Sodium thiosulfate attenuates glial-mediated neuroinflammation in degenerative neurological diseases.

BACKGROUND: Sodium thiosulfate (STS) is an industrial chemical which has also been approved for the treatment of certain rare medical conditions. These include cyanide poisoning and calciphylaxis in hemodialysis patients with end-stage kidney disease. Here, we investigated the anti-inflammatory activity of STS in our glial-mediated neuroinflammatory model. METHODS: Firstly, we measured glutathione (GSH) and hydrogen sulfide (H2S, SH(-)) levels in glial cells after treatment with sodium hydrosulfide (NaSH) or STS. We also measured released levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) from them. We used two cell viability assays, MTT and lactate dehydrogenase (LDH) release assays, to investigate glial-mediated neurotoxicity and anti-inflammatory effects of NaSH or STS. We also employed Western blot to examine activation of intracellular inflammatory pathways. RESULTS: We found that STS increases H2S and GSH expression in human microglia and astrocytes. When human microglia and astrocytes are activated by lipopolysaccharide (LPS)/interferon-γ (IFNγ) or IFNγ, they release materials that are toxic to differentiated SH-SY5Y cells. When the glial cells were treated with NaSH or STS, there was a significant enhancement of neuroprotection. The effect was concentration-dependent and incubation time-dependent. Such treatment reduced the release of TNFα and IL-6 and also attenuated activation of P38 MAPK and NFκB proteins. The compounds tested were not harmful when applied directly to all the cell types. CONCLUSIONS: Although NaSH was somewhat more powerful than STS in these in vitro assays, STS has already been approved as an orally available treatment. STS may therefore be a candidate for treating neurodegenerative disorders that have a prominent neuroinflammatory component.

The amyloid cascade-inflammatory hypothesis of Alzheimer disease: implications for therapy.

The amyloid cascade hypothesis is widely accepted as the centerpiece of Alzheimer disease (AD) pathogenesis. It proposes that abnormal production of beta amyloid protein (Abeta) is the cause of AD and that the neurotoxicity is due to Abeta itself or its oligomeric forms. We suggest that this, in itself, cannot be the cause of AD because demonstrating such toxicity requires micromolar concentrations of these Abeta forms, while their levels in brain are a million times lower in the picomolar range. AD probably results from the inflammatory response induced by extracellular Abeta deposits, which later become enhanced by aggregates of tau. The inflammatory response, which is driven by activated microglia, increases over time as the disease progresses. Disease-modifying therapeutic attempts to date have failed and may continue to do so as long as the central role of inflammation is not taken into account. Multiple epidemiological and animal model studies show that NSAIDs, the most widely used antiinflammatory agents, have a substantial sparing effect on AD. These studies provide a proof of concept regarding the anti-inflammatory approach to disease modification. Biomarker studies have indicated that early intervention may be necessary. They have established that disease onset occurs more than a decade before it becomes clinically evident. By combining biomarker and pathological data, it is possible to define six phases of disease development, each separated by about 5 years. Phase one can be identified by decreases in Abeta in the CSF, phase 2 by increases of tau in the CSF plus clear evidence of Abeta brain deposits by PET scanning, phase 3 by slight decreases in brain metabolic rate by PET-FDG scanning, phase 4 by slight decreases in brain volume by MRI scanning plus minimal cognitive impairment, phase 5 by increased scanning abnormalities plus clinical diagnosis of AD, and phase 6 by advanced AD requiring institutional care. Utilization of antiinflammatory agents early in the disease process remains an overlooked therapeutic opportunity. Such agents, while not preventative, have the advantage of being able to inhibit the consequences of both Abeta and tau aggregation. Since there is more than a decade between disease onset and cognitive decline, a window of opportunity exists to introduce truly effective disease-modifying regimens. Taking advantage of this opportunity is the challenge for the future.

Mechanisms of GABA release from human astrocytes.

We have previously demonstrated that human astrocytes are GABAergic cells. Throughout the adult human brain, they express the GABA synthesizing enzyme GAD 67, the GABA metabolizing enzyme GABA-T, and the GABA(A) and GABA(B) receptors. GABA modulates the actions of microglia, indicating an important role for astrocytes beyond that of influencing neurotransmitter function. Here we report on the mechanisms by which astrocytes release GABA. Astrocytes were found to express the mRNA and protein for multiple GABA transporters, and multiple receptors for glutamate, GABA, and glycine. In culture, untreated human astrocytes maintained an intracellular GABA level of 2.32 mM. They exported GABA into the culture medium so that an intracellular-extracellular gradient of 3.64 fold was reached. Inhibitors of the GABA transporters GAT1, GAT2, and GAT3, significantly reduced this export in a Ca(2+)-independent fashion. Intracellular GABA levels were enhanced by treatment with the GABA-T inhibitors gabaculine or vigabatrin. Treatment with glutamate increased GABA release in a concentration-dependent fashion. This was partially inhibited by blockers of N-methyl-D-aspartate and kainate receptors. Conversely, glycine and D-serine, co-agonists of NMDA receptors, enhanced the GABA release. GABA release was accompanied by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and was reduced by adding the Ca(2+) chelator, BAPTA-AM to the medium. These data indicate that astrocytes continuously synthesize GABA and that there are multiple mechanisms which can mediate its release. Each of these may play a role in the physiological functioning of astrocytes.

Saliva Diagnosis as a Disease Predictor.

BACKGROUND: Saliva, the most readily available body fluid, is the product of genes which are in constant activity throughout life. Measurement of saliva can predict the onset of some diseases years before their accumulation in vulnerable tissues causes clinical signs to appear. The purpose of this study was is to demonstrate current applications of saliva analysis and to predict and prevent disease progression. METHODS: We measured levels of Abeta42, C-reactive proteins (CRPs), and tumornecrosis factors (TNFs) in saliva from both healthy and fatal diseased cases such as cancer, Alzheimer's disease (AD), and coronary heart disease by ELISA-mediated techniques. We also immunostained human tissue sections with antibodies specific to these proteins to demonstrate the data are comparable. RESULTS: We found all the proteins expressed constantly in saliva from healthy controls but increased in diseased cases. This was accompanied by data from immunohistochemistry. It was also found that these proteins wereexpressed in high amounts in some healthy controls, which reflects high risk for the onset of diseases such as AD and heart diseases. CONCLUSIONS: It is concluded that measuring changes in essential gene products in saliva can predict onset of fatal diseases and open the door to effective protection measures, thus preventing premature death.

Lrrk2 phosphorylates alpha synuclein at serine 129: Parkinson disease implications.

Mutations in the alpha synuclein gene (SNCA) are the most potent cause of autosomal dominant Parkinson disease (PD) while mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause. We hypothesized that a direct interaction may exist between their protein products. Here we show that full-length Lrrk2 or fragments containing its kinase domain have a significant capacity to phosphorylate recombinant alpha synuclein (Asyn) at serine 129. Such phosphorylated Asyn is the major component of pathological deposits in PD. We further show that the G2019S mutation in Lrrk2, which is the most common genetic determinant of PD, has a significantly greater capacity than wild-type Lrrk2 to phosphorylate Asyn. This suggests that the G2019S mutant protein may cause PD by generating pathological levels of phosphorylated Asyn. Controlling Lrrk2 Asyn phosphokinase activity may be an approach to disease modifying therapy for PD and other synucleinopathies.

Enduring involvement of tau, beta-amyloid, alpha-synuclein, ubiquitin and TDP-43 pathology in the amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam (ALS/PDC).

Guam ALS/PDC is a severe tangle forming disorder endemic to Guam with features overlapping such neurodegenerative disorders as Alzheimer disease (AD), Parkinson disease (PD), progressive supranuclear palsy (PSP), ALS, corticobasal degeneration (CBD) and pallido-ponto-nigral degeneration (PPND). Since the prevalence is declining, we examined brain tissue from 35 clinically diagnosed Chamorro patients with ALS/PDC and two Chamorro controls autopsied between 1946 and 2006, to determine if distinct variations in the pathology could be identified up to this time. Although the age at autopsy increased by 4.5-5 years per decade, we identified no qualitative differences in pathological deposits with antibodies against tau, ubiquitin, A beta, alpha-synuclein and TDP-43, indicating that these more recently identified proteins have been involved in the neuropathogenesis over the past 6 decades. Tau and TDP-43 positive neuronal, oligodendroglial and astrocytic inclusions involving multiple nerve fiber tracts occurred in both the ALS and PDC types, reinforcing the concept that these forms are part of the same disorder. The results obtained may help to define the commonality of the Guam disease with other tangle forming disorders and may help in monitoring the epidemiological changes that are taking place.

Glial reactions in Parkinson's disease.

Dopaminergic neurons of the substantia nigra are particularly vulnerable to oxidative and inflammatory attack. Such processes may play a crucial role in the etiology of Parkinson disease (PD). Since glia are the main generators of these processes, the possibility that PD may be caused by glial dysfunction needs to be considered. This review concentrates on glial reactions in PD. Reactive astrocytes and reactive microglia are abundant in the substantia nigra (SN) of PD cases indicating a robust inflammatory state. Glia normally serve neuroprotective roles but, given adverse stimulation, they may contribute to damaging chronic inflammation. Microglia, the phagocytes of brain, may be the main contributors since they can produce large numbers of superoxide anions and other neurotoxins. Their toxicity towards dopaminergic neurons has been demonstrated in tissue culture and various animal models of PD. The MPTP and alpha-synuclein models are of particular interest. Years after exposure to MPTP, inflammation has been observed in the SN. This has established that an acute insult to the SN can result in a sustained local inflammation. The alpha-synuclein model indicates that an endogenous protein can induce inflammation, and, when overexpressed, can lead to autosomal dominant PD. Less is known about the role of astrocytes than microglia, but they are known to secrete both inflammatory and anti-inflammatory molecules and may play a role in modulating microglial activity. Oligodendrocytes do not seem to play a role in promoting inflammation although, like neurons, they may be damaged by inflammatory processes. Further research concerning glial reactions in PD may lead to disease-modifying therapeutic approaches.

Alzheimer's Disease Can Be Spared by Nonsteroidal Anti-Inflammatory Drugs.

Alzheimer's disease (AD) is characterized by deposits of amyloid-ß protein (Aß) in brain which become foci of inflammation. Neurons are destroyed by this inflammatory process, leading to the cognitive deficits which define AD clinical onset. Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) can ameliorate this destructive process if they are started well before clinical signs develop. Biomarker studies indicate that the disease process starts at least a decade before cognitive deficits appear. This pre-clinical onset explains the NSAID effect. It also opens a window of opportunity for preventive treatment that can be met with a simple diagnostic test. Salivary levels of Aß42 may fulfill that need. They can be measured by a simple ELISA test we have developed using commercially available reagents. By this ELISA test, normal controls, who are not at risk for AD, have levels of Aß42 close to 20 pg/ml. AD cases, as well as high level controls, secrete levels in the range of 40-85 pg/ml. Widespread application of this test to detect high level controls, followed by NSAID consumption, could substantially reduce the prevalence of AD.

The role of anti-inflammatory agents in Parkinson's disease.

There is ample and increasing evidence, from studies of human pathology, animal models and tissue culture, that chronic inflammation occurs in the basal ganglia in patients with Parkinson's disease. In such inflammatory states, activated glia can produce large quantities of free radicals and other neurotoxic materials. Dopaminergic neurons appear to be particularly vulnerable to these neurotoxins. The anti-inflammatory drugs that are presently in wide use act on peripheral players in the inflammatory process. Many experiments are under way to find agents that inhibit more potent contributors, such as the activated microglia or terminal complement proteins. Whether such drugs will slow the process of Parkinson's disease or reduce the high risk of dementia in such patients remains to be determined in future work.

A review of human diseases caused or exacerbated by aberrant complement activation.

Complement is the backbone of our innate immune system. It is of ancient evolutionary origin, being traced back to horseshoe crabs 350 million years ago. It consists today of more than 25 proteins which must work together like clockwork to distinguish friend from foe. Self-attack by the complement system can occur whenever it fails to do so. This failure has been reported to occur in an estimated 22 human diseases. A significant number of these are chronic degenerative neurological disorders. In some, there is overwhelming evidence that complement self-attack causes the disease. In many others, it is considered only to contribute to the overall pathology. Finding effective therapeutic agents should be a high priority for medical research. To date, the monoclonal antibody eculizumab is the only approved agent. Molecules under development include other monoclonal antibodies directed at C5, C3, and properdin, various aptamers to C3, and small molecules that are orally available.

A Method for Diagnosing Alzheimer's Disease Based on Salivary Amyloid-ß Protein 42 Levels.

We have developed a non-invasive method of diagnosing Alzheimer's disease (AD), which can also predict the risk of its future onset. It is based on measuring salivary levels of amyloid-ß protein terminating at position 42 (Aß42). Brain deposits of this peptide are characteristic of AD. Biomarker studies indicate that such brain deposits commence a decade or more prior to clinical onset of the disease. We report here that Aß42 is produced in all peripheral organs tested, thus establishing the generality of its production. We used this information to develop simple and sensitive tests to determine salivary Aß42 levels. The levels were first stabilized by adding thioflavin S as an anti-aggregation agent and sodium azide as an anti-bacterial agent. We then quantitated the Aß42 in a series of samples with ELISA type tests. Control cases showed almost identical levels of salivary Aß42 regardless of sex or age. All AD cases secreted levels of Aß42 more than double those of controls. Individuals at elevated risk of developing AD secreted levels comparable to the AD cases. The results establish that salivary Aß42 levels can be used to diagnose AD as well as to predict the risk of its future onset.

LRRK2 expression in normal and pathologic human brain and in human cell lines.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal-dominant late-onset Parkinson disease. We report that by reverse transcriptase-polymerase chain reaction, the mRNA of LRRK2 is expressed in soluble extracts of human brain, liver, and heart and in cultured human astrocytes, microglia, and oligodendroglia as well as in human neuroblastoma cell lines. We find by Western blotting using a polyclonal antibody of the leucine-rich repeat kinase 2 protein (Lrrk2) specific for C-terminal residues 2,511-2,527 that an apparent full-length protein and several of its fractions are expressed in soluble extracts of normal human brain. By immunocytochemistry, the antibody recognizes neurons, and more weakly astrocytes and microglia, in normal brain tissue. It intensely labels Lewy bodies in Parkinson disease and related neurodegenerative disorders. It also labels a subset of neurofibrillary tangles in Alzheimer disease and the Parkinsonism dementia complex of Guam (PDCG). It labels thorn-shaped astrocytes and oligodendroglial coiled bodies in PDCG; oligodendroglial inclusions in multiple system atrophy; Pick bodies in Pick disease; nuclear and cytoplasmic inclusions in Huntington disease; and intraneuronal and glial inclusions in amyotrophic lateral sclerosis. In summary, LRRK2 is constitutively expressed in neurons and also in glial cells of human brain. It strongly associates with pathological inclusions in several neurodegenerative disorders.

Inflammation, anti-inflammatory agents and Alzheimer disease: the last 12 years.

Two basic discoveries have spurred research into inflammation as a driving force in the pathology of Alzheimer disease (AD). The first was the identification of activated microglia in association with the lesions. The second was the finding that rheumatoid arthritics were relatively spared from the disease. These findings spurred the first pilot trial of a classical NSAID in the treatment of AD. This trial showed promise for indomethacin as a useful therapeutic agent but appropriate follow up trials have not been done. However, more than 20 epidemiological studies have since been conducted showing a sparing effect for antiinflammatories in AD, including four which specifically addressed the use of classical NSAIDs. Other key findings linking inflammation to AD pathology are the identification of activated complement fragments, including the membrane attack complex, as well as inflammatory cytokines in association with the lesions. In vitro, activated microglia release factors which are toxic to neurons, and these can be partially blocked by NSAIDs. Future directions should include a search for other inflammatory mediators in AD and exploitation of current knowledge to improve available treatments.

Inflammation, Antiinflammatory Agents, and Alzheimer's Disease: The Last 22 Years.

Two basic discoveries spurred research into inflammation as a driving force in the pathogenesis of Alzheimer's disease (AD). The first was the identification of activated microglia in association with the lesions. The second was the discovery that rheumatoid arthritics, who regularly consume anti-inflammatory agents, were relatively spared from the disease. These findings led to an exploration of the inflammatory pathways that were involved in AD pathogenesis. A pivotal advance was the discovery that amyloid-ß protein (Aß) activated the complement system. This focused attention on anti-inflammatories as blockers of complement activation. More than 15 epidemiological studies have since showed a sparing effect of non-steroidal anti-inflammatory drugs (NSAIDs) in AD. A consistent finding has been that the longer the NSAIDs were used prior to clinical diagnosis, the greater the sparing effect. The reason has since emerged from studies of biomarkers such as amyloid-ß (Aß) levels in the cerebrospinal fluid and Aß deposits in brain. They have established that the onset of AD commences at least a decade before cognitive decline permits clinical diagnosis. Such biomarker studies have revealed that a huge window of opportunity exists when application of NSAIDs, other anti-inflammatory agents, or complement activation blockers, could arrest further progress of AD, thus eliminating its manifestation. It can be anticipated that this principle will apply to many other chronic neurodegenerative diseases. Neuroinflammation, discovered in AD more than 30 years ago, has now become a major field of brain research today. Inhibiting it may be the key to successful treatment of many chronic neurological disorders.

Quercetin, not caffeine, is a major neuroprotective component in coffee.

Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson's disease and Alzheimer's disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-γ or interferon-γ released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-α and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFκB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson's disease and Alzheimer's disease.

Lack of support for bexarotene as a treatment for Alzheimer's disease.

Bexarotene has been reported to reduce brain amyloid-ß (Aß) levels and to improve cognitive function in transgenic mouse models of Alzheimer's disease (AD). Four groups failed to fully replicate the primary results but the original authors claimed overall support for the general conclusions. Because of its potential clinical importance, the current work studied the effects of bexarotene using two animal species and highly relevant paradigms. Rats were tested for the ability of bexarotene to prevent changes induced by an Aß challenge in the form intracerebroventricular (i.c.v) administration of 7PA2 conditioned medium (7PA2 CM) which contains high levels of Aß species. Bexarotene had no effect on the long-term potentiation of evoked extracellular field excitatory postsynaptic potentials induced by i.c.v. 7PA2 CM. It also had no effect following subcutaneous administration of 2, 5, 10 and 15 mg/kg on behavioral/cognitive impairment using an alternating-lever cyclic-ratio schedule of operant responding in the rat. The effects of bexarotene were further tested using the APPSwFILon, PSEN1*M146L*L286V transgenic mouse model of AD, starting at the time Aß deposits first begin to develop. Mice were sacrificed after 48 days of exposure to 100 mg bexarotene per day. No significant difference between test and control mice was found using a water-maze test, and no significant difference in the number of Aß deposits in cerebral cortex, using two different antibodies, was apparent. These results question the potential efficacy of bexarotene for AD treatment, even if instigated in the preclinical period prior to the onset of cognitive deficits reported for human AD. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

Inflammation, the complement system and the diseases of aging.

Inflammation is characteristic of neurodegenerative diseases of aging. Neuropathological evidence of activated microglia and activated astrocytes in lesioned areas, combined with epidemiological evidence of sparing of Alzheimer's disease (AD), Parkinson's disease (PD) and age-related macular degeneration (AMD) in long-term users of anti-inflammatory agents, indicates that inflammation is autodestructive of neurons. Locally produced autodestructive molecules include the membrane attack complex (MAC) of complement and oxygen-free radicals. Stimulation is provided by a variety of inflammatory cytokines. Agents which reduce the intensity of inflammation should have broad spectrum application in degenerative diseases of aging.

Abeta immunotherapy and other means to remove amyloid.

The amyloid cascade hypothesis postulates that accumulation of beta-amyloid (Abeta) plays a key role in the development of Alzheimer's disease (AD). Accordingly, much effort has gone into reducing the amyloid burden, especially in transgenic mice expressing mutations in human amyloid precursor protein. Such mice develop amyloid plaques but not neurofibrillary tangles. Immunization with Abeta and other inflammatory stimuli, inhibitors of Abeta formation, cholesterol lowering agents, beta-sheet breaker peptides, antioxidants and various miscellaneous agents have been found to reduce the more soluble Abeta in such transgenic mice. Whether they would affect the more consolidated, cross-linked Abeta of AD and, if they did, whether that would really prove an effective treatment for the disease remains for future research to determine.

The possible role of complement activation in Alzheimer disease.

Molecular pathological studies of Alzheimer disease (AD) brain have revealed the presence of a spectrum of inflammatory mediators. Epidemiological studies have indicated that the use of anti-inflammatory agents, especially non-steroidal anti-inflammatory drugs (NSAIDs), results in a substantially reduced risk of contracting the disease. It is possible that well targeted anti-inflammatory agents will also be useful in treating established AD. Inhibitors of cyclooxygenase-2 have been unsuccessful in this regard, and traditional NSAIDs have produced mixed results. The complement system, which is strongly activated in AD brain, is an attractive target for therapeutic intervention, particularly through inhibition of the autodestructive action of the membrane attack complex. The complement system works in conjunction with activated microglia, which express high levels of complement receptors. Overactive microglia secrete many toxic materials. Inhibition of microglial activation is another potential therapeutic target.

Pharmacologic approaches to the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease for which no cure or effective treatment presently exists. Many different types of drugs have been tested; most are based on various hypotheses of mechanisms for neuronal death, including oxidative damage, loss of trophic factor support, glutamate-mediated excitotoxicity, and chronic inflammation. The discovery that a small percentage of ALS cases are familial and involve mutation in a superoxide dismutase gene (SOD1) led to the development of transgenic mouse models presently widely used for testing possible drugs. Mutations in the vascular endothelial growth factor gene (VEGF) also appear to be involved. Riluzole, an inhibitor of glutamate release and the only agent presently approved for clinical use, only extends survival by a few months. A number of trophic factors, anti-inflammatory agents, and inhibitors of oxidative stress have been reported to prolong survival in mouse models and some are now in clinical trials. Gene transfer of VEGF or glial cell-line derived neurotrophic factor, anti-inflammatory COX-2 inhibitors, and minocycline have had particularly promising results in mice. No breakthrough has yet occurred and present thinking is that combinations of drugs may be required to slow the multifactorial neurodegeneration process effectively.