Exosomes are emerging as effective therapeutic tools for various pathologies. These extracellular vesicles can bypass biological barriers, including the blood-brain barrier, and can serve as powerful drug and gene therapy transporters. However, the progress of therapy development is impeded by several challenges, including insufficient data on exosome trafficking and biodistribution and the difficulty to image deep brain structures in vivo. Herein, we established a method for noninvasive in vivo neuroimaging and tracking of exosomes, based on glucose-coated gold nanoparticle (GNP) labeling and computed tomography imaging. Labeling of exosomes with the GNPs was achieved directly, as opposed to the typical and less efficient indirect labeling mode through parent cells. On the mechanistic level, we found that the glucose-coated GNPs were uptaken into MSC-derived exosomes via an active, energy-dependent mechanism that is mediated by the glucose transporter GLUT-1 and involves endocytic proteins. Next, we determined optimal parameters of size and administration route; we demonstrated that 5 nm GNPs enabled improved exosome labeling and that intranasal, compared to intravenous, administration led to superior brain accumulation and thus enhanced in vivo neuroimaging. Furthermore, using a mouse model of focal brain ischemia, we noninvasively tracked intranasally administered GNP-labeled exosomes, which showed increased accumulation at the lesion site over 24 h, as compared to nonspecific migration and clearance from control brains over the same period. Thus, this exosome labeling technique can serve as a powerful diagnostic tool for various brain disorders and could potentially enhance exosome-based treatments for neuronal recovery.

Exosomes, nanovesicles that are secreted by different cell types, enable intercellular communication at local or distant sites. Alhough they have been found to cross the blood brain barrier, their migration and homing abilities within the brain remain unstudied. We have recently developed a method for longitudinal and quantitative in vivo neuroimaging of exosomes based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with gold nanoparticles as labeling agents.Here, we used this technique to track the migration and homing patterns of intranasally administrated exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) in different brain pathologies, including stroke, autism, Parkinson's disease, and Alzheimer's disease. We found that MSC-exo specifically targeted and accumulated in pathologically relevant murine models brains regions up to 96 h post administration, while in healthy controls they showed a diffuse migration pattern and clearance by 24 h. The neuro-inflammatory signal in pathological brains was highly correlated with MSC-exo accumulation, suggesting that the homing mechanism is inflammatory-driven. In addition, MSC-exo were selectively uptaken by neuronal cells, but not glial cells, in the pathological regions. Taken together, these findings can significantly promote the application of exosomes for therapy and targeted drug delivery in various brain pathologies.

Lung endothelial damage is a characteristic morphological feature of ischemia-reperfusion (I/R) injury, although the molecular steps involved in the loss of endothelial integrity are still poorly understood. We tested the hypothesis that the activation of vascular endothelial growth factor (VEGF) cell signaling would be responsible for the increase in lung vascular permeability seen early after the onset of I/R in rats. Furthermore, we hypothesized that the I/R-induced pulmonary edema would be significantly attenuated in rats by the activation of the stress protein response. Pretreatment with Ad Flk-1, an adenovirus encoding for the soluble VEGF receptor type II, prevented I/R-mediated increase in lung vascular permeability in rats. Furthermore, the I/R-induced lung injury was significantly decreased by prior activation of the stress protein response with geldanamycin or pyrrolidine dithiocarbamate. In vitro studies demonstrated that VEGF caused an increase in protein permeability across primary cultures of bovine macro- and microvascular lung endothelial cell monolayers that were associated with a phosphorylation of VE- and E-cadherin and the formation of actin stress fibers. Activation of the stress protein response prevented the VEGF-mediated changes in protein permeability across these cell monolayers and reduced the phosphorylation of VE-and E-cadherins, as well as the formation of actin stress fibers in these cells.

Neuroinflammation contributes to amyotrophic lateral sclerosis (ALS) progression. TLR4, a transmembrane protein that plays a central role in activation of the innate immune system, has been shown to induce microglial activation in ALS models. TLR4 is up-regulated in the spinal cords of hSOD1G93A mice. We aimed to examine the effects of specific TLR4 inhibition on disease progression and survival in the hSOD1G93A mouse model of ALS. Immunologic effect of TLR4 inhibition in vitro was measured by the effect of TAK-242 treatment on LPS-induced splenocytes proliferation. hSOD1G93A transgenic mice were treated with TAK-242, a selective TLR4 inhibitor, or vehicle. Survival, body weight, and motor behavior were monitored. To evaluate in vivo immunologic modifications associated with TAK-242 treatment, we measured serum IL-1β in the plasma, as well as IL-1β and TNF-α mRNAs in the spinal cord in wild-type mice and in TAK-242-treated and vehicle-treated early symptomatic hSOD1G93A mice. Immunohistochemical analysis of motor neurons, astrocytes, and microglial reactivity in the spinal cords were performed on symptomatic (100 days old) TAK-242-treated and vehicle-treated hSOD1G93A mice. In vitro, splenocytes taken from 100 days old hSOD1G93A mice showed significantly increased proliferation when exposed to LPS (p = 0.0002), a phenomenon that was reduced by TAK-242 (p = 0.0179). TAK-242 treatment did not attenuate body weight loss or significantly affect survival. However, TAK-242-treated hSOD1G93A mice showed temporary clinical delay in disease progression evident in the ladder test and hindlimb reflex measurements. Plasma IL-1β levels were significantly reduced in TAK-242-treated compared to vehicle-treated hSOD1G93A mice (p = 0.0023). TAK-242 treatment reduced spinal cord astrogliosis and microglial activation and significantly attenuated spinal cord motor neuron loss at early disease stage (p = 0.0259). Compared to wild-type animals, both IL-1β and TNF-α mRNAs were significantly upregulated in the spinal cords of hSOD1G93A mice. Spinal cord analysis in TAK-242-treated hSOD1G93A mice revealed significant attenuation of TNF-α mRNA (p = 0.0431), but no change in IL-1β mRNA. TLR4 inhibition delayed disease progression, attenuated spinal cord astroglial and microglial reaction, and reduced spinal motor neuron loss in the ALS hSOD1G93A mouse model. However, this effect did not result in increased survival. To our knowledge, this is the first report on TAK-242 treatment in a neurodegenerative disease model. Further studies are warranted to assess TLR4 as a therapeutic target in ALS.

We introduce a novel visual-stimuli four-arm maze (ViS4M) equipped with spectrally- and intensity-controlled LED emitters and dynamic grayscale objects that relies on innate exploratory behavior to assess color and contrast vision in mice. Its application to detect visual impairments during normal aging and over the course of Alzheimer’s disease (AD) is evaluated in wild-type (WT) and transgenic APPSWE/PS1∆E9 murine models of AD (AD+) across an array of irradiance, chromaticity, and contrast conditions. Substantial color and contrast-mode alternation deficits appear in AD+ mice at an age when hippocampal-based memory and learning is still intact. Profiling of timespan, entries and transition patterns between the different arms uncovers variable AD-associated impairments in contrast sensitivity and color discrimination, reminiscent of tritanomalous defects documented in AD patients. Transition deficits are found in aged WT mice in the absence of alternation decline. Overall, ViS4M is a versatile, controlled device to measure color and contrast-related vision in aged and diseased mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common form of dementia in the elderly. Caspases, a family of cysteine proteases, are major mediators of apoptosis and inflammation. Caspase-6 is considered to be an up-stream modulator of AD pathogenesis as active caspase-6 is abundant in neuropil threads, neuritic plaques, and neurofibrillary tangles of AD brains. In order to further elucidate the role of caspase-6 activity in the pathogenesis of AD, we produced a double transgenic mouse model, combining the 5xFAD mouse model of AD with caspase-6 knock out (C6-KO) mice. Behavioral examinations of 5xFAD/C6-KO double transgenic mice showed improved performance in spatial learning, memory, and anxiety/risk assessment behavior, as compared to 5xFAD mice. Hippocampal mRNA expression analyses showed significantly reduced levels of inflammatory mediator TNF-α, while the anti-inflammatory cytokine IL-10 was increased in 5xFAD/C6-KO mice. A significant reduction in amyloid-β plaques could be observed and immunohistochemistry analyses showed reduced levels of activated microglia and astrocytes in 5xFAD/C6-KO, compared to 5xFAD mice. Together, these results indicate a substantial role for caspase-6 in the pathology of the 5xFAD model of AD and suggest further validation of caspase-6 as a potential therapeutic target for AD. cleavage into smaller subunits that form the active caspase heterodimer [6]. Caspases have been implicated in numerous diseases as their function leads to apoptosis and cell death [7,8]. Elucidating their roles in those diseases has been the subject of many years of research around the world.Caspase-6 belongs to the pro-apoptotic executioner caspase group and is activated by a series of upstream activators, amongst which are caspase-3 and caspase-1 [9,10]. Caspase-6 was shown to mediate several non-apoptotic processes, such as facilitating axonal pruning during development [11,12]. Cleavage by caspase-6 of the mutant huntingtin gene was shown to be a prerequisite for neuronal dysfunction and degeneration, and therefore, exacerbates neurodegeneration in Huntington's disease [8,13]. Caspase-6 activity is associated with AD pathological lesions and is present at the early stages of tangle formation [14]. The pro-apoptotic protein p53 is increased in AD brains and p53 directly up-regulates the transcription of caspase-6 [15], resulting in a significant increase in caspase-6 mRNA in human AD brains. Caspase-6 cleaves the APP at a specific site, creating the toxic small peptide C31, a potent inducer of apoptosis in cultured neuroblastoma cells [4]. Thus, caspase-6 is considered to be an up-stream modulator of AD pathogenesis, and as a result, a viable therapeutic target for the treatment of AD [8].Caspase-6 knock-out (KO) neurons are protected against excitotoxicity, trophic factor deprivation, and myelin induced axonal degeneration [16]. Caspase-6 KO mice were first described by Uribe et al. [16]. Research using these mice showed a reduction in the inflammatory response and...

Background: Alzheimer's Disease (AD) is associated with impairments in key brain Mitogen- Activated Protein Kinase (MAPK) signaling cascades including the p38, c-Jun N-terminal kinase (JNK), ERK and Akt pathways. Apolipoprotein E4 (ApoE4) is the most prevalent genetic risk factor of AD. Objectives: To investigate the extent to which the MAPK signaling pathway plays a role in mediating the pathological effects of apoE4 and can be reversed by experimental manipulations. Methods: Measurements of total level and activation of MAPK signaling pathway factors, obtained utilizing immunoblot assay of hippocampal tissues from naïve and viral-treated apoE3 and apoE4 targeted replacement mice. Methods: Measurements of total level and activation of MAPK signaling pathway factors, obtained utilizing immunoblot assay of hippocampal tissues from naïve and viral-treated apoE3 and apoE4 targeted replacement mice. Results: ApoE4 mice showed robust activation of the stress related p38 and JNK pathways and a corresponding decrease in Akt activity, which is coupled to activation of GSK3β and tau hyperphosphorylation. There was no effect on the ERK pathway. We have previously shown that the apoE4- related pathology, namely; accumulation of Aβ, hyper-phosphorylated tau, synaptic impairments and decreased VEGF levels can be reversed by up-regulation of VEGF level utilizing a VEGF-expressing adeno-associated virus. Utilizing this approach, we assessed the extent to which the AD-hallmark and synaptic pathologies of apoE4 are related to the corresponding MAPK signaling effects. This revealed that the reversal of the apoE4-driven pathology via VEGF treatment was associated with a reversal of the p38 and Akt related effects. Conclusion: Taken together, these results suggest that the p38 and Akt pathways play a role in mediating the AD-related pathological effects of apoE4 in the hippocampus.

Research into stroke is driven by frustration over the limited available therapeutics. Targeting a single aspect of this multifactorial disease contributes to the therapeutic boundaries. To overcome this, we devised a novel multifactorial-cocktail treatment, using lentiviruses encoding excitatory amino acid transporter 2 (EAAT2(, glutamate dehydrogenase 2 (GDH2), and nuclear factor E2-related factor 2 (Nrf2) genes, that acts synergistically to address the effected excito-oxidative axis. Here, we used the vasoconstrictor endothelin-1 (ET-1) to induce focal ischemic injury in mice by direct injection into the striatum. Mice treated with the mixture of these three genes show significant improvement in body balance, motor coordination, and decreased motor asymmetry compared to each gene separately. These results demonstrate that overexpression of the combined EAAT2, GDH2, and NRF2 genes can provide neuroprotection after ischemic injury.