Repurposing drugs against Alzheimer's disease: can the anti-multiple sclerosis drug fingolimod (FTY720) effectively tackle inflammation processes in AD?

Therapeutic approaches providing effective medication for Alzheimer's disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models and in humans suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals or in elderly humans before onset of disease symptoms. However, a pharmacological treatment that can reverse memory deficits in AD patients was thus far not identified. Importantly, AD disease-related dysfunctions have increasingly been associated with neuro-inflammatory mechanisms and searching for anti-inflammatory medication to treat AD seems promising. Like for other diseases, repurposing of FDA-approved drugs for treatment of AD is an ideally suited strategy to reduce the time to bring such medication into clinical practice. Of note, the sphingosine-1-phosphate analogue fingolimod (FTY720) was FDA-approved in 2010 for treatment of multiple sclerosis patients. It binds to the five different isoforms of Sphingosine-1-phosphate receptors (S1PRs) that are widely distributed across human organs. Interestingly, recent studies in five different mouse models of AD suggest that FTY720 treatment, even when starting after onset of AD symptoms, can reverse synaptic deficits and memory dysfunction in these AD mouse models. Furthermore, a very recent multi-omics study identified mutations in the sphingosine/ceramide pathway as a risk factor for sporadic AD, suggesting S1PRs as promising drug target in AD patients. Therefore, progressing with FDA-approved S1PR modulators into human clinical trials might pave the way for these potential disease modifying anti-AD drugs.

Anti-Inflammatory Treatment with FTY720 Starting after Onset of Symptoms Reverses Synaptic Deficits in an AD Mouse Model.

Therapeutic approaches providing effective medication for Alzheimer's disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals long before onset of disease symptoms, while a pharmacological treatment that can reverse synaptic and memory deficits in AD mice was thus far not identified. Repurposing food and drug administration (FDA)-approved drugs for treatment of AD is a promising way to reduce the time to bring such medication into clinical practice. The sphingosine-1 phosphate analog fingolimod (FTY720) was approved recently for treatment of multiple sclerosis patients. Here, we addressed whether fingolimod rescues AD-related synaptic deficits and memory dysfunction in an amyloid precursor protein/presenilin-1 (APP/PS1) AD mouse model when medication starts after onset of symptoms (at five months). Male mice received intraperitoneal injections of fingolimod for one to two months starting at five to six months. This treatment rescued spine density as well as long-term potentiation in hippocampal cornu ammonis-1 (CA1) pyramidal neurons, that were both impaired in untreated APP/PS1 animals at six to seven months of age. Immunohistochemical analysis with markers of microgliosis (ionized calcium-binding adapter molecule 1; Iba1) and astrogliosis (glial fibrillary acid protein; GFAP) revealed that our fingolimod treatment regime strongly down regulated neuroinflammation in the hippocampus and neocortex of this AD model. These effects were accompanied by a moderate reduction of Aß accumulation in hippocampus and neocortex. Our results suggest that fingolimod, when applied after onset of disease symptoms in an APP/PS1 mouse model, rescues synaptic pathology that is believed to underlie memory deficits in AD mice, and that this beneficial effect is mediated via anti-neuroinflammatory actions of the drug on microglia and astrocytes.

Peripheral Tumor Necrosis Factor-Alpha (TNF-α) Modulates Amyloid Pathology by Regulating Blood-Derived Immune Cells and Glial Response in the Brain of AD/TNF Transgenic Mice.

Increasing evidence has suggested that systemic inflammation along with local brain inflammation can play a significant role in Alzheimer's disease (AD) pathogenesis. Identifying key molecules that regulate the crosstalk between the immune and the CNS can provide potential therapeutic targets. TNF-α is a proinflammatory cytokine implicated in the pathogenesis of systemic inflammatory and neurodegenerative diseases, such as rheumatoid arthritis (RA) and AD. Recent studies have reported that anti-TNF-α therapy or RA itself can modulate AD pathology, although the underlying mechanism is unclear. To investigate the role of peripheral TNF-α as a mediator of RA in the pathogenesis of AD, we generated double-transgenic 5XFAD/Tg197 AD/TNF mice that develop amyloid deposits and inflammatory arthritis induced by human TNF-α (huTNF-α) expression. We found that 5XFAD/Tg197 mice display decreased amyloid deposition, compromised neuronal integrity, and robust brain inflammation characterized by extensive gliosis and elevated blood-derived immune cell populations, including phagocytic macrophages and microglia. To evaluate the contribution of peripheral huTNF-α in the observed brain phenotype, we treated 5XFAD/Tg197 mice systemically with infliximab, an anti-huTNF-α antibody that does not penetrate the blood-brain barrier and prevents arthritis. Peripheral inhibition of huTNF-α increases amyloid deposition, rescues neuronal impairment, and suppresses gliosis and recruitment of blood-derived immune cells, without affecting brain huTNF-α levels. Our data report, for the first time, a distinctive role for peripheral TNF-α in the modulation of the amyloid phenotype in mice by regulating blood-derived and local brain inflammatory cell populations involved in ß-amyloid clearance.SIGNIFICANCE STATEMENT Mounting evidence supports the active involvement of systemic inflammation, in addition to local brain inflammation, in Alzheimer's disease (AD) progression. TNF-α is a pluripotent cytokine that has been independently involved in the pathogenesis of systemic inflammatory rheumatoid arthritis (RA) and AD. Here we first demonstrate that manipulation of peripheral TNF-α in the context of arthritis modulates the amyloid phenotype by regulating immune cell trafficking in the mouse brain. Our study suggests that additionally to its local actions in the AD brain, TNF-α can also indirectly modulate amyloid pathology as a regulator of peripheral inflammation. Our findings may have significant implications in the treatment of RA patients with anti-TNF-α drugs and in the potential use of TNF-targeted therapies for AD.

A delay in vesicle endocytosis by a C-terminal fragment of N-cadherin enhances Aß synaptotoxicity.

Synaptotoxic Aß oligomers are thought to play a major role in the early pathology of Alzheimer´s disease (AD). However, the molecular mechanisms involved in Aß-induced synaptic dysfunction and synapse damage remain largely unclear. Previously, Aß synaptotoxicity has been reported to be enhanced by increased levels of a C-terminal fragment of the synaptic adhesion molecule N-cadherin that is generated by proteolytic shedding of the extracellular domains [1]. To address the molecular mechanisms involved in this process, we have now studied the functional synaptic changes induced by C-terminal fragments (CTF1) of synaptic adhesion proteins. We used synaptophysin-pHluorin (SypHy) fluorescence imaging to monitor synaptic vesicle exo- and endocytosis in cultures of mouse cortical neurons. We increased the levels of C-terminal fragments of synaptic adhesion proteins by pharmacologically inhibiting γ-secretase, which further degrades CTF1 fragments. We found that this intervention caused a delay in synaptic vesicle endocytosis. A similar effect was induced by overexpression of N-cadherin CTF1, but not by overexpression of Neurexin3ß CTF1. Based on these observations, we further studied whether directly modulating synaptic vesicle endocytosis enhances Aß synaptotoxicity. We pharmacologically induced a delayed synaptic vesicle endocytosis by a low concentration of the endocytosis inhibitor dynasore. This treatment enhanced synaptoxicity of Aß oligomers as indicated by a reduced frequency of miniature postsynaptic currents. In conclusion, we propose that delayed endocytosis results in prolonged exposure of synaptic vesicle membranes to the extracellular space, thus enabling enhanced vesicle membrane binding of Aß oligomers. This might in turn promote the endocytic uptake of toxic Aß oligomers and might thus play an important role in intracellular Aß-mediated synaptotoxicity in AD.

Golgi-Cox impregnation combined with fluorescence staining of amyloid plaques reveals local spine loss in an Alzheimer mouse model.

BACKGROUND: Spine loss is a hallmark of Alzheimer´s and other neurodegenerative diseases, and testing candidate therapeutic drugs needs quantitative analysis of dendritic spine densities. Golgi-Cox impregnation of neurons is a classical method to visualize dendritic spines in diseased brains. Importantly, at early disease stages spine loss occurs locally in the vicinity of amyloid plaques, and concomitant fluorescence labeling of amyloid plaques is required to detect local spine damage. NEW METHOD: Because Golgi-Cox impregnation is done on unsectioned brains, whereas fluorescence staining is performed on sectioned material, the combination is technically challenging. We have now developed a novel combination of Golgi-Cox impregnation with methoxy-X04 fluorescence labeling of plaques that is performed on unsectioned brains. RESULTS: We used this new combination method to quantify dendritic spine densities in mouse hippocampal CA1 pyramidal neurons. Comparison of neurons from wildtype and APP/PS1 mice revealed local spine loss in the vicinity of amyloid plaques in both male and female APP/PS1 mice. COMPARISON WITH EXISTING METHOD: Golgi-Cox impregnation of neurons combined with methoxy-X04 staining of amyloid plaques is a highly reliable, easy-to-use method for permanent visualization of spines as compared to the technically more sophisticated and less stable fluorescence imaging of spines. CONCLUSION: Our novel combination method will be highly useful for testing potential therapeutic drugs in Alzheimer mouse models.