We present millimeter to centimeter-sized injectable neural scaffolds based on macroporous cryogels. The polymer-scaffolds are made from alginate and carboxymethyl-cellulose by a novel simple one-pot cryosynthesis. They allow for surgical sterility by means of autoclaving, and present native laminin as an attachment motive for neural adhesion and neurite
Rapid remodeling of the actin cytoskeleton in the pre- and/or post-synaptic compartments is responsible for the regulation of neuronal plasticity,which is an important process for learning and memory. Cofilin1 plays an essential role in these processes and a dysregulation of its activity was associated with the cognitive decline observed during normal aging and Alzheimer's disease (AD). To understand the mechanism(s) regulating Cofilin1 activity we evaluated changes occurring with regard to Cofilin1 and its up-stream regulators Lim kinase-1 (LIMK1) and Slingshot phosphatase-1 (SSH1) in (i) human AD brain, (ii) 1-, 4-, and 10-months old APP/PS1 mice, (iii) wildtype 3-, 8-, 12-, 18- and 26-months old mice, as well as in cellular models including (iv) mouse primary cortical neurons (PCNs, cultured for 5, 10, 15 and 20 days in vitro) and (v) mouse embryonic fibroblasts (MEF). Interestingly,we found an increased Cofilin1 phosphorylation/inactivation with age and AD pathology, both in vivo and in vitro. These changes were associated with a major inactivation of SSH1. Interestingly, inhibition of ã-secretase activity with Compound-E (10 ìM) prevented Cofilin1 phosphorylation/inactivation through an increase of SSH1 activity in PCNs. Similarly, MEF cells double knock-out for ã-secretase catalytic subunits presenilin-1 and -2(MEFDKO) showed a strong decrease of both Cofilin1 and SSH1 phosphorylation,which were rescued by the over expression of human ã-secretase. Together, these results shed new light in understanding the molecular mechanisms promoting Cofilin1 dysregulation, both during aging and AD. They further have the potential to impact the development of therapies to safely treat AD.
Polydimethylsiloxane (PDMS) and SU-8 are currently two very commonly used polymeric materials in the microfluidics field for biological applications. However, there is a pressing need to find a simple, reliable, irreversible bonding method between these two materials for their combined use in innovative integrated microsystems. In this paper, we attempt to investigate the aminosilane-mediated irreversible bonding method for PDMS and SU-8 with X-Ray Photoelectron Spectroscopy (XPS) surface analysis and bonding strength tests. Additionally, the selected bonding method was applied in fabricating a microelectrode array (MEA) device, including microfluidic features, which allows electrophysiological observations on compartmentalized neuronal cultures. As there is a growing trend towards microfluidic devices for neuroscience research, this type of integrated microdevice, which can observe functional alterations on compartmentalized neuronal culture, can potentially be used for neurodegenerative disease research and pharmaceutical development.
The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.
The adipocyte plasma membrane-associated protein APMAP is expressed in the brain where it associates with γ-secretase, a protease responsible for the generation of the amyloid-β peptides (Aβ) implicated in the pathogenesis of Alzheimer’s disease (AD). In this study, behavioral investigations revealed spatial learning and memory deficiencies in our newly generated mouse line lacking the protein APMAP. In a mouse model of AD, the constitutive deletion of APMAP worsened the spatial memory phenotype and led to increased Aβ production and deposition into senile plaques. To investigate at the molecular level the neurobiological functions of APMAP (memory and Aβ formation) and a possible link with the pathological hallmarks of AD (memory impairment and Aβ pathology), we next developed a procedure for the high-grade purification of cellular APMAP protein complexes. The biochemical characterization of these complexes revealed a series of new APMAP interactomers. Among these, the heat shock protein HSPA1A and the cation-dependent mannose-6-phosphate receptor (CD-M6PR) negatively regulated APP processing and Aβ production, while clusterin, calnexin, arginase-1, PTGFRN and the cation-independent mannose-6-phosphate receptor (CI-M6PR/IGF2R) positively regulated APP and Aβ production. Several of the newly identified APMAP interactomers contribute to the autophagy-lysosome system, further supporting an emergent agreement that this pathway can modulate APP metabolism and Aβ generation. Importantly, we have also demonstrated increased alternative splicing of APMAP and lowered levels of the Aβ controllers HSPA1A and CD-M6PR in human brains from neuropathologically verified AD cases.Electronic supplementary materialThe online version of this article (10.1186/s40478-019-0660-3) contains supplementary material, which is available to authorized users.
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