Monica S. Guzman

Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission.

A novel mouse model that eliminates cholinergic neurotransmission in the striatum while leaving glutamate release intact reveals differential effects on cocaine-induced behavior and dopaminergic responses.

ChAT–ChR2–EYFP Mice Have Enhanced Motor Endurance But Show Deficits in Attention and Several Additional Cognitive Domains

Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT–ChR2–EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT–ChR2–EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.

Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on transcriptional regulation and control of locomotor behavior.

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to “turn down” neuronal circuits controlling locomotion.

Elimination of the vesicular acetylcholine transporter in the forebrain causes hyperactivity and deficits in spatial memory and long-term potentiation

Basal forebrain cholinergic neurons, which innervate the hippocampus and cortex, have been implicated in many forms of cognitive function. Immunolesion-based methods in animal models have been widely used to study the role of acetylcholine (ACh) neurotransmission in these processes, with variable results. Cholinergic neurons have been shown to release both glutamate and ACh, making it difficult to deduce the specific contribution of each neurotransmitter on cognition when neurons are eliminated. Understanding the precise roles of ACh in learning and memory is critical because drugs that preserve ACh are used as treatment for cognitive deficits. It is therefore important to define which cholinergic-dependent behaviors could be improved pharmacologically. Here we investigate the contributions of forebrain ACh on hippocampal synaptic plasticity and cognitive behavior by selective elimination of the vesicular ACh transporter, which interferes with synaptic storage and release of ACh. We show that elimination of vesicular ACh transporter in the hippocampus results in deficits in long-term potentiation and causes selective deficits in spatial memory. Moreover, decreased cholinergic tone in the forebrain is linked to hyperactivity, without changes in anxiety or depression-related behavior. These data uncover the specific contribution of forebrain cholinergic tone for synaptic plasticity and behavior. Moreover, these experiments define specific cognitive functions that could be targeted by cholinergic replacement therapy.

The Transient Receptor Potential Melastatin 2 (TRPM2) Channel Contributes to β-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment

In Alzheimers disease, accumulation of soluble oligomers of β-amyloid peptide is known to be highly toxic, causing disturbances in synaptic activity and neuronal death. Multiple studies relate these effects to increased oxidative stress and aberrant activity of calcium-permeable cation channels leading to calcium imbalance. The transient receptor potential melastatin 2 (TRPM2) channel, a Ca2+-permeable nonselective cation channel activated by oxidative stress, has been implicated in neurodegenerative diseases, and more recently in amyloid-induced toxicity. Here we show that the function of TRPM2 is augmented by treatment of cultured neurons with β-amyloid oligomers. Aged APP/PS1 Alzheimers mouse model showed increased levels of endoplasmic reticulum stress markers, protein disulfide isomerase and phosphorylated eukaryotic initiation factor 2α, as well as decreased levels of the presynaptic marker synaptophysin. Elimination of TRPM2 in APP/PS1 mice corrected these abnormal responses without affecting plaque burden. These effects of TRPM2 seem to be selective for β-amyloid toxicity, as ER stress responses to thapsigargin or tunicamycin in TRPM2−/− neurons was identical to that of wild-type neurons. Moreover, reduced microglial activation was observed in TRPM2−/−/APP/PS1 hippocampus compared with APP/PS1 mice. In addition, age-dependent spatial memory deficits in APP/PS1 mice were reversed in TRPM2−/−/APP/PS1 mice. These results reveal the importance of TRPM2 for β-amyloid neuronal toxicity, suggesting that TRPM2 activity could be potentially targeted to improve outcomes in Alzheimers disease. SIGNIFICANCE STATEMENT Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress sensing calcium-permeable channel that is thought to contribute to calcium dysregulation associated with neurodegenerative diseases, including Alzheimers disease. Here we show that oligomeric β-amyloid, the toxic peptide in Alzheimers disease, facilitates TRPM2 channel activation. In mice designed to model Alzheimers disease, genetic elimination of TRPM2 normalized deficits in synaptic markers in aged mice. Moreover, the absence of TRPM2 improved age-dependent spatial memory deficits observed in Alzheimers mice. Our results reveal the importance of TRPM2 for neuronal toxicity and memory impairments in an Alzheimers mouse model and suggest that TRPM2 could be targeted for the development of therapeutic agents effective in the treatment of dementia.

Forebrain Deletion of the Vesicular Acetylcholine Transporter Results in Deficits in Executive Function, Metabolic, and RNA Splicing Abnormalities in the Prefrontal Cortex

One of the key brain regions in cognitive processing and executive function is the prefrontal cortex (PFC), which receives cholinergic input from basal forebrain cholinergic neurons. We evaluated the contribution of synaptically released acetylcholine (ACh) to executive function by genetically targeting the vesicular acetylcholine transporter (VAChT) in the mouse forebrain. Executive function was assessed using a pairwise visual discrimination paradigm and the 5-choice serial reaction time task (5-CSRT). In the pairwise test, VAChT-deficient mice were able to learn, but were impaired in reversal learning, suggesting that these mice present cognitive inflexibility. Interestingly, VAChT-targeted mice took longer to reach criteria in the 5-CSRT. Although their performance was indistinguishable from that of control mice during low attentional demand, increased attentional demand revealed striking deficits in VAChT-deleted mice. Galantamine, a cholinesterase inhibitor used in Alzheimers disease, significantly improved the performance of control mice, but not of VAChT-deficient mice on the 5-CSRT. In vivo magnetic resonance spectroscopy showed altered levels of two neurochemical markers of neuronal function, taurine and lactate, suggesting altered PFC metabolism in VAChT-deficient mice. The PFC of these mice displayed a drastic reduction in the splicing factor heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), whose cholinergic-mediated reduction was previously demonstrated in Alzheimers disease. Consequently, several key hnRNPA2/B1 target transcripts involved in neuronal function present changes in alternative splicing in VAChT-deficient mice, including pyruvate kinase M, a key enzyme involved in lactate metabolism. We propose that VAChT-targeted mice can be used to model and to dissect the neurochemical basis of executive abnormalities.

Increased prion protein processing and expression of metabotropic glutamate receptor 1 in a mouse model of Alzheimer's disease

Prion protein (PrPC), a glycosylphosphatidylinositol‐anchored protein corrupted in prion diseases, has been shown recently to interact with group I metabotropic glutamate receptors (mGluRs). Moreover, both PrPC and mGluRs were proposed to function as putative receptors for β‐amyloid in Alzheimers disease. PrPC can be processed in neurons via α or β‐cleavage to produce PrPC fragments that are neuroprotective or toxic, respectively. We found PrPC α‐cleavage to be 2–3 times higher in the cortex of APPswe/PS1dE9 mice, a mouse model of Alzheimers disease. A similar age‐dependent increase was observed for PrPC β‐cleavage. Moreover, we observed considerable age‐dependent increase in cortical expression of mGluR1, but not mGluR5. Exposure of cortical neuronal cultures to β‐amyloid oligomers upregulated mGluR1 and PrPC α‐cleavage, while activation of group I mGluRs increased PrPC shedding from the membrane, likely due to increased levels of a disintegrin and metalloprotease10, a key disintegrin for PrPC shedding. Interestingly, a similar increase in a disintegrin and metalloprotease10 was detected in the cortex of 9‐month‐old APPswe/PS1dE9 animals. Our experiments reveal novel and complex processing of PrPC in connection with mGluR overexpression that seems to be triggered by β‐amyloid peptides.

Mice with selective elimination of striatal acetylcholine release are lean, show altered energy homeostasis and changed sleep/wake cycle

Cholinergic neurons are known to regulate striatal circuits; however, striatal‐dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)D2‐Cre‐flox/flox mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChTD2‐Cre‐flox/flox mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChTD2‐Cre‐flox/flox mice showed increased physical activity. Interestingly, VAChTD2‐Cre‐flox/flox mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity.

Regulation of Cognitive Processing by Hippocampal Cholinergic Tone

Abstract Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric diseases. Here we tested how information processing is regulated by cholinergic tone in genetically modified mice targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine release. We measured long‐term potentiation of Schaffer collateral‐CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of paired associates learning task (PAL). Acquisition of information in the mouse PAL task correlated to levels of hippocampal VAChT, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signaling. Disrupted forebrain cholinergic signaling also affected working memory, a result reproduced by selectively decreasing VAChT in the hippocampus. In contrast, spatial memory was relatively preserved, whereas reversal spatial memory was sensitive to decreased hippocampal cholinergic signaling. This work provides a refined roadmap of how synaptically secreted acetylcholine influences distinct behaviors and suggests that distinct forms of cognitive processing may be regulated in different ways by cholinergic activity.

Deletion of the Vesicular Acetylcholine Transporter from Pedunculopontine/laterodorsal tegmental neurons modifies gait

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinsons disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long‐term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1‐Cre‐flox/flox). VAChTEn1‐Cre‐flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire‐hang and grip‐force tests. Young VAChTEn1‐Cre‐flox/flox mice (2–5 months‐old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1‐Creflox/flox mice (13–16 months‐old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT‐deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients.