Susanna Mantovani

The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization

C3a is a key complement activation fragment, yet its neutrophil-expressed receptor (C3aR) still has no clearly defined role. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to explore the role of C3aR in acute tissue injuries. C3aR deficiency worsened intestinal injury, which corresponded with increased numbers of tissue-infiltrating neutrophils. Circulating neutrophils were significantly increased in C3aR−/− mice after intestinal ischemia, and C3aR−/− mice also mobilized more circulating neutrophils after granulocyte colony-stimulating factor infusion compared with WT mice, indicating a specific role for C3aR in constraining neutrophil mobilization in response to intestinal injury. In support of this role, C3aR−/− mice reconstituted with WT bone marrow reversed IR pathology back to WT levels. Complement C5a receptor (C5aR) antagonism in C3aR−/− mice also rectified the worsened pathology after intestinal IR injury but had no effect on circulating neutrophils, highlighting the opposing roles of C3a and C5a in disease pathogenesis. Finally, we found that using a potent C3a agonist to activate C3aR in vivo reduced neutrophil mobilization and ameliorated intestinal IR pathology in WT, but not C3aR−/−, mice. This study identifies a role for C3aR in regulating neutrophil mobilization after acute intestinal injury and highlights C3aR agonism as a potential treatment option for acute, neutrophil-driven pathologies.

Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.

Transgenic fatal familial insomnia mice indicate prion infectivity-independent mechanisms of pathogenesis and phenotypic expression of disease.

Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.

Immunopurification of Pathological Prion Protein Aggregates

Background Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrPC) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrPSc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrPC molecules. A great deal of effort has been devoted to developing protocols for purifying PrPSc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrPSc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases. Principal Findings We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrPSc and mutant PrP aggregates at electrophoretic homogeneity. PrPSc purified from prion-infected mice was able to seed misfolding of PrPC in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons. Significance The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.

Altered expression of metabolic proteins and adipokines in patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the loss of upper cortical and lower motor neurons. ALS causes death within 2-5years of diagnosis. Diet and body mass index influence the clinical course of disease, however there is limited information about the expression of metabolic proteins and fat-derived cytokines (adipokines) in ALS. In healthy controls and subjects with ALS, we have measured levels of proteins and adipokines that influence metabolism. We find altered levels of active ghrelin, gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), lipocalin-2, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and 8 (IL-8), and tumor necrosis factor alpha (TNFα) in the plasma of ALS patients relative to controls. We also observe a positive correlation between the expression of plasma nerve growth factor (NGF) relative to disease duration, and an inverse correlation between plasma glucagon and the ALS functional rating scale-revised (ALSFRS-R). Further studies are required to determine whether altered expression of metabolic proteins and adipokines contribute to motor neuron vulnerability and how these factors act to modify the course of disease.

Motor deficits associated with Huntington's disease occur in the absence of striatal degeneration in BACHD transgenic mice

Huntingtons disease (HD) is an incurable neurodegenerative condition characterized by progressive motor and cognitive dysfunction, and depletion of neurons in the striatum. Recently, BACHD transgenic mice expressing the full-length human huntingtin gene have been generated, which recapitulate some of the motor and cognitive deficits seen in HD. In this study, we carried out a series of extensive behavioural and neuropathological tests on BACHD mice, to validate this mouse for preclinical research. Transgenic C57BL/6J BACHD and litter-matched wild-type mice were examined in a battery of motor and cognitive function tests at regular intervals up to 12 months of age. Brains from these mice were also analysed for signs of neurodegeneration and striatal and cortical volume sizes compared using anatomic 16.4T magnetic resonance imaging (MRI) brain scans. BACHD mice showed progressive motor impairments on rotarod and balance beam tests starting from 3 months of age, were hypoactive in the open field tests starting from 6 months of age, however, showed no alterations in gait and grip strength at any age. Surprisingly, despite these distinct motor deficits, no signs of neuronal loss, gliosis or blood-brain barrier degeneration were observed in the striatum of 12-month-old mice. MRI brain scans confirmed no reduction in striatal or cortical volumes at 12 months of age, and BACHD mice had a normal lifespan. These results demonstrate that classical Huntingtons-like motor impairments seen in this transgenic model, do not occur due to degeneration of the striatum, and thus caution against the use of this model for preclinical studies into HD.

Cell Type-Specific Neuroprotective Activity of Untranslocated Prion Protein

Background A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. Principal Findings Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. Significance These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

Co-ordinated expression of innate immune molecules during mouse neurulation.

The innate immune system is the first line of defence against pathogens and infection. Recently, it has become apparent that many innate immune factors have roles outside of immunity and there is growing evidence that these factors play important functional roles during the development of a range of model organisms. Several studies have documented developmental expression of individual factors of the toll-like receptor and complement systems, and we recently demonstrated a key role for complement C5a receptor (C5aR1) signalling in neural tube closure in mice. Despite these emerging studies, a comprehensive expression analysis of these molecules in embryonic development is lacking. In the current study, we therefore, examined the expression of key innate immune factors in the early development period of neurulation (7.5-10.5dpc) in mice. We found that complement factor genes were differentially expressed during this period of murine development. Interestingly, the expression patterns we identified preclude activation of the classical and alternative pathways and formation of the membrane attack complex. Additionally, several other classes of innate immune molecules were expressed during the period of neurulation, including toll-like receptors (TLR-2, -3, -4 and -9), receptor for advanced glycation end-products (RAGE), and their signalling adapters (TRAF-4, TRAF-6, TAK-1 and MyD88). Taken together, this study highlights a number of innate immune factors as potential novel players in early embryonic development.

Inhibition of IL-1β Signaling Normalizes NMDA-Dependent Neurotransmission and Reduces Seizure Susceptibility in a Mouse Model of Creutzfeldt–Jakob Disease

Creutzfeldt–Jakob disease (CJD) is a neurodegenerative disorder caused by prion protein (PrP) misfolding, clinically recognized by cognitive and motor deficits, electroencephalographic abnormalities, and seizures. Its neurophysiological bases are not known. To assess the potential involvement of NMDA receptor (NMDAR) dysfunction, we analyzed NMDA-dependent synaptic plasticity in hippocampal slices from Tg(CJD) mice, which model a genetic form of CJD. Because PrP depletion may result in functional upregulation of NMDARs, we also analyzed PrP knock-out (KO) mice. Long-term potentiation (LTP) at the Schaffer collateral–commissural synapses in the CA1 area of ∼100-d-old Tg(CJD) mice was comparable to that of wild-type (WT) controls, but there was an inversion of metaplasticity, with increased GluN2B phosphorylation, which is indicative of enhanced NMDAR activation. Similar but less marked changes were seen in PrP KO mice. At ∼300 d of age, the magnitude of LTP increased in Tg(CJD) mice but decreased in PrP KO mice, indicating divergent changes in hippocampal synaptic responsiveness. Tg(CJD) but not PrP KO mice were intrinsically more susceptible than WT controls to focal hippocampal seizures induced by kainic acid. IL-1β-positive astrocytes increased in the Tg(CJD) hippocampus, and blocking IL-1 receptor signaling restored normal synaptic responses and reduced seizure susceptibility. These results indicate that alterations in NMDA-dependent glutamatergic transmission in Tg(CJD) mice do not depend solely on PrP functional loss. Moreover, astrocytic IL-1β plays a role in the enhanced synaptic responsiveness and seizure susceptibility, suggesting that targeting IL-1β signaling may offer a novel symptomatic treatment for CJD. SIGNIFICANCE STATEMENT Dementia and myoclonic jerks develop in individuals with Creutzfeldt–Jakob disease (CJD), an incurable brain disorder caused by alterations in prion protein structure. These individuals are prone to seizures and have high brain levels of the inflammatory cytokine IL-1β. Here we show that blocking IL-1β receptors with anakinra, the human recombinant form of the endogenous IL-1 receptor antagonist used to treat rheumatoid arthritis, normalizes hippocampal neurotransmission and reduces seizure susceptibility in a CJD mouse model. These results link neuroinflammation to defective neurotransmission and the enhanced susceptibility to seizures in CJD and raise the possibility that targeting IL-1β with clinically available drugs may be beneficial for symptomatic treatment of the disease.

The Role of C5a Receptor Signaling in Endotoxin-Induced Miscarriage and Preterm Birth

Complement factor 5a (C5a), a potent pro‐inflammatory mediator of the complement system, has been implicated in fetal rejection throughout gestation, from miscarriage to preterm birth. This study aimed to investigate the role of the principal C5a receptor, C5aR1 (CD88), in both miscarriage and preterm birth, in a bacterial endotoxin (lipopolysaccharide; LPS) murine model.