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Advances in the understanding of the endogenous cannabinoid system have led to several therapeutic indications for new classes of compounds that enhance cannabinergic responses. Endocannabinoid levels are elevated during pathogenic conditions, and inhibitors of endocannabinoid inactivation promote such on-demand responses. The endocannabinoids anandamide and 2-arachidonoyl glycerol have been implicated in protective signaling against excitotoxic episodes, including seizures. To better understand modulatory pathways that can exploit such responses, we used the new generation compound AM6701 that blocks both the anandamide-deactivating enzyme fatty acid amide hydrolase (FAAH) and the 2-arachidonoyl glycerol-deactivating enzyme monoacylglycerol lipase (MAGL) with equal potency. Also studied was the structural isomer AM6702 which is 44-fold more potent for inhibiting FAAH versus MAGL. When applied before and during kainic acid (KA) exposure to cultured hippocampal slices, AM6701 protected against the resulting excitotoxic events of calpain-mediated cytoskeletal damage, loss of presynaptic and postsynaptic proteins, and pyknotic changes in neurons. The equipotent inhibitor was more effective than its close relative AM6702 at protecting against the neurodegenerative cascade assessed in the slice model. In vivo, AM6701 was also the more effective compound for reducing the severity of KA-induced seizures and protecting against behavioral deficits linked to seizure damage. Corresponding with the behavioral improvements, cytoskeletal and synaptic protection was elicited by AM6701, as found in the KA-treated hippocampal slice model. It is proposed that the influence of AM6701 on FAAH and MAGL exerts a synergistic action on the endocannabinoid system, thereby promoting the protective nature of cannabinergic signaling to offset excitotoxic brain injury. [ABSTRACT FROM AUTHOR]

Spontaneous pneumothorax is a commonly encountered problem in the Emergency Department. Patients are often treated without further investigation for an underlying etiology. We present a patient who was unable to completely resolve a pneumothorax and was found to have lymphangioleiomyomatosis (LAM), a rare cystic lung disease. In the past, LAM was difficult to diagnose and had a mortality of 100% after 10 years, but now there is a 71% survival after 10 years. Recent research has led to increased characterization of the pathology and radiographic findings. This article briefly presents the case and discusses the etiology, diagnosis, and treatment of LAM. [ABSTRACT FROM AUTHOR]

Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced 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, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ(1-38) peptide corresponded with decreased levels of Aβ(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, 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 controlled strategy against protein accumulation disorders to enhance protein clearance, promote synaptic integrity, and slow the progression of dementia. [ABSTRACT FROM AUTHOR]

Unhealthy lifestyle choices, poor diet, and aging can have negative influences on cognition, gradually increasing the risk for mild cognitive impairment (MCI) and the continuum comprising early dementia. Aging is the greatest risk factor for age-related dementias such as Alzheimer's disease, and the aging process is known to be influenced by life events that can positively or negatively affect age-related diseases. Remarkably, life experiences that make the brain vulnerable to dementia, such as seizure episodes, neurotoxin exposures, metabolic disorders, and trauma-inducing events (e.g. traumatic injuries or mild neurotrauma from a fall or blast exposure), have been associated with negative effects on proteostasis and synaptic integrity. Functional compromise of the autophagy-lysosomal pathway, a major contributor to proteostasis, has been implicated in Alzheimer's disease, Parkinson's disease, obesity-related pathology, Huntington's disease, as well as in synaptic degeneration which is the best correlate of cognitive decline. Correspondingly, pharmacological and non-pharmacological strategies that positively modulate lysosomal proteases are recognized as synaptoprotective through degradative clearance of pathogenic proteins. Here, we discuss life-associated vulnerabilities that influence key hallmarks of brain aging and the increased burden of age-related dementias. Additionally, we discuss exercise and diet among the lifestyle strategies that regulate proteostasis as well as synaptic integrity, leading to evident prevention of cognitive deficits during brain aging in pre-clinical models., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ben A. Bahr, Ph.D. is listed as inventor on U.S. Patent 8,163,953 (Compounds for lysosomal modulation and methods of use) and U.S. Patent 10,702,571 (lysosomal enhancing compounds)., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Excitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N-methyl-d-aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology-specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24-h post-insult. Egr-1 and NOR-1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA-induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up-regulated pathogenic genes, down-regulated pro-survival genes) and opposing compensatory responses (down-regulated pathogenic genes, up-regulated pro-survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF-κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF-κB inhibitor MG-132 indeed altered NMDA-mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi-target treatment strategies and implications for predictive medicine., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Explosive shockwaves, and other types of blast exposures, are linked to injuries commonly associated with military service and to an increased risk for the onset of dementia. Neurological complications following a blast injury, including depression, anxiety, and memory problems, often persist even when brain damage is undetectable. Here, hippocampal explants were exposed to the explosive 1,3,5-trinitro-1,3,5-triazinane (RDX) to identify indicators of blast-induced changes within important neuronal circuitries. Highly controlled detonations of small, 1.7-gram RDX spherical charges reduced synaptic markers known to be downregulated in cognitive disorders, but without causing overt neuronal loss or astroglial responses. In the absence of neuromorphological alterations, levels of synaptophysin, GluA1, and synapsin IIb were significantly diminished within 24 hr, and these synaptic components exhibited progressive reductions following blast exposure as compared to their stable maintenance in control explants. In contrast, labeling of the synapsin IIa isoform remained unaltered, while neuropilar staining of other markers decreased, including synapsin IIb and neural cell adhesion molecule (NCAM) isoforms, along with evidence of NCAM proteolytic breakdown. NCAM 180 displayed a distinct decline after the RDX blasts, whereas NCAM 140 and NCAM 120 exhibited smaller or no deterioration, respectively. Interestingly, the extent of synaptic marker reduction correlated with AT8-positive tau levels, with tau pathology stochastically found in CA1 neurons and their dendrites. The decline in synaptic components was also reflected in the size of evoked postsynaptic currents recorded from CA1 pyramidals, which exhibited a severe and selective reduction. The identified indicators of blast-mediated synaptopathy point to the need for early biomarkers of explosives altering synaptic integrity with links to dementia risk, to advance strategies for both cognitive health and therapeutic monitoring., (© 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Patients with frontotemporal dementia (FTD) resulting from granulin (GRN) haploinsufficiency have reduced levels of progranulin and exhibit dysregulation in inflammatory and lysosomal networks. Microglia produce high levels of progranulin, and reduction of progranulin in microglia alone is sufficient to recapitulate inflammation, lysosomal dysfunction, and hyperproliferation in a cell-autonomous manner. Therefore, targeting microglial dysfunction caused by progranulin insufficiency represents a potential therapeutic strategy to manage neurodegeneration in FTD. Limitations of current progranulin-enhancing strategies necessitate the discovery of new targets. To identify compounds that can reverse microglial defects in Grn-deficient mouse microglia, we performed a compound screen coupled with high throughput sequencing to assess key transcriptional changes in inflammatory and lysosomal pathways. Positive hits from this initial screen were then further narrowed down based on their ability to rescue cathepsin activity, a critical biochemical readout of lysosomal capacity. The screen identified nor-binaltorphimine dihydrochloride (nor-BNI) and dibutyryl-cAMP, sodium salt (DB-cAMP) as two phenotypic modulators of progranulin deficiency. In addition, nor-BNI and DB-cAMP also rescued cell cycle abnormalities in progranulin-deficient cells. These data highlight the potential of a transcription-based platform for drug screening, and advance two novel lead compounds for FTD.

The endosomal-lysosomal pathways and related autophagic processes are responsible for proteostasis, involving complexes between lysosomes and autophagosomes. Lysosomes are a key component of homeostasis, involved in cell signaling, metabolism, and quality control, and they experience functional compromise in metabolic diseases, aging, and neurodegenerative diseases. Many genetic mutations and risk factor genes associated with proteinopathies, as well as with metabolic diseases like diabetes, negatively influence endocytic trafficking and autophagic clearance. In contrast, health-improving exercise induces autophagy-lysosomal degradation, perhaps promoting efficient digestion of injured organelles so that undamaged organelles ensure cellular healthiness. Reductions in lysosomal hydrolases are implicated in Alzheimer's, Parkinson's, and lysosomal storage diseases, as well as obesity-related pathology, and members of the cathepsin enzyme family are involved in clearing both Aβ42 and α-synuclein. Upregulation of cathepsin hydrolases improves synaptic and memory functions in models of dementia and in exercising humans, thus identifying lysosomal-related systems as vital for healthy cognitive aging., (© 2020 Elsevier Inc. All rights reserved.)

FAAH inhibitors offer safety advantages by augmenting the anandamide levels "on demand" to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed "precovalent" FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many neurodegenerative disorders have lysosomal impediments, and the list of proposed treatments targeting lysosomes is growing. We investigated the role of lysosomes in Alzheimer's disease (AD) and other age-related disorders, as well as in a strategy to compensate for lysosomal disturbances. Comprehensive immunostaining was used to analyze brains from wild-type mice vs. amyloid precursor protein/presenilin-1 (APP/PS1) mice that express mutant proteins linked to familial AD. Also, lysosomal modulation was evaluated for inducing synaptic and behavioral improvements in transgenic models of AD and Parkinson's disease, and in models of mild cognitive impairment (MCI). Amyloid plaques were surrounded by swollen organelles positive for the lysosome-associated membrane protein 1 (LAMP1) in the APP/PS1 cortex and hippocampus, regions with robust synaptic deterioration. Within neurons, lysosomes contain the amyloid β 42 (Aβ42) degradation product Aβ38, and this indicator of Aβ42 detoxification was augmented by Z-Phe-Ala-diazomethylketone (PADK; also known as ZFAD) as it enhanced the lysosomal hydrolase cathepsin B (CatB). PADK promoted Aβ42 colocalization with CatB in lysosomes that formed clusters in neurons, while reducing Aβ deposits as well. PADK also reduced amyloidogenic peptides and α-synuclein in correspondence with restored synaptic markers, and both synaptic and cognitive measures were improved in the APP/PS1 and MCI models. These findings indicate that lysosomal perturbation contributes to synaptic and cognitive decay, whereas safely enhancing protein clearance through modulated CatB ameliorates the compromised synapses and cognition, thus supporting early CatB upregulation as a disease-modifying therapy that may also slow the MCI to dementia continuum.

Engulfment and cell motility protein 1 (ELMO1) is part of a guanine nucleotide exchange factor for Ras-related C3 botulinum toxin substrate (Rac), and ELMO1 polymorphisms were identified to be associated with diabetic nephropathy in genome-wide association studies. We generated a set of Akita Ins2C96Y diabetic mice having 5 graded cardiac mRNA levels of ELMO1 from 30% to 200% of normal and found that severe dilated cardiomyopathy develops in ELMO1-hypermorphic mice independent of renal function at age 16 weeks, whereas ELMO1-hypomorphic mice were completely protected. As ELMO1 expression increased, reactive oxygen species indicators, dissociation of the intercalated disc, mitochondrial fragmentation/dysfunction, cleaved caspase-3 levels, and actin polymerization increased in hearts from Akita mice. Cardiomyocyte-specific overexpression in otherwise ELMO1-hypomorphic Akita mice was sufficient to promote cardiomyopathy. Cardiac Rac1 activity was positively correlated with the ELMO1 levels, and oral administration of a pan-Rac inhibitor, EHT1864, partially mitigated cardiomyopathy of the ELMO1 hypermorphs. Disrupting Nox4, a Rac-independent NADPH oxidase, also partially mitigated it. In contrast, a pan-NADPH oxidase inhibitor, VAS3947, markedly prevented cardiomyopathy. Our data demonstrate that in diabetes mellitus ELMO1 is the "rate-limiting" factor of reactive oxygen species production via both Rac-dependent and Rac-independent NADPH oxidases, which in turn trigger cellular signaling cascades toward cardiomyopathy.

Organophosphates account for many of the world's deadliest poisons. They inhibit acetylcholinesterase causing cholinergic crises that lead to seizures and death, while survivors commonly experience long-term neurological problems. Here, we treated brain explants with the organophosphate compound paraoxon and uncovered a unique mechanism of neurotoxicity. Paraoxon-exposed hippocampal slice cultures exhibited progressive declines in synaptophysin, synapsin II, and PSD-95, whereas reduction in GluR1 was slower and NeuN and Nissl staining showed no indications of neuronal damage. The distinctive synaptotoxicity was observed in dendritic zones of CA1 and dentate gyrus. Interestingly, declines in synapsin II dendritic labeling correlated with increased staining for β1 integrin, a component of adhesion receptors that regulate synapse maintenance and plasticity. The paraoxon-induced β1 integrin response was targeted to synapses, and the two-fold increase in β1 integrin was selective as other synaptic adhesion molecules were unchanged. Additionally, β1 integrin-cofilin signaling was triggered by the exposure and correlations were found between the extent of synaptic decline and the level of β1 integrin responses. These findings identified organophosphate-mediated early and lasting synaptotoxicity which can explain delayed neurological dysfunction later in life. They also suggest that the interplay between synaptotoxic events and compensatory adhesion responses influences neuronal fate in exposed individuals.

Background: Ageing is a highly complex process marked by a temporal cascade of events, which promote alterations in the normal functioning of an individual organism. The triggers of normal brain ageing are not well understood, even less so the factors which initiate and steer the neuronal degeneration, which underpin disorders such as dementia. A wealth of data on how nutrients and diets may support cognitive function and preserve brain health are available, yet the molecular mechanisms underlying their biological action in both normal ageing, age-related cognitive decline, and in the development of neurodegenerative disorders have not been clearly elucidated., Objectives: This review aims to summarise the current state of knowledge of vulnerabilities that predispose towards dysfunctional brain ageing, highlight potential protective mechanisms, and discuss dietary interventions that may be used as therapies. A special focus of this paper is on the impact of nutrition on neuroprotection and the underlying molecular mechanisms, and this focus reflects the discussions held during the 2nd workshop 'Nutrition for the Ageing Brain: Functional Aspects and Mechanisms' in Copenhagen in June 2016. The present review is the most recent in a series produced by the Nutrition and Mental Performance Task Force under the auspice of the International Life Sciences Institute Europe (ILSI Europe)., Conclusion: Coupling studies of cognitive ageing with studies investigating the effect of nutrition and dietary interventions as strategies targeting specific mechanisms, such as neurogenesis, protein clearance, inflammation, and non-coding and microRNAs is of high value. Future research on the impact of nutrition on cognitive ageing will need to adopt a longitudinal approach and multimodal nutritional interventions will likely need to be imposed in early-life to observe significant impact in older age., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Threshold shock-impulse levels required to induce cellular injury and cumulative effects upon single and/or multiple exposures are not well characterized. Currently, there are few in vitro experimental models with blast pressure waves generated by using real explosives in the laboratory for investigating the effects of primary blast-induced traumatic brain injury. An in vitro indoor experimental platform is developed using real military explosive charges to accurately represent battlefield blast exposure and to probe the effects of primary explosive blast on dissociated neurons and tissue slices. Preliminary results indicate that physical insults altered membrane permeability, impacted cellular viability, created axonal beadings, and led to synaptic protein loss in hippocampal slice cultures. Injuries from blast under the conditions that were examined did not appear to cause immediate or sustained damage to the cells. Three consecutive primary blasts failed to disrupt the overall cellular integrity in the hippocampal slice cultures and produced a unique type of pathology comprised with distinct reduction in synaptic proteins before cellular deterioration set in. These observed changes might add to the challenges in regard to enhancing our understanding of the complex biochemical and molecular mechanisms caused by primary blast-induced injury.

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 to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca 2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

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., (Copyright © 2017. Published by Elsevier Ltd.)

Background/aims: Limited data are available regarding the undergraduate dermatology clinical clerkship curriculum in the United States. Our primaryaim is to assess medical students' perspectives of the dermatology clinical clerkship., Methods: A multicenter survey study was conducted, which included four California dermatology academic programs. A 17-item questionnaire was designed to investigate medical student perception with regard tothe overall educational value of the various teaching aspects of the dermatology clinical clerkship., Results: A total of 152 medical student surveys were completed. Over half of the medical students felt proficient in diagnosing the most commondermatologic conditions. Eighty-seven percent of medical students were very satisfied with the dermatology clerkship. Ninety-one percent of students felt the length of the clerkship was appropriate., Conclusions: The vast majority of medical students reported a high level of proficiency in the treatment and diagnosis of common skin disorders. In contrast, our findings suggest that medical students may not begaining sufficient hands-on experience in conducting certain dermatologic procedures following the dermatology clerkship. Overall, medical studentperception of the dermatology clinical clerkship was mostly positive.

Cysteine protease inhibitors have long been part of drug discovery programs for Alzheimer's disease (AD), traumatic brain injury (TBI), and other disorders. Select inhibitors reduce accumulating proteins and AD pathology in mouse models. One such compound, Z-Phe-Aladiazomethylketone (PADK), exhibits a very weak IC 50 (9-11 μM) towards cathepsin B (CatB), but curiously PADK causes marked up-regulation of the Aβ-degrading CatB and improves spatial memory. Potential therapeutic and weak inhibitor E64d (14 μM IC 50 ) also up-regulates CatB. PADK and E64d were compared regarding the blockage of calcium-induced cytoskeletal deterioration in brain samples, monitoring the 150-kDa spectrin breakdown product (SBDP) known to be produced by calpain. PADK had little to no effect on SBDP production at 10-100 μM. In contrast, E64d caused a dose-dependent decline in SBDP levels with an IC 50 of 3-6 μM, closely matching its reported potency for inhibiting μ-calpain. Calpain also cleaves the cytoskeletal organizing protein gephyrin, producing 49-kDa (GnBDP49) and 18-kDa (GnBDP18) breakdown products. PADK had no apparent effect on calcium-induced gephyrin fragments whereas E64d blocked their production. E64d also protected the parent gephyrin in correspondence with reduced BDP levels. The findings of this study indicate that PADK's positive and selective effects on CatB are consistent with human studies showing exercise elevates CatB and such elevation correlates with improved memory. On the other hand, E64d exhibits both marginal CatB enhancement and potent calpain inhibition. This dual effect may be beneficial for treating AD. Alternatively, the potent action on calpain-related pathology may explain E64d's protection in AD and TBI models.

The anticholinesterase paraoxon (Pxn) is an organophosphate (OP) and the active metabolite of the insecticide parathion. It potently inhibits the enzyme acetylcholinesterase and leads to enhanced glutamate release, diminished GABA uptake, oxidative damage, and neurodegeneration. The resulting increased levels of acetylcholine can trigger seizures and cause neuronal and excitotoxic damage in the brain. The brain susceptibility related to anticholinesterase toxins extends beyond potential brain damage and death from toxic levels of the agent. Asymptomatic low-level exposure to such toxins can also leave the brain vulnerable or even cause it to exhibit neurological problems later in life. The actions of Pxn and similar neurotoxins have been studied in order to examine the events associated with anticholinesterase toxicity in the brain. A recent study demonstrated that Pxn exposure initiates a pathogenic cascade involving seizure events and subsequent signs of damage including unique presynaptic vulnerability and associated behavioral deficits. In addition, Pxn-mediated synaptotoxicity is also associated with enhanced production of oxidative stress as well as integrin adhesion responses. These findings provide a better understanding of the molecular events involved in Pxn toxicity.

The anticholinesterase paraoxon (Pxn) is related to military nerve agents that increase acetylcholine levels, trigger seizures, and cause excitotoxic damage in the brain. In rat hippocampal slice cultures, high-dose Pxn was applied resulting in a presynaptic vulnerability evidenced by a 64% reduction in synapsin IIb (syn IIb) levels, whereas the postsynaptic protein GluR1 was unchanged. Other signs of Pxn-induced cytotoxicity include the oxidative stress-related production of stable 4-hydroxynonenal (4-HNE)-protein adducts. Next, the Pxn toxicity was tested for protective effects by the fatty acid amide hydrolase (FAAH) inhibitor AM5206, a compound linked to enhanced repair signaling through the endocannabinoid pathway. The Pxn-mediated declines in syn IIb and synaptophysin were prevented by AM5206 in the slice cultures. To test if the protective results in the slice model translate to an in vivo model, AM5206 was injected i.p. into rats, followed immediately by subcutaneous Pxn administration. The toxin caused a pathogenic cascade initiated by seizure events, leading to presynaptic marker decline and oxidative changes in the hippocampus and frontal cortex. AM5206 exhibited protective effects including the reduction of seizure severity by 86%, and improving balance and coordination measured 24 h post-insult. As observed in hippocampal slices, the FAAH inhibitor also prevented the Pxn-induced loss of syn IIb in vivo. In addition, the AM5206 compound reduced the 4-HNE modifications of proteins and the β1 integrin activation events both in vitro and in vivo. These results indicate that Pxn exposure produces oxidative and synaptic toxicity that leads to the behavioral deficits manifested by the neurotoxin. In contrast, the presence of FAAH inhibitor AM5206 offsets the pathogenic cascade elicited by the Pxn anticholinesterase.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer's disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5-1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways.

Sadly many military veterans, who left home to serve their country honorably, return from service with permanent life-changing injuries. It is easy to remember our debt to those who have incurred such visible injuries, and all too easy to forget the invisible wounds that afflict so many of our military servicemen and women. Brain injuries can be invisible during initial medical evaluations and are often caused by military explosives that create blast shockwaves of varying intensity. One of the most common types of traumatic brain injury (TBI) linked to military service is blast-induced neurotrauma. To better understand this type of injury, a recently published study subjected rat brain slice cultures to detonations of RDX military explosives, resulting in reduced levels of specific synaptic markers. Such alterations have in fact been linked to depressive behavior, anxiety, and cognitive rigidity, and the blast-induced synaptic modifications may underlie the behavioral changes in those TBI sufferers who do not exhibit measurable brain damage. This research has the potential to improve diagnoses by identifying indicators of synapse integrity for the assessment of subtle synaptopathogenesis linked to blast-induced neurotrauma., Competing Interests: Conflict of Interest: No conflicts declared

Explosives create shockwaves that cause blast-induced neurotrauma, one of the most common types of traumatic brain injury (TBI) linked to military service. Blast-induced TBIs are often associated with reduced cognitive and behavioral functions due to a variety of factors. To study the direct effects of military explosive blasts on brain tissue, we removed systemic factors by utilizing rat hippocampal slice cultures. The long-term slice cultures were briefly sealed air-tight in serum-free medium, lowered into a 37°C water-filled tank, and small 1.7-gram assemblies of cyclotrimethylene trinitramine (RDX) were detonated 15cm outside the tank, creating a distinct shockwave recorded at the culture plate position. Compared to control mock-treated groups of slices that received equal submerge time, 1-3 blast impacts caused a dose-dependent reduction in the AMPA receptor subunit GluR1. While only a small reduction was found in hippocampal slices exposed to a single RDX blast and harvested 1-2days later, slices that received two consecutive RDX blasts 4min apart exhibited a 26-40% reduction in GluR1, and the receptor subunit was further reduced by 64-72% after three consecutive blasts. Such loss correlated with increased levels of HDAC2, a histone deacetylase implicated in stress-induced reduction of glutamatergic transmission. No evidence of synaptic marker recovery was found at 72h post-blast. The presynaptic marker synaptophysin was found to have similar susceptibility as GluR1 to the multiple explosive detonations. In contrast to the synaptic protein reductions, actin levels were unchanged, spectrin breakdown was not detected, and Fluoro-Jade B staining found no indication of degenerating neurons in slices exposed to three RDX blasts, suggesting that small, sub-lethal explosives are capable of producing selective alterations to synaptic integrity. Together, these results indicate that blast waves from military explosive cause signs of synaptic compromise without producing severe neurodegeneration, perhaps explaining the cognitive and behavioral changes in those blast-induced TBI sufferers that have no detectable neuropathology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In several animal models of motor neuron disease, degeneration begins in the periphery. Clarifying the possible role of Schwann cells remains a priority. We recently showed that terminal Schwann cells (TSCs) exhibit abnormalities in postnatal mice that express mutations of the SOD1 enzyme found in inherited human motor neuron disease. TSC abnormalities appeared before disease-related denervation commenced and the extent of TSC abnormality at P30 correlated with the extent of subsequent denervation. Denervated neuromuscular junctions (NMJs) were also observed that lacked any labeling for TSCs. This suggested that SOD1 TSCs may respond differently than wildtype TSCs to denervation which remain at denervated NMJs for several months. In the present study, the response of SOD1 TSCs to experimental denervation was examined. At P30 and P60, SC-specific S100 labeling was quickly lost from SOD1 NMJs and from preterminal regions. Evidence indicates that this loss eventually becomes complete at most SOD1 NMJs before reinnervation occurs. The loss of labeling was not specific for S100 and did not depend on loss of activity. Although some post-denervation labeling loss occurred at wildtype NMJs, this loss was never complete. Soon after denervation, large cells appeared near SOD1 NMJ bands which colabeled for SC markers as well as for activated caspase-3 suggesting that distal SOD1 SCs may experience cell death following denervation. Denervated SOD1 NMJs viewed 7 days after denervation with the electron microscope confirmed the absence of TSCs overlying endplates. These observations demonstrate that SOD1 TSCs and distal SCs respond abnormally to denervation. This behavior can be expected to hinder reinnervation and raises further questions concerning the ability of SOD1 TSCs to support normal functioning of motor terminals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Exfoliative cheilitis is a condition of unknown etiology characterized by hyperkeratosis and scaling of vermilion epithelium with cyclic desquamation. It remains largely refractory to treatment, including corticosteroid therapy, antibiotics, antifungals, and immunosuppressants., Objective: We sought to evaluate the safety and efficacy of excimer laser therapy and narrowband ultraviolet B therapy in female patients with refractory exfoliative cheilitis., Methods: We reviewed the medical records of two female patients who had been treated unsuccessfully for exfoliative cheilitis. We implemented excimer laser therapy, followed by hand-held narrowband UVB treatments for maintenance therapy, and followed them for clinical improvement and adverse effects., Results: Both patients experienced significant clinical improvement with minimal adverse effects with excimer laser therapy 600-700 mJ/cm 2 twice weekly for several months. The most common adverse effects were bleeding and burning, which occurred at higher doses. The hand-held narrowband UVB unit was also an effective maintenance tool., Limitations: Limitations include small sample size and lack of standardization of starting dose and dose increments., Conclusion: Excimer laser therapy is a well-tolerated and effective treatment for refractory exfoliative cheilitis with twice weekly laser treatments of up to 700 mJ/cm 2 . Transitioning to the hand-held narrowband UVB device was also an effective maintenance strategy.

Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.

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 identified 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 α-synuclein species related to Parkinson's disease and other α-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 Aβ peptides of Alzheimer's disease and tau species of tauopathies. Enhancing autophagy-lysosomal efficiency may provide a therapeutic avenue for diverse types of proteinopathies, including the most common neurodegenerative disorders of the elderly.

In this unit we describe an overlay brain slice culture assay for studying migration of transgenic neurospheres derived from human embryonic stem cells (hESC). Neuronal progenitor cells were generated from hESC by derivation of embryoid bodies and rosettes. Rosettes were transfected using the PiggyBac transposon system with either control plasmids (GFP) or plasmid encoding a gene important for migration of neuronal progenitor cells, Doublecortin (DCX). Transfected cells were subsequently grown in low-adhesion plates to generate transgenic human neurospheres (t-hNS). Organotypic slice cultures were prepared from postnatal rat forebrain and maintained using the interface method, before transfected t-hNS were overlaid below the cortex of each hemisphere. After 1 to 5 days, forebrain slices were fixed and processed for immunofluorescence. The distance at which cells migrated from the center of neurospheres to the host forebrain tissue was measured using Image J software. This protocol provides details for using the slice culture method for studying migration and integration of human neuronal cells into the host brain tissue., (Copyright © 2014 John Wiley & Sons, Inc.)

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Recently, the neurodegenerative component of multiple sclerosis has come under focus particularly because permanent disability in patients correlates well with neurodegeneration; and observations in both humans and multiple sclerosis animal models highlight neurodegeneration of retinal ganglion cells as an early event. After myelin oligodendrocyte glycoprotein immunization of Brown Norway rats, significant retinal ganglion cell loss precedes the onset of pathologically defined autoimmune optic neuritis. To study the role calcium and calpain activation may play in mediating early degeneration, manganese-enhanced magnetic resonance imaging was used to monitor preclinical calcium elevations in the retina and optic nerve of myelin oligodendrocyte glycoprotein-immunized Brown Norway rats. Calcium elevation correlated with an increase in calpain activation during the induction phase of optic neuritis, as revealed by increased calpain-specific cleavage of spectrin. The relevance of early calpain activation to neurodegeneration during disease induction was addressed by performing treatment studies with the calpain inhibitor calpeptin. Treatment not only reduced calpain activity but also protected retinal ganglion cells from preclinical degeneration. These data indicate that elevation of retinal calcium levels and calpain activation are early events in autoimmune optic neuritis, providing a potential therapeutic target for neuroprotection.

Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak cathepsin B and L inhibitor Z-Phe-Ala-diazomethylketone (PADK) enhances lysosomal cathepsin levels at low concentrations, thereby eliciting protective clearance of PHF-τ and Aβ42 in the hippocampus and other brain regions. Here, a class of positive modulators is established with compounds decoupled from the cathepsin inhibitory properties. We utilized PADK as a departure point to develop nonpeptidic structures with the hydroxyethyl isostere. The first-in-class modulators SD1002 and SD1003 exhibit improved levels of cathepsin up-regulation but almost complete removal of cathepsin inhibitory properties as compared to PADK. Isomers of the lead compound SD1002 were synthesized, and the modulatory activity was determined to be stereoselective. In addition, the lead compound was tested in transgenic mice with results indicating protection against AD-type protein accumulation pathology.

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.

The oligomerization of the amyloid-β protein (Aβ) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aβ and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aβ42 form of the protein. The mass spectrum of a mixture of PADK and Aβ42 clearly shows that PADK binds directly to Aβ42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aβ42 dodecamers, but also removes preformed Aβ42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aβ42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aβ42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.

Synaptic pathology in Alzheimer's disease brains is thought to involve soluble Aβ42 peptide. Here, sterile incubation in PBS caused small Aβ42 oligomer formation as well as heterogeneous, 6E10-immunopositive aggregates of 80-100kDa. 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 experimental strategy with which to address synaptic compromise produced by the different Aβ 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 80-600nM for 7days, the Aβ42 preparations caused dose-dependent and aggregation-dependent declines in α-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, Aβ42 induced only trace cellular degeneration that was CA1 specific. The 37°C preparation was less effective at decreasing synaptic markers, corresponding with its reduced levels of Aβ42 monomers and dimers. Aβ42 dimers decayed significantly faster at 37°C than 23°C, and more rapidly than monomers at either temperature. These findings indicate that Aβ42 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 Aβ complexes that form amyloid plaques are protective through the sequestration of oligomeric species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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 Asn(534) and Lys(542). 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(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 Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 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 Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ 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 Aβ(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 Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.

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 24h 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., (Copyright 2009 Elsevier Inc. All rights reserved.)

Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.

Aims: This review posits that fatty acid amide hydrolase (FAAH) inhibition has therapeutic potential against neuropathological states including traumatic brain injury; Alzheimer's, Huntington's, and Parkinson's diseases; and stroke., Main Methods: This proposition is supported by data from numerous in vitro and in vivo experiments establishing metabolic and pharmacological contexts for the neuroprotective role of the endogenous cannabinoid ("endocannabinoid") system and selective FAAH inhibitors., Key Findings: The systems biology of endocannabinoid signaling involves two main cannabinoid receptors, the principal endocannabinoid lipid mediators N-arachidonoylethanolamine ("anandamide") (AEA) and 2-arachidonoyl glycerol (2-AG), related metabolites, and the proteins involved in endocannabinoid biosynthesis, biotransformation, and transit. The endocannabinoid system is capable of activating distinct signaling pathways on-demand in response to pathogenic events or stimuli, thereby enhancing cell survival and promoting tissue repair. Accumulating data suggest that endocannabinoid system modulation at discrete targets is a promising pharmacotherapeutic strategy for treating various medical conditions. In particular, neuronal injury activates cannabinoid signaling in the central nervous system as an intrinsic neuroprotective response. Indirect potentiation of this salutary response through pharmacological inhibition of FAAH, an endocannabinoid-deactivating enzyme, and consequent activation of signaling pathways downstream from cannabinoid receptors have been shown to promote neuronal maintenance and function., Significance: This therapeutic modality has the potential to offer site- and event-specific neuroprotection under conditions where endocannabinoids are being produced as part of a physiological protective mechanism. In contrast, direct application of cannabinoid receptor agonists to the central nervous system may activate CB receptors indiscriminately and invite unwanted psychotrophic effects., (Copyright 2009 Elsevier Inc. All rights reserved.)

Protein accumulation leads to CNS effects in Alzheimer's disease, frontotemporal dementia, and other age-related disorders. Common mechanisms may contribute to the progressive pathology in the different protein accumulation disorders, and synergistic toxicity between dissimilar protein structures may also be involved. Among several avenues being pursued to reduce proteins prone to oligomerization and/or aggregation, a lysosomal avenue has been described that regulates the lysosomal system's broad clearance capability. Lysosomes are the primary site for protein clearance, to remove old and misfolded proteins and maintain cellular homeostasis. Small-molecule lysosomal modulators trigger a feedback response in vitro and in vivo, resulting in marked up-regulation of cathepsins and other lysosomal enzymes without any indications of synaptic pathology, behavioral abnormalities, or major organ malfunctions. For the characterization and screening of lysosomal modulatory drugs, the hippocampal slice model of protein accumulation has proved very useful. The model exhibits experimentally-induced phosphorylated tau species, paired helical filament deposits, ubiquitinated inclusions, and protein oligomers, thus providing a valuable tool to study the associated sequelae underlying progressive cellular and synaptic compromise. In the absence of modulatory drugs, the protein accumulation events lead to microtubule destabilization, transport failure, and synaptic decline. When lysosomal modulators are administered to slices with pre-existing deposits, protein accumulations are reduced causing normalization of tau chemistry, restoration of tubulin structures and tubulin-binding proteins, and recovery of synaptic composition. Thus, positive modulators of the lysosomal system represent first-in-class drugs, providing a suitable strategy to enhance protein clearance, promote synaptic health, and slow the progression of proteinopathies.

During the last decade, the interest in stem and progenitor cells, and their applications in spinal cord injuries have steadily increased. However, little is known about proliferation and cell death mechanisms in these cells after transplantation to the spinal cord. The aim of the present project was to study cell turn-over, i.e. total cell number, with time course of proliferation and cell death, in human neural precursor cells (NPCs) after transplantation to the injured rat spinal cord. Immunodeficient rats were subjected to lateral clip compression injuries, transplanted with neurospheres of human forebrain-derived NPCs two weeks after lesion, and sacrificed after 6 h, 1, 3, 10, or 21 days. Cell death was assessed by quantifying human cells immunoreactive for active caspase-3 and calpain 1-dependent fodrin breakdown products (FBDP). The results showed that after an initial drop, the number of implanted cells increased over time after transplantation. Cell proliferation was substantial, with 34% of human cells being immunoreactive for proliferating cell nuclear antigen at 6 h, but which declined over the next few days. The fractions of caspase-3-, and FBDP-immunoreactive cells were remarkably low, together representing 18% of all human cells at 6 h, and rapidly decreasing the next few days. Our results show that already 10 days after spinal cord transplantation of human NPCs as intact neurospheres, the number of human cells exceeded the initially implanted, which was the result of marked cell proliferation in combination with a low rate of apoptotic and non-apoptotic cell death taking place early after transplantation.

AMPAkines are positive modulators of AMPA receptors, and previous work has shown that these compounds can facilitate synaptic plasticity and improve learning and memory in both animals and humans; thus, their role in the treatment of cognitive impairment is worthy of investigation. In this study, we have utilized an organotypic slice model in which chloroquine-induced lysosomal dysfunction produces many of the pathogenic attributes of Alzheimer's disease. Our previous work demonstrated that synaptic AMPA receptor function is impaired in hippocampal slice cultures exhibiting lysosomal dysfunction leading to protein accumulation. The present study investigated the effect of the AMPAkine CX516 on AMPAR-mediated synaptic transmission as well as the CX516 induced modification of single channel AMPA receptor properties in this organotypic slice-culture model. In whole cell recordings from CA1 pyramidal neurons in chloroquine-treated slices we observed a significant decrease in AMPAR-mediated mEPSC frequency and amplitude indicating synaptic dysfunction. Following application of CX516, these parameters returned to nearly normal levels. Similarly, we report chloroquine-induced impairment of AMPAR single channel properties (decreased probability of opening and mean open time), and significant recovery of these properties following CX516 administration. These results suggest that AMPA receptors may be potential pharmaceutical targets for the treatment of neurodegenerative diseases, and highlights AMPAkines, in particular, as possible therapeutic agents.

The calcitonin gene-related peptide (CGRP) is a neuropeptide involved in vasodilation and other physiological functions throughout the body. The receptor for CGRP has been cloned and well studied, but the mechanism of CGRP receptor desensitization has not been fully elucidated. In the present study, we evaluated the kinetics for agonist-mediated desensitization of the adenylate cyclase response in human neuroblastoma SK-N-MC cells. Distinct CGRP receptor agonists were used, including alpha and beta isoforms of CGRP, the linearized derivative cys(Et)2,7 alphaCGRP, adrenomedullin, and adrenomedullin 2. betaCGRP was 4-600 times more potent at desensitizing the cAMP production as compared to the other receptor-activating ligands, and all of the desensitization effects were blocked by a CGRP receptor antagonist. Although the different agonists vary in their ability to induce functional desensitization, a pretreatment/washout sequence with each peptide was able to reduce the activation potency of the other members of the calcitonin/CGRP peptide family. Next we tested whether the desensitizing effects of the distinct peptides involve protein kinase C (PKC) or protein kinase A (PKA). A PKC inhibitor, Ro 31-8220, concentration-dependently reduced the desensitization induced by the 5 CGRP receptor agonists, while having little effect on their desensitization potencies. PKA inhibitors KT-5720 and H-89, on the other hand, showed little effect on the induced level of desensitization. The findings indicate that functional desensitization is produced by distinct peptides acting through the active site of CGRP receptors, and involves the activation of PKC as a common component necessary to achieve maximal desensitization of receptor signaling.

We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic post-synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post-synaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main post-synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4-7. An engineered deletion construct of GRIP1a/b (GRIP1a4-7), which both contains the aforementioned common region and binds to gephyrin, targets to the post-synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the post-synaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post-synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.