Intracellular Aβ Accumulation Research Articles

The Antiproteinopathy, Antioxidant, and Antiapoptotic Effects of Methylene Blue and 4-Phenylbutyric Acid Alone, and in Combination on Familial Alzheimer's Disease PSEN1 I416T Cholinergic-Like Neurons.

Familial Alzheimer's disease (FAD) is a chronic neurological condition that progresses over time. Currently, lacking a viable treatment, the use of multitarget medication combinations has generated interest as a potential FAD therapy approach. In this study, we examined the effects of 4-phenylbutyric acid (4-PBA) and methylene blue (MB) either separately or in combination on PSEN1 I416T cholinergic-like neuron cells (ChLNs), which serve as a model for FAD. We found that MB was significantly efficient at reducing the accumulation of intracellular Aβ, phosphorylation of TAU Ser202/Thr205, and increasing Δψm, whereas 4-PBA was significantly efficient at diminishing oxidation of DJ-1Cys106-SH, expression of TP53, and increasing ACh-induced Ca2+ influx. Both agents were equally effective at blunting phosphorylated c-JUN at Ser63/Ser73 and activating caspase 3 (CASP3) into cleaved caspase 3 (CC3) on mutant cells. Combination of MB and 4-PBA at middle (0.1, 1) concentration significantly reduced iAβ, p-TAU, and oxDJ-1 and augmented the ACh-induced Ca2+ influx compared to combined agents at low (0.05, 0.5) or high (0.5, 5) concentration. However, combined MB and 4-PBA were efficient only at dropping DJ-1Cys106-SO3 and increasing ACh-induced Ca2+ inward in mutant ChLNs. Our data show that the reagents MB and 4-PBA alone possess more than one action (e.g., antiamyloid, antioxidant, anti-TAU, antiapoptotic, and ACh-induced Ca2+ influx enhancers), that in combination might cancel or diminish each other. Together, these results strongly argue that MB and 4-PBA might protect PSEN1 I416T ChLNs from Aβ-induced toxicity by working intracellularly as anti-Aβ and anti-Tau agents, improving Δψm and cell survival, and extracellularly, by increasing ACh-induced Ca2+ ion influx. MB and 4-PBA are promising drugs with potential for repurposing in familial AD.

Lack of cellular prion protein causes Amyloid β accumulation, increased extracellular vesicle abundance, and changes to exosome biogenesis proteins.

Alzheimer's disease (AD) progression is closely linked to the propagation of pathological Amyloid β (Aβ), a process increasingly understood to involve extracellular vesicles (EVs), namely exosomes. The specifics of Aβ packaging into exosomes remain elusive, although evidence suggests an ESCRT (Endosomal Sorting Complex Required for Transport)-independent origin to be responsible in spreading of AD pathogenesis. Intriguingly, PrPC, known to influence exosome abundance and bind oligomeric Aβ (oAβ), can be released in exosomes via both ESCRT-dependent and ESCRT-independent pathways, raising questions about its role in oAβ trafficking. Thus, we quantified Aβ levels within EVs, cell medium, and intracellularly, alongside exosome biogenesis-related proteins, following deletion or overexpression of PrPC. The same parameters were also evaluated in the presence of specific exosome inhibitors, namely Manumycin A and GW4869. Our results revealed that deletion of PrPC increases intracellular Aβ accumulation and amplifies EV abundance, alongside significant changes in cellular levels of exosome biogenesis-related proteins Vps25, Chmp2a, and Rab31. In contrast, cellular expression of PrPC did not alter exosomal Aβ levels. This highlights PrPC's influence on exosome biogenesis, albeit not in direct Aβ packaging. Additionally, our data confirm the ESCRT-independent exosome release of Aβ and we show a direct reduction in Chmp2a levels upon oAβ challenge. Furthermore, inhibition of opposite exosome biogenesis pathway resulted in opposite cellular PrPC levels. In conclusion, our findings highlight the intricate relationship between PrPC, exosome biogenesis, and Aβ release. Specifically, they underscore PrPC's critical role in modulating exosome-associated proteins, EV abundance, and cellular Aβ levels, thereby reinforcing its involvement in AD pathogenesis.

Liquid-State Fermented Ganoderma lucidum GANO99 Regulates Gut Microbiota and Concomitantly Modulates the Behavioral Deficits and Neurohistopathological Hallmarks of Alzheimer’s Disease in a Preclinical Transgenic Mouse Model of Alzheimer’s Disease

Amyloid beta (Aβ) is a neuropathological hallmark of Alzheimer’s disease (AD). Oxidative stress promotes intracellular accumulation of Aβ in patients with AD. The effects of liquid-state fermentation products of Ganoderma lucidum without mycelia on AD improvement or prevention have not been reported. Therefore, this study aimed to determine whether the liquid-state fermented products of G. lucidum GANO99 can alter AD pathology in the brains of APPNL-G-F/NL-G-F model mice. Our results suggest that these products promote the expression of superoxide dismutase and inhibit the expression of Aβ and glial fibrillary acidic protein, thereby affecting behavioral, learning, and memory disorders in AD mice. In contrast, G. lucidum GANO99 may regulate the gut microbiota to improve AD pathology. Furthermore, these liquid-state fermented products increased the abundance of intestinal probiotics Bifidobacterium in AD mice. Clinical evidence suggests that these lactic acid bacteria can alleviate AD. Overall, the liquid-state fermented products of G. lucidum GANO99 can act as symbionts by reducing the fermentative role of intestinal microbes and chemo-heterotrophy in AD mice.

AMYLOID‐Β AND PHOSPHORYLATED‐TAU ACCUMULATION IN RETROSPLENIAL CORTEX OF YOUNG ALZHEIMER’S MODEL

AbstractBackgroundOur aim in the present study was to evaluate molecular and functional alterations in the retrosplenial cortex (RSC) of very young Alzheimer’s transgenic mice (3xTg‐AD). The RSC is a cortical area that functions as a key component of the core network of brain regions involved in cognitive functions such as episodic memory, navigation, and planning.MethodElectrophysiology: Local field potentials were recorded using borosilicate electrode (2‐6 MΩ). Integrated theta‐band power was calculated in 5 s binds over a period of 2‐10 min and the mean spectrum was taken as the grand mean of each animal.Immunohistochemistry: For immunofluorescence, antibodies: pS396 and BAM10 were used. The brain areas were visualized using an epifluorescence microscope (Axioplan2, Zeiss).ResultOur results show significant accumulation of intracellular amyloid‐β (Aβ) and hyperphosphorylated tau (pTau) in principal neurons from 1‐month‐old 3xTg‐AD mice, which correlates with GSK3β activation and tau phosphorylation at serine 396. Coincidently, oscillatory activity from the RSC is altered in the young 3xTg‐AD mice. Specifically, we found that theta frequency is significantly higher in the transgenic animals.ConclusionDespite the wide consensus that Aβ deposition in a variety of AD transgenic mice, including 3xTg‐AD, is detected after several months of age, this study shows the first evidence of intracellular Aβ accumulation in pyramidal cells as early as 30 days of age, which does not seem to be related to any cognitive compromise. Thus, early Aβ and pTau accumulation was correlated with altered oscillatory activity from the RSC. In sum, our results indicate that very early accumulation of intracellular Aβ may impact the excitability of the RSC network, possibly due to changes in pTau throughout GSK3β activation.

Single walled carbon nanotube optical reporter for amyloid beta detection

AbstractBackgroundAmyloid beta (Aß) extracellular deposits are one of the pathohistological hallmarks of Alzheimer’s Disease (AD). In recent years intracellular accumulation of Aß has been linked with early pathogenic mechanisms. Recent reports now associate intracellular Aβ accumulation, and lysosomal disfunction as an early event in the disease, preceding extracellular amyloid‐deposits. Today methods to visualize intracellular Aβ accumulation rely on antibody staining or pre‐labeled Aβ. There is an outstanding need for live‐imaging tools capable of monitoring Aβ dynamics at the cellular level.MethodWe have developed an optical sensor that can monitor Aß in live cells transiently. Single wall carbon nanotubes (SWCNT) present advantages as optical sensors owing to their near‐infrared emission, as well as photostability and high‐sensitivity. SWCNT emission spectra (wavelength ranges of 1100‐1150nm) is within the biological transparency window (1000‐1700nm) allowing for optimal imaging in live samples. SWCNT were non‐covalently biofunctionalized; by which we confer selectivity to Aβ.To obtain intracellular detection we used two cell lines: monocytes (THP‐1) and differentiated SH‐SY5Y. Cells were incubated with functionalized nanotubes. We utilized near‐infrared spectroscopy and hyper‐spectral microscopy to obtain spectral response from live‐cells.ResultWe developed and characterized a new class of bio‐functionalized intrinsically photoluminescent SWCNT sensor that can detect Aß via shifting of their intrinsic near‐infrared emission wavelength. Our biofunctionalized sensors specifically recognize Aβ in the solution phase, showing a monochromatic center wavelength decrease in a dose‐dependent fashion. The reporters show high specificity compared to competing analytes and in serum samples. In cell models, nanosensors accumulate intracellularly and probe Aβ introduced to the cells specifically. Finally, initial through‐skull near‐IR imaging in‐vivo in an AD model (5XFAD), following intracranial injection, show significant decrease in central wavelength in AD mice compared to wild type.ConclusionThe SWCNT sensor provide a framework for dynamically investigating Aβ in live samples. We believe utilizing the nanosensors can enhance the study of Aβ ‐pathogenic mechanism, structure and molecular function and consequently improve and accelerate effective amyloid‐targeted therapies.

Most dystrophic neurites in the common 5xFAD Alzheimer mouse model originate from axon terminals

How dystrophic neurites form around amyloid plaques is a key aspect of understanding the early pathophysiology of Alzheimer's disease. At present, three hypotheses prevail: (1) dystrophies result from extracellular amyloid-beta (Aβ) toxicity; (2) dystrophies results from accumulation of Aβ into distal neurites; and (3) dystrophies represent blebbing of the somatic membrane of a neuron with high Aβ load. We utilized a unique feature of the common 5xFAD AD mouse model to test these hypotheses. Cortical layer 5 pyramidal neurons show intracellular APP and Aβ accumulation before amyloid plaque formation while dentate granule cells in these mice show no APP accumulation at any age. However, the dentate gyrus shows amyloid plaques by 3 months of age. By a careful confocal microscopic analysis we found no evidence of severe degeneration in amyloid laden layer 5 pyramidal neurons in contrast to hypothesis 3. Using injecting red fluorescent marker into lateral entorhinal projection neurons in 5xFAD mice with endogenous green fluorescent protein (GFP) in dentate granule cells we could demonstrate that all dystrophies is outer molecular layer originate from the axon terminal of entorhinal projection neurons. Immunostaining with vesicular glutamate transporter supported the axonal nature of the dystrophies in the acellular dentate molecular layer. We observed few small dystrophies in the GFP labeled granule cell dendrites. In general GFP labeled dendrites appear normal around the amyloid plaques. These findings favor hypothesis 2 as the most likely mechanism of dystrophic neurite formation.

Long-term effects of amyloid-beta deposits in human iPSC-derived astrocytes

Growing evidence indicates that astrocytes are tightly connected to Alzheimer's disease (AD) pathogenesis. However, the way in which astrocytes participate in AD initiation and progression remains to be clarified. Our previous data show that astrocytes engulf large amounts of aggregated amyloid-beta (Aβ) but are unable to successfully degrade the material. In this study, we aimed to evaluate how intracellular Aβ-accumulation affects the astrocytes over time. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ-fibrils and then cultured further for one week or ten weeks in Aβ-free medium. Cells from both time points were analyzed for lysosomal proteins and astrocyte reactivity markers and the media were screened for inflammatory cytokines. In addition, the overall health of cytoplasmic organelles was investigated by immunocytochemistry and electron microscopy. Our data demonstrate that long-term astrocytes retained frequent Aβ-inclusions that were enclosed within LAMP1-positive organelles and sustained markers associated with reactivity. Furthermore, Aβ-accumulation resulted in endoplasmic reticulum and mitochondrial swelling, increased secretion of the cytokine CCL2/MCP-1 and formation of pathological lipid structures. Taken together, our results provide valuable information of how intracellular Aβ-deposits affect astrocytes, and thereby contribute to the understanding of the role of astrocytes in AD progression.

An intracerebroventricular injection of AΒ (1–42) modifies temporal profiles of spatial memory performance and oxidative status in the temporal cortex rat

Alzheimer's dementia (AD) is a neurodegenerative disorder that causes memory loss and dementia in older adults. Intracellular accumulation of Aβ causes an imbalance in the oxidative status and cognitive dysfunctions. Besides oxidative stress and loss of memory, Alzheimer's patients show dysfunction of the circadian rhythms. The objective of this work was to evaluate the consequences of an intracerebroventricular injection of Aβ (1–42) on temporal patterns of cognitive performance, as well as on lipid peroxidation, protein oxidation and total antioxidant capacity levels, in the rat temporal cortex. Holtzman male rats from control and Aβ-injected groups were used in this study. We found that MDA, protein carbonyls and total antioxidant capacity levels displayed day-night oscillations in the rat temporal cortex and spatial memory performance also varied rhythmically. An intracerebroventricular injection of Aβ (1–42) modified temporal patterns of cognitive performance as well as daily profiles of parameters of oxidative stress. Thus, elevated levels of Aβ aggregates induces alterations in daily rhythmicity of parameters of oxidative stress and, consequently, would affect cellular clock activity, affecting the spatial memory performance in the AD.

Identification of a dual Aβ‐tau disaggregator for the treatment of Alzheimer’s disease

AbstractBackgroundThe accumulation of misfolded protein aggregates is connected with neurodegenerative diseases such as Alzheimer’s disease (AD), and Parkinson’s disease. Because of the accumulation of amyloid β (Aβ) plaques and tau tangles (NFTs) in the brain, neurons slowly degenerate and lose their functions in AD. Recently, many clinical trials for AD therapy with single‐target drugs, especially targeted for Aβ or tau aggregates, have failed. As a therapeutic strategy for AD, we therefore propose to introduce a multifunctional anti‐AD agent to disaggregate the Aβ plaques and NFTs.MethodHit compound was selected with disaggregation of Aβ and tau aggregates in various concentrations and half maximal effective concentration by thioflavin T (ThT) fluorescence assay. In vitro study, we examined the effects of our hit compound on the accumulation of intracellular Aβ or tau in over‐expressing cells. In vivo study, we administrated orally our hit compound to AD mouse model for 12 weeks (100mg/kg by weekly). Analysis of Aβ and tau was performed by molecular biological and histological experiments.ResultOur results show that the hit compound had dual disaggregation capacity for Aβ and tau aggregates. We observed significant decrease of Aβ and NFTs in hit compound treated AD mice compared to vehicle treated. Also, we found the restoration of cognitive decline in compound treated AD mice.ConclusionThe study demonstrate that our hit compound was identified as dual functional drug for Aβ and NFTs disaggregator. These findings may help to design the next generations of dual or selective disaggregators.

Reduced CSF Aβ42 and Aβ42/Aβ40 ratio during early cerebral amyloid deposition in the AppNL‐F knock‐in mouse model of Alzheimer’s disease

AbstractBackgroundThe concentration of Aβ42 in cerebrospinal fluid (CSF) is decreased at least a decade before cognitive symptoms caused by Alzheimer’s disease (AD) manifest. This drop in concentration has been suggested to occur before abnormal levels of fibrillar Aβ in the brain are reached, but the underlying cause is not well understood. The present study aims to explore the translational potential of the App NL‐F knock‐in mouse model, and compare it to transgenic App‐overexpressing 3xTg mice, for investigation of the early events in Aβ pathology that associates with reduced CSF Aβ42 in preclinical AD.MethodCSF and brain tissue was collected from 3‐18 months old App NL‐F knock‐in mice (n=56) and 3‐24 months old 3xTg mice (n=74). CSF Aβ40 and Aβ42 concentrations were measured using Single Molecule Array (Simoa) technology and immunohistochemistry was performed on brain sections for detection of cerebral Aβ deposition.ResultIn App NL‐F knock‐in mice, CSF Aβ40 remained unchanged while CSF Aβ42 and Aβ42/Aβ40 ratio were reduced from 12 months of age after which a plateau was reached (Fig.1). The initial reduction coincided with early deposition of cortical Aβ42 in the brain that gradually increased with age (Fig.2). Accordingly, in the whole study population, CSF Aβ42 and Aβ42/Aβ40 ratio showed a moderate negative hyperbolic association with cortical Aβ42 load (Fig.3). In comparison, increased CSF Aβ40 and Aβ42 was observed in 3xTg mice prior to Aβ plaque deposition, although the Aβ42/Aβ40 ratio remained stable. These changes coincided with the accumulation of cerebral intracellular Aβ. Once Aβ plaques started to deposit in the brain from 12 months of age, a subsequent reduction of CSF Aβ42 and Aβ42/Aβ40 was observed (Fig.4).ConclusionIn line with clinical findings, CSF Aβ42 and Aβ42/Aβ40 ratio are reduced in App NL‐F knock‐in mice and seem to reflect pathological processes that occur slightly before deposition of amyloid in the brain is widespread. This reduction quickly starts to stabilize towards a plateau, although accumulation of cerebral amyloid steadily continues to increase. In contrast to 3xTg mice, no initial increase in CSF Aβ40 and Aβ42 was observed and further studies are needed to elucidate these differences and their translational implication.

In silico screening of existing FDA approved drugs for spermine synthase inhibition as a therapeutic approach in Alzheimer’s disease

AbstractBackgroundAutophagy dysfunction may lead to amyloid‐β (Aβ) secretion and directly influence the accumulation of intracellular Aβ and generation of extracellular Aβ plaque. Natural polyamine, spermidine present in all living organisms, is critically involved in cellular homeostasis maintenance and influences many biological processes, namely cell growth and proliferation, DNA and RNA stabilization, tissue regeneration, regulation of translation, and enzymatic modulation. Brain spermidine/polyamine levels decrease during aging and various neurodegenerative disorders leading to impairment in autophagy. Spermine synthase is an enzyme involved in biosynthesis of polyamines. It catalyzes the formation of spermine from spermidine. Thus, we focused to repurpose USFDA approved drugs to inhibit spermine synthase which may increase the spermidine levels in the brain.MethodSchrodinger Suite's Maestro graphical user interface was used to carry out computational simulation experiments. Spermidine structure and US‐FDA licensed molecules compounds were downloaded. The molecules were configured using LigPrep tool in maestro software of Schrodinger. Spermine synthase structure (3C6K) bound to 5‐methylthioadenosine (MTA) was downloaded from Protein Data Bank (PDB). After the protein was minimized, the 2D interaction of the ligand‐protein interaction was conducted to observe the amino acid interactions. Spermidine and USFDA licensed molecules were docked using the Schrodinger Glide module. In the present study, the MMGBSA tool is utilized to calculate the prediction of the top compounds. The MD simulation was performed on Schrodinger's Desmond module.ResultFor increasing spermidine pool in the brain by inhibiting spermine synthase, Ribavirin, Sapropterin and Theobromine were found to cross the BBB and have good inhibitory interactions with the enzyme in an in‐silico system. RMSD values for all these three drugs were not satisfactory, but other parameters like protein‐ligand contact‐interaction fraction, 2D interaction diagram and protein‐Ligand contacts through different amino acids present in 3C6K showed satisfactory results.ConclusionOur study showed that ribavirin, sapropterin and theobromine were able to show inhibitory interactions with the spermine synthase. Further, in vitro and in vivo studies may be done to search for the ability of these molecules in the treatment of cognitive dysfunction.

Retinal Vasculopathy in Alzheimer's Disease.

The retina has been increasingly investigated as a site of Alzheimer’s disease (AD) manifestation for over a decade. Early reports documented degeneration of retinal ganglion cells and their axonal projections. Our group provided the first evidence of the key pathological hallmarks of AD, amyloid β-protein (Aβ) plaques including vascular Aβ deposits, in the retina of AD and mild cognitively impaired (MCI) patients. Subsequent studies validated these findings and further identified electroretinography and vision deficits, retinal (p)tau and inflammation, intracellular Aβ accumulation, and retinal ganglion cell-subtype degeneration surrounding Aβ plaques in these patients. Our data suggest that the brain and retina follow a similar trajectory during AD progression, probably due to their common embryonic origin and anatomical proximity. However, the retina is the only CNS organ feasible for direct, repeated, and non-invasive ophthalmic examination with ultra-high spatial resolution and sensitivity. Neurovascular unit integrity is key to maintaining normal CNS function and cerebral vascular abnormalities are increasingly recognized as early and pivotal factors driving cognitive impairment in AD. Likewise, retinal vascular abnormalities such as changes in vessel density and fractal dimensions, blood flow, foveal avascular zone, curvature tortuosity, and arteriole-to-venule ratio were described in AD patients including early-stage cases. A rapidly growing number of reports have suggested that cerebral and retinal vasculopathy are tightly associated with cognitive deficits in AD patients and animal models. Importantly, we recently identified early and progressive deficiency in retinal vascular platelet-derived growth factor receptor-β (PDGFRβ) expression and pericyte loss that were associated with retinal vascular amyloidosis and cerebral amyloid angiopathy in MCI and AD patients. Other studies utilizing optical coherence tomography (OCT), retinal amyloid-fluorescence imaging and retinal hyperspectral imaging have made significant progress in visualizing and quantifying AD pathology through the retina. With new advances in OCT angiography, OCT leakage, scanning laser microscopy, fluorescein angiography and adaptive optics imaging, future studies focusing on retinal vascular AD pathologies could transform non-invasive pre-clinical AD diagnosis and monitoring.

Cardiometabolic Modification of Amyloid Beta in Alzheimer's Disease Pathology.

In recent years, several studies have suggested that cardiometabolic disorders, such as diabetes, obesity, hypertension, and dyslipidemia, share strong connections with the onset of neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease (AD). However, establishing a definitive link between medical disorders with coincident pathophysiologies is difficult due to etiological heterogeneity and underlying comorbidities. For this reason, amyloid β (Aβ), a physiological peptide derived from the sequential proteolysis of amyloid precursor protein (APP), serves as a crucial link that bridges the gap between cardiometabolic and neurodegenerative disorders. Aβ normally regulates neuronal synaptic function and repair; however, the intracellular accumulation of Aβ within the brain has been observed to play a critical role in AD pathology. A portion of Aβ is believed to originate from the brain itself and can readily cross the blood-brain barrier, while the rest resides in peripheral tissues that express APP required for Aβ generation such as the liver, pancreas, kidney, spleen, skin, and lungs. Consequently, numerous organs contribute to the body pool of total circulating Aβ, which can accumulate in the brain and facilitate neurodegeneration. Although the accumulation of Aβ corresponds with the onset of neurodegenerative disorders, the direct function of periphery born Aβ in AD pathophysiology is currently unknown. This review will highlight the contributions of individual cardiometabolic diseases including cardiovascular disease (CVD), type 2 diabetes (T2D), obesity, and non-alcoholic fatty liver disease (NAFLD) in elevating concentrations of circulating Aβ within the brain, as well as discuss the comorbid association of Aβ with AD pathology.

Aging induces abnormal accumulation of Aβ in extracellular vesicle and/or intraluminal membrane vesicle-rich fractions in nonhuman primate brain

Aβ metabolism in the brain is mediated by endocytosis, one part of the intracellular membrane trafficking system. We previously showed that aging attenuates the interaction of dynein with dynactin, which disrupts the endosomal/lysosomal trafficking pathway involved in Aβ metabolism, resulting in intracellular accumulation of Aβ. Several studies have shown that in Alzheimer's disease (AD), intraneuronal accumulation of Aβ precedes extracellular Aβ depositions. However, it is unclear what accounts for this transition from intracellular to extracellular depositions. Accumulating evidence suggest that autophagy has an important role in AD pathology, and we observed that autophagy-related protein levels began to decrease before amyloid plaque formation in cynomolgus monkey brains. Surprisingly, experimental induction of autophagosome formation in Neuro2a cells significantly increased intracellular Aβ and decreased extracellular release of Aβ, accompanied by the prominent reduction of extracellular vesicle (EV) secretion. RNAi study confirmed that EV secretion affected intracellular and extracellular Aβ levels, and siRNA-induced downregulation of autophagosome formation enhanced EV secretion to ameliorate intracellular Aβ accumulation induced by dynein knockdown. In aged cynomolgus monkeys, Aβ levels in EV/intraluminal membrane vesicle (ILV)-rich fractions isolated from temporal lobe parenchyma were drastically increased. Moreover, EV/ILV marker proteins overlapped spatially with amyloid plaques. These findings suggest that EV would be an important carrier of Aβ in brain and abnormal accumulation of Aβ in EVs/ILVs may be involved in the transition of age-dependent Aβ pathology.

A regional and cellular analysis of the early intracellular and extracellular accumulation of Aβ in the brain of 5XFAD mice

Intracellular Aβ (iAβ) expression, extracellular Aβ (eAβ) plaque formation and microglial reactivity are characteristic neuropathological events of Alzheimer’s disease (AD) and have been detected in several transgenic mouse models of this disease. In this work we decided to investigate the early (2–7 months of age) development of these phenomena at both regional and cellular levels in 5XFAD mice, a severe transgenic mouse model of AD. We demonstrated that 1) Aβ pathology develops in many but not all brain regions, 2) iAβ is transient and almost always followed by eAβ in grey matter regions, and the respective levels are roughly proportional, and 3) in about 1/3 of the grey matter regions with Aβ pathology and in several white matter regions, eAβ plaques can appear where no iAβ-positive structures were detected. We also showed that male and female mice share a similar regional and cellular pattern of Aβ pathology development that is more prominent in females. Early iAβ is associated to the activation of microglia, while subsequent formation of eAβ plaques is associated with markedly increased density of microglial cells that acquire a characteristic clustered phenotype. Present analysis is relevant to set a reference for pathophysiological studies and to define specific targets for the test of therapeutic interventions in this widely used AD transgenic model.

Changes in neuronal excitability and synaptic transmission in nucleus accumbens in a transgenic Alzheimer\u2019s disease mouse model

Several previous studies showed that hippocampus and cortex are affected in Alzheimer’s disease (AD). However, other brain regions have also been found to be affected and could contribute with new critical information to the pathophysiological basis of AD. For example, volumetric studies in humans have shown a significant atrophy of the striatum, particularly in the nucleus Accumbens (nAc). The nAc is a key component of the limbic reward system and it is involved in cognition and emotional behaviors such as pleasure, fear, aggression and motivations, all of which are affected in neurodegenerative diseases such as AD. However, its role in AD has not been extensively studied. Therefore, using an AD mouse model, we investigated if the nAc was affected in 6 months old transgenic 2xTg (APP/PS1) mice. Immunohistochemistry (IHC) analysis in 2xTg mice showed increased intraneuronal Aβ accumulation, as well as occasional extracellular amyloid deposits detected through Thioflavin-S staining. Interestingly, the intracellular Aβ pathology was associated to an increase in membrane excitability in dissociated medium spiny neurons (MSNs) of the nAc. IHC and western blot analyses showed a decrease in glycine receptors (GlyR) together with a reduction in the pre- and post-synaptic markers SV2 and gephyrin, respectively, which correlated with a decrease in glycinergic miniature synaptic currents in nAc brain slices. Additionally, voltage-clamp recordings in dissociated MSNs showed a decrease in AMPA- and Gly-evoked currents. Overall, these results showed intracellular Aβ accumulation together with an increase in excitability and synaptic alterations in this mouse model. These findings provide new information that might help to explain changes in motivation, anhedonia, and learning in the onset of AD pathogenesis.

Remodeling of projections from ventral hippocampus to prefrontal cortex in Alzheimer's mice.

Emotional dysregulation often accompanies cognitive deficits in Alzheimer's disease (AD). The hippocampus, most notably damaged by AD pathology, is classified into the cognition-bound posterior and emotion-bound anterior hippocampi. Since the anterior hippocampus or its rodent counterpart, the ventral hippocampus (VH), sends dense afferents to the prefrontal cortex (PFC) and the basolateral amygdala (BLA), the two structures implicated in fear responses, we investigated whether these afferents are modified in 3xTg AD model mice. An anterograde dextrin tracer injected into VH revealed that axons in PFC were more ramified in 3xTg than wild-type (WT) mice, with the synaptic density reduced. The VH projections to BLA were not affected. Intracellular accumulation of amyloid β (Aβ) or Aβ-like immunoreactivity was found in PFC and BLA neurons alike. Behaviorally, in the 2-way active avoidance test, the frequency of chamber change was higher, with the test performance better, in 3xTg than WT mice, suggesting a distorted contextual fear in the 3xTg group. Given the essential involvement of parts of PFC in contextual fear responses and that of BLA in fear responses in general, the observed remodeling of VH-to-PFC afferents and the accumulation of intracellular Aβ in BLA and PFC pyramidal cells might exercise critical influences on enhanced avoidance behavior in 3xTg mice.

Enhancing microtubule stabilization rescues cognitive deficits and ameliorates pathological phenotype in an amyloidogenic Alzheimer\u2019s disease model

In Alzheimer’s disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aβ) deposits. However, the effect of microtubule stabilizing agents on Aβ pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aβ accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aβ levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aβ pathology.

Near-Infrared Responsive Dopamine/Melatonin-Derived Nanocomposites Abrogating in Situ Amyloid β Nucleation, Propagation, and Ameliorate Neuronal Functions.

Alzheimer's disease (AD) is one of the common causes of dementia and mild cognitive impairments, which is progressively expanding among the elderly population worldwide. A short Amyloid-β (Aβ) peptide generated after amyloidogenic processing of amyloid precursor protein exist as intermolecular β-sheet rich oligomeric, protofibriler, and fibrillar structures and believe to be toxic species which instigate neuronal pathobiology in the brain and deposits as senile plaque. Enormous efforts are being made to develop an effective anti-AD therapy that can target Aβ processing, aggregation, and propagation and provide a synergistic neuroprotective effect. However, a nanodrug prepared from natural origin can confer a multimodal synergistic chemo/photothermal inhibition of Aβ pathobiology is not yet demonstrated. In the present work, we report a dopamine-melatonin nanocomposite (DM-NC), which possesses a synergistic near-infrared (NIR) responsive photothermal and pharmacological modality. The noncovalent interaction-mediated self-assembly of melatonin and dopamine oxidative intermediates leads to the evolution of DM-NCs that can withstand variable pH and peroxide environment. NIR-activated melatonin release and photothermal effect collectively inhibit Aβ nucleation, self-seeding, and propagation and can also disrupt the preformed Aβ fibers examined using in vitro Aβ aggregation and Aβ-misfolding cyclic amplification assays. The DM-NCs display a higher biocompatibility to neuroblastoma cells, suppress the AD-associated generation of intracellular reactive oxygen species, and are devoid of any negative impact on the axonal growth process. In okadaic acid-induced neuroblastoma and ex vivo midbrain slice culture-based AD model, DM-NCs exposure suppresses the intracellular Aβ production, aggregation, and accumulation. Therefore, this nature-derived nanocomposite demonstrates a multimodal NIR-responsive synergistic photothermal and pharmacological modality for effective AD therapy.

Herpes Simplex Virus Type-1 Infection Impairs Adult Hippocampal Neurogenesis via Amyloid-β Protein Accumulation

We previously reported that Herpes simplex virus type-1 (HSV-1) infection of cultured neurons triggered intracellular accumulation of amyloid-β protein (Aβ) markedly impinging on neuronal functions. Here, we demonstrated that HSV-1 affects in vitro and in vivo adult hippocampal neurogenesis by reducing neural stem/progenitor cell (NSC) proliferation and their neuronal differentiation via intracellular Aβ accumulation. Specifically, cultured NSCs were more permissive for HSV-1 replication than mature neurons and, once infected, they exhibited reduced proliferation (assessed by 5'-bromo-deoxyuridine incorporation, Ki67 immunoreactivity, and Sox2 mRNA expression) and impaired neuronal differentiation in favor of glial phenotype (evaluated by immunoreactivity for the neuronal marker MAP2, the glial marker glial fibrillary astrocyte protein, and the expression of the proneuronal genes Mash1 and NeuroD1). Similarly, impaired adult neurogenesis was observed in the subgranular zone of hippocampal dentate gyrus of an in vivo model of recurrent HSV-1 infections, that we recently set up and characterized, with respect to mock-infected mice. The effects of HSV-1 on neurogenesis did not depend on cell death and were due to Aβ accumulation in infected NSCs. Indeed, they were: (a) reverted, in vitro, by the presence of either β/γ-secretase inhibitors preventing Aβ production or the specific 4G8 antibody counteracting the action of intracellular Aβ; (b) not detectable, in vivo, in HSV-1-infected amyloid precursor protein knockout mice, unable to produce and accumulate Aβ. Given the critical role played by adult neurogenesis in hippocampal-dependent memory and learning, our results suggest that multiple virus reactivations in the brain may contribute to Alzheimer's disease phenotype by also targeting NSCs. Stem Cells 2019;37:1467-1480.