Epicutaneous allergen immunotherapy induces a profound and selective modulation in skin dendritic-cell subsets.

BACKGROUND: Epicutaneous immunotherapy (EPIT) protocols have recently been developed to restore tolerance in patients with food allergy. The mechanisms by which EPIT protocols promote desensitization rely on a profound immune deviation of pathogenic T- and B-cell responses. OBJECTIVE: To date, little is known about the contribution of skin dendritic cells (skDCs) to T-cell remodeling and EPIT efficacy. METHODS: We capitalized on a preclinical model of food allergy to ovalbumin (OVA) to characterize the phenotype and functions of OVA+ skDCs throughout the course of EPIT. RESULTS: Our results showed that both Langerhans cells and dermal conventional cDC1 and cDC2 subsets retained their ability to capture OVA in the skin and to migrate toward the skin-draining lymph nodes during EPIT. However, their activation/maturation status was significantly impaired, as evidenced by the gradual and selective reduction of CD86, CD40, and OVA protein expression in respective subsets. Phenotypic changes during EPIT were also characterized by a progressive diversification of single-cell gene signatures within each DC subset. Interestingly, we observed that OVA+ Langerhans cells progressively lost their capacity to prime CD4+ TEFF cells, but gained regulatory T-cell stimulatory properties. In contrast, cDC1 were inefficient in priming CD4+ TEFF cells or in reactivating TMEM cells in vitro, whereas cDC2 retained moderate stimulatory properties, and progressively biased type 2 immunity toward type 1 and type 17 responses. CONCLUSIONS: Our results therefore emphasize that the acquisition of distinct phenotypic and functional specializations by skDCs during EPIT is at the cornerstone of the desensitization process.

Synaptic strength is bidirectionally controlled by opposing activity-dependent regulation of Nedd4-1 and USP8.

The trafficking of AMPA receptors (AMPARs) to and from synapses is crucial for synaptic plasticity. Previous work has demonstrated that AMPARs undergo activity-dependent ubiquitination by the E3 ubiquitin ligase Nedd4-1, which promotes their internalization and degradation in lysosomes. Here, we define the molecular mechanisms involved in ubiquitination and deubiquitination of AMPARs. We report that Nedd4-1 is rapidly redistributed to dendritic spines in response to AMPAR activation and not in response to NMDA receptor (NMDAR) activation in cultured rat neurons. In contrast, NMDAR activation directly antagonizes Nedd4-1 function by promoting the deubiquitination of AMPARs. We show that NMDAR activation causes the rapid dephosphorylation and activation of the deubiquitinating enzyme (DUB) USP8. Surface AMPAR levels and synaptic strength are inversely regulated by Nedd4-1 and USP8. Strikingly, we show that homeostatic downscaling of synaptic strength is accompanied by an increase and decrease in Nedd4-1 and USP8 protein levels, respectively. Furthermore, we show that Nedd4-1 is required for homeostatic loss of surface AMPARs and downscaling of synaptic strength. This study provides the first mechanistic evidence for rapid and opposing activity-dependent control of a ubiquitin ligase and DUB at mammalian CNS synapses. We propose that the dynamic regulation of these opposing forces is critical in maintaining synapses and scaling them during homeostatic plasticity.

Molecular imaging insights into neurodegeneration: focus on α-synuclein radiotracers.

Neurodegenerative diseases characterized by the presence of α-synuclein-a hallmark of pathologic inclusions termed Lewy bodies-include Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy. Although motor symptoms are related to the altered presynaptic dopaminergic function in these diseases, the appearance of α-synuclein inclusions precedes the involvement of the nigrostriatal dopaminergic pathway. Hence, the most accurate and earliest definition of premotor Parkinson's disease ought to rely on imaging α-synuclein rather than dopaminergic changes. Moreover, dopaminergic imaging has been controversial in monitoring the effects of investigational disease-modifying drugs. For these clinical trials, intense interest in longitudinally imaging α-synuclein as the primary pathologic process has led to efforts toward developing a suitable radiotracer for this key protein. An overview of the present α-synuclein radiotracer development scenario is presented here.

Hippocampal neuronal cells that accumulate α-synuclein fragments are more vulnerable to Aß oligomer toxicity via mGluR5--implications for dementia with Lewy bodies.

BACKGROUND: In dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aß) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aß and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aß in the hippocampus. RESULTS: We generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aß, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aß oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aß oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aß in α-syn-expressing neuronal cells. CONCLUSIONS: Together, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aß might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB.

Differential effects of UCHL1 modulation on alpha-synuclein in PD-like models of alpha-synucleinopathy.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn) within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg) and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy) in control cells, in cells over expressing a-syn UCH-L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/aggregated a-syn species in the PD brain.

Alterations in mGluR5 expression and signaling in Lewy body disease and in transgenic models of alpha-synucleinopathy--implications for excitotoxicity.

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are neurodegenerative disorders of the aging population characterized by the abnormal accumulation of alpha-synuclein (alpha-syn). Previous studies have suggested that excitotoxicity may contribute to neurodegeneration in these disorders, however the underlying mechanisms and their relationship to alpha-syn remain unclear. For this study we proposed that accumulation of alpha-syn might result in alterations in metabotropic glutamate receptors (mGluR), particularly mGluR5 which has been linked to deficits in murine models of PD. In this context, levels of mGluR5 were analyzed in the brains of PD and DLB human cases and alpha-syn transgenic (tg) mice and compared to age-matched, unimpaired controls, we report a 40% increase in the levels of mGluR5 and beta-arrestin immunoreactivity in the frontal cortex, hippocampus and putamen in DLB cases and in the putamen in PD cases. In the hippocampus, mGluR5 was more abundant in the CA3 region and co-localized with alpha-syn aggregates. Similarly, in the hippocampus and basal ganglia of alpha-syn tg mice, levels of mGluR5 were increased and mGluR5 and alpha-syn were co-localized and co-immunoprecipitated, suggesting that alpha-syn interferes with mGluR5 trafficking. The increased levels of mGluR5 were accompanied by a concomitant increase in the activation of downstream signaling components including ERK, Elk-1 and CREB. Consistent with the increased accumulation of alpha-syn and alterations in mGluR5 in cognitive- and motor-associated brain regions, these mice displayed impaired performance in the water maze and pole test, these behavioral alterations were reversed with the mGluR5 antagonist, MPEP. Taken together the results from study suggest that mGluR5 may directly interact with alpha-syn resulting in its over activation and that this over activation may contribute to excitotoxic cell death in select neuronal regions. These results highlight the therapeutic importance of mGluR5 antagonists in alpha-synucleinopathies.

A pathologic cascade leading to synaptic dysfunction in alpha-synuclein-induced neurodegeneration.

Several neurodegenerative diseases are typified by intraneuronal alpha-synuclein deposits, synaptic dysfunction, and dementia. While even modest alpha-synuclein elevations can be pathologic, the precise cascade of events induced by excessive alpha-synuclein and eventually culminating in synaptotoxicity is unclear. To elucidate this, we developed a quantitative model system to evaluate evolving alpha-synuclein-induced pathologic events with high spatial and temporal resolution, using cultured neurons from brains of transgenic mice overexpressing fluorescent-human-alpha-synuclein. Transgenic alpha-synuclein was pathologically altered over time and overexpressing neurons showed striking neurotransmitter release deficits and enlarged synaptic vesicles; a phenotype reminiscent of previous animal models lacking critical presynaptic proteins. Indeed, several endogenous presynaptic proteins involved in exocytosis and endocytosis were undetectable in a subset of transgenic boutons ("vacant synapses") with diminished levels in the remainder, suggesting that such diminutions were triggering the overall synaptic pathology. Similar synaptic protein alterations were also retrospectively seen in human pathologic brains, highlighting potential relevance to human disease. Collectively the data suggest a previously unknown cascade of events where pathologic alpha-synuclein leads to a loss of a number of critical presynaptic proteins, thereby inducing functional synaptic deficits.

Progressive accumulation of amyloid-beta oligomers in Alzheimer's disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins.

The cognitive impairment in patients with Alzheimer's disease is closely associated with synaptic loss in the neocortex and limbic system. Although the neurotoxic effects of aggregated amyloid-beta oligomers in Alzheimer's disease have been studied extensively in experimental models, less is known about the characteristics of these aggregates across the spectrum of Alzheimer's disease. In this study, postmortem frontal cortex samples from controls and patients with Alzheimer's disease were fractionated and analyzed for levels of oligomers and synaptic proteins. We found that the levels of oligomers correlated with the severity of cognitive impairment (blessed information-memory-concentration score and mini-mental state examination) and with the loss of synaptic markers. Reduced levels of the synaptic vesicle protein, vesicle-associated membrane protein-2, and the postsynaptic protein, postsynaptic density-95, correlated with the levels of oligomers in the various fractions analyzed. The strongest associations were found with amyloid-beta dimers and pentamers. Co-immunoprecipitation and double-labeling experiments supported the possibility that amyloid-beta and postsynaptic density-95 interact at synaptic sites. Similarly, in transgenic mice expressing high levels of neuronal amyloid precursor protein, amyloid-beta co-immunoprecipitated with postsynaptic density-95. This was accompanied by a decrease in the levels of the postsynaptic proteins Shank1 and Shank3 in patients with Alzheimer's disease and in the brains of amyloid precursor protein transgenic mice. In conclusion, this study suggests that the presence of a subpopulation of amyloid-beta oligomers in the brains of patients with Alzheimer's disease might be related to alterations in selected synaptic proteins and cognitive impairment.

Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

The Us3 protein kinase of herpes simplex virus 1 blocks apoptosis and induces phosporylation of the Bcl-2 family member Bad.

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. The herpes simplex virus 1 (HSV-1) Us3 open-reading frame encodes a serine/threonine protein kinase that participates in the inhibition of apoptosis induced by virus infection and other stress agents. Previous studies have shown that Us3 counteracts the virus-induced activation of caspase-3 by acting at a premitochondrial stage. Using stable transfectants that express Us3 under the control of constitutive or inducible promoters we demonstrate that apoptosis induced by treatment with anti-Fas antibody and sorbitol is blocked when Us3 is expressed at levels comparable to those achieved during virus infection. Expression of Us3 correlated with phosphorylation of Bad, a BH3-only proapoptotic Bcl-2 family member that is also a target for growth factor-induced cellular kinases. Bad was phosphorylated by Us3 in in vitro kination assays. These results point to a strategy for viral inhibition of apoptosis based on functional inactivation of a critical component of the cellular death machinery.