A role for the classical complement pathway in hippocampal dendritic injury and hippocampal dependent memory deficits in a model of acquired epilepsy

Status epilepticus (SE) triggers pathological changes to hippocampal dendrites that may promote epileptogenesis. The microtubule associated protein 2 (Map2) helps stabilize microtubules of the dendritic cytoskeleton. Recently, we reported a substantial decline in Map2 that coincided with robust microglia accumulation in the CA1 hippocampal region after an episode of SE. A spatial correlation between Map2 loss and reactive microglia was also reported in human cortex from refractory epilepsy. New evidence supports that microglia are guided by proteins of the classical complement pathway (C1q and C3) to prune dendritic structures. Furthermore, components of complement have been shown to be upregulated in human and experimental epilepsy. Thus, to identify a potential role of the classical complement pathway in SE-induced Map2 and microglial changes, we characterized the spatiotemporal profile of these events. We used immunohistochemistry to determine the distribution of Map2 and the microglia marker IBA1 in the hippocampus after pilocarpine-induced SE from 4 hours to 35 days. We found a decline in Map2 immunoreactivity in the CA1 area that reached minimal levels at 14 days post-SE and partially increased thereafter. In contrast, maximal microglia accumulation occurred in the CA1 area at 14 days post-SE. We then mapped the spatiotemporal profile of C1q using immunohistochemistry at 3-35 days after SE, where substantial Map2 and microglial alterations were observed. We used western blot to determine the levels of C3 and its cleavage products. C1q and C3 were both increased in the hippocampus at 14 days after SE, when Map2 and microglia changes were most profound. Our data indicate that SE-induced Map2 and microglial changes parallel each other’s spatiotemporal profiles. These findings also suggest a potential role for the classical complement pathway in SE-induced Map2-microglial interactions

Identification of a resilient mouse facial motoneuron population following target disconnection by injury or disease

Background: When nerve transection is performed on adult rodents, a substantial population of neurons survives short-term disconnection from target, and the immune system supports this neuronal survival, however long-term survival remains unknown. Understanding the effects of permanent axotomy on cell body survival is important as target disconnection is the first pathological occurrence in fatal motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Objective: The goal of this study was to determine if facial motoneurons (FMN) could survive permanent target disconnection up to 26 weeks post-operation (wpo) after facial nerve axotomy (FNA). In addition, the potentially additive effects of immunodeficiency and motoneuron disease on post-axotomy FMN survival were examined. Methods: This study included three wild type (WT) mouse strains (C57BL/6J, B6SJL, and FVB/NJ) and three experimental models (RAG-2-/-: immunodeficiency; mSOD1: ALS; Smn-/-/SMN2+/+: SMA). All animals received a unilateral FNA, and FMN survival was quantified at early and extended post-operative timepoints. Results: In the C57BL/6J WT group, FMN survival significantly decreased at 10 wpo (55 ± 6%), and then remained stable out to 26 wpo (47 ± 6%). In the RAG-2-/- and mSOD1 groups, FMN death occurred much earlier at 4 wpo, and survival plateaued at approximately 50% at 10 wpo. The SMA model and other WT strains also exhibited approximately 50% FMN survival after FNA. Conclusion: These results indicate that immunodeficiency and motoneuron disease accelerate axotomy-induced neuron death, but do not increase total neuron death in the context of permanent target disconnection. This consistent finding of a target disconnection-resilient motoneuron population is prevalent in other peripheral nerve injury models and in neurodegenerative disease models as well. Characterization of the distinct populations of vulnerable and resilient motoneurons may reveal new therapeutic approaches for injury and disease

C5aR1 antagonism suppresses inflammatory glial responses and alters cellular signaling in an Alzheimer’s disease mouse model

Alzheimer's disease (AD) is the leading cause of dementia in older adults, and the need for effective, sustainable therapeutic targets is imperative. The complement pathway has been proposed as a therapeutic target. C5aR1 inhibition reduces plaque load, gliosis, and memory deficits in animal models, however, the cellular bases underlying this neuroprotection were unclear. Here, we show that the C5aR1 antagonist PMX205 improves outcomes in the Arctic48 mouse model of AD. A combination of single cell and single nucleus RNA-seq analysis of hippocampi derived from males and females identified neurotoxic disease-associated microglia clusters in Arctic mice that are C5aR1-dependent, while microglial genes associated with synapse organization and transmission and learning were overrepresented in PMX205-treated mice. PMX205 also reduced neurotoxic astrocyte gene expression, but clusters associated with protective responses to injury were unchanged. C5aR1 inhibition promoted mRNA-predicted signaling pathways between brain cell types associated with cell growth and repair, while suppressing inflammatory pathways. Finally, although hippocampal plaque load was unaffected, PMX205 prevented deficits in short-term memory in female Arctic mice. In conclusion, C5aR1 inhibition prevents cognitive loss, limits detrimental glial polarization while permitting neuroprotective responses, as well as leaving most protective functions of complement intact, making C5aR1 antagonism an attractive therapeutic strategy for AD

Impact of peripheral immune status on central molecular responses to facial nerve axotomy

When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease

The Role of the Medial Prefrontal Cortex in Regulating Social Familiarity-Induced Anxiolysis

Overcoming specific fears and subsequent anxiety can be greatly enhanced by the presence of familiar social partners, but the neural circuitry that controls this phenomenon remains unclear. To overcome this, the social interaction (SI) habituation test was developed in this lab to systematically investigate the effects of social familiarity on anxiety-like behavior in rats. Here, we show that social familiarity selectively reduced anxiety-like behaviors induced by an ethological anxiogenic stimulus. The anxiolytic effect of social familiarity could be elicited over multiple training sessions and was specific to both the presence of the anxiogenic stimulus and the familiar social partner. In addition, socially familiar conspecifics served as a safety signal, as anxiety-like responses returned in the absence of the familiar partner. The expression of the social familiarity-induced anxiolysis (SFiA) appears dependent on the prefrontal cortex (PFC), an area associated with cortical regulation of fear and anxiety behaviors. Inhibition of the PFC, with bilateral injections of the GABAA agonist muscimol, selectively blocked the expression of SFiA while having no effect on SI with a novel partner. Finally, the effect of D-cycloserine, a cognitive enhancer that clinically enhances behavioral treatments for anxiety, was investigated with SFiA. D-cycloserine, when paired with familiarity training sessions, selectively enhanced the rate at which SFiA was acquired. Collectively, these outcomes suggest that the PFC has a pivotal role in SFiA, a complex behavior involving the integration of social cues of familiarity with contextual and emotional information to regulate anxiety-like behavior

The Role of Complement C3 in the Hippocampal Pathology of Status Epilepticus

Epilepsy is comorbid with cognitive and psychiatric dysfunctions. This pathophysiology, associated with hippocampal synaptodendritic structural and functional changes, is exacerbated by prolonged seizures (status epilepticus; SE). We found a correlation between hippocampal dendritic loss and microgliosis after SE, along with hyperactivation of the classical complement pathway (C1q-C3). These paralleled increased seizure frequency and memory deficits in a rat model of SE and acquired epilepsy. C1q leads to C3 cleavage into biologically active fragments C3a and C3b. Evidence suggests that C1q and C3b contribute to synaptic stripping by microglia in the developing brain and neurodegenerative disorders. Thus, we hypothesized that SE-induced C3 activation may alter hippocampal synaptic protein levels thereby promoting memory deficits. To test the hypothesis, different groups of wild type (WT) or C3 deficient (C3KO) mice were injected with pilocarpine (350mg/kg) to induce SE or saline (controls): WT-C, WT-SE, C3KO-C, and C3KOSE. At two weeks after SE, mice were subjected to novel object recognition (NOR) to evaluate recognition memory, and Barnes maze (BM) to measure hippocampal-dependent spatial learning and memory. Following behavioral testing, mice were sacrificed and hippocampi collected at either 2 or 5 weeks after SE to measure changes in C3 protein levels and levels of synaptic proteins including PSD95, Vglut1, and Vgat. As a method of verifying our findings, we used a second model of pilocarpine-induced SE in male Sprague Dawley rats. Starting at 7 days after SE, rats were treated with cobra venom factor (CVF; 100ng/g, i.p.) or vehicle (veh) every third day. On days 14-15 rats were subjected to open field and NOR to measure anxiety and recognition memory. On day 16, rats were sacrificed and hippocampi collected for western blotting. WT and C3KO mice were able to reach stage 4.5-6 seizures after pilocarpine injections. In NOR trial 1, exploration time for both objects was similar in all groups (p\u3e .05). In trial 2, WT-C and C3KO-C mice spent more time exploring the novel object than the familiar one (p\u3c .05) while WT-SE mice explored both objects equally (p\u3e .05). Interestingly, C3KO-SE mice spent more time with the novel object similar to controls (p\u3e .05), suggesting that the deficit in object recognition memory induced by SE was attenuated in C3KO mice. Similarly, veh- and CVF-treated control rats spent more time exploring the novel object during trial 2 (p \u3c .05). The veh-treated SE rats did not show significant preference for the novel object versus familiar (p\u3e .05), whereas the CVFtreated SE rats explored the novel object significantly more than the familiar (p\u3c .05). These findings support that C3 inhibition after SE prevents recognition memory deficits. Furthermore, there was a reduction in synaptic proteins PSD95 and Vgat in the SE-veh group compared to the C-veh group. This difference was not observed in the C-CVF and SE-CVF groups, suggesting that blocking C3 after SE is neuroprotective against hippocampal synaptic loss. Taken together, these findings are the first to show an association between C3 activation and hippocampal and cognitive deficits in two rodent models of SE and acquired TLE. We found that depletion of C3 is sufficient to attenuate SE-induced deficits in NOR-evaluated recognition memory and changes in the levels of an inhibitory synaptic protein. In conclusion, our data suggest that SE-induced complement C3 activation contributes to hippocampal synaptic remodeling and impairments in recognition memory, and that the complement C3 may be a potential therapeutic target for the memory comorbidities associated with SE. Future studies will determine the effect of C3 inhibition on spontaneous recurrent seizures, and whether C3-guided and microglial-dependent phagocytosis is an underlying mechanism for the SE-induced epileptogenic synaptic remodeling

The good, the bad, and the opportunities of the complement system in neurodegenerative disease

The complement cascade is a critical effector mechanism of the innate immune system that contributes to the rapid clearance of pathogens and dead or dying cells, as well as contributing to the extent and limit of the inflammatory immune response. In addition, some of the early components of this cascade have been clearly shown to play a beneficial role in synapse elimination during the development of the nervous system, although excessive complement-mediated synaptic pruning in the adult or injured brain may be detrimental in multiple neurogenerative disorders. While many of these later studies have been in mouse models, observations consistent with this notion have been reported in human postmortem examination of brain tissue. Increasing awareness of distinct roles of C1q, the initial recognition component of the classical complement pathway, that are independent of the rest of the complement cascade, as well as the relationship with other signaling pathways of inflammation (in the periphery as well as the central nervous system), highlights the need for a thorough understanding of these molecular entities and pathways to facilitate successful therapeutic design, including target identification, disease stage for treatment, and delivery in specific neurologic disorders. Here, we review the evidence for both beneficial and detrimental effects of complement components and activation products in multiple neurodegenerative disorders. Evidence for requisite co-factors for the diverse consequences are reviewed, as well as the recent studies that support the possibility of successful pharmacological approaches to suppress excessive and detrimental complement-mediated chronic inflammation, while preserving beneficial effects of complement components, to slow the progression of neurodegenerative disease

C5aR1 signaling promotes region‐ and age‐dependent synaptic pruning in models of Alzheimer's disease

IntroductionSynaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD.MethodsA combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD.ResultsGenetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP).DiscussionReduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD.HighlightsC5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease

Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

BackgroundThe complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer's disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation.MethodsTo elucidate the effects of C5a-C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7-10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states.ResultsArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a-C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a-C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers.ConclusionC5a-C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD