Forebrain Cholinergic Neurons Research Articles

Electroacupuncture regulates Rab5a-mediating NGF transduction to improve learning and memory ability in the early stage of AD mice.

Nerve growth factor (NGF) loss is a potential factor for the degeneration of basal forebrain cholinergic neurons (BFCNs) in Alzheimer's disease (AD), and Rab5a is a key regulatory molecule of NGF signaling transduction. Here, we investigated the changes of Rab5a in 5 × FAD mice and further explored the mechanism of Electroacupuncture (EA) treatment in improving cognition in the early stage of AD. The total Rab5a and Rab5a-GTP in 5-month-old 5 × FAD mice and wild-type mice were detected using WB and IP technologies. 5 × FAD mice were treated with EA at the Bai hui (DU20) and Shen ting (DU24) acupoints for 4 weeks and CRE/LOXP technology was used to confirm the role of Rab5a in AD mediated by EA stimulation. The Novel Object Recognition and Morris water maze tests were used to evaluate the cognitive function of 5 × FAD mice. The Nissl, immunohistochemistry, and Thioflavin S staining were used to observe pathological morphological changes in the basal forebrain circuit. The Golgi staining was used to investigate the synaptic plasticity of the basal forebrain circuit and WB technology was used to detect the expression levels of cholinergic-related and NGF signal-related proteins. The total Rab5a was unaltered, but Rab5a-GTP increased and the rab5a-positive early endosomes appeared enlarged in the hippocampus of 5 × FAD mice. Notably, EA reduced Rab5a-GTP in the hippocampus in the early stage of 5 × FAD mice. EA could improve object recognition memory and spatial learning memory by reducing Rab5a activity in the early stage of 5 × FAD mice. Moreover, EA could reduce Rab5a activity to increase NGF transduction and increase the levels of phosphorylated TrkA, AKT, and ERK in the basal forebrain and hippocampus, and increase the expression of cholinergic-related proteins, such as ChAT, vAchT, ChT1, m1AchR, and m2AchR in the basal forebrain and ChAT, m1AchR, and m2AchR in the hippocampus, improving synaptic plasticity in the basal forebrain hippocampal circuit in the early stage of 5 × FAD mice. Rab5a hyperactivation is an early pathological manifestation of 5 × FAD mice. EA could suppress Rab5a-GTP to promote the transduction of NGF signaling, and enhance the synaptic plasticity of the basal forebrain hippocampal circuit improving cognitive impairment in the early stage of 5 × FAD mice.

Acetylcholine Engages Distinct Amygdala Microcircuits to Gate Internal Theta Rhythm.

Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in the basolateral amygdala (BLA) in both humans and rodents have been shown to underlie emotional memory, yet their mechanism remains unclear. Here, using brain slice electrophysiology in male and female mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potentials that depend upon M3 muscarinic receptor activation of cholecystokinin (CCK) interneurons (INs) without the need for external glutamate signaling. Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, providing a functional SOM→CCK IN circuit connection gating BLA theta. Parvalbumin (PV) INs, which can drive BLA oscillations in baseline states, are not involved in the generation of ACh-induced theta, highlighting that ACh induces a cellular switch in the control of BLA oscillatory activity and establishes an internally BLA-driven theta oscillation through CCK INs. Theta activity is more readily evoked in BLA over the cortex or hippocampus, suggesting preferential activation of the BLA during high ACh states. These data reveal a SOM→CCK IN circuit in the BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.

Brain histamine improves colonic hyperpermeability through the basal forebrain cholinergic neurons, adenosine A2B receptors and vagus nerve in rats

Intestinal barrier dysfunction, leaky gut, is implicated in various diseases, including irritable bowel syndrome (IBS) and neurodegenerative conditions like Alzheimer's disease. Our recent investigation revealed that basal forebrain cholinergic neurons (BFCNs), critical for cognitive function, receive signals from butyrate and orexin, playing a role in regulating intestinal barrier function through adenosine A2B signaling and the vagus. This study explores the involvement and function of brain histamine, linked to BFCNs, in the regulation of intestinal barrier function. Colonic permeability, assessed by quantifying absorbed Evans blue in rat colonic tissue, showed that histamine did not affect increased colonic permeability induced by LPS when administered subcutaneously. However, intracisternal histamine administration improved colonic hyperpermeability. Elevating endogenous histamine levels in the brain with SKF91488, a histamine N-methyltransferase inhibitor, also improved colonic hyperpermeability. This effect was abolished by intracisternal chlorpheniramine, an histamine H1 receptor antagonist, not ranitidine, an H2 receptor antagonist. The SKF91488-induced improvement in colonic hyperpermeability was blocked by vagotomy, intracisternal pirenzepine (suppressing BFCNs activity), or alloxazine (an adenosine A2B receptor antagonist). Additionally, intracisternal chlorpheniramine injection eliminated butyrate-induced improvement in colonic hyperpermeability. These findings suggest that brain histamine, acting via the histamine H1 receptor, regulates intestinal barrier function involving BFCNs, adenosine A2B signaling, and the vagus. Brain histamine appears to centrally regulate intestinal barrier function influenced by butyrate, differentiating its actions from peripheral histamine in conditions like IBS, where mast cell-derived histamine induces leaky gut. Brain histamine emerges as a potential pharmacological target for diseases associated with leaky gut, such as dementia and IBS.

Current Progress on Central Cholinergic Receptors as Therapeutic Targets for Alzheimer's Disease.

Acetylcholine (ACh) is ubiquitously present in the nervous system and has been involved in the regulation of various brain functions. By modulating synaptic transmission and promoting synaptic plasticity, particularly in the hippocampus and cortex, ACh plays a pivotal role in the regulation of learning and memory. These procognitive actions of ACh are mediated by the neuronal muscarinic and nicotinic cholinergic receptors. The impairment of cholinergic transmission leads to cognitive decline associated with aging and dementia. Therefore, the cholinergic system has been of prime focus when concerned with Alzheimer's disease (AD), the most common cause of dementia. In AD, the extensive destruction of cholinergic neurons occurs by amyloid-β plaques and tau protein-rich neurofibrillary tangles. Amyloid-β also blocks cholinergic receptors and obstructs neuronal signaling. This makes the central cholinergic system an important target for the development of drugs for AD. In fact, centrally acting cholinesterase inhibitors like donepezil and rivastigmine are approved for the treatment of AD, although the outcome is not satisfactory. Therefore, identification of specific subtypes of cholinergic receptors involved in the pathogenesis of AD is essential to develop future drugs. Also, the identification of endogenous rescue mechanisms to the cholinergic system can pave the way for new drug development. In this article, we discussed the neuroanatomy of the central cholinergic system. Further, various subtypes of muscarinic and nicotinic receptors involved in the cognition and pathophysiology of AD are described in detail. The article also reviewed primary neurotransmitters that regulate cognitive processes by modulating basal forebrain cholinergic projection neurons.

Effects of Aging and Nerve Growth Factor on Neuropeptide Expression and Cholinergic Innervation of the Rat Basolateral Amygdala.

The basolateral amygdala (BLA) contains interneurons that express neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), both of which are involved in the regulation of functions and behaviors that undergo deterioration with aging. There is considerable evidence that, in some brain areas, the expression of NPY and VIP might be modulated by acetylcholine. Importantly, the BLA is one of the brain regions that has one of the densest cholinergic innervations, which arise mainly from the basal forebrain cholinergic neurons. These cholinergic neurons depend on nerve growth factor (NGF) for their survival, connectivity, and function. Thus, in this study, we sought to determine if aging alters the densities of NPY- and VIP-positive neurons and cholinergic varicosities in the BLA and, in the affirmative, if those changes might rely on insufficient trophic support provided by NGF. The number of NPY-positive neurons was significantly reduced in aged rats, whereas the number of VIP-immunoreactive neurons was unaltered. The decreased NPY expression was fully reversed by the infusion of NGF in the lateral ventricle. The density of cholinergic varicosities was similar in adult and old rats. On the other hand, the density of cholinergic varicosities is significantly higher in old rats treated with NGF than in adult and old rats. Our results indicate a dissimilar resistance of different populations of BLA interneurons to aging. Furthermore, the present data also show that the BLA cholinergic innervation is particularly resistant to aging effects. Finally, our results also show that the reduced NPY expression in the BLA of aged rats can be related to changes in the NGF neurotrophic support.

Cholinergic Basal Forebrain Connectivity to the Basolateral Amygdala Modulates Food Intake.

Obesity results from excessive caloric input associated with overeating and presents a major public health challenge. The hypothalamus has received significant attention for its role in governing feeding behavior and body weight homeostasis. However, extrahypothalamic brain circuits also regulate appetite and consumption by altering sensory perception, motivation, and reward. We recently discovered a population of basal forebrain cholinergic (BFc) neurons that regulate appetite suppression. Through viral tracing methods in the mouse model, we found that BFc neurons densely innervate the basolateral amygdala (BLA), a limbic structure involved in motivated behaviors. Using channelrhodopsin-assisted circuit mapping, we identified cholinergic responses in BLA neurons following BFc circuit manipulations. Furthermore, in vivo acetylcholine sensor and genetically encoded calcium indicator imaging within the BLA (using GACh3 and GCaMP, respectively) revealed selective response patterns of activity during feeding. Finally, through optogenetic manipulations in vivo, we found that increased cholinergic signaling from the BFc to the BLA suppresses appetite and food intake. Together, these data support a model in which cholinergic signaling from the BFc to the BLA directly influences appetite and feeding behavior.

Reward contingency gates selective cholinergic suppression of amygdala neurons.

Basal forebrain cholinergic neurons modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, and memory. It is unknown, however, whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of secondary roles in the processing of external stimuli. Using fluorescent imaging, we found that cholinergic neurons are active during behavioral responding for a reward - even prior to reward delivery and in the absence of discrete stimuli. Photostimulation of basal forebrain cholinergic neurons, or their terminals in the basolateral amygdala (BLA), selectively promoted conditioned responding (licking), but not unconditioned behavior nor innate motor outputs. In vivo electrophysiological recordings during cholinergic photostimulation revealed reward-contingency-dependent suppression of BLA neural activity, but not prefrontal cortex. Finally, ex vivo experiments demonstrated that photostimulation of cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling, while also facilitating firing in BLA GABAergic interneurons. Taken together, we show that the neural and behavioral effects of basal forebrain cholinergic activation are modulated by reward contingency in a target-specific manner.

Reward contingency gates selective cholinergic suppression of amygdala neurons

Basal forebrain cholinergic neurons modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, and memory. It is unknown, however, whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of secondary roles in the processing of external stimuli. Using fluorescent imaging, we found that cholinergic neurons are active during behavioral responding for a reward – even prior to reward delivery and in the absence of discrete stimuli. Photostimulation of basal forebrain cholinergic neurons, or their terminals in the basolateral amygdala (BLA), selectively promoted conditioned responding (licking), but not unconditioned behavior nor innate motor outputs. In vivo electrophysiological recordings during cholinergic photostimulation revealed reward-contingency-dependent suppression of BLA neural activity, but not prefrontal cortex. Finally, ex vivo experiments demonstrated that photostimulation of cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling, while also facilitating firing in BLA GABAergic interneurons. Taken together, we show that the neural and behavioral effects of basal forebrain cholinergic activation are modulated by reward contingency in a target-specific manner.

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SIp) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can 'learn' the association between a naive tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24 hr later (recall). In the NBM/SIp cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SIp cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SIa cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

Accelerated neurodegeneration of basal forebrain cholinergic neurons in HIV-1 gp120 transgenic mice: Critical role of the p75 neurotrophin receptor

Human Immunodeficiency Virus-1 (HIV) infection of the brain induces HIV-associated neurocognitive disorders (HAND). The set of molecular events employed by HIV to drive cognitive impairments in people living with HIV are diverse and remain not completely understood. We have shown that the HIV envelope protein gp120 promotes loss of synapses and decreases performance on cognitive tasks through the p75 neurotrophin receptor (p75NTR). This receptor is abundant on cholinergic neurons of the basal forebrain and contributes to cognitive impairment in various neurological disorders. In this study, we examined cholinergic neurons of gp120 transgenic (gp120tg) mice for signs of degeneration. We observed that the number of choline acetyltransferase-expressing cells is decreased in old (12–14-month-old) gp120tg mice when compared to age matched wild type. In the same animals, we observed an increase in the levels of pro-nerve growth factor, a ligand of p75NTR, as well as a disruption of consolidation of extinction of conditioned fear, a behavior regulated by cholinergic neurons of the basal forebrain. Both biochemical and behavioral outcomes of gp120tg mice were rescued by the deletion of the p75NTR gene, strongly supporting the role that this receptor plays in the neurotoxic effects of gp120. These data indicate that future p75NTR-directed pharmacotherapies could provide an adjunct therapy against synaptic simplification caused by HIV.

Analogs of α-conotoxin PnIC selectively inhibit α7β2- over α7-only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and β2 subunits (α7β2-nAChR subtype). Basal forebrain cholinergic neurons express α7β2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-β associated with early Alzheimer's disease. Additional work indicates that α7β2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7β2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7β2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7β2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7β2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7β2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7β2-nAChR and detailed investigations of their physiological roles.

A human stem cell model of development and pathology of basal forebrain cholinergic neurons

AbstractBackgroundBasal forebrain cholinergic neurons (BFCNs) in the nucleus basalis regulate attention, memory, learning and processing of information related to cognitive function throughout the lifespan. Degeneration of BFCNs is strongly correlated with cognitive function and degeneration of the BFCN projection system is an early event in Alzheimer’s disease (AD) pathogenesis and in Down syndrome (DS, trisomy 21) that is characterized by intellectual impairment at birth and AD pathology in middle age. Understanding the vulnerability of BFCN neurons will help us understand the underlying mechanisms of neurodegeneration in both AD and DS.MethodWe generated BFCNs from trisomy 21 induced pluripotent stem cells (iPSCs) and isogenic controls through addition of morphogens and growth factors following developmental principles. The efficiency of the protocol was assessed through immunofluorescence for choline acetyl transferase (ChAT).ResultWe established a protocol to generate BFCNs from control and trisomy 21 iPSCs through a stepwise process of 1) ventralization of neural progenitors with SHH or SHH agonist, 2) regulation of LHX8 expression with NGF and, 3) regulation of ISLET1 expression and BFCN survival with BMP9. The protocol generated approximately 60% BFCNs from both trisomy 21 and control iPSCs.ConclusionFew differentiation protocols have been established to derive BFCNs from iPSCs. Our iPSC model enables analysis of pathological markers, identification of disrupted molecular pathways, manipulation of conditions to affect neurodegeneration and screening of potential therapeutics in human BFCNs.

Etiological contributions of adolescent binge alcohol on onset of Alzheimer’s disease‐associated basal forebrain neuropathology in 5xFAD mice and post‐mortem human AUD brain

AbstractBackgroundHeavy alcohol use is an etiological factor for development of dementia and may contribute to onset of Alzheimer’s disease (AD). Both alcohol use disorder (AUD) and AD share common underlying neuropathology, including basal forebrain cholinergic neuron degeneration and proinflammatory neuroimmune activation, and evidence suggest these systems are particularly sensitive to adolescent alcohol exposure. Using the 5xFAD mouse model of AD, we tested the hypothesis that adolescent intermittent ethanol (AIE), which models human adolescent binge drinking, would accelerate onset of AD‐associated pathology in the adult basal forebrain.MethodIn Experiment 1, non‐transgenic and 5xFAD male and female mice received AIE treatment (5.0 g/kg, i.g. 2‐days on/2‐days off; postnatal day [P]30 – P55) and basal forebrain tissue collected for immunohistochemistry and gene expression analysis on P100. In Experiment 2, 5xFAD female mice received AIE treatment as described above followed by glycyrrhizic acid (GA; 150 mg/L), a high mobility group box 1 (HMGB1) inhibitor, in drinking water from P56 – P100. At study conclusion, basal forebrain tissue was collected for immunohistochemistry and gene expression analysis on P100. In Experiment 3, AD‐associated neuropathology was assessed in the post‐mortem human basal forebrain of individuals with AUD relative to age‐matched moderate drinking CONs.ResultIn Experiment 1, AIE accelerated loss of basal forebrain cholinergic neurons and heterogeneous nuclear ribonucleoprotein (hnRNP) expression in 5xFAD females, but not males, relative to age‐matched 5xFAD CONs. This was accompanied by a female‐specific accelerated accumulation of plaque markers (e.g., Aβ1‐42, Thioflavin‐S, HMGB1) as well as microglial activation and proinflammatory neuroimmune signaling genes (e.g., HMGB1, RAGE) in the adult basal forebrain. In Experiment 2, post‐AIE treatment with the HMGB1 inhibitor GA reversed AIE‐induced accelerated loss of cholinergic neurons, accumulation of plaque markers, and upregulation of microglial activation markers and proinflammatory signaling molecules. In Experiment 3, AUD individuals exhibited decreased cholinergic and hnRNP markers and increased Aβ1‐42 immunoreactivity as well as cholinergic neuron co‐expression with Aβ1‐42 that negatively correlated with an adolescent age of drinking onset.ConclusionThese data reveal that adolescent binge ethanol exposure may be an etiological factor contributing to onset of AD‐associated neuropathology in the adult basal forebrain through HMGB1‐neuroimmune signaling.

Utilization of the novel adeno‐associated virus PHP.eB serotype to modulate Intersectin 1 expression in the Ts65Dn mouse model of Down syndrome

AbstractBackgroundDown Syndrome (DS) and Alzheimer’s disease (AD) include cholinergic neurodegeneration which parallels cognitive decline. Triplicated genes in DS are thought to play a role in basal forebrain cholinergic neuron (BFCN) pathology; one avenue to assess triplicated genes’ role is to normalize their expression in DS mouse models by stereotaxic injection of adeno‐associated viruses (AAVs) carrying silencing constructs such as single hairpin mRNA (shRNA). However, stereotaxic surgeries have drawbacks particularly when targeting areas lying deep within the brain. The introduction of the AAV‐PHP.eB serotype, which can cross the blood‐brain‐barrier when introduced into peripheral vasculature, has been shown to vary in efficiency depending on mouse background. Here, we investigated tropism of the AAV‐PHP.eB serotype in the Ts65Dn mouse, the most commonly used DS mouse model. We then used AAV‐PHP.eB to assess our ability to globally modulate expression of intersectin 1 (ITSN1), a protein triplicated in DS that may relate to BFCN dysfunction and memory impairments.MethodsWe retro‐orbitally injected N = 16 4‐4.5 month‐old male mice, (C57Bl, Ts65Dn trisomic and normosomic) with AAV‐PHP.eB‐CamKII‐GFP‐scrmble‐shRNA, to assess tropism and optimize virus concentration (2, 5, and 10µL). Four weeks post injection we imaged for GFP expression in 50µm coronal sections. To knock down ITSN1 globally, we injected N = 58 Ts65Dn mice (3 months, balanced by sex, genotype, virus) with either 5μl AAV‐PHP.eB‐U6‐scrmble‐GFP (control) or AAV‐PHP.eB‐U6‐shITSN1‐GFP (ITSN1); mice were behaviorally tested at 7 months and euthanized at 8 months followed by tissue collection.ResultsMice injected with AAV‐PHP.eB‐CamKII‐GFP‐scrmble‐shRNA showed GFP expression 4 weeks following injection, with robust expression visible with 5µL virus. Mice subsequently injected with control and ITSN1 AAVs showed significant effects of ITSN1 knockdown, including increased tremors, increased latency in the radial arm water maze – suggesting impaired spatial reference learning and memory – decreased brain weight, and altered weight change.ConclusionThe AAV‐PHP.eB serotype shows successful CNS tropism in the Ts65Dn mouse model, with 5µL of AAV providing the best efficiency throughout key brain regions affected in DS and AD. Furthermore, the AAV‐PHP.eB serotype can be used to modulate expression of genes triplicated in DS to investigate their mechanistic role in neurodegeneration both molecularly and behaviorally.

Circuit mechanism for suppression of frontal cortical ignition during NREM sleep

Conscious perception is greatly diminished during sleep, but the underlying circuit mechanism is poorly understood. We show that cortical ignition-a brain process shown to be associated with conscious awareness in humans and non-human primates-is strongly suppressed during non-rapid-eye-movement (NREM) sleep in mice due to reduced cholinergic modulation and rapid inhibition of cortical responses. Brain-wide functional ultrasound imaging and cell-type-specific calcium imaging combined with optogenetics showed that activity propagation from visual to frontal cortex is markedly reduced during NREM sleep due to strong inhibition of frontal pyramidal neurons. Chemogenetic activation and inactivation of basal forebrain cholinergic neurons powerfully increased and decreased visual-to-frontal activity propagation, respectively. Furthermore, although multiple subtypes of dendrite-targeting GABAergic interneurons in the frontal cortex are more active during wakefulness, soma-targeting parvalbumin-expressing interneurons are more active during sleep. Chemogenetic manipulation of parvalbumin interneurons showed that sleep/wake-dependent cortical ignition is strongly modulated by perisomatic inhibition of pyramidal neurons.

Alzheimer’s disease linked amyloid beta 1‐42 exerts product feedback inhibition on gamma‐secretase impairing downstream signaling cascades

AbstractBackgroundΓ‐secretases are proteolytic switches at the membrane regulating multiple signaling cascades. Their dysfunction, resulting in enhanced generation of longer amyloid β (Aβ) peptides from the amyloid precursor protein (APP), leads to neurodegeneration in the context of Alzheimer’s disease (AD), while their inhibition causes neurodegenerative phenotypes in mice and cognitive worsening in AD patients treated with γ‐secretase inhibitors. The accumulation of Aβ in the brain is the earliest pathological hallmark of AD. Based on the proven affinity of Aβ peptides for γ‐secretases, we hypothesized that elevations in Aβ levels would promote an inhibitory‐feedback mechanism on γ‐secretases and impair downstream cell signaling events.MethodWe conducted rigorous kinetic analyses of γ‐secretase activity in the presence of a series of Aβ and p3 peptides, by quantifying the levels of intracellular domains generated from different γ‐secretase substrates in cell‐free assays. In addition, we determined the effects of these peptides on endogenous γ‐secretase activity in living neurons, using a ratiometric FRET‐based reporter and western blot analysis of the levels of immediate γ‐secretase substrates. Furthermore, we assessed the impact of Aβ and p3 peptides on γ‐secretase‐mediated, p75‐ and TrkA‐dependent downstream signaling via immunostaining for an apoptotic marker: cleaved caspase 3. Finally, we evaluated the impact of Aβ peptides on APP processing in synaptosome fractions derived from mouse brains.ResultOur analyses showed that human Aβ42 inhibited γ‐secretase activity and accordingly caused accumulation of unprocessed γ‐secretase substrates in neuronal cells, i.e. CTFs of APP, p75 and pan‐cadherin. Remarkably, neither murine Aβ42 nor human p3 (17‐42) peptides exerted the inhibition. In TrkA signaling deficient PC12 cells and basal forebrain cholinergic neurons, Aβ1‐42‐mediated inhibition of γ‐secretase led to the accumulation of unprocessed p75‐CTFs and potentiated p75‐dependent cell death, mimicking the effects of γ‐secretase inhibitors.ConclusionWe demonstrate that the pathologically relevant human Aβ1‐42 exerts product feedback inhibition on γ‐secretases, leading to dysregulation of downstream cellular signaling. These findings provide a novel conceptual framework for investigations of Aβ toxicity in the context of γ‐secretase‐dependent homeostatic signaling and raise the possibility that Aβ42‐mediated inactivation of these enzymes contributes to AD development.

Age-induced nitrative stress decreases retrograde transport of proNGF via TrkA and increases proNGF retrograde transport and neurodegeneration via p75NTR.

Axonal transport of pro nerve growth factor (proNGF) is impaired in aged basal forebrain cholinergic neurons (BFCNs), which is associated with their degeneration. ProNGF is neurotrophic in the presence of its receptor tropomyosin-related kinase A (TrkA) but induces apoptosis via the pan-neurotrophin receptor (p75NTR) when TrkA is absent. It is well established that TrkA is lost while p75NTR is maintained in aged BFCNs, but whether aging differentially affects transport of proNGF via each receptor is unknown. Nitrative stress increases during aging, but whether age-induced nitrative stress differentially affects proNGF transport via TrkA versus p75NTR has not yet been studied. Answering these questions is essential for developing an accurate understanding of the mechanisms contributing to age-induced loss of proNGF transport and BFCN degeneration. In this study, fluorescence microscopy was used to analyze axonal transport of quantum dot labeled proNGF in rat BFCNs in vitro. Receptor specific effects were studied with proNGF mutants that selectively bind to either TrkA (proNGF-KKE) or p75NTR (proNGF-Δ9-13). Signaling factor activity was quantified via immunostaining. Young BFCNs transported proNGF-KKE but not proNGF-Δ9-13, and proNGF transport was not different in p75NTR knockout BFCNs compared to wildtype BFCNs. These results indicate that young BFCNs transport proNGF via TrkA. In vitro aging increased transport of proNGF-Δ9-13 but decreased transport of proNGF-KKE. Treatment with the nitric oxide synthase inhibitor L-NAME reduced retrograde transport of proNGF-Δ9-13 in aged BFCNs while increasing retrograde transport of proNGF-KKE but did not affect TrkA or p75NTR levels. ProNGF-Δ9-13 induced greater pro-apoptotic signaling and neurodegeneration and less pro-survival signaling relative to proNGF-KKE. Together, these results indicate that age-induced nitrative stress decreases proNGF transport via TrkA while increasing proNGF transport via p75NTR. These transport deficits are associated with decreased survival signaling, increased apoptotic signaling, and neurodegeneration. Our findings elucidate the receptor specificity of age-and nitrative stress-induced proNGF transport deficits. These results may help to rescue the neurotrophic signaling of proNGF in aging to reduce age-induced loss of BFCN function and cognitive decline.

Maternal choline supplementation protects against age-associated cholinergic and GABAergic basal forebrain neuron degeneration in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease

Down syndrome (DS) is a genetic disorder caused by triplication of human chromosome 21. In addition to intellectual disability, DS is defined by a premature aging phenotype and Alzheimer's disease (AD) neuropathology, including septohippocampal circuit vulnerability and degeneration of basal forebrain cholinergic neurons (BFCNs). The Ts65Dn mouse model recapitulates key aspects of DS/AD pathology, namely age-associated atrophy of BFCNs and cognitive decline in septohippocampal-dependent behavioral tasks. We investigated whether maternal choline supplementation (MCS), a well-tolerated treatment modality, protects vulnerable BFCNs from age- and genotype-associated degeneration in trisomic offspring. We also examined the effect of trisomy, and MCS, on GABAergic basal forebrain parvalbumin neurons (BFPNs), an unexplored neuronal population in this DS model. Unbiased stereological analyses of choline acetyltransferase (ChAT)-immunoreactive BFCNs and parvalbumin-immunoreactive BFPNs were conducted using confocal z-stacks of the medial septal nucleus and the vertical limb of the diagonal band (MSN/VDB) in Ts65Dn mice and disomic (2N) littermates at 3–4 and 10–12 months of age. MCS trisomic offspring displayed significant increases in ChAT-immunoreactive neuron number and density compared to unsupplemented counterparts, as well as increases in the area of the MSN/VDB occupied by ChAT-immunoreactive neuropil. MCS also rescued BFPN number and density in Ts65Dn offspring, a novel rescue of a non-cholinergic cell population. Furthermore, MCS prevented age-associated loss of BFCNs and MSN/VDB regional area in 2N offspring, indicating genotype-independent neuroprotective benefits. These findings demonstrate MCS provides neuroprotection of vulnerable BFCNs and non-cholinergic septohippocampal BFPNs, indicating this modality has translational value as an early life therapy for DS, as well as extending benefits to the aging population at large.

Focal acetylcholinergic modulation of the human midcingulo-insular network during attention: Meta-analytic neuroimaging and behavioral evidence.

The basal forebrain cholinergic neurons provide acetylcholine to the cortex via large projections. Recent molecular imaging work in humans indicates that the cortical cholinergic innervation is not uniformly distributed, but rather may disproportionately innervate cortical areas relevant to supervisory attention. In this study, we therefore reexamined the spatial relationship between acetylcholinergic modulation and attention in the human cortex using meta-analytic strategies targeting both pharmacological and non-pharmacological neuroimaging studies. We found that pharmaco-modulation of acetylcholine evoked both increased activity in the anterior cingulate and decreased activity in the opercular and insular cortex. In large independent meta-analyses of non-pharmacological neuroimaging research, we demonstrate that during attentional engagement these cortical areas exhibit (1) task-related co-activation with the basal forebrain, (2) task-related co-activation with one another, and (3) spatial overlap with dense cholinergic innervations originating from the basal forebrain, as estimated by multimodal positron emission tomography and magnetic resonance imaging. Finally, we provide meta-analytic evidence that pharmaco-modulation of acetylcholine also induces a speeding of responses to targets with no apparent tradeoff in accuracy. In sum, we demonstrate in humans that acetylcholinergic modulation of midcingulo-insular hubs of the ventral attention/salience network via basal forebrain afferents may coordinate selection of task relevant information, thereby facilitating cognition and behavior.

Chronic neuroinflammation during aging leads to cholinergic neurodegeneration in the mouse medial septum

BackgroundLow-grade, chronic inflammation in the central nervous system characterized by glial reactivity is one of the major hallmarks for aging-related neurodegenerative diseases like Alzheimer’s disease (AD). The basal forebrain cholinergic neurons (BFCN) provide the primary source of cholinergic innervation of the human cerebral cortex and may be differentially vulnerable in various neurodegenerative diseases. However, the impact of chronic neuroinflammation on the cholinergic function is still unclear.MethodsTo gain further insight into age-related cholinergic decline, we investigated the cumulative effects of aging and chronic neuroinflammation on the structure and function of the septal cholinergic neurons in transgenic mice expressing interleukin-6 under the GFAP promoter (GFAP-IL6), which maintains a constant level of gliosis. Immunohistochemistry combined with unbiased stereology, single cell 3D morphology analysis and in vitro whole cell patch-clamp measurements were used to validate the structural and functional changes of BFCN and their microglial environment in the medial septum.ResultsStereological estimation of MS microglia number displayed significant increase across all three age groups, while a significant decrease in cholinergic cell number in the adult and aged groups in GFAP-IL6 mice compared to control. Moreover, we observed age-dependent alterations in the electrophysiological properties of cholinergic neurons and an increased excitability profile in the adult GFAP-IL6 group due to chronic neuroinflammation. These results complimented the significant decrease in hippocampal pyramidal spine density seen with aging and neuroinflammation.ConclusionsWe provide evidence of the significant impact of both aging and chronic glial activation on the cholinergic and microglial numbers and morphology in the MS, and alterations in the passive and active electrophysiological membrane properties of septal cholinergic neurons, resulting in cholinergic dysfunction, as seen in AD. Our results indicate that aging combined with gliosis is sufficient to cause cholinergic disruptions in the brain, as seen in dementias.