Bryce Vissel

Background: L-Dopa remains the primary treatment for Parkinson’s disease (PD), but chronic administration frequently leads to L-Dopa-induced dyskinesia (LID). While D1 and D2 medium spiny neuron (MSN) specific structural changes on the spine level have been observed in the striatum of PD and LID, studying microglia mediated synapse loss has not been done to date. Methods: Here we generated novel reporter mice by crossing floxed PSD95c(mCherry/eGFP) mice with D1-Cre and D2-Cre lines, producing D1-PSD95-EGFP and D2-PSD95-EGFP strains for MSN-specific synapse visualization. Using the 6-OHDA mouse model of PD and LID we assessed microglia mediated MSN subtype specific synapse loss in these mice while PLX3397 was used to investigate effects of microglia depletion and repopulation on LID development and synapse loss. Results: Both D1- and D2-MSNs exhibited significant PSD95 synapse loss in PD, with D1-MSN loss further exacerbated in LID. Microglia displayed increased phagocytic activity and accumulated PSD95 material within lysosomes, particularly in LID. PLX3397-mediated microglial depletion reduced LID severity and preserved D1-MSN synapses. A depletion and repopulation paradigm attenuated LID severity, preserved D1-MSN synapses, and reduced synaptic material within microglia. Conclusions: Microglia-mediated synapse loss in MSN subtypes contributes to PD and LID pathogenesis. Pharmacological microglial depletion and repopulation mitigate synapse loss and dyskinesia, highlighting microglial turnover as a promising therapeutic strategy for LID.

Diphtheria toxin receptor (DTR)–based depletion models are widely used to study microglial and macrophage function, yet the extent to which diphtheria toxin (DT) produces off-target effects remains incompletely defined. Here, we examined tolerability, behavioural outcomes, and cellular responses following intracerebroventricular (i.c.v.) DT administration in wild-type (WT) and CD11b-DTR mice. Mice received bilateral i.c.v. infusions of DT or vehicle over a 10-day period and were assessed for survival, motor and cognitive behaviour, myeloid cell changes, and neuropathology. Unexpectedly, DT induced dose-dependent mortality in WT mice, demonstrating that toxicity can occur independently of DTR expression. CD11b-DTR mice exhibited greater susceptibility, with reduced survival and the emergence of illness at lower DT doses. Behavioural testing revealed significant dose-dependent impairments in rotarod performance and Y-maze spontaneous alternation in both genotypes, while open-field mobility was largely preserved among animals. Region-specific analysis of myeloid cells in CD11b-DTR mice showed robust depletion in the midbrain at higher DT doses, whereas hippocampal cell numbers remained unchanged with marked morphological signs of activation. These findings indicate that DT-mediated myeloid cell responses vary across brain regions, potentially reflecting differential toxin exposure following ventricular delivery. Consistent with this, focal abnormalities in the brain—including ventriculitis, meningoencephalitis and spongiotic changes—were observed in a subset of clinically affected DT-treated animals, whereas peripheral organs were largely unremarkable and haematological changes were infrequent. Together, these data demonstrate that i.c.v. DT administration can induce mortality, behavioural dysfunction, and focal CNS pathology in both WT and CD11b-DTR mice, with transgene expression amplifying susceptibility. Our findings highlight the need for careful dose optimisation, appropriate DT-treated controls, and cautious interpretation of behavioural phenotypes when employing this model.

Neuroinflammatory pathways are emerging therapeutic targets for neurological conditions such as Parkinson’s disease (PD). Wnt-3a may exert anti-inflammatory effects via canonical pathway activation and β-catenin stabilization while dysregulation of the Wnt/β-catenin pathway has been implicated in the degeneration of dopamine neurons in PD. However, canonical pathway stimulation via application of Wnt-3a to protect against inflammation and dopaminergic degeneration has not been explored. We found Wnt-3a alone had no effect on pro-inflammatory TNF-α or IL-1β release from homeostatic primary microglia, however co-administration with LPS significantly increased TNF-α release beyond that seen with LPS alone. This exacerbation in TNF-α levels was not mediated by the NFκB pathway or activation of β-catenin. Canonical pathway inhibition via DKK1 showed no changes in TNF-α levels, however both SP600125 and U723122 were able to block Wnt-3a + LPS induced TNF-α release, implicating non-canonical pathways. Meanwhile, infusion of Wnt-3a in vivo did not alter dopaminergic or microglial populations in MPTP lesioned animals. Together, these findings suggest Wnt-3a may enhance pro-inflammatory TNF-α release via non-canonical signaling in inflammatory conditions, with minimal effect on homeostatic microglia. This demonstrates the importance of cellular context when identifying potential therapies for neurodegenerative diseases where neuroinflammation is a critical mediator of pathology.

Background: The cannabinoid receptor 2 (CB ₂ ) is involved in regulating immune responses, yet its specific function in microglia remains poorly defined. This study aimed to generate and validate a microglia-specific, inducible CB ₂ knockout mouse model incorporating reporter genes to enable precise detection of CB ₂ expression and CB ₂ knockout. Methods: A novel floxed CB ₂ mouse line was generated, incorporating GFP and tdTomato reporter genes driven by the Cnr2 promoter to indicate CB ₂ expression and CB ₂ knockout, respectively. This line was crossed with Cx3cr1 or Tmem119 tamoxifen-inducible Cre lines to achieve macrophage- or microglia-specific CB ₂ knockout, respectively. Behavioural testing, in vitro assays, sequencing and in vivo immunofluorescence were used to assess the efficiency and specificity of CB ₂ knockout as well as potential off-target effects. Results: The floxed allele did not alter breeding or motor behaviour in mice, nor CB ₂ function. CB ₂ expression, indicated by GFP, followed expected patterns across tissues and conditions. Sequencing revealed both DNA and RNA of the floxed allele was as anticipated. Tamoxifen-induced Cre activity successfully initiated tdTomato expression exclusively in microglia of tamoxifen treated, Cre positive mice, validating the specificity and inducibility of CB ₂ knockout. Microglial tdTomato expression confirmed successful CB ₂ knockout in 9.3% of TmemCB ₂ and 91.7% of Cx3CB ₂ microglia. Peripheral tdTomato expression persisted beyond 3 weeks post-tamoxifen in Cx3CB ₂ mice but was minimal in TmemCB ₂ mice. Conclusion: This novel microglia-specific, inducible CB ₂ knockout model is the first to combine a floxed CB ₂ allele with reporter genes, an essential advancement given the lack of reliable CB ₂ antibodies. The findings demonstrate the model’s specificity and effectiveness, while highlighting important considerations regarding Cre-mediated effects and recombination specificity. Furthermore, the floxed mouse can be crossed with any Cre line to study CB ₂ expression and function in various tissues. This model provides a powerful platform for advancing understanding of CB ₂ roles in microglia and supports future exploration of CB ₂ -targeted therapeutic strategies.

Background: Neuroinflammatory pathways are emerging therapeutic targets for neurological conditions such as Parkinson’s disease (PD). Studies have indicated Wnt-3a, a member of the wingless type MMTV integration (Wnt) signalling cascade, may exert anti-inflammatory effects via canonical pathway activation and β-catenin stabilisation. Furthermore, dysregulation of the Wnt/β-catenin pathway has been implicated in the degeneration of dopamine neurons in PD, however, stimulation of the canonical pathway via application of Wnt-3a to protect against inflammation and dopaminergic degeneration has not been explored. Methods: Primary microglial cultures were stimulated with lipopolysaccharide (LPS) for 24 hours with or without Wnt-3a. TNF-α levels were measured via ELISA while changes in NFkB inflammatory pathway proteins and phosphorylated and non-phosphorylated β-catenin were analysed via capillary western blot. To assess Wnt pathway involvement, cultures were treated with DKK1 (β-catenin canonical pathway inhibitor), SP600125 (Wnt/Pcp pathway inhibitor) or U73122 (Wnt/Ca ²⁺ pathway inhibitor). Finally, C57BL/6 mice received continuous intracerebroventricular infusion of Wnt-3a via osmotic pumps to investigate the effects of Wnt-3a on dopaminergic neuron survival and on microglial numbers in the MPTP model of PD. Results: Wnt-3a alone had no effect on TNF-α release from microglia. However, when co-administered with LPS, there was a significant increase in cytokine release beyond that seen with LPS alone. Protein analysis revealed that this exacerbation in TNF-α levels was not due to alterations in the NFkB pathway or differences in activation of β-catenin. Furthermore, DKK1 treated cells showed no changes in TNF-α levels, however both SP600125 and U723122 were able to block Wnt-3a + LPS induced TNF-α release, implicating the non-canonical pathways. Meanwhile Wnt-3a in vivo did not alter dopaminergic or microglial populations in the substantia nigra in MPTP lesioned animals. Conclusion: Together, these results suggest a pro-inflammatory response to Wnt-3a in an inflammatory context with little or no effect on resting microglia. Importantly, this outcome was independent of the β-catenin canonical pathway, revealing that Wnt-3a can increase pro-inflammatory TNF-α release via non-canonical signaling in an inflammatory environment. This demonstrates the importance of cellular context when identifying potential therapies for neurodegenerative diseases where neuroinflammation is a critical mediator of pathology.

Background: Finding successful therapies for individuals with Alzheimer's disease (AD) remains an ongoing challenge. One contributing factor is that the mouse models commonly used in preclinical research primarily mimic the familial form of AD, whereas the vast majority of human cases are sporadic. Accordingly, for a sporadic mouse model of AD, incorporating the multifactorial aspects of the disease is of utmost importance.Methods: In the current study, we exposed humanized Aβ knock-in mice (hAβ-KI) to weekly low-dose lipopolysaccharide (LPS) injections until 24 weeks of age and compared the development of AD pathologies to the familial AD mouse model known as the J20 mice.Results: At the early time point of 24 weeks, hAβ-KI mice and J20 mice exhibited spatial memory impairments in the Barnes maze. Strikingly, both hAβ-KI mice and J20 mice showed significant loss of dendritic spines when compared to WT controls, despite the absence of Aβ plaques in hAβ-KI mice at 24 weeks of age. Glial cell numbers remained unchanged in hAβ-KI mice compared to WT, and LPS exposure in hAβ-KI mice did not result in memory deficits and failed to exacerbate any other examined AD pathology.Conclusion: The study highlights the potential of hAβ-KI mice as a model for sporadic AD, demonstrating early cognitive deficits and synaptic alterations despite no evidence of Aβ plaque formation. These findings underscore the importance of considering multifactorial influences in sporadic AD pathogenesis and the need for innovative models to advance our understanding and treatment strategies for this complex disease.

Introduction: Developing effective treatment for Alzheimer’s disease (AD) remains a challenge. This can be partially attributed to the fact that the mouse models used in preclinical research largely replicate familial form of AD, while majority of human cases are sporadic; both forms differ widely in the onset and origin of pathology, therefore requiring specific/targeted treatments.Methods: In this study, we aimed to model sporadic AD in mice by combining two of the many risk factors that are strongly implicated in AD: ApoE4, a major genetic risk factor, together with an inflammatory stimuli. Accordingly, we subjected ApoE4 knock in (KI) mice, expressing humanized ApoE4, to low doses of Lipopolysaccharide (LPS) injections (i.p, weekly, for 4 months).Results: We assessed these animals for behavioral impairments at 6 months of age using Open Field, Y-maze, and Barnes Maze Test. LPS induced hypoactivity was observed in the Open Field and Y-maze test, whereas spatial learning and memory was intact. We then quantified differences in dendritic spine density, which is a strong correlate of AD. ApoE4KI mice showed a significant reduction in the number of spines after treatment with LPS, whereas there were no obvious differences in the total number of microglia and astrocytes.Discussion: To conclude, in the current study the APoEe4 risk gene increases the vulnerability of hippocampal neurons to inflammation induced spine loss, laying a foundation for an early sporadic AD mouse model.

Introduction: Accurate modelling of molecular changes in Alzheimer’s disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting.Methods: Here, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice.Results: We observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD.Discussion: Our findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD.

L‐Dopa, while treating motor symptoms of Parkinson's disease, can lead to debilitating L‐Dopa‐induced dyskinesias, limiting its use. To investigate the causative relationship between neuro‐inflammation and dyskinesias, we assessed if striatal M1 and M2 microglia numbers correlated with dyskinesia severity and whether the anti‐inflammatories, minocycline and indomethacin, reverse these numbers and mitigate against dyskinesia. In 6‐OHDA lesioned mice, we used stereology to assess numbers of striatal M1 and M2 microglia populations in non‐lesioned (naïve) and lesioned mice that either received no L‐Dopa (PD), remained non‐dyskinetic even after L‐Dopa (non‐LID) or became dyskinetic after L‐Dopa treatment (LID). We also assessed the effect of minocycline/indomethacin treatment on striatal M1 and M2 microglia and its anti‐dyskinetic potential via AIMs scoring. We report that L‐Dopa treatment leading to LIDs exacerbates activated microglia numbers beyond that associated with the PD state; the severity of LIDs is strongly correlated to the ratio of the striatal M1 to M2 microglial numbers; in non‐dyskinetic mice, there is no M1/M2 microglia ratio increase above that seen in PD mice; and reducing M1/M2 microglia ratio using anti‐inflammatories is anti‐dyskinetic. Parkinson's disease is associated with increased inflammation, but this is insufficient to underpin dyskinesia. Given that L‐Dopa‐treated non‐LID mice show the same ratio of M1/M2 microglia as PD mice that received no L‐Dopa, and, given minocycline/indomethacin reduces both the ratio of M1/M2 microglia and dyskinesia severity, our data suggest the increased microglial M1/M2 ratio that occurs following L‐Dopa treatment is a contributing cause of dyskinesias.