confirmed the specific binding of A beta Os and STI1 to the PrP and showed that STI1 efficiently inhibited A beta O binding to PrP in vitro (IC50 of similar to 70 nM) and also decreased A beta O binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented A beta O-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to A beta O-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both A beta O binding to PrPC and PrPC-dependent A beta O toxicity were inhibited by TPR2A, the PrPC-interacting domain of STI1. Additionally, PrPC-STI1 engagement activated alpha 7 nicotinic acetylcholine receptors, which participated in neuroprotection against A beta O-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrPC ligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset A beta O-induced toxicity

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein.

Stress-inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase-3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1-mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild-type astrocytes by 3-fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia-mediated neuronal death in a prion protein-dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience