Intraneuronal Amyloid Research Articles

Early oxidative stress and DNA damage in Aβ-burdened hippocampal neurons in an Alzheimer’s-like transgenic rat model

Oxidative stress is a key contributor to AD pathology. However, the earliest role of pre-plaque neuronal oxidative stress, remains elusive. Using laser microdissected hippocampal neurons extracted from McGill-R-Thy1-APP transgenic rats we found that intraneuronal amyloid beta (iAβ)-burdened neurons had increased expression of genes related to oxidative stress and DNA damage responses including Ercc2, Fancc, Sod2, Gsr, and Idh1. DNA damage was further evidenced by increased neuronal levels of XPD (Ercc2) and γH2AX foci, indicative of DNA double stranded breaks (DSBs), and by increased expression of Ercc6, Rad51, and Fen1, and decreased Sirt6 in hippocampal homogenates. We also found increased expression of synaptic plasticity genes (Grin2b (NR2B), CamkIIα, Bdnf, c-fos, and Homer1A) and increased protein levels of TOP2β. Our findings indicate that early accumulation of iAβ, prior to Aβ plaques, is accompanied by incipient oxidative stress and DSBs that may arise directly from oxidative stress or from maladaptive synaptic plasticity.

Targeting mitochondria for healthy brain aging: a case study of non‐psychoactive cannabinoids to rescue mitochondrial dysfunction in Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) arises from complex and multifaceted pathogenic mechanisms. Although many alterations occur in the aging brain that are likely to contribute to AD, there is growing evidence that mitochondrial inefficiency and dysfunction are one of the pathophysiological changes contributing to neurodegeneration in AD. The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway could be used to identify novel AD drug candidates that act by preserving mitochondrial function.MethodsAiming to identify AD drug candidates from natural products that preserve mitochondrial function, we have focused on phytochemical libraries with medicinal properties. We used a novel preclinical drug‐screening platform based on phenotypic assays that mimic different cellular toxicities and stresses associated with the AD brain that are characterized by mitochondrial dysfunction. To elucidate the molecular mechanisms for mitochondrial regulation, we harnessed an inducible, mitochondrially‐depleted neuronal cell line and employed the combined techniques of live‐cell imaging, super‐resolution microscopy, real‐time mitochondrial metabolic analysis, biochemical assays, immunoblotting, and qPCR.ResultsUsing this combined approach, we discovered that cannabinol (CBN), a non‐psychoactive cannabinoid, is a novel and potent neuroprotector. We demonstrated that not only does CBN protect neurons from oxytosis/ferroptosis in a manner that is dependent on mitochondria, it does so independently of cannabinoid CB1/CB2 receptors. Specifically, CBN alleviates intraneuronal amyloid toxicity, directly targets mitochondria, and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis in neuronal cells. These neuroprotective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP‐activated protein kinase (AMPK) signaling which help to maintain mitochondrial homeostasis.ConclusionsThis study highlights the potential of mitochondrially‐targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction for healthy brain aging, as well as opportunities for the discovery and development of future therapeutics for Alzheimer’s disease and other age‐associated neurological disorders.

Serum and Fecal Markers of Intestinal Inflammation and Intestinal Barrier Permeability Are Elevated in Parkinson's Disease.

Parkinson’s disease (PD) is characterized by alpha-synuclein misfolding with subsequent intraneuronal amyloid formation and accumulation, low grade neuroinflammatory changes, and selective neurodegeneration. Available evidence suggests that the pathology usually begins in the gut and olfactory mucosa, spreading to the brain via the vagus and olfactory nerves, by a prion-like mechanism. A causal relationship has not been established, but gut dysbiosis is prevalent in PD and may lead to intestinal inflammation and barrier dysfunction. Additionally, epidemiological data indicate a link between inflammatory bowel diseases and PD. Calprotectin and zonulin are markers of intestinal inflammation and barrier permeability, respectively. We evaluated their serum and fecal levels in 22 patients with sporadic PD and 16 unmatched healthy controls. Mean calprotectin was higher in PD, both in serum (14.26 mcg/ml ± 4.50 vs. 5.94 mcg/ml ± 3.80, p = 0.0125) and stool (164.54 mcg/g ± 54.19 vs. 56.19 mcg/g ± 35.88, p = 0.0048). Mean zonulin was also higher in PD serum (26.69 ng/ml ± 3.55 vs. 19.43 ng/ml ± 2.56, p = 0.0046) and stool (100.19 ng/ml ± 28.25 vs. 37.3 ng/ml ± 13.26, p = 0.0012). Calprotectin was above the upper reference limit in 19 PD serums and 6 controls (OR = 10.56, 95% CI = 2.17–51.42, p = 0.0025) and in 20 PD stool samples and 4 controls (OR = 30, 95% CI = 4.75–189.30, p = 0.000045). Increased zonulin was found only in the stool samples of 8 PD patients. Despite the small sample size, our findings are robust, complementing and supporting other recently published results. The relation between serum and fecal calprotectin and zonulin levels and sporadic PD warrants further investigation in larger cohorts.

The Relevance of Amyloid β-Calmodulin Complexation in Neurons and Brain Degeneration in Alzheimer’s Disease

Intraneuronal amyloid β (Aβ) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer’s disease (AD)-affected brains, intraneuronal Aβ oligomers can derive from Aβ peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aβ neurotoxicity in AD. However, the identification of primary Aβ-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aβ peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aβ concentrations in the low nanomolar range. In turn, the concentration of Aβ-CaM complexes within neurons will increase as a function of time after the induction of Aβ production, and free Aβ will rise sharply when accumulated Aβ exceeds all available CaM. Thus, Aβ-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aβ; a point that has been overlooked until now. In this review, we address the implications of Aβ-CaM complexation in the formation of neurotoxic Aβ oligomers, in the alteration of intracellular calcium homeostasis induced by Aβ, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aβ.

The 5xFAD Mouse Model of Alzheimer's Disease Displays Early Stunting of Neuronal Development in the Dentate Gyrus: a Potential Novel Treatment Window for Dementias Associated with Amyloidosis?

Approximately 5.8 million people over the age of 65 are living with Alzheimer's Disease (AD), and the number of individuals afflicted with AD is projected to double by 2050. Hallmarks of AD are intraneural plaques of amyloid-beta and neurofibrillary tangles with concomitant and progressive decline in neuro-cognitive function. Currently, there is no cure for AD, which stems partly from the sporadic nature of the disease and “after-the-fact” treatments which have failed to remediate disease progression. The goal of our lab is to elucidate early markers of AD onset prior to overt disease occurrence. To this end, we are using the 5XFAD transgenic mouse model which displays an aggressive form of amyloidosis and associated dementias, which develop because of insertion of the five most common genetic mutations associated with aberrant amyloid processing found in AD. Typically, intraneural amyloid plaques develop at 2-4 months of age in 5xFAD mice and cognitive deficits are observed at 4 to 6 months of age. The objective of our current study was to determine characteristics of hippocampal neuronal arborization in 5xFAD mice and non-transgenic (WT) littermates at at 15 days, 1-month, and 3-months of age, which is thought to be a pre-symptomatic period. Mice were euthanized in strict accordance with AALAC guidelines and approved protocols. Isolated brains from male and female 5xFAD and WT mice were fixed and Golgi stained (Rapid Stain Golgi kit, FDNeurotechnologies). Fixed tissue was sectioned by a cryostat (100 um), and slides were imaged using a Leica DM6000 at 40X. Images were then reassembled in Z-stacks and analyzed with FIJI. A Sholl analysis was performed on the hippocampal subregions of the dentate gyrus (DG), cortical area 1 (CA1), and cortical area 3 (CA3) where dendritic length was the primary endpoint. Results showed that there was no shortening at 15 days and 3-months in 5XFAD versus WT, but there was a shortening of neurites in the 5XFAD versus WT at 1-month of age, mainly within the DG (p = 0.04969; N = 6). At 1-month, the dentate gyrus neurons were 108.2μm (p = 0.04969; N = 6), or 5% shorter, with observed simplification of arborization. The other hippocampal subregions did not display significant alterations in neuronal development. These results show that at an extreme early age, 5xFAD mice already show a lack of neuronal development that occurs prior to intraneuronal amyloid plaque appearance, especially in a hippocampal subregion known to be vulnerable to AD-related neuronal loss. This subregion is especially vulnerable due to the fact that amyloid beta accumulates in this region initially, which could lead to the noted shortening in neuronal length. The DG receives input from the entorhinal cortex and is relayed to CA3 via mossy fibers, which is imperative for long-term potentiation and spatial memory. This suggests that the so-called pre-symptomatic period will have to be re-evaluated. Furthermore, our data suggest that a potential window for treatments have been found which may aid in developing novel therapeutic regimens to slow or prevent progression of AD symptomology associated with amyloidosis.

Identification of intraneuronal amyloid beta oligomers in locus coeruleus neurons of Alzheimer's patients and their potential impact on inhibitory neurotransmitter receptors and neuronal excitability.

Amyloid β-oligomers (AβO) are potent modulators of Alzheimer's pathology, yet their impact on one of the earliest brain regions to exhibit signs of the condition, the locus coeruleus (LC), remains to be determined. Of particular importance is whether AβO impact the spontaneous excitability of LC neurons. This parameter determines brain-wide noradrenaline (NA) release, and thus NA-mediated brain functions, including cognition, emotion and immune function, which are all compromised in Alzheimer's patients. Therefore, the aim of the study was to determine the expression profile of AβO in the LC of Alzheimer's patients and to probe their potential impact on the molecular and functional correlates of LC excitability, using a mouse model of increased Aβ production (APP-PSEN1). Immunohistochemistry and confocal microscopy, using AβO-specific antibodies, confirmed LC AβO expression both intraneuronally and extracellularly in both Alzheimer's and APP-PSEN1 samples. Patch clamp electrophysiology recordings revealed that APP-PSEN1 LC neuronal hyperexcitability accompanied this AβO expression profile, arising from a diminished inhibitory effect of GABA due to impaired expression and function of the GABA-A receptor (GABAA R) α3 subunit. This altered LC α3-GABAA R expression profile overlapped with AβO expression in samples from both APP-PSEN1 mice and Alzheimer's patients. Finally, strychnine-sensitive glycine receptors (GlyRs) remained resilient to Aβ-induced changes and their activation reversed LC hyperexcitability. The data suggest a close association between AβO and α3-GABAA Rs in the LC of Alzheimer's patients, and their potential to dysregulate LC activity, thereby contributing to the spectrum of pathology of the LC-NA system in this condition.

Early intraneuronal amyloid triggers neuron-derived inflammatory signaling in APP transgenic rats and human brain

Chronic inflammation during Alzheimer's disease (AD) is most often attributed to sustained microglial activation in response to amyloid-β (Aβ) plaque deposits and cell death. However, cytokine release and microgliosis are consistently observed in AD transgenic animal models devoid of such pathologies, bringing into question the underlying processes that may be at play during the earliest AD-related immune response. We propose that this plaque-independent inflammatory reaction originates from neurons burdened with increasing levels of soluble and oligomeric Aβ, which are known to be the most toxic amyloid species within the brain. Laser microdissected neurons extracted from preplaque amyloid precursor protein (APP) transgenic rats were found to produce a variety of potent immune factors, both at the transcript and protein levels. Neuron-derived cytokines correlated with the extent of microglial activation and mobilization, even in the absence of extracellular plaques and cell death. Importantly, we identified an inflammatory profile unique to Aβ-burdened neurons, since neighboring glial cells did not express similar molecules. Moreover, we demonstrate within disease-vulnerable regions of the human brain that a neuron-specific inflammatory response may precede insoluble Aβ plaque and tau tangle formation. Thus, we reveal the Aβ-burdened neuron as a primary proinflammatory agent, implicating the intraneuronal accumulation of Aβ as a significant immunological component in the AD pathogenesis.

In vitro study of the mechanism of intraneuronal β-amyloid aggregation in Alzheimer’s disease

Alzheimer’s disease (AD) is atrophy of brain cells that lead to decline in the mental capacity and memory. This study investigated the mechanism which postulates that intraneuronal accumulation of amyloid aggregates for pathogenesis of AD. The PC12 cell line was used to examine the amyloid beta (Aβ) aggregation in different stages. It was found that dot-blot filter retardation assay for Ub-CTF was 0.25 and 0.2 µM for SS-CTF. In addition, incubation of SS-CTF with 200 µM Aβ-42 then bounded with an antibody directed against Aβ. It was suggested that most bound Aβ-42 in the oligomeric form. Confocal microscope showed that stained with DAPI (blue) in the neuritic plaques, APP-GFP (green) and specific monoclonal M78 (red). Aß oligomeric taken up by neurons and accumulation of misfolded Aß aggregates continue in a perinuclear location. Fluorescence intensities correlate with the priming effect observed on the Aβ (p < .001). It was concluded that a new amyloid hypothesis is promising in therapy development to reduce the incidence of disease by inhibition of intraneuronal amyloid aggregation.

Age-Related Intraneuronal Aggregation of Amyloid-β in Endosomes, Mitochondria, Autophagosomes, and Lysosomes.

This work provides new insight into the age-related basis of Alzheimer’s disease (AD), the composition of intraneuronal amyloid (iAβ), and the mechanism of an age-related increase in iAβ in adult AD-model mouse neurons. A new end-specific antibody for Aβ45 and another for aggregated forms of Aβ provide new insight into the composition of iAβ and the mechanism of accumulation in old adult neurons from the 3xTg-AD model mouse. iAβ levels containing aggregates of Aβ45 increased 30-50-fold in neurons from young to old age and were further stimulated upon glutamate treatment. iAβ was 8 times more abundant in 3xTg-AD than non-transgenic neurons with imaged particle sizes following the same log-log distribution, suggesting a similar snow-ball mechanism of intracellular biogenesis. Pathologically misfolded and mislocalized Alz50 tau colocalized with iAβ and rapidly increased following a brief metabolic stress with glutamate. AβPP-CTF, Aβ45, and aggregated Aβ colocalized most strongly with mitochondria and endosomes and less with lysosomes and autophagosomes. Differences in iAβ by sex were minor. These results suggest that incomplete carboxyl-terminal trimming of long Aβs by gamma-secretase produced large intracellular deposits which limited completion of autophagy in aged neurons. Understanding the mechanism of age-related changes in iAβ processing may lead to application of countermeasures to prolong dementia-free health span.

New Alzheimer\u2019s disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid \u03b2 oligomers

Oligomers of intracellular amyloid β protein (Aβ) are strongly cytotoxic and play crucial roles in synaptic transmission and cognitive function in Alzheimer’s disease (AD). However, there is currently no AD model mouse in which to specifically analyze the function of Aβ oligomers only. We have now developed a novel AD model mouse, an Aβ-GFP transgenic mouse (Aβ-GFP Tg), that expresses the GFP-fused human Aβ1-42 protein, which forms only Aβ oligomers within neurons throughout their life. The fusion proteins are expressed mainly in the hippocampal CA1-CA2 region and cerebral cortex, and are not secreted extracellularly. The Aβ-GFP Tg mice exhibit increased tau phosphorylation, altered spine morphology, decreased expressions of the GluN2B receptor and neuroligin in synaptic regions, attenuated hippocampal long-term potentiation, and impaired object recognition memory compared with non-Tg littermates. Interestingly, these dysfunctions have already appeared in 2–3-months-old animals. The Aβ-GFP fusion protein is bioactive and highly toxic, and induces the similar synaptic dysfunctions as the naturally generated Aβ oligomer derived from postmortem AD patient brains and synthetic Aβ oligomers. Thus, Aβ-GFP Tg mouse is a new tool specialized to analyze the function of Aβ oligomers in vivo and to find subtle changes in synapses in early symptoms of AD.

Efficacy of Cannabinoids in a Pre-Clinical Drug-Screening Platform for Alzheimer's Disease.

Finding a therapy for Alzheimer's disease (AD) is perhaps the greatest challenge for modern medicine. The chemical scaffolds of many drugs in the clinic today are based upon natural products from plants, yet Cannabis has not been extensively examined as a source of potential AD drug candidates. Here, we determine if a number of non-psychoactive cannabinoids are neuroprotective in a novel pre-clinical AD and neurodegeneration drug-screening platform that is based upon toxicities associated with the aging brain. This drug discovery paradigm has yielded several compounds in or approaching clinical trials for AD. Eleven cannabinoids were assayed for neuroprotection in assays that recapitulate proteotoxicity, loss of trophic support, oxidative stress, energy loss, and inflammation. These compounds were also assayed for their ability to remove intraneuronal amyloid and subjected to a structure-activity relationship analysis. Pairwise combinations were assayed for their ability to synergize to produce neuroprotective effects that were greater than additive. Nine of the 11 cannabinoids have the ability to protect cells in four distinct phenotypic neurodegeneration screening assays, including those using neurons that lack CB1 and CB2 receptors. They are able to remove intraneuronal Aβ, reduce oxidative damage, and protect from the loss of energy or trophic support. Structure-activity relationship (SAR) data show that functional antioxidant groups such as aromatic hydroxyls are necessary but not sufficient for neuroprotection. Therefore, there is a need to focus upon CB1 agonists that have these functionalities if neuroprotection is the goal. Pairwise combinations of THC and CBN lead to a synergistic neuroprotective interaction. Together, these results significantly extend the published data by showing that non-psychoactive cannabinoids are potential lead drug candidates for AD and other neurodegenerative diseases.

Chronic Hippocampal Expression of Notch Intracellular Domain Induces Vascular Thickening, Reduces Glucose Availability, and Exacerbates Spatial Memory Deficits in a Rat Model of Early Alzheimer.

The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aβ) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and 1H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aβ in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aβ pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.

Early long-term administration of the CSF1R inhibitor PLX3397 ablates microglia and reduces accumulation of intraneuronal amyloid, neuritic plaque deposition and pre-fibrillar oligomers in 5XFAD mouse model of Alzheimer\u2019s disease

BackgroundBesides the two main classical features of amyloid beta aggregation and tau-containing neurofibrillary tangle deposition, neuroinflammation plays an important yet unclear role in the pathophysiology of Alzheimer’s disease (AD). Microglia are believed to be key mediators of neuroinflammation during AD and responsible for the regulation of brain homeostasis by balancing neurotoxicity and neuroprotective events. We have previously reported evidence that neuritic plaques are derived from dead neurons that have accumulated intraneuronal amyloid and further recruit Iba1-positive cells, which play a role in either neuronal demise or neuritic plaque maturation or both.MethodsTo study the impact of microglia on neuritic plaque development, we treated two-month-old 5XFAD mice with a selective colony stimulation factor 1 receptor (CSF1R) inhibitor, PLX3397, for a period of 3 months, resulting in a significant ablation of microglia. Directly after this treatment, we analyzed the amount of intraneuronal amyloid and neuritic plaques and performed behavioral studies including Y-maze, fear conditioning and elevated plus maze.ResultsWe found that early long-term PLX3397 administration results in a dramatic reduction of both intraneuronal amyloid as well as neuritic plaque deposition. PLX3397 treated young 5XFAD mice also displayed a significant decrease of soluble fibrillar amyloid oligomers in brain lysates, a depletion of soluble pre-fibrillar oligomers in plasma and an improvement in cognitive function measured by fear conditioning tests.ConclusionsOur findings demonstrate that CSF1R signaling, either directly on neurons or mediated by microglia, is crucial for the accumulation of intraneuronal amyloid and formation of neuritic plaques, suggesting that these two events are serially linked in a causal pathway leading to neurodegeneration and neuritic plaque formation. CSF1R inhibitors represent potential preventative or therapeutic approach that target the very earliest stages of the formation of intraneuronal amyloid and neuritic plaques.

Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis.

Alzheimer’s disease (AD) is a neurodegenerative syndrome classically depicted by the parenchymal accumulation of extracellular amyloid beta plaques. However, recent findings suggest intraneuronal amyloid beta (iAβ1–42) accumulation precedes extracellular deposition. Furthermore, the pathologic increase in iAβ1–42 has been implicated in dysregulation of cellular mechanisms critically important in axonal transport. Owing to neuronal cell polarity, retrograde and anterograde axonal transport are essential trafficking mechanism necessary to convey membrane bound neurotransmitters, neurotrophins, and endosomes between soma and synaptic interfaces. Although iAβ1–42 disruption of axonal transport has been implicated in dysregulation of neuronal synaptic transmission, the role of iAβ1–42 and its influence on signal transduction involving the mitogen-activated protein kinase (MAPK) and morphogenetic signaling axis are unknown. Our biochemical characterization of intracellular amyloid beta accumulation on MAPK and morphogenetic signaling have revealed increased iAβ1–42 expression leads to significant reduction in ERK 1/2 phosphorylation and increased bone morphogenetic protein 2 dependent Smad 1/5/8 phosphorylation. Furthermore, rescue of iAβ1–42 mediated attenuation of MAPK signaling can be accomplished with the small molecule PLX4032 as a downstream enhancer of the MAPK pathway. Consequently, our observations regarding the dysregulation of these gatekeepers of neuronal viability may have important implications in understanding the iAβ1–42 mediated effects observed in AD.

Neuronally-directed effects of RXR activation in a mouse model of Alzheimer\u2019s disease

Alzheimer’s disease (AD) is characterized by extensive neuron loss that accompanies profound impairments in memory and cognition. We examined the neuronally directed effects of the retinoid X receptor agonist bexarotene in an aggressive model of AD. We report that a two week treatment of 3.5 month old 5XFAD mice with bexarotene resulted in the clearance of intraneuronal amyloid deposits. Importantly, neuronal loss was attenuated by 44% in the subiculum in mice 4 months of age and 18% in layer V of the cortex in mice 8 months of age. Moreover, bexarotene treatment improved remote memory stabilization in fear conditioned mice and improved olfactory cross habituation. These improvements in neuron viability and function were correlated with significant increases in the levels of post-synaptic marker PSD95 and the pre-synaptic marker synaptophysin. Moreover, bexarotene pretreatment improved neuron survival in primary 5XFAD neurons in vitro in response to glutamate-induced excitotoxicity. The salutary effects of bexarotene were accompanied by reduced plaque burden, decreased astrogliosis, and suppression of inflammatory gene expression. Collectively, these data provide evidence that bexarotene treatment reduced neuron loss, elevated levels of markers of synaptic integrity that was linked to improved cognition and in an aggressive model of AD.

Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

Age is, by far, the greatest risk factor for Alzheimer's disease (AD), yet few AD drug candidates have been generated that target pathways specifically associated with the aging process itself. Two ubiquitous features of the aging brain are the intracellular accumulation of aggregated proteins and inflammation. As intraneuronal amyloid protein is detected before markers of inflammation, we argue that old, age-associated, aggregated proteins in neurons can induce inflammation, resulting in multiple forms of brain toxicities. The consequence is the increased risk of old, age-associated, neurodegenerative diseases. As most of these diseases are associated with the accumulation of aggregated proteins, it is possible that any therapeutic that reduces intracellular protein aggregation will benefit all.-Currais, A., Fischer, W., Maher, P., Schubert, D. Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

Rescue of Early bace-1 and Global DNA Demethylation by S-Adenosylmethionine Reduces Amyloid Pathology and Improves Cognition in an Alzheimer's Model.

General DNA hypomethylation is associated with Alzheimer’s disease (AD), but it is unclear when DNA hypomethylation starts or plays a role in AD pathology or whether DNA re-methylation would rescue early amyloid-related cognitive impairments. In an APP transgenic mouse model of AD-like amyloid pathology we found that early intraneuronal amyloid beta build-up is sufficient to unleash a global and beta-site amyloid precursor protein cleaving enzyme 1 (bace-1) DNA demethylation in AD-vulnerable brain regions. S-adenosylmethionine administration at these early stages abolished this hypomethylation, diminished the amyloid pathology and restored cognitive capabilities. To assess a possible human significance of findings, we examined the methylation at 12 CpGs sites in the bace-1 promoter, using genome-wide DNA methylation data from 740 postmortem human brains. Thus, we found significant associations of bace-1 promoter methylation with β-amyloid load among persons with AD dementia, and PHFtau tangle density. Our results support a plausible causal role for the earliest amyloid beta accumulation to provoke DNA hypomethylation, influencing AD pathological outcomes.

Intraneuronal Amyloid Beta Accumulation Disrupts Hippocampal CRTC1-Dependent Gene Expression and Cognitive Function in a Rat Model of Alzheimer Disease.

In Alzheimer disease (AD), the accumulation of amyloid beta (Aβ) begins decades before cognitive symptoms and progresses from intraneuronal material to extracellular plaques. To date, however, the precise mechanism by which the early buildup of Aβ peptides leads to cognitive dysfunction remains unknown. Here, we investigate the impact of the early Aβ accumulation on temporal and frontal lobe dysfunction. We compared the performance of McGill-R-Thy1-APP transgenic AD rats with wild-type littermate controls on a visual discrimination task using a touchscreen operant platform. Subsequently, we conducted studies to establish the biochemical and molecular basis for the behavioral alterations. It was found that the presence of intraneuronal Aβ caused a severe associative learning deficit in the AD rats. This coincided with reduced nuclear translocation and genomic occupancy of the CREB co-activator, CRTC1, and decreased production of synaptic plasticity-associated transcripts Arc, c-fos, Egr1, and Bdnf. Thus, blockade of CRTC1-dependent gene expression in the early, preplaque phase of AD-like pathology provides a molecular basis for the cognitive deficits that figure so prominently in early AD.

Long-term dantrolene treatment reduced intraneuronal amyloid in aged Alzheimer triple transgenic mice.

In this study, we investigated the long-term treatment of dantrolene on amyloid and tau neuropathology, brain volume, and cognitive function in aged triple transgenic Alzheimer (3xTg-AD) mice. Fifteen-month old 3xTg-AD mice and wild-type controls were treated with oral dantrolene (5 mg/kg) or vehicle control twice a week for 6 months. Learning and memory were examined using the Morris Water Maze at 21 and 22 months of age. After the behavioral testing, hippocampal and cortical brain volumes were calculated with magnetic resonance imaging and motor function was evaluated using the rotorod. The amyloid burden and tau neurofibrillary tangles in the hippocampus were determined using immunohistochemistry. We found that dantrolene significantly decreased the intraneuronal amyloid accumulation by as much as 76% compared with its corresponding vehicle control, together with a trend to reduce phosphorylated tau in the hippocampus. No significant differences could be detected in hippocampal or cortical brain volume, motor function or cognition among all experimental groups, indicating that the mice were still presymptomatic for Alzheimer disease. Thus, presymptomatic and long-term dantrolene treatment significantly decreased the intraneuronal amyloid burden in aged 3xTg-AD mice before significant changes in brain volume, or cognition.

Cognitive and emotional alterations in young Alzheimer's disease (3xTgAD) mice: Effects of neonatal handling stimulation and sexual dimorphism

Alzheimer disease is the most common neurodegenerative disorder and cause of senile dementia. It is characterized by an accelerated memory loss, and alterations of mood, reason, judgment and language. The main neuropathological hallmarks of the disorder are β-amyloid (βA) plaques and neurofibrillary Tau tangles. The triple transgenic 3xTgAD mouse model develops βA and Tau pathologies in a progressive manner which mimicks the pattern that takes place in the human brain with AD, and showing cognitive alterations characteristic of the disease. The present study intended to examine whether 3xTgAD mice of both sexes present cognitive, emotional and other behavioral alterations at the early age of 4 months, an age in which only some intraneuronal amyloid accumulation is found. Neonatal handling (H) is an early-life treatment known to produce profound and long-lasting behavioral and neurobiological effects in rodents, as well as improvements in cognitive functions. Therefore, we also aimed at evaluating the effects of H on the behavioral/cognitive profile of 4-month-old male and female 3xTgAD mice. The results indicate that, (1) 3xTgAD mice present spatial learning/memory deficits and emotional alterations already at the early age of 4 months, (2) there exists sexual dimorphism effects on several behavioral variables at this age, (3) neonatal handling exerts a preventive effect on some cognitive (spatial learning) and emotional alterations appearing in 3xTgAD mice already at early ages, and 4) H treatment appears to produce stronger positive effects in females than in males in several spatial learning measures and in the open field test.