R e s e a R c h a R t i c l e1 8 1 6jci.org Volume 126Number 5 May 2016 26, 54, 55). We assayed the level of GTP-Rab5 in brains of 12-monthold Ts65Dn and 2N mice following a published protocol (56). As previously reported (28), the level of full-length APP in Ts65Dn was approximately 1.5-fold than in 2N samples ( We next tested whether the increase in App gene dose in Ts65Dn BFCNs was responsible for enlargement of Rab5 + endosomes (Figure 1). By immunoblotting, the APP siRNA caused an approximately 30% reduction in the level of full-length APP, as compared with control siRNA ( Figure 2E). Rab5 + puncta in BFCNs treated with either the APP siRNA or control siRNA were analyzed (Figure 2, F and G) as in Figure 1. Large, sometimes lobulated Rab5 + puncta were seen in cultures treated with the control siRNA, whereas these structures were typically smaller and rounded in cultures treated with the APP siRNA ( Figure 2G). Treatment with the APP siRNA significantly reduced the size of Rab5 + puncta in Ts65Dn neurons to a value equivalent to that in 2N neurons ( Figure 2F). Thus, increased App gene dose is necessary for increased Rab5 activation and for early endosome enlargement in Ts65Dn neurons.Full-length APP and β-CTF caused enlargement of early endosomes in PC12 cells. To determine how increased APP expression caused an increase in Rab5 activation, we asked which APP product(s) were responsible (Supplemental Figure 1A). We transfected PC12M cells with full-length APP-GFP, C99-GFP (β-CTF), C83-GFP (α-CTF), or AICD-GFP and examined endosomes by live cell imaging (Supplemental Figure 1B). Bright foci of GFP + intracellular structures were present in PC12M cells that overexpressed APP-GFP or C99-GFP. In contrast, cells expressing C83-GFP or AICD-GFP showed diffuse, hazy signals for GFP, with occasional foci in C83-GFP cells. In APP-GFP and C99-GFP cells, the GFP + intracellular structures were, on average, approximately 2 μm 2 (Supplemental Figure 1E). GFP signals in C83-GFP or AICD-GFP cells contained speckled small puncta within the haze, as well as a small number of larger bright puncta, as seen in cells expressing C99-GFP and APP-GFP (Supplemental Figure 1B). However, the average puncta size in C83-GFP and AICD-GFP cells was approximately 1.2 and 1.3 μm 2 , respectively. Thus, overexpressing APP and β-CTF, but not α-CTF or AICD, routinely induced formation of enlarged, bright intracellular structures. We also tested two APP mutants: APP M596V and APP SWE . APP M596V , which abolishes β-secretase cleavage, prevents production of β-CTF (57); APP SWE enhances β-secretase cleavage to increase the level of β-CTF (57). Both induced the formation of enlarged intracellular structures (Supplemental Figure 1C).We examined colocalization of APP or C99 with Rab5 in cotransfection experiments; APP-mCherry with GFP-Rab5 WT ( Figure 3B); C99-GFP with mCherry-Rab5 WT ( Figure 3C); and Rab5 + endosomes (26, 28, 37) was correlated with reduced endosomal trafficking and signaling of nerve growth factor (NGF), leading to degeneration...

BackgroundThe aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.Methodology/Principal FindingsWe analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.ConclusionsTherefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease.

Traumatic brain injury (TBI) is one of the leading causes of death of young people in the developed world. In the United States alone, 1.7 million traumatic events occur annually accounting for 50,000 deaths. The etiology of TBI includes traffic accidents, falls, gunshot wounds, sports, and combat-related events. TBI severity ranges from mild to severe. TBI can induce subtle changes in molecular signaling, alterations in cellular structure and function, and/or primary tissue injury, such as contusion, hemorrhage, and diffuse axonal injury. TBI results in blood-brain barrier (BBB) damage and leakage, which allows for increased extravasation of immune cells (i.e., increased neuroinflammation). BBB dysfunction and impaired homeostasis contribute to secondary injury that occurs from hours to days to months after the initial trauma. This delayed nature of the secondary injury suggests a potential therapeutic window. The focus of this article is on the (1) pathophysiology of TBI and (2) potential therapies that include biologics (stem cells, gene therapy, peptides), pharmacological (anti-inflammatory, antiepileptic, progrowth), and noninvasive (exercise, transcranial magnetic stimulation). In final, the review briefly discusses membrane/lipid rafts (MLR) and the MLR-associated protein caveolin (Cav). Interventions that increase Cav-1, MLR formation, and MLR recruitment of growth-promoting signaling components may augment the efficacy of pharmacologic agents or already existing endogenous neurotransmitters and neurotrophins that converge upon progrowth signaling cascades resulting in improved neuronal function after injury.

A better understanding of the cellular physiological role that plasma membrane lipids, fatty acids and sterols play in various cellular systems may yield more insight into how cellular and whole organ function is altered during the ageing process. Membrane lipid rafts (MLRs) within the plasma membrane of most cells serve as key organizers of intracellular signalling and tethering points of cytoskeletal components. MLRs are plasmalemmal microdomains enriched in sphingolipids, cholesterol and scaffolding proteins; they serve as a platform for signal transduction, cytoskeletal organization and vesicular trafficking. Within MLRs are the scaffolding and cholesterol binding proteins named caveolin (Cav). Cavs not only organize a multitude of receptors including neurotransmitter receptors (NMDA and AMPA receptors), signalling proteins that regulate the production of cAMP (G protein‐coupled receptors, adenylyl cyclases, phosphodiesterases (PDEs)), and receptor tyrosine kinases involved in growth (Trk), but also interact with components that modulate actin and tubulin cytoskeletal dynamics (e.g. RhoGTPases and actin binding proteins). MLRs are essential for the regulation of the physiology of organs such as the brain, and age‐related loss of cholesterol from the plasma membrane leads to loss of MLRs, decreased presynaptic vesicle fusion, and changes in neurotransmitter release, all of which contribute to different forms of neurodegeneration. Thus, MLRs provide an active membrane domain that tethers and reorganizes the cytoskeletal machinery necessary for membrane and cellular repair, and genetic interventions that restore MLRs to normal cellular levels may be exploited as potential therapeutic means to reverse the ageing and neurodegenerative processes.