Pharmacological Stimulation of Phagocytosis Enhances Amyloid Plaque Clearance; Evidence from a Transgenic Mouse Model of ATTR Neuropathy

Hereditary ATTR V30M amyloidosis is a lethal autosomal dominant sensorimotor and autonomic neuropathy caused by deposition of aberrant transthyretin (TTR). Immunohistochemical examination of sural nerve biopsies in patients with amyloidotic neuropathy show co-aggregation of TTR with several proteins; including apolipoprotein E, serum amyloid P and components of the complement cascade. Complement activation and macrophages are increasingly recognized to play a crucial role in amyloidogenesis at the tissue bed level. In the current study we test the effect of two C5a receptor agonists and a C5a receptor antagonist (PMX53) on disease phenotype in ATTR V30M mice. Our results indicate that amyloid deposition was significantly reduced following treatment with the C5a receptor agonists, while treatment with the antagonist resulted in a significant increase of amyloid load. Administration of the C5a receptor agonists triggered increased recruitment of phagocytic cells resulting in clearance of amyloid deposits

C1q ablation exacerbates amyloid deposition: A study in a transgenic mouse model of ATTRV30M amyloid neuropathy.

ATTRV30M amyloid neuropathy is a lethal autosomal dominant sensorimotor and autonomic neuropathy, caused by deposition of amyloid fibrils composed of aberrant transthyretin (TTR). Ages of onset and penetrance exhibit great variability and genetic factors have been implicated. Complement activation co-localizes with amyloid deposits in amyloidotic neuropathy and is possibly involved in the kinetics of amyloidogenesis. A candidate gene approach has recently identified C1q polymorphisms to correlate with disease onset in a Cypriot cohort of patients with ATTRV30M amyloid neuropathy. In the current study we use a double transgenic mouse model of ATTRV30M amyloid neuropathy in which C1q is ablated to elucidate further a possible modifier role for C1q. Amyloid deposition is found to be increased by 60% in the absence of C1q. Significant up regulation is also recorded in apoptotic and cellular stress markers reflecting extracellular toxicity of pre-fibrillar and fibrillar TTR. Our data further indicate that in the absence of C1q there is marked reduction of macrophages in association with amyloid deposits and thus less effective phagocytosis of TTR

C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aβ amyloid and preserves memory in a mouse model of familial Alzheimer's disease.

According to the amyloid hypothesis of Alzheimer's disease (AD) the deposition of prefibrillar and fibrillar Aβ peptide sets off the pathogenic cascades of neuroinflammation and neurodegeneration that lead to synaptic and neuronal loss resulting in cognitive decline. Various approaches to reduce amyloid load by reducing production of the Aβ peptide or enhancing amyloid clearance by primary or secondary immunization have not proven successful in clinical trials. Interfering with the normal function of secretases and suboptimal timing of Aβ peptide removal have been put forward as possible explanations. Complement, an innate component of the immune system, has been found to modulate disease pathology and in particular neuronal loss in the AD mouse model but its mechanism of action is complex. C1Q has been shown to facilitate phagocytosis of Aβ peptide but its Ablation attenuates neuroinflammation. Experiments in AD mouse models show that inhibition of complement component C5a reduces amyloid deposition and alleviates neuroinflammation. Phagocytes including microglia, monocytes and neutrophils carry C5a receptors. Here, a widely used mouse model of AD, 5XFAD, was intermittently treated with the oral C5a receptor agonist EP67 and several neuronal and neuroinflammatory markers as well as memory function were assessed. EP67 treatment enhanced phagocytosis, resulting in a significant reduction of both fibrillar and non-fibrillar Aβ, reduced astrocytosis and preserved synaptic and neuronal markers as well as memory function. Timely and phasic recruitment of the innate immune system offers a new therapeutic avenue of treating pre-symptomatic Alzheimer disease

Expression of stress markers.

<p>The expression of BiP protein measured by immunoblotting (<i>A</i>) in stomach tissue of both V30M and V30M C1q KO animals in all three age groups depicting an increased expression in V30M C1q KO mice of all ages. The same was observed by immunofluorescence (<i>B</i>). Furthermore, the presence of the 4-HNE was quantified via immunoblotting (<i>C</i>) in stomach tissues of both V30M and V30M C1q KO animals in all three age groups, again indicating the higher expression of 4-HNE in V30M C1q KO mice. The same was observed by immunofluorescence (<i>D</i>). A&C n = 15/age group/line, data presented as mean ± 1SD. <i>B&D</i> Scale bar = 150mm.</p

hTTR levels in serum and stomach.

<p>hTTR immunoblotting exhibiting a single band at 14KDa (<i>A</i>). hTTR levels measured via immunoblotting in serum of V30M and V30M C1q KO animals of all three age groups indicating higher hTTR in the V30M mice and a gradual decrease in the amount of hTTR present as the animals age (<i>B</i>). hTTR levels measured via immunoblotting in stomach tissue of the V30M and V30M C1q KO animals exhibit a severe decrease in the second age group (<i>C</i>). <i>B&C</i> n = 15/age group/line. Data presented as mean ± 1SD.</p

Model of observed C1q ablation effects.

<p>Classical and alternative pathway activation lead to the hydrolysis of C3 initiating the formation of the membrane attack complex (MAC) and production of the chemoattractant C5a. C1q and C5a recruit and activate phagocytic cells such as macrophages through receptors located on their surfaces (<i>A</i>). In the absence of C1q, properdin expression increases along with the entire alternative pathway and the terminal MAC complex. Concurrently, the presence of CD68 positive phagocytes was decreased along with the expression of C5a anaphylatoxin and its receptor CD88 (<i>B</i>). As a result, amyloid deposition increases following C1q ablation (<i>b</i>) versus the original transgenic model (<i>a</i>).</p

Expression of complement markers.

<p>The expression of Properdin (<i>A</i>) and C5b-9 (<i>B</i>) measured via immunoblotting in both V30M and V30M C1q KO animals stomach tissue in all three age groups exhibiting an age related increase of both markers, as well as a higher expression in the V30M C1q KO animals (n = 15/age group/line, data presented as mean ± 1SD).</p

Expression of CD88 and C5a.

<p>The expression of the CD88 receptor (<i>A</i>) and its ligand C5a (<i>B</i>) were measured by immunoblotting in stomach tissues of both V30M and V30M C1q KO animals in all three age groups. Both markers exhibit an astounding reduction in the V30M C1q KO mice. A&B n = 15/age group/line, data presented as mean ± 1SD.</p