Dorit Trudler

Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria

Phenylketonuria (PKU) is characterized by phenylalanine accumulation and progressive mental retardation caused by an unknown mechanism. We demonstrate that at pathological concentrations, phenylalanine self-assembles into fibrils with amyloid-like morphology and well-ordered electron diffraction. These assemblies are specifically recognized by antibodies, show cytotoxicity that can be neutralized by the antibodies and are present in the hippocampus of model mice and in parietal cortex brain tissue from individuals with PKU. This is, to our knowledge, the first demonstration that a single amino acid can form amyloid-like deposits, suggesting a new amyloidosis-like etiology for PKU.

Toll-Like Receptors Expression and Signaling in Glia Cells in Neuro-Amyloidogenic Diseases: Towards Future Therapeutic Application

Toll-like receptors (TLRs) are known to be expressed by innate immune response cells and to play a critical role in their activation against foreign pathogens. It was recently suggested that TLRs have an important role in the crosstalk between neurons and glial cells in the central nervous system (CNS). TLR signaling was reported to be associated with a yin-yang effect in the CNS. While TLR signaling was linked to neurogenesis, it was also found to be involved in the pathogenesis of neurodegenerative diseases. This paper will focus on TLR signaling in glial cells in neurodegenerative diseases such as Alzheimers disease, prion diseases, amyotrophic lateral sclerosis, and Parkinsons disease. Understanding the pattern of TLR signaling in the glial cells may lead to the identification of new targets for therapeutic application.

γ-Secretase component presenilin is important for microglia β-amyloid clearance.

The cleavage of amyloid precursor protein by γ‐secretase is an important aspect of the pathogenesis of Alzheimers disease. γ‐Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the γ‐secretase catalytic site. Our aim was to investigate whether γ‐secretase can be important for microglial phagocytosis of Alzheimers disease β‐amyloid.

DJ‐1 deficiency triggers microglia sensitivity to dopamine toward a pro‐inflammatory phenotype that is attenuated by rasagiline

DJ‐1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ‐1 mutations that result in gene deficiency are linked to increased risk of Parkinsons disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ‐1 deficiency may increase microglial neurotoxicity. We found that down‐regulation of DJ‐1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro‐inflammatory cytokines such as IL‐1β and IL‐6. Furthermore, we discovered that DJ‐1‐deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro‐inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ‐1‐deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro‐inflammation in neurodegenerative diseases. Further studies of DJ‐1‐mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention.

Naphthoquinone-tyrptophan reduces neurotoxic Aβ*56 levels and improves cognition in Alzheimer's disease animal model.

An increasing body of evidence indicates a role for oligomers of the amyloid-β peptide (Aβ) in the neurotoxicity of this peptide and the pathology of Alzheimers disease (AD). Several neurotoxic oligomeric forms of Aβ have been noted ranging from the larger Amyloid β-Derived Diffusible Ligands (ADDLs) to smaller trimers and dimers of Aβ. More recently a dodecameric form of Aβ with a 56 kDa molecular weight, denoted Aβ*56, was shown to cause memory impairment in AD model mice. Here, we present for the first time a potential therapeutic strategy for AD that targets the early stages in the formation of neurotoxic Aβ*56 oligomers using a modified quinone-Tryptophan small molecule N-(3-chloro-1,4-dihydro-1,4-dioxo-2-naphthalenyl)-L-Tryptophan (Cl-NQTrp). Using NMR spectroscopy we show that this compound binds the aromatic recognition core of Aβ and prevents the formation of oligomers. We assessed the effect of Cl-NQTrp in vivo in transgenic flies expressing Aβ(1-42) in their nervous system. When these flies were fed with Cl-NQTrp a marked alleviation of their Aβ-engendered reduced life span and defective locomotion was observed. Finally, intraperitoneal injection of Cl-NQTrp into an aggressive AD mouse model reduced the level of the Aβ*56 species in their brain and reversed their cognitive defects. Further experiments should assess whether this is a direct effect of the drug in the brain or an indirect peripheral effect. This is the first demonstration that targeted reduction of Aβ*56 results in amelioration of AD symptoms. This second generation of tryptophan-modified naphthoquinones could therefore serve as potent disease modifying therapeutic for AD.

Astrocytes from old Alzheimer's disease mice are impaired in Aβ uptake and in neuroprotection.

In Alzheimers disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aβ42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aβ clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aβ uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.

Preconditioning to mild oxidative stress mediates astroglial neuroprotection in an IL-10-dependent manner

Oxidative stress plays an important role in the pathogenesis of various brain insults, including stroke. Astroglia are the main glial cells that play a fundamental role in maintaining the homeostasis of the CNS. They are important for protection from injury and aid the brain in functional recovery after injuries. It has been shown that the brain can be prepared to withstand an oxidative stress insult by a process known as preconditioning. We used primary astroglial cell culture to investigate whether preconditioning to mild oxidative stress and glucose deprivation (OSGD) can increase both astroglia survival and neuroprotective features. We found that preconditioning astroglia to mild OSGD increases astroglial survival of a second insult through activation of the NF-E2-related factor-2 (Nrf-2) pathway. Moreover, we found that Nrf-2 is highly expressed in adult brain astroglia and that preconditioning to OSGD in vivo, such as in a murine model of ischemic stroke, leads to a significant increase in astroglial Nrf-2 expression. Furthermore, we discovered an increase in neuroprotection, as measured by increased neuronal cell survival, following OSGD in the presence of medium from astroglia exposed to a mild OSGD condition. Interestingly, we discovered a significant increase in astroglial secretion of the anti-inflammatory cytokine IL-10 vs. the pro-inflammatory cytokine IL-1β in mild vs. severe oxidative stress, respectively. We demonstrated that preconditioning astroglia to mild oxidative stress increases neuroprotection in an IL-10-dependent manner. By using tert-butylhydroquinone (tBHQ), a known specific activator of Nrf-2, we found that Nrf-2 can enhance IL-10 expression. Further studies of Nrf-2-mediated cellular pathways in astroglia through IL-10 may provide useful insights into the development of therapeutic interventions following oxidative stress insults such as ischemic stroke.

DJ‐1 Deficiency Impairs Autophagy and Reduces Alpha‐synuclein Phagocytosis by Microglia

Parkinsons disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.