IntroductionEarly synaptic failure is widely considered a primary driver of cognitive decline in Alzheimer's disease (AD), and previous studies have suggested that dopaminergic signaling may contribute to hippocampal synaptic dysfunction. Among dopaminergic receptors, dopamine D3 receptors (D3Rs) have emerged as important modulators of synaptic plasticity and cognitive processes, but their role in AD-related synaptic impairment remains unclear. The aim of this study was to determine whether pharmacological blockade of D3Rs could restore memory deficits and hippocampal synaptic dysfunction in preclinical models of AD.MethodsBehavioral studies, including novel object recognition (NOR), novel object location (NOL), and open-field tests, were performed to evaluate recognition memory, spatial memory, locomotor activity, and anxiety-related behavior in two mechanistically distinct mouse models of AD: triple-transgenic 3xTg-AD mice and α7 nicotinic acetylcholine receptor knockout (α7KO) mice. Electrophysiological recordings in hippocampal slices were used to assess AMPA/NMDA ratio, basal synaptic transmission, and long-term potentiation (LTP). qPCR and western blot analyses were performed to evaluate hippocampal D3R mRNA and protein expression, respectively. Pharmacological treatments included the selective D3R antagonist NGB-2904 and cariprazine, a clinically approved antipsychotic with high affinity for D3Rs.ResultsRecognition and spatial memory deficits were rescued by NGB-2904 and cariprazine in both AD models, without affecting locomotor activity or anxiety-related behavior. No sex-dependent differences were observed in the behavioral response to treatment. Electrophysiological recordings revealed a reduced AMPA/NMDA ratio and impaired LTP in both models, while basal synaptic transmission was selectively reduced in 3xTg-AD mice. D3R-targeting compounds restored synaptic transmission and plasticity. Inhibition of PKA prevented the rescue of LTP induced by D3R blockade, suggesting the involvement of the cAMP/PKA signaling pathway. Both models displayed reduced hippocampal D3R mRNA expression and protein levels, suggesting that the residual population of D3Rs might represent a viable target.ConclusionTogether, these findings demonstrate that D3R-targeting compounds rescue synaptic plasticity and memory deficits in two mechanistically distinct AD models. These results support a role for dopaminergic signaling in early synaptic dysfunction and highlights D3R modulation as a relevant pathway for further investigation in AD-related cognitive impairment.

Astrocytes regulate hippocampal excitatory transmission by controlling glutamate dynamics, yet the contribution of cholinergic signaling to these processes remains poorly understood. Here, we investigated the role of astrocytic α7 nicotinic acetylcholine receptors (α7-nAChRs) in regulating glutamate homeostasis, synaptic plasticity, and memory flexibility in the hippocampus. Using ultrastructural analyses, functional imaging of glutamate, calcium, and sodium, electrophysiology, and behavioral approaches, we studied α7-nAChR knockout mice (α7KO) and an AAV-based mouse model enabling astrocyte-specific re-expression of α7-nAChRs (α7KI-astro). Electron microscopy revealed abundant α7nAChR expression in astrocytic processes at excitatory synapses in the hippocampal CA1 region. In α7KO mice, astrocytic calcium signaling and glutamate uptake were impaired. These alterations of glutamate dynamics were accompanied by reduced expression and altered localization of the glutamate transporter GLT-1, retraction of perisynaptic astrocytic processes, and deficits in synaptic fatigue, vesicle recycling, long-term depression, and memory flexibility. In α7KI-astro mice, calcium signaling, glutamate dynamics, synaptic plasticity, and memory flexibility were restored. Similarly, pharmacological enhancement of GLT-1 activity with ceftriaxone rescued both synaptic and behavioral deficits. Together, these findings identify astrocytic α7-nAChRs as critical regulators of glutamate homeostasis and hippocampal function, revealing a cholinergic mechanism through which astrocytes influence synaptic plasticity and cognitive flexibility.

Although post-traumatic stress disorder (PTSD) occurs more in women than in men, how sex influences trauma susceptibility remains largely unknown. We developed the arousal-based individual screening (AIS) model, which identifies mice as susceptible/resilient to PTSD-like phenotypes, based on changes in startle reactivity induced by 24-hour-restraint. To test the hypothesis that sex drives trauma susceptibility/resilience, we applied a multidisciplinary approach involving electrophysiological, structural, and synaptoproteomic analyses of the hippocampus in susceptible and resilient mice of both sexes. Female mice were more susceptible to the trauma than male mice and exhibited long-lasting PTSD-like phenotypes. Long-term potentiation (LTP) was impaired in hippocampal slices of both male and female susceptible mice, whereas short-term presynaptic forms of plasticity and vesicle recycling remained unchanged. Increased apical dendritic length and augmented basal dendritic spine density of pyramidal neurons were found in CA1 of male susceptible mice, while decreased dendritic length of granule neurons was uncovered in the dentate gyrus of female resilient mice. Although minor synaptoproteomic changes were observed, bioinformatic analysis suggested sex- and susceptibility/resilience-dependent profiles. Notably, several pathways involving RHO Family GTPases were found to be upregulated exclusively in susceptible male mice. Accordingly, the Rac1/Rac3 GTPases inhibitor EHop-016 rescued the hippocampal LTP impairment in susceptible male mice but not in susceptible female mice. Our findings suggest that the AIS model mirrors sex differences in PTSD susceptibility/resilience highlighting associated functional, molecular and structural alterations. This model may represent a critical first step for studying sex-dependent pathophysiological mechanisms subserving PTSD susceptibility and for sex-tailored drug development.

Protein post-translational modifications are critical regulators of synaptic function, and their disruption is implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Among these modifications, S-palmitoylation, catalyzed by zinc finger DHHC domain-containing (zDHHC) S-acyltransferases, has been reported to modulate the localization and activity of proteins crucial for synaptic plasticity and amyloid-β (Aβ) metabolism. We analyzed zDHHC expression and protein S-palmitoylation levels in hippocampi from 3×Tg-AD mice and post-mortem AD patient samples using western blotting and acyl-biotin exchange assays. To evaluate the therapeutic potential of modulating S-palmitoylation, we performed in vivo zDHHC inhibition through chronic intranasal administration of the S-palmitoylation inhibitor 2-bromopalmitate (2-BP) or locked nucleic acid-based antisense oligonucleotides targeting zDHHC7. Additionally, hippocampal zDHHC silencing was achieved using lentiviral vectors carrying short hairpin RNA. Palmitoyl-proteome analysis was conducted on zDHHC7-silenced hippocampi to identify specific targets involved in AD pathogenesis. We observed a significant increase in zDHHC7 expression and S-palmitoylation of synaptic proteins in the hippocampi of both 3×Tg-AD mice and AD patients. Chronic intranasal administration of 2-BP restored synaptic plasticity, reduced hippocampal Aβ deposition, improved cognitive function, and extended lifespan in both male and female 3×Tg-AD mice. Similarly, hippocampal silencing of zDHHC7 prevented cognitive deficits in the same model. Palmitoyl-proteome analysis identified several zDHHC7 targets potentially involved in neurodegeneration. Importantly, hippocampal protein S-palmitoylation levels showed a significant inverse correlation with cognitive performance as measured by the Mini-Mental State Examination in patients. These findings indicate that aberrant S-palmitoylation plays a critical role in the synaptic dysfunction and cognitive impairments observed in AD. Targeting zDHHC enzymes represents a novel and promising therapeutic approach to address cognitive decline in AD patients by mitigating pathological S-palmitoylation.

Alpha7 nicotinic acetylcholine receptors (α7-nAChRs) are ionotropic, Ca2+-permeable receptors highly expressed in brain regions involved in memory formation, such as the hippocampus. Their activation induces cation influx and neuronal depolarization, which in turn promotes glutamate release—highlighting a crucial interplay between cholinergic and glutamatergic signaling in the healthy brain. Interestingly, the genetic deletion of α7-nAChRs in mice (α7-KO mice) leads to an Alzheimer’s disease (AD)-like phenotype characterized by aberrant amyloid-β accumulation, tau phosphorylation, and neuroinflammation in aged (>12 months) mice. However, glutamatergic transmission in these mice prior to the onset of the AD-like phenotype has been poorly investigated. We thus studied molecular and functional properties of glutamatergic transmission in 4–6-months-old α7-KO mice, representing a prodromal phase of the AD-like neuropathology. We found that hippocampal CA1 neurons in brain slices from α7-KO mice showed a reduced frequency of the spontaneous excitatory post-synaptic currents (sEPSCs) compared to those of wild-type (WT) mice. On the contrary, the amplitude of sEPSCs was not affected, although in α7-KO neurons these currents displayed a longer rise time than in wild-type. CA1 neurons from α7-KO mice also exhibited a significantly smaller evoked NMDA currents than WT neurons, whereas AMPA currents were unaffected. From a molecular point of view, hippocampal neurons of α7-KO mice exhibited an increased expression of the pre-synaptic protein Synapsin-1 and of the NMDA subunits GluN2A and GluN2B, but no modifications in the expression of AMPA receptor subunits (GluA1 and GluA2) were found. Importantly, selective re-expression of the α7-nAChRs in neurons of α7-KO mice restored the evoked NMDA current amplitude and the rise time of sEPSCs, but it did not rescue the frequency of sEPSCs, thus suggesting that post-synaptic integrity depends on neuronal α7-nAChRs.

This commentary critically examines the long-standing emphasis on amyloid-β (Aβ)-based therapies in Alzheimer's disease (AD), despite numerous clinical trial failures. It highlights the urgency to reassess research methodologies and challenges the initiation of anti-Aβ trials in preclinical stages of the disease without conclusive proofs of their safety and efficacy. Instead, a comprehensive approach that considers Aβ's physiological roles and addresses AD complex nature is suggested, encouraging the idea that clinical trial failures may result from targeting the wrong mechanism. Evidence-based scientific research is needed to advance with AD treatment, moving beyond the current conception of Aβ hypothesis.

Dopamine D3 receptors (D3Rs) modulate neuronal activity in several brain regions including the hippocampus. Although previous studies reported that blocking D3Rs exerts pro-cognitive effects, their involvement in hippocampal synaptic function and memory in the healthy and aged brain has not been thoroughly investigated. We demonstrated that in adult wild type (WT) mice, D3R pharmacological blockade or genetic deletion as in D3 knock out (KO) mice, converted the weak form of long-term potentiation (LTP1) into the stronger long-lasting LTP (LTP2) via the cAMP/PKA pathway, and allowed the formation of long-term memory. D3R effects were mainly mediated by post-synaptic mechanisms as their blockade enhanced basal synaptic transmission (BST), AMPAR-mediated currents, mEPSC amplitude, and the expression of the post-synaptic proteins PSD-95, phospho(p)GluA1 and p-CREB. Consistently, electron microscopy revealed a prevalent expression of D3Rs in post-synaptic dendrites. Interestingly, with age, D3Rs decreased in axon terminals while maintaining their levels in post-synaptic dendrites. Indeed, in aged WT mice, blocking D3Rs reversed the impairment of LTP, BST, memory, post-synaptic protein expression, and PSD length. Notably, aged D3-KO mice did not exhibit synaptic and memory deficits. In conclusion, we demonstrated the fundamental role of D3Rs in hippocampal synaptic function and memory, and their potential as a therapeutic target to counteract the age-related hippocampal cognitive decline.

Background: Soluble aggregates of oligomeric forms of tau protein (oTau) have been associated with impairment of synaptic plasticity and memory in Alzheimer's disease. However, the molecular mechanisms underlying the synaptic and memory dysfunction induced by elevation of oTau are still unknown. Methods: This work used a combination of biochemical, electrophysiological and behavioral techniques. Biochemical methods included analysis of phosphorylation of the cAMP-responsive element binding (CREB) protein, a transcriptional factor involved in memory, histone acetylation, and expression immediate early genes c-Fos and Arc. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated both short-term spatial memory and associative memory. These phenomena were examined following oTau elevation. Results: Levels of phospho-CREB, histone 3 acetylation at lysine 27, and immediate early genes c-Fos and Arc, were found to be reduced after oTau elevation during memory formation. These findings led us to explore whether up-regulation of various components of the nitric oxide (NO) signaling pathway impinging onto CREB is capable of rescuing oTau-induced impairment of plasticity, memory, and CREB phosphorylation. The increase of NO levels protected against oTau-induced impairment of LTP through activation of soluble guanylyl cyclase. Similarly, the elevation of cGMP levels and stimulation of the cGMP-dependent protein kinases (PKG) re-established normal LTP after exposure to oTau. Pharmacological inhibition of cGMP degradation through inhibition of phosphodiesterase 5 (PDE5), rescued oTau-induced LTP reduction. These findings could be extrapolated to memory because PKG activation and PDE5 inhibition rescued oTau-induced memory impairment. Finally, PDE5 inhibition re-established normal elevation of CREB phosphorylation and cGMP levels after memory induction in the presence of oTau. Conclusions: Up-regulation of CREB activation through agents acting on the NO cascade might be beneficial against tau-induced synaptic and memory dysfunctions.

Protein post-translational modifications (PTM) play a crucial role in the modulation of synaptic function and their alterations are involved in the onset and progression of neurodegenerative disorders. S-palmitoylation is a PTM catalyzed by zinc finger DHHC domain containing (zDHHC) S-acyltransferases that affects both localization and activity of proteins regulating synaptic plasticity and amyloid-β (Aβ) metabolism. Here, we found significant increases of both zDHHC7 expression and protein S-palmitoylation in hippocampi of both 3×Tg-AD mice and post-mortem Alzheimer’s disease (AD) patients. Chronic intranasal administration of the S-palmitoylation inhibitor 2-bromopalmitate counteracted synaptic plasticity and cognitive deficits, reduced the Aβ deposition in the hippocampus and extended the lifespan of both male and female 3×Tg-AD mice. Moreover, hippocampal silencing of zDHHC7 prevented the onset of cognitive deficits in the same experimental model. We also identified a FoxO1-mediated epigenetic mechanism inducing zDHHC7 expression, which was triggered by brain insulin resistance in 3×Tg-AD mice. Finally, in hippocampi of AD patients S-palmitoylation levels of Beta-Secretase 1 were associated with Aβ 1 to 42 load and they inversely correlated with Mini Mental State Examination scores. Our data reveal a key role of both zDHHC7 overexpression and protein hyperpalmitoylation in the onset and progression of AD-related alterations of synaptic plasticity and memory.

Growing evidence points to altered metabolic signals as a risk factor for Alzheimer’s disease (AD). The high fatty acid levels and brain insulin resistance (BIR) found in AD brains may impinge on protein S-palmitoylation, which is a post-translational modification critically involved in the regulation of neuronal protein localization and synaptic function. Our previous findings highlighted the critical role of aberrant palmitoylation in BIR-dependent memory impairment (Spinelli et al., 2017). We analyzed both the expression of palmitoyl-transferase enzymes (zDHHCs) in mouse AD brains and S-palmitoylation levels of key proteins in human AD brains. We also tested the effect of chronic intranasal injection of the palmitoylation inhibitor 2-bromopalmitate (2BP) on a large cohort of male and female 3xTg-AD mice, starting from 3 months of age, by performing cognitive (novel object recognition and object displacement tests), electrophysiological (LTP), immunohistochemical (Abeta deposition) and molecular analyses (Abeta measurement by ELISA). Finally, we investigated the effects of lentiviral particles-mediated zDHHC7 or zDHHC21 silencing in the hippocampus of 3xTg-AD. We found increased levels of zDHHC7 and zDHHC21 in the hippocampus of 3xTg-AD mice and aberrant palmitoylation of key enzymes triggering beta amyloid aggregation in human AD brains. 2BP delayed the onset of memory deficits in both male and female 3xTg-AD mice. More importantly, 2BP significantly enhanced cognitive performances in 6-, 9- and 12-month-old animals. Accordingly, electrophysiological analyses on hippocampal brain slices from 2BP-treated 3xTg-AD mice revealed greater long-term potentiation at CA3-CA1 synapses. In addition, both 2BP-treated male and female mice showed lower Abeta deposition in hippocampus and a significant extension of lifespan. Finally, genetic inhibition of palmitoyl transferase zDHHC7 reverted the aberrant palmitoylation of proteins involved in synaptic plasticity and APP metabolism, and counteracted the onset of neurodegeneration and cognitive deficits in 3xTg-AD mice. This is the first preclinical study on the effect of 2BP treatment on AD-related cognitive decline. Our data indicate that aberrant palmitoylation plays a critical role in the onset and progression of AD and unveil the potential druggable role of zDHHC enzymes in dementia-related neurodegenerative disorders.

Dopamine D3 receptors (D3-Rs) are involved in several functions such as reward and social behavior and they have been proposed as a therapeutical target for addiction, schizophrenia, and Parkinson’s disease. However, the role of D3-Rs in cognition has been poorly investigated, with few studies demonstrating that the activation of D3-Rs impairs attention and working memory. Results obtained in our laboratory suggested that D3 inhibition exerts a pro-cognitive effect on long term potentiation (LTP) and recognition memory on healthy wild-type (WT) mice. Based on these findings, here, we aimed to investigate whether the positive effect shown by D3 inhibition at the synapse could be exploited to counteract the synaptic plasticity and memory impairment found in aged and AD mice. We used D3 knock out (D3-KO), WT at 18-22 months of age as a physiological model of aging, the 3XTg transgenic mouse model of AD and a7 KO mice, recently found to present an AD-like phenotype. The pharmacological inhibition of D3-Rs was obtained with the selective antagonist NGB-2904. To test whether the pharmacological inhibition of D3-Rs or their genetic deletion could rescue LTP and memory impairment, we performed electrophysiological recordings at CA3-CA1 synapses in hippocampal slices and behavioral experiments of novel object recognition, novel object location and Morris Water Maze. We found that D3-Rs antagonism rescued the impairment of LTP, and different forms of memory in aged WT mice. Consistently, aged D3 KO mice showed no alteration of LTP and memory, suggesting that the genetic deletion of D3 receptors prevented the synaptic plasticity and memory decline that occurs with aging. The pharmacological inhibition of D3-Rs was also able to rescue the memory impairment in both 3XTg and a7 KO mice. In conclusion, our findings suggest that the pro-cognitive effect exerted by D3-Rs inhibition or genetic deletion could represent a viable therapeutic strategy to treat the cognitive dysfunction occurring during aging and AD.

Amyloid precursor protein (APP) is a transmembrane protein expressed at the synapse throughout life. In absence of APP, extracellular Aß- and tau-oligomers no longer impair memory and its synaptic surrogate, long-term potentiation (LTP). Synapses include pre- and post-synaptic compartments. However, the relative role of pre- vs. post-synaptic APP at the CA3-CA1 hippocampal synapse in the Aß- and tau-oligomer-induced damage of memory and LTP is not know. We used a combination of gene editing, electrophysiological, behavioral and biochemical techniques to investigate the contribution of the pre- and post-synaptic APP in oligomer induced impairment of memory and LTP. Specific ablation of APP expression in the post-synaptic neuron did not alter the negative effects of Aß and tau oligomers on LTP and memory. In contrast, APP-KO in the presynaptic neuron mimicked and occluded the negative effects of Aß and tau oligomers on LTP and memory, suggesting that presynaptic APP mediates the synapto-toxic effect of Aß and tau. Further investigation demonstrated that pre-synaptic APP deletion (but not post-synaptic) mimicked and occluded the Aß and tau oligomer induced reduction of neurotransmitter vesicle availability during tetanic stimulation and increase of refilling rate after depletion of the readily-releasable pool. Moreover, the increase in refilling rate after deletion of presynaptic APP was dependent upon intracellular calcium. Indeed, intracellular calcium homeostasis was affected both in basal conditions and after activity in full APP-KO mice, likely due to decrease in the levels of inositol 1,4,5-trisphosphate receptor, ryanodine receptor and the calcium pump, SERCA3. These data support the view that Aß and tau oligomers affect synaptic function and memory through pre-synaptic APP.

The failure of cholinergic transmission and the increase of amyloid-β peptide (Aβ) and tau protein are key players in Alzheimer’s disease (AD). The interaction between α7 nicotinic acetylcholine receptors (α7-nAChRs) and Aβ has been previously investigated, but few studies have focused on the crosstalk between α7-nAChRs and tau. We previously reported that Aβ and tau might act independently or concomitantly to impair synaptic plasticity and memory, converging onto common targets such as amyloid precursor protein (APP). Here we investigated the role of α7-nAChRs on tau-mediated impairment of synaptic plasticity and memory, tau neuropathology, and their interplay with APP. We used WT (C57Bl6/J) and α7 KO at different ages. 3XTg models of AD were used as positive controls. We performed electrophysiology to investigate synaptic transmission and plasticity at the CA3-CA1 synapses from hippocampal slices, and behavioral studies (Novel Object Recognition and Location, Fear Conditioning) to evaluate memory. We used western blot for the expression of APP, tau (Ser199, Ser396, Thr205) and GSK-3β (Ser9) phosphorylation. Formation of tau paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) were evaluated by immunohistochemistry and Bielschowsky staining. Immunoreactivity for NeuN was measured to assess neuronal loss. We found that α7 KO mice presented an AD phenotype characterized by the impairment of Long-Term Potentiation and Paired Pulse Facilitation, and the reduction of different types of memory starting at 12 months of age. This age-dependent phenotype was paralleled by an increase of APP expression, hyperphosphorylation of tau, and a decrease of GSK-3β (Ser9) phosphorylation. α7 KO hippocampi presented a marked tau neuropathology with an increase of PHF-1 immunoreactivity, the presence of PHFs and NFTs, and neuronal loss. In summary, the lack of α7-nAChRs was sufficient to trigger an AD-like phenotype characterized by a marked tau pathology. Our data also suggest that the concomitant increase of APP expression might contribute to tau neurotoxicity.

Gene transcription mechanisms leading to synaptic plasticity and memory formation, including phosphorylation of the transcription factor CREB, are perturbed in Alzheimer’s disease (AD). In this regard, cyclic guanosine monophosphate (cGMP), which activates cGMP-dependent protein kinases that, in turn, phosphorylate CREB, has been implicated in the memory and synaptic plasticity impairment occurring in the disease. Sildenafil (Viagra ® ), the well-known FDA approved inhibitor of phosphodiesterase 5 (PDE5I), the enzyme that degrades cGMP, has shown efficacy in AD animal models (Zuccarello et al, Biochem Pharmacol. 2020) and was recently significantly associated with a reduced risk of AD in an unbiased study including 7.23 million individuals (Fang et al, Nat. Aging 2020). Following the development of a library of small molecules inhibiting phosphodiesterase 5 (PDE5), compound efficacy was tested in different animal models of Aβ and tau elevation. Specifically, these animals were tested using a combination of biochemical (western blotting of proteins involved with gene transcription machinery), electrophysiological [analysis of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation], and behavioral (assessment of spatial and associative memory through radial arm water maze and fear conditioning) techniques. Structure activity relationship (SAR) analysis of existing PDEI scaffolds led to the design and synthesis of compound 7a, a quinoline customized for AD with improved physiochemical properties compared to existing inhibitors. The molecule has outstanding inhibitory activity against PDE5 (IC 50 = 0.27nM) and selectivity against all other PDE isoforms (PDE5/PDEs > 1000). It also crosses the blood brain barrier (AUC 0-t ratio = 0.41) and has similar T max values in the brain and plasma, indicating that its distribution to the brain is fast. Moreover, similar to other PDE5Is, 7a re-established normal LTP and both spatial and associative memory after Aβ and tau elevation. Finally, the compound re-established normal increase of CREB phosphorylation and cGMP levels after memory induction in the presence of Aβ and tau. Up-regulation of CREB activation through PDE5 inhibitors might be beneficial against Aβ and tau-induced synaptic and memory dysfunctions.

BACKGROUND:Object recognition task (ORT) is a widely used behavioral paradigm to assess memory in rodent models, due to its easy technical execution, the lack of aversive stressful stimuli, and the possibility to repeat the test on the same animals. However, mouse exploration might be strongly influenced by a variety of variables.OBJECTIVE:To study whether innate preferences influenced exploration in male and female wild type mice and the Alzheimer's disease (AD) model 3xTg.METHODS:We first evaluated how object characteristics (material, size, and shape) influence exploration levels, latency, and exploration modality. Based on these findings, we evaluated whether these innate preferences biased the results of ORT performed in wild type mice and AD models.RESULTS:Assessment of Exploration levels, i.e., the time spent in exploring a certain object in respect to the total exploration time, revealed an innate preference for objects made in shiny materials, such as metal and glass. A preference for bigger objects characterized by higher affordance was also evident, especially in male mice. When performing ORT, exploration was highly influenced by these innate preferences. Indeed, both wild type and AD mice spent more time in exploring the metal object, regardless of its novelty. Furthermore, the use of objects with higher affordance such as the cube was a confounding factor leading to "false" results that distorted ORT interpretation.CONCLUSION:When designing exploration-based behavioral experiments aimed at assessing memory in healthy and AD mice, object characteristics should be carefully evaluated to improve scientific outcomes and minimize possible biases.

EDITORIAL article Front. Mol. Neurosci., 14 March 2022Sec.Brain Disease Mechanisms https://doi.org/10.3389/fnmol.2022.876224

The nitric oxide (NO)/cGMP pathway has been extensively studied for its pivotal role in synaptic plasticity and memory processes, resulting in an increase of cAMP response element-binding (CREB) phosphorylation, and consequent synthesis of plasticity-related proteins. The NO/cGMP/CREB signaling is downregulated during aging and neurodegenerative disorders and is affected by Amyloid-β peptide (Aβ) and tau protein, whose increase and deposition is considered the key pathogenic event of Alzheimer's disease (AD). On the other hand, in physiological conditions, the crosstalk between the NO/cGMP/PKG/CREB pathway and Aβ ensures long-term potentiation and memory formation. This review summarizes the current knowledge on the interaction between the NO/cGMP/PKG/CREB pathway and Aβ in the healthy and diseased brain, offering a new perspective to shed light on AD pathophysiology. We will focus on the synaptic mechanisms underlying Aβ physiological interplay with cGMP pathway and how this balance is corrupted in AD, as high levels of Aβ interfere with NO production and cGMP molecular signaling leading to cognitive impairment. Finally, we will discuss results from preclinical and clinical studies proposing the increase of cGMP signaling as a therapeutic strategy in the treatment of AD.

Implantation of guide cannulas is a widely used technique to access specific brain areas. Although commercially available, the need to personalize these implants and the high cost prompted us to design open-source customized devices taking advantage of 3D printing technology. Our cannulas consisted in a 3D-printed head mount designed according to the Paxinos coordinates to reach the CA1 area of the hippocampus. To cut guide cannulas to the proper length, we designed and realized an original 3D-printed linear motion apparatus. Polylactic acid thermoplastic polymer was used as printing material. Homemade or commercial cannulas were implanted in 4- to 6-month-old wild-type mice and intrahippocampal injections of amyloid-β peptide at different concentrations were performed. In vivo behavioral studies of novel object recognition indicated that results obtained with homemade versus commercial devices were comparable. Methylene blue injections and Nissl staining confirmed the correct localization of cannulas in the CA1 area of mouse hippocampus. Our method allows a fast manufacturing of hippocampal cannulas preserving the required precision at very low cost. Furthermore, this system can be easily modified to produce cannulas to target other brain areas. In conclusion, 3D printing might be used as a useful and versatile technology to realize open-source customized devices in neuroscience laboratories.

Growing evidence points to altered metabolic signals as a risk factor for Alzheimer’s disease (AD). The high fatty acid levels and brain insulin resistance (BIR) found in AD brains may impinge on protein palmitoylation (P-S-palm), a post-translational modification critically involved in the regulation of neuronal protein localization and synaptic function. Our previous findings highlighted the critical role of aberrant palmitoylation in BIR-dependent memory impairment (Spinelli et al., 2017). We tested the effect of chronic intranasal injection of the palmitoylation inhibitor 2-bromopalmitate (2BP) on a large cohort of male and female 3xTg-AD mice, starting from 3 months of age, by performing cognitive (novel object recognition and object displacement tests), electrophysiological (LTP), immunohistochemical (Abeta deposition) and molecular analyses (Abeta measurement by ELISA). We also analyzed the palmitoyl-proteome (by acyl biotin exchange assay and mass-spectrometry) in the hippocampus of 9-month-old wild type, 3xTg-AD and 2BP-treated 3xTg-AD mice. 2BP delayed the onset of memory deficits in both male and female 3xTg-AD mice. More importantly, 2BP significantly enhanced cognitive performances in 6-, 9- and 12-month-old animals. Accordingly, electrophysiological analyses on hippocampal brain slices from 2BP-treated 3xTg-AD mice revealed greater long-term potentiation at CA3-CA1 synapses. In addition, 2BP mice showed lower Abeta deposition in hippocampus. Finally, palmitoyl-proteome analysis revealed a large number of proteins involved in APP and tau metabolism, synaptic plasticity and brain metabolism aberrantly palmitoylated in the AD mouse model and reverted by 2BP. Our data indicate that aberrant palmitoylation plays a critical role in the onset and progression of AD and reveal novel targets of protein palmitoylation potentially involved in the development of neurodegeneration and dementia. This is also the first preclinical study on the effect of 2BP on AD-related cognitive decline.

Depression is a risk factor for the development of Alzheimer’s disease (AD). A neurobiological and clinical continuum exists between AD and depression, with neuroinflammation and oxidative stress being involved in both diseases. Second-generation antidepressants, in particular selective serotonin reuptake inhibitors (SSRIs), are currently investigated as neuroprotective drugs in AD. By employing a non-transgenic AD model, obtained by intracerebroventricular (i.c.v.) injection of amyloid-β (Aβ) oligomers in 2-month-old C57BL/6 mice, we recently demonstrated that the SSRI fluoxetine (FLX) and the multimodal antidepressant vortioxetine (VTX) reversed the depressive-like phenotype and memory deficits induced by Aβ oligomers rescuing the levels of transforming growth factor-β1 (TGF-β1). Aim of our study was to test FLX and VTX for their ability to prevent oxidative stress in the hippocampus of Aβ-injected mice, a brain area strongly affected in both depression and AD. The long-term intraperitoneal (i.p.) administration of FLX (10 mg/kg) or VTX (5 and 10 mg/kg) for 24 days, starting 7 days before Aβ injection, was able to prevent the over-expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (Nox2) induced by Aβ oligomers. Antidepressant pre-treatment was also able to rescue the mRNA expression of glutathione peroxidase 1 (Gpx1) antioxidant enzyme. FLX and VTX also prevented Aβ-induced neurodegeneration in mixed neuronal cultures treated with Aβ oligomers. Our data represent the first evidence that the long-term treatment with the antidepressants FLX or VTX can prevent the oxidative stress phenomena related to the cognitive deficits and depressive-like phenotype observed in a non-transgenic animal model of AD.