Constitutive activation of WASp leads to abnormal cytotoxic cells with increased granzyme B and degranulation response to target cells.

X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood; however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from 2 patients with XLN harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased granzyme B content and elevated degranulation and IFN-γ production when compared with healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28-coated beads, respectively. WASpL272P mouse T cells had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8+ T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to lymphocyte function associated antigen-1 engagement. When compared with WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggest that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.

The Proteome of the Dentate Terminal Zone of the Perforant Path Indicates Presynaptic Impairment in Alzheimer Disease.

Synaptic dysfunction is an early pathogenic event in Alzheimer disease (AD) that contributes to network disturbances and cognitive decline. Some synapses are more vulnerable than others, including the synapses of the perforant path, which provides the main excitatory input to the hippocampus. To elucidate the molecular mechanisms underlying the dysfunction of these synapses, we performed an explorative proteomic study of the dentate terminal zone of the perforant path. The outer two-thirds of the molecular layer of the dentate gyrus, where the perforant path synapses are located, was microdissected from five subjects with AD and five controls. The microdissected tissues were dissolved and digested by trypsin. Peptides from each sample were labeled with different isobaric tags, pooled together and pre-fractionated into 72 fractions by high-resolution isoelectric focusing. Each fraction was then analyzed by liquid chromatography-mass spectrometry. We quantified the relative expression levels of 7322 proteins, whereof 724 showed significantly altered levels in AD. Our comprehensive data analysis using enrichment and pathway analyses strongly indicated that presynaptic signaling, such as exocytosis and synaptic vesicle cycle processes, is severely disturbed in this area in AD, whereas postsynaptic proteins remained unchanged. Among the significantly altered proteins, we selected three of the most downregulated synaptic proteins; complexin-1, complexin-2 and synaptogyrin-1, for further validation, using a new cohort consisting of six AD and eight control cases. Semi-quantitative analysis of immunohistochemical staining confirmed decreased levels of complexin-1, complexin-2 and synaptogyrin-1 in the outer two-thirds of the molecular layer of the dentate gyrus in AD. Our in-depth proteomic analysis provides extensive knowledge on the potential molecular mechanism underlying synaptic dysfunction related to AD and supports that presynaptic alterations are more important than postsynaptic changes in early stages of the disease. The specific synaptic proteins identified could potentially be targeted to halt synaptic dysfunction in AD.

Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS): A Rare Treatable Neuroradiological Spectrum.

Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a rare CNS inflammatory disorder involving the pons and other parts of the brainstem. It is characterised by a combination of brainstem symptoms and classical magnetic resonance imaging (MRI) features of bilateral, symmetrical punctate, perivascular enhancement of pontine lesions. Another hallmark feature of this rare disease is the responsiveness to corticosteroid treatment. As the corticosteroid treatment is tapered, the symptoms exacerbate and worsen the clinical outcome. Clinicians and radiologists should be aware of this infrequent inflammatory disorder and should always be considered as a differential diagnosis. Herein, we report the case of a 17-year-old female with a similar clinicoradiological spectrum as CLIPPERS.

Overexpression of SNX3 Decreases Amyloid-ß Peptide Production by Reducing Internalization of Amyloid Precursor Protein.

BACKGROUND: Sorting nexins (SNXs) have diverse functions in protein sorting and membrane trafficking. Recently, single-nucleotide polymorphisms in SNX3 were found to be associated with Alzheimer disease. However, it remains unknown whether SNX3 participates in amyloid (A)ß peptide production. OBJECTIVE: To examine the role of SNX3 in Aß production and APP processing. METHODS: The effect of increased expression of SNX3 was studied in HEK293T cells. Aß peptides were measured by immunoassay. Protein-protein association was analyzed by a bimolecular fluorescence complementation (BiFC) assay. APP uptake was measured with an α-bungarotoxin-binding assay, and flow cytometry was used to measure cell surface APP levels. RESULTS: We found that overexpression of SNX3 in HEK293T cells decreases the levels of secreted Aß and soluble N-terminal APP fragments (sAPPß). The reduction correlated with a decreased association of APP with BACE1, as revealed by BiFC. This effect may, in part, be explained by a reduced internalization of APP; SNX3 overexpression reduced APP internalization as determined by an α-bungarotoxin-binding assay, and caused increased APP levels on the cell surface, as shown by flow cytometry. In addition, SNX3 overexpression increased the cellular levels of full-length APP. CONCLUSION: These results provide evidence that SNX3 regulates Aß production by influencing the internalization of APP.

Exercise and BDNF reduce Aß production by enhancing α-secretase processing of APP.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by aggregation of toxic forms of amyloid ß peptide (Aß). Treatment strategies have largely been focused on inhibiting the enzymes (ß- and γ-secretases) that liberate Aß from the amyloid precursor protein (APP). While evidence suggests that individuals who exercise regularly are at reduced risk for AD and studies of animal models demonstrate that running can ameliorate brain Aß pathology and associated cognitive deficits, the underlying mechanisms are unknown. However, considerable evidence suggests that brain-derived neurotrophic factor (BDNF) mediates beneficial effects of exercise on neuroplasticity and cellular stress resistance. Here, we tested the hypothesis that BDNF promotes non-amyloidogenic APP processing. Using a transgenic mouse model of Alzheimer's disease and cultured human neural cells, we demonstrate that exercise and BDNF reduce production of toxic Aß peptides through a mechanism involving enhanced α-secretase processing of APP. This anti-amyloidogenic APP processing involves subcellular redistribution of α-secretase and an increase in intracellular neuroprotective APP peptides capable of binding and inhibiting ß-secretase. Moreover, our results suggest that BDNF's ability to promote neurite outgrowth is primarily exerted through pathways other than APP processing. Exercise and other factors that enhance BDNF signaling may therefore have both therapeutic and prophylactic value in the battle against AD. Read the Editorial Highlight for this article on page 191.

Extracellular Vesicle-Associated Aß Mediates Trans-Neuronal Bioenergetic and Ca2+-Handling Deficits in Alzheimer's Disease Models.

Alzheimer's Disease (AD) is an age-related neurodegenerative disorder in which aggregation-prone neurotoxic amyloid ß-peptide (Aß) accumulates in the brain. Extracellular vesicles (EVs) are small 50-150 nanometer membrane vesicles that have recently been implicated in the prion-like spread of self-aggregating proteins. Here we report that EVs isolated from AD patient CSF and plasma, from the plasma of two AD mouse models, and from the medium of neural cells expressing familial AD presenilin 1 mutations, destabilize neuronal Ca2+ homeostasis, impair mitochondrial function, and sensitize neurons to excitotoxicity. EVs contain a relatively low amount of Aß but have an increased Aß42/ Aß40 ratio; the majority of Aß is located on the surface of the EVs. Impairment of lysosome function results in increased generation EVs with elevated Aß42 levels. EVs may mediate transcellular spread of pathogenic Aß species and that impair neuronal Ca2+ handling and mitochondrial function, and may thereby render neurons vulnerable to excitotoxicity.

Endogenous APP accumulates in synapses after BACE1 inhibition.

BACE1-mediated cleavage of APP is a pivotal step in the production of the Alzheimer related Aß peptide and inhibitors of BACE1 are currently in clinical development for the treatment of Alzheimer disease (AD). While processing and trafficking of APP has been extensively studied in non-neuronal cells, the fate of APP at neuronal synapses and in response to reduced BACE1 activity has not been fully elucidated. Here we examined the consequence of reduced BACE1 activity on endogenous synaptic APP by monitoring N- and C-terminal APP epitopes by immunocytochemistry. In control rodent primary hippocampal neuron cultures, labeling with antibodies directed to N-terminal APP epitopes showed a significant overlap with synaptic vesicle markers (SV2 or synaptotagmin). In contrast, labeling with antibodies directed to C-terminal epitopes of APP showed only a limited overlap with these proteins. In neurons derived from BACE1-deficient mice, and in control neurons treated with a BACE1 inhibitor, both the N-terminal and the C-terminal APP labeling overlapped significantly with synaptic vesicle markers. Moreover, BACE1 inhibition increased the proximity between the APP C-terminus and SV2 as shown by a proximity ligation assay. These results, together with biochemical observations, indicate that BACE1 can regulate the levels of full-length APP at neuronal synapses.