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Previous reports showed that brain Aβ amyloidosis can be induced in animal models by exogenous administration of pre-formed aggregates. To date, only intra-peritoneal and intra-venous administrations are described as effective means to peripherally accelerate brain Aβ amyloidosis by seeding. Here, we show that cerebral accumulation of Aβ can be accelerated after exposing mouse models of Alzheimer’s disease (AD) to Aβ seeds by different peripheral routes of administration, including intra-peritoneal and intra-muscular. Interestingly, animals receiving drops of brain homogenate laden with Aβ seeds in the eyes were efficiently induced. On the contrary, oral administration of large quantities of brain extracts from aged transgenic mice and AD patients did not have any effect in brain pathology. Importantly, pathological induction by peripheral administration of Aβ seeds generated a large proportion of aggregates in blood vessels, suggesting vascular transport. This information highlights the role of peripheral tissues and body fluids in AD-related pathological changes.

There was a typo in author Andrew Wahba’s name in the initial online publication. The original article has been corrected.

In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-β (infants) subgroups. Neuronal maturation is greater in SHH-β than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1^+/− cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context–specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1. Expression of Arrb1, which encodes β-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed REST^TG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1. These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-β than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.

Diffuse Intrinsic Pontine Glioma (DIPG) is a fatal pediatric cancer. Immunotherapy, and specifically Natural Killer (NK) cell therapy has been investigated for glioma and other brain malignancies. Indeed, our group is the first to demonstrate safety of loco-regional infusion of ex vivo expanded and activated NK cells in a Phase-I trial of patients with recurrent/refractory fourth ventricular tumors. But their effectiveness against DIPG has not been assessed. Here, we show that NK cells have cytolytic activity against DIPG cells in vitro. In vivo, DIPG tumors implanted in the forebrain were lysed by intratumoral injections of ex vivo expanded NK cells. In addition, NK cells infused through the fourth ventricle trafficked to and promoted shrinkage of DIPG tumors in their orthotopic location. In work from other investigators, epigenetic modulation of tumors by Histone Deacetylate Inhibitors (HDACIs) is being investigated as a potential therapeutic approach for this disease. However, the effect of HDACIs on immune cell activity has not been examined. We observed that pre-treatment of NK cells with the HDACI, MS-275, enhanced NK cell mediated cytolysis of DIPG cells in vitro. This was associated with significantly enhanced expression of genes controlling motility and cytolysis. The ability of MS-275 primed NK cells to traffic and promote cytolysis of tumors implanted proximal and distal to the site of NK cell introduction will be discussed. In summary, we suggest that the use of epigenetic therapy as an ex vivo primer of NK cells could increase the effectiveness of NK cell therapy against DIPG tumors.

Introduction: Sex differences in ischemic stroke are seen in clinical and experimental studies. Men have a higher risk of stroke throughout most of the life-span. Women have greater stroke mortality than men, however a recent multi-continental meta-analysis showed lower mortality in women when adjusted for age, pre-morbid function, stroke severity and other confounding factors. In experimental models, middle-aged female mice have larger strokes, likely due to the loss of estrogen. Less is known about outcomes in very aged animals. Hypothesis: Inflammatory responses are key determinants of stroke outcome and there are important sex differences in the immune response in aged mice. Methods: Male and female C57BL/6 mice (21-22 months) were subjected to 60 min middle cerebral artery occlusion (n=28-32), or sham surgery (n=16). Neutrophils and T cells were quantified in brain and blood at 24 h and 15 days post-stroke by flow cytometry. Functional outcomes were assessed at day 3, 7 and 14. Results: Male mortality was significantly higher than in females (41% vs 16%, P+ T cells in the blood compared to females at day 15 (73±5% of CD3 ⁺ T cells vs 52±3%, PConclusion: Higher mortality and hemorrhagic transformation rates were seen in aged males compared to females after ischemic stroke. This was associated with higher levels of neutrophils/Tregs in the brain and CD8 ⁺ T cells in the blood suggesting a greater stroke-induced immune response in aged males.

Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

Natural forms of prion diseases frequently originate by oral (p.o.) infection. However, quantitative information on the gastro-intestinal (GI) absorption of prions (i.e. the bioavailability and subsequent biodistribution) is mostly unknown. The main goal of this study was to evaluate the fate of prions after oral administration, using highly purified radiolabeled PrP^Sc. The results showed a bi-phasic reduction of PrP^Sc with time in the GI, except for the ileum and colon which showed sustained increases peaking at 3–6 hr, respectively. Plasma and whole blood ¹²⁵I-PrP^Sc reached maximal levels by 30 min and 3 hr, respectively, and blood levels were constantly higher than plasma. Upon crossing the GI-tract ¹²⁵I-PrP^Sc became associated to blood cells, suggesting that binding to cells decreased the biological clearance of the agent. Size-exclusion chromatography revealed that oligomeric ¹²⁵I-PrP^Sc were transported from the intestinal tract, and protein misfolding cyclic amplification showed that PrP^Sc in organs and blood retained the typical prion self-replicating ability. Pharmacokinetic analysis found the oral bioavailability of ¹²⁵I-PrP^Sc to be 33.6%. Interestingly, ¹²⁵I-PrP^Sc reached the brain in a quantity equivalent to the minimum amount needed to initiate prion disease. Our findings provide a comprehensive and quantitative study of the fate of prions upon oral infection.

Prions are composed of the misfolded prion protein (PrP^Sc) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP^Sc aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP^Sc aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP^Sc aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP^Sc aggregates and the incubation periods for the strains studied. The relative presence of PrP^Sc in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP^Sc aggregates in prion-induced neurodegeneration.