Submicromolar Aß42 Reduces Hippocampal Glutamate Receptors and Presynaptic Markers in an Aggregation-Dependent Manner

Synaptic pathology in Alzheimer's disease brains is thought to involve soluble Aß42 peptide. Here, sterile in-cubation in PBS caused small Aß42 oligomer formation as well as heterogeneous, 6E10-immunopositive ag-gregates of 80–100 kDa. The high molecular weight aggregates (H-agg) formed in a time-dependent manner over an extended 30-day period. Interestingly, an inverse relationship between dimeric and H-agg formation was more evident when incubations were performed at 37 °C as compared to 23 °C, thus providing an exper-imental strategy with which to address synaptic compromise produced by the different Aß aggregates. H-agg species formed faster and to higher levels at 37 °C compared to 23 °C, and the two aggregate preparations were evaluated in hippocampal slice cultures, a sensitive system for monitoring synaptic integrity. Applied daily at 80–600 nM for 7 days, the Aß42 preparations caused dose-dependent and aggregation-dependent declines in a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits as well as in presynaptic components. Unlike the synaptic effects, Aß42 induced only trace cellular degeneration that was CA1 speci?c. The 37 °C preparation was less effective at decreasing synaptic markers, corresponding with its reduced levels of Aß42 monomers and dimers. Aß42 dimers decayed signi?cantly faster at 37 °C than 23 °C, and more rapidly than monomers at either temperature. These ?ndings indicate that Aß42 can self-aggregate into potent synaptotoxic oligomers as well as into larger aggregates that may serve to neutralize the toxic formations. These results will add to the growing debate concerning whether high molecular weight Aß complexes that form amyloid plaques are protective through the sequestration of oligomeric species

A New Generation Fatty Acid Amide Hydrolase Inhibitor Protects Against Kainate-Induced Excitotoxicity

Endocannabinoids, including anandamide (AEA), have been implicated in neuroprotective on-demand responses. Related to such a response to injury, an excitotoxic kainic acid (KA) injection (i.p.) was found to increase AEA levels in the brain. To modulate the endocannabinoid response during events of excitotoxicity in vitro and in vivo, we utilized a new generation compound (AM5206) that selectively inhibits the AEA deactivating enzyme fatty acid amide hydrolase (FAAH). KA caused calpain-mediated spectrin breakdown, declines in synaptic markers, and disruption of neuronal integrity in cultured hippocampal slices. FAAH inhibition with AM5206 protected against the neurodegenerative cascade assessed in the slice model 24 h postinsult. In vivo, KA administration induced seizures and the same neurodegenerative events exhibited in vitro. When AM5206 was injected immediately after KA in rats, the seizure scores were markedly reduced as were levels of cytoskeletal damage and synaptic protein decline. The pre- and postsynaptic proteins were protected by the FAAH inhibitor to levels comparable to those found in healthy control brains. These data support the idea that endocannabinoids are released and converge on pro-survival pathways that prevent excitotoxic progression

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid ß peptide (Aß) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aß(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aß pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1?E9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aß immunostaining as well as Aß(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aß deposition in 20-22-month APPswe/PS1?E9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aß(1-38) occurs as Aß(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aß truncation. Associated with Aß clearance Associated with Aß clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aß(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aß pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders

Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and a-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator ZPhe-Ala-diazomethylketone(PADK). Such positive modulators of the lysosomal system have been studied in thewell-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Activecathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulationreduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, a-secretase, and b-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Ab) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Ab1–38 peptide corresponded with decreased levels of Ab1–42, supporting the lysosome’s antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomalsystem, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlledstrategy against protein accumulation disorders to enhance protein clearance, promote synaptic integrity, and slow the progression of dementia

Cleavage of the Vesicular Glutamate Transporters Under Excitotoxic Conditions

Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn534 and Lys542. The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels

Nuclear Translocation and Calpain-Dependent Reduction of Bcl-2 After Neonatal Cerebral Hypoxia–Ischemia

Apoptosis-related mechanisms are important in the pathophysiology of hypoxic–ischemic injury in the neonatal brain. Caspases are the major executioners of apoptosis, but there are a number of upstream players that influence the cell death pathways. The Bcl-2 family proteins are important modulators of mitochondrial permeability, working either to promote or prevent apoptosis. In this study we focused on the anti-apoptotic Bcl-2 protein after neonatal cerebral hypoxia–ischemia (HI) in 8-day-old rats. Bcl-2 translocated to nuclei and accumulated there over the first 24 h of reperfusion after HI, as judged by immunohistochemistry and immuno-electron microscopy. We also found that the total level of Bcl-2 decreased after HI in vivo and after ionophore challenge in cultured human neuroblastoma (IMR-32) cells in vitro. Furthermore, the Bcl-2 reduction was calpain-dependent, because it could be prevented by the calpain inhibitor CX295 both in vivo and in vitro, suggesting cross-talk between excitotoxic and apoptotic mechanisms

A Single Pathway Targets Several Health Challenges of the Elderly

New avenues to modulate the autophagy–lysosomal route of protein clearance have the potential to help treat several disease states to which the elderly are particularly vulnerable. Two recent papers identi?ed distinct ways to tap into the lysosomal degradation pathway of autophagy to reduce age-related protein accumulation events. Shoji-Kawata et al. (Nature 2013;494:201–206) describe a new autophagy-inducing peptide, Tat-Beclin 1, that enhances the clearance of polyglutamine aggregates related to Huntington’s disease and, interestingly, suppresses viral and bacterial infections. Savolainen et al. (Neurobiol Dis 2014;68:1–15) describe a prolyl oligopeptidase inhibitor that reduces a-synuclein species related to Parkinson’s disease and other a-synucleinopathies, and this inhibitor caused a concomitant increase in autophagic activation markers. Previous studies have also linked the autophagy–lysosomal pathway to the protective clearing of the A ß peptides of Alzheimer’s disease and tau species of tauopathies. Enhancing autophagy–lysosomal ef?ciency may provide a therapeutic avenue for diverse types of proteinopathies, including the most common neurodegenerative disorders of the elderly

Endo-lysosomal dysfunction: a converging mechanism in neurodegenerative diseases

Endo-lysosomal pathways are essential in maintaining protein homeostasis in the cell. Numerous genes in the endo-lysosomal pathways have been found to associate with neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). Mutations of these genes lead to dysfunction in multiple steps of the endo-lysosomal network: autophagy, endocytic trafficking and lysosomal degradation, resulting in accumulation of pathogenic proteins. Although the exact pathogenic mechanism varies for different disease-associated genes, dysfunction of the endo-lysosomal pathways represents a converging mechanism shared by these diseases. Therefore, strategies that correct or compensate for endo-lysosomal dysfunction may be promising therapeutic approaches to treat neurodegenerative diseases

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly