Chronic inflammation has emerged as a clear pathological hallmark of AD. While some inflammatory responses can damage the brain, others have potent beneficial effects. A better understanding of specific molecular pathways in activated glial cells may help reconcile the apparently contradictory outcomes of inflammatory responses.

To learn how to direct the inflammatory machinery to exert beneficial functions, scientists in the Gladstone Institute of Neurological Disease led by Dr. Lennart Mucke and Dr. Li Gan are dissecting the inflammatory pathways. These studies will provide the basis for the development of better therapies for AD patients and contribute to ending the scourge of AD and possibly other diseases.

Some components of the inflammatory response to central nervous system (CNS) degeneration. Pathogenic triggers, such as accumulation of abnormal proteins in cells or extracellular spaces, elicit cellular stress responses and can result in progressive degeneration and eventual death of neurons. Many soluble factors are involved in promoting or inhibiting these processes. For example, cytokines and other mediators of the innate immune response are released by astrocytes and microglia to orchestrate defense mechanisms and initiate the removal or sequestration of the pathogenic triggers. Some of the molecules and receptors involved in the recognition of abnormal proteins and degenerating cells are illustrated. Receptors on glial cells recognize ligands and initiate inflammatory responses. C1qRp, C1q receptor for phagocytosis; CR3, complement receptor 3; FcR, Fc receptor; PSR, phosphatidyl serine receptor; SR, scavenger receptor.

In AD, the brain regions responsible for learning, memory, and emotional behaviors undergo severe biochemical and structural changes resulting from the accumulation of abnormal proteins. Although the exact cause of AD is unknown, most studies of AD pathogenesis have focused on two pathological hallmarks of the disease: senile plaques and neurofibrillary tangles, both of which involve abnormal protein deposits. Senile plaques are extracellular deposits of fibrils and other aggregates of amyloid beta (Aβ) peptides, which result from the proteolytic cleavage of the amyloid precursor protein (APP). Neurofibrillary tangles are intracellular aggregates of an abnormal form of the microtubule-associated protein tau. Considerable evidence supports the hypothesis that Aβ accumulation in the brain triggers a complex pathological cascade leading to neuronal dysfunction and ultimately dementia.

Inflammation in AD: A Double-Edged Sword

A key event in this cascade is an inflammatory response involving glial cells, a specialized type of injury-responsive brain cell. Normally, glial cells nourish and communicate with neurons. In AD brains, the accumulation of Aβ peptides and the presence of injured neurons appear to trigger glial cell activation and proliferation in an attempt to return the brain to a normal state. Glial cells produce inflammatory cytokines that summon other immune cells into action. In addition, complement, a multiprotein network that is part of the immune system, is also activated.

What is the purpose of the inflammatory response? In many tissues, it is the body's first defense against infection. Most of the time, inflammation is a lifesaver. A complex combination of cells and secreted proteins marshal a defensive attack against disease-causing bacteria, viruses, and parasites. In other situations, inflammation promotes wound repair. However, if inflammation continues for too long or turns against the body's own proteins, it can lead to a host of diseases, including rheumatoid arthritis and ulcerative colitis.

Inflammatory responses and neurodegeneration. Inflammatory responses associated with neurodegeneration are probably attempts to remove the pathogenic trigger and to inhibit the neurodegenerative process. However, uncontrolled or chronic inflammation may promote the process. MHC, major histocompatibility complexes; NK, natural killer; TCR, T-cell receptor (arrow), stimulation (tee), inhibition.

The inflammatory response triggered by the accumulation of Aβ results in removal of Aβ aggregates and thereby may decrease the harmful effects of chronic Aβ accumulation. For example, when aged transgenic mice expressing human APP were vaccinated with Aβ, amyloid plaques were drastically reduced in size and number, possibly through uptake and digestion by microglia. Clinical trials of Aβ immunization were halted because some patients developed meningoencephalitis, a pathological inflammation of the brain and the thin sheets of tissue that enwrap it. However, intensive efforts have been made to design immunization regimens that avoid these detrimental side effects, and new clinical trials are well under way.

On the other hand, there also is evidence that Aβ-induced inflammation can damage the central nervous system. In cultured cells, Aβ stimulates microglia to release cytokines and other neurotoxic factors. Supporting the idea that chronic inflammation has a damaging effect in AD, circumstantial evidence suggests that nonsteroidal anti-inflammatory drugs such as ibuprofen may reduce the risk of developing the disease.

Because the inflammatory response can either promote or counteract the neurodegenerative processes in AD, it is critical to pinpoint its role in specific clinically relevant scenarios.

Change in distribution and overall decrease in Aβ deposits in human APP (hAPP)/TGF-β1 mice. Sagittal brain sections of mice transgenic for human APP (A) and hAPP/TGF-β1 (B) were immunostained for human Aβ deposition, and antibody binding was visualized with the immunoperoxidase method. Aβ deposition in the hippocampal stratum oriens and in the molecular layer of the dentate gyrus (red arrowheads) is much more prominent in the hAPP brain (A) than in the hAPP/TGF-β1 brain (B). The latter showed dense staining in the hippocampal fissure and the meninges (green arrowheads). Scale bar indicates 500 mm (A and B).

TGF-β1 and the Clearance of Aβ

Dr. Mucke and his colleagues have unraveled how transforming growth factor β1 (TGF-β1), a key cytokine regulator of the brain's responses to injury and inflammation, promotes the removal of Aβ aggregates. In transgenic mouse models overexpressing human APP and carrying mutations linked to familial AD, elevated production of TGF-β1 reduced the number of amyloid plaques by 67% and the overall Aβ load by 50%, and also decreased the number of plaque-associated distortions of neuronal branches called neuritic dystrophy. Plaque reduction in these mice was associated with a robust activation of microglia and an increase in inflammatory mediators. In cell culture, recombinant TGF-β1 directly promoted Aβ clearance by microglia. These findings provide strong evidence that TGF-β1 can reduce the accumulation of Aβ in the brain.

Red Wine and Microglial Activation

Focusing on the interaction of microglia and Aβ, Dr. Gan and colleagues showed that Aβ can trigger a molecular pathway in microglia that makes these cells attack neurons with poisonous chemicals. Working with cell cultures, they showed that this type of toxicity could be blocked with a compound called resveratrol, a natural component of red wine. Resveratrol blocks a key mediator in the toxic pathway called NF-κB. In the absence of resveratrol, Aβ activated NF-κB in microglia, turning them into powerful neuron-killing machines. However, in the presence of resveratrol or of other molecules that block NF-κB, microglia were well behaved, and Aβ was unable to harm the neurons. Thus, the study pinpoints NF-κB as a key AD drug target and singles out resveratrol as a promising therapeutic intervention.

Components of the inflammatory response in microglia-mediated Aβ toxicity. Accumulation of Aβ leads to activation of signaling pathways in microglia, such as the NF-κB pathway, which regulates the transcription of microglial factors, such as cathepsin B, that contribute to neurodegeneration. Blocked arrows indicate inhibitory effects of resveratrol.

Cathepsin B and Aβ Toxicity

In other studies, Dr. Gan has highlighted the benefits of the inflammatory responses for treating AD. She and her colleagues showed that cathepsin B (CatB), a cysteine proteinase, is an inflammatory factor that is triggered by Aβ stimulation in microglia. Active CatB has been observed in association with neuritic plaques in AD brains. Interestingly, Dr. Gan's team recently discovered that upregulation of CatB may play a protective role by destroying small as well as large clusters of Aβ.

Dr. Gan's team used genetic engineering to inactivate CatB in transgenic mice that produce high levels of human Aβ in the brain. With CatB inactivated, the mice had increased Aβ levels, more plaques, and more severe neuronal deficits. The scientists then used gene therapy to express CatB in the brain of aged transgenic mice and found that even the preexisting plaques were markedly reduced. In complementary experiments, carried out in cell-free conditions in the test tube, Dr. Gan's team was able to show that CatB effectively truncates the most dangerous Aβ variety, generating shorter Aβ peptides that result in fewer plaques and are less toxic.

CatB gene transfer reduces both diffuse and compact amyloid plaques in aged (12—15-month-old) hAPP mice. Photomicrographs of Lenti-CatB-injected (upper panels) and uninjected (lower panels) hippocampus after immunostaining with 3D6 antibody to detect Aβ deposits (A) and with thioflavin-S to detect neuritic plaques, which are associated with damaged neuronal processes (B). Arrows indicate the injection site.

Active cathepsin B has been observed in association with neuritic plaques in AD brains. Cathepsin B can degrade a variety of extracellular matrix proteins and associated signal transduction molecules, thereby triggering apoptosis (“programmed” cell death) and contributing to neuronal and synaptic loss in AD.

Interestingly, extracellular cathepsin B has been implicated in various diseases involving tissue-remodeling states, such as rheumatoid arthritis and tumor metastasis. Potent cathepsin B inhibitors were developed to target those devastating diseases. Studies by Dr. Gan and her colleagues suggest that these agents may also have therapeutic value for AD.

Conclusion

Inflammation is a complex phenomenon that can be beneficial or harmful. Insights from the research at Gladstone will point to new therapeutic strategies that harness inflammation for the benefit of patients with AD and other neurodegenerative diseases.