Claudia Balducci

Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein

Inability to form new memories is an early clinical sign of Alzheimer’s disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ1–42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ1–42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrPC) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ1–42 oligomers interact with PrPC, with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ1–42 oligomers are responsible for cognitive impairment in AD and that PrPC is not required.

Reduced anxiety and improved stress coping ability in mice lacking NPY-Y2 receptors.

Neuropeptide Y (NPY) has been implicated in the pathophysiology of certain mood disorders, including depression and anxiety. It is, however, not known which of the five cloned NPY receptors mediate these functions. We investigated the effect of Y2 receptor deletion on anxiety and stress‐related behaviours. In the elevated plus maze, Y2 knock out (Y2−/−) mice showed a 2.7‐fold higher frequency of entering into, and spent 3.8 times more time within, the open arms compared to controls, while entries into the closed arms did not differ. Similarly Y2−/− mice entered the central area of the open field 1.7 times more frequently and also spent 1.8 times more time there. In the light/dark test Y2−/− mice had a 4.8‐fold lower latency to enter the lit area but stayed there 2.6 times longer than control mice. Y2−/− mice displayed 3.2‐fold less immobility in the forced swim test, indicating improved stress coping ability. Y2 receptors are predominantly located presynaptically where they mediate feedback inhibition of neurotransmitter release. Deletion of these receptors may result in enhanced release of NPY, GABA and/or glutamate in brain areas linked to the manifestation of anxiety, and stress‐related behaviour such as the amygdala. Taken together, deletion of the Y2 receptor has revealed an important role of Y2 receptors in the generation of anxiety‐related and stress‐related behaviours in mice.

Dissociable Contribution of 5-HT1A and 5-HT2A Receptors in the Medial Prefrontal Cortex to Different Aspects of Executive Control such as Impulsivity and Compulsive Perseveration in Rats

Serotonin (5-HT) receptors are increasingly recognized as major targets for cognitive enhancement in schizophrenia. Several lines of evidence suggest a pathophysiological role for glutamate NMDA receptors in the prefrontal cortex in schizophrenia and associated disorders in attention and executive functioning. We investigated how the interactions between 5-HT1A and 5-HT2A and glutamate NMDA receptor mechanisms in the medial prefrontal cortex (mPFC) contribute to the control of different aspects of attentional performance. Rats were trained on a five-choice serial reaction time (5-CSRT) task, which provides indices of attentional functioning (percentage of correct responses), executive control (measured by anticipatory and perseverative responses), and speed. The competitive NMDA receptor antagonist CPP (50 ng/side) was infused directly into the mPFC 5 min after infusion of either 8-OH-DPAT (30 and 100 ng/side) or M100907 (100 and 300 ng/side) into the same brain area. Impairments in attentional functioning induced by CPP were completely abolished by both doses of 8-OH-DPAT or M100907. In addition, M100907 abolished the CPP-induced anticipatory responding but had no effects on perseverative over-responding, while 8-OH-DPAT reduced the perseverative over-responding but had no effects on anticipatory responding induced by CPP. The selective 5-HT1A receptor antagonist WAY100635 (30 ng/side) antagonized the effects of 8-OH-DPAT (100 ng/side). 8-OH-DPAT at 30 ng/side reduced the latency of correct responses in controls and CPP-injected rats and lowered the percentage of omissions in CPP-injected rats. The data show that 5-HT1A and 5-HT2A receptors in the mPFC exert opposing actions on attentional functioning and demonstrate a dissociable contribution of 5-HT1A and 5-HT2A receptors in the mPFC to different aspects of executive control such as impulsivity and compulsive perseveration.

Neuropeptide Y gene therapy decreases chronic spontaneous seizures in a rat model of temporal lobe epilepsy

Temporal lobe epilepsy remains amongst the most common and drug refractory of neurological disorders. Gene therapy may provide a realistic therapeutic approach alternative to surgery for intractable focal epilepsies. To test this hypothesis, we applied here a gene therapy approach, using a recombinant adeno-associated viral (rAAV) vector expressing the human neuropeptide Y (NPY) gene, to a progressive and spontaneous seizure model of temporal lobe epilepsy induced by electrical stimulation of the temporal pole of the hippocampus, which replicates many features of the human condition. rAAV-NPY or a control vector lacking the expression cassette (rAAV-Empty) was delivered into the epileptic rat hippocampi at an early progressive stage of the disease. Chronic epileptic rats were video-EEG monitored to establish pre-injection baseline recordings of spontaneous seizures and the effect of rAAV-NPY versus rAAV-Empty vector injection. Both non-injected stimulated controls and rAAV-empty injected rats showed a similar progressive increase of spontaneous seizure frequency consistent with epileptogenesis. The delivery of rAAV-NPY in epileptic rat brain leads to a remarkable decrease in the progression of seizures as compared to both control groups and this effect was correlated with the NPY over-expression in the hippocampus. Moreover, spontaneous seizure frequency was significantly reduced in 40% of treated animals as compared to their pre-injection baseline. Our data show that this gene therapy strategy decreases spontaneous seizures and suppresses their progression in chronic epileptic rats, thus representing a promising new therapeutic strategy.

An N-terminal Fragment of the Prion Protein Binds to Amyloid-β Oligomers and Inhibits Their Neurotoxicity in Vivo

Background: The cellular prion protein (PrPC) could be a toxicity-transducing receptor for amyloid-β (Aβ) oligomers. Results: N1, a naturally occurring fragment of PrPC, binds Aβ oligomers, inhibits their polymerization into fibrils, and suppresses their neurotoxic effects in vitro and in vivo. Conclusion: N1 binds tightly to Aβ oligomers and blocks their neurotoxicity. Significance: Administration of exogenous N1 or related peptides may represent an effective therapy for Alzheimer disease. A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide in the brain. Considerable evidence suggests that soluble Aβ oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aβ oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aβ polymerization into amyloid fibrils. The ability of N1 to bind Aβ oligomers is influenced by positively charged residues in two sites (positions 23–31 and 95–105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aβ oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aβ-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aβ oligomer toxicity and represent an entirely new class of therapeutic agents for AD.

Mutant Prion Protein Expression Causes Motor and Memory Deficits and Abnormal Sleep Patterns in a Transgenic Mouse Model

A familial form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 prion protein (PrP) mutation. Tg(CJD) mice expressing the mouse homolog of this mutant PrP synthesize a misfolded form of the mutant protein, which is aggregated and protease resistant. These mice develop clinical and pathological features reminiscent of CJD, including motor dysfunction, memory impairment, cerebral PrP deposition, and gliosis. Tg(CJD) mice also display electroencephalographic abnormalities and severe alterations of sleep-wake patterns strikingly similar to those seen in a human patient carrying the D178N/V129 mutation. Neurons in these mice show swelling of the endoplasmic reticulum (ER) with intracellular retention of mutant PrP, suggesting that ER dysfunction could contribute to the pathology. These results establish a transgenic animal model of a genetic prion disease recapitulating cognitive, motor, and neurophysiological abnormalities of the human disorder. Tg(CJD) mice have the potential for giving greater insight into the spectrum of neuronal dysfunction in prion diseases.

Multifunctional Liposomes Reduce Brain β-Amyloid Burden and Ameliorate Memory Impairment in Alzheimer's Disease Mouse Models

Alzheimers disease is characterized by the accumulation and deposition of plaques of β-amyloid (Aβ) peptide in the brain. Given its pivotal role, new therapies targeting Aβ are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aβ assemblies and evaluated their efficiency in reducing the Aβ burden in Alzheimers disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood–brain barrier targeting and with phosphatidic acid for Aβ binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aβ assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aβ1–42 (−33%), assessed by ELISA, and the number and total area of plaques (−34%) detected histologically. Also, brain Aβ oligomers were reduced (−70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [11C]Pittsburgh compound B (PIB). The reduction of brain Aβ was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aβ aggregates and promote peptide removal across the blood–brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimers disease.

Blocking ADAM10 synaptic trafficking generates a model of sporadic Alzheimer’s disease

We describe here an innovative, non-transgenic animal model of Alzheimers disease. This model mimics early stages of sporadic disease, which represents the vast majority of cases. The model was obtained by interfering with the complex between a disintegrin and metalloproteinase domain containing protein 10 (ADAM10), the main α-secretase candidate, and its partner, synapse-associated protein 97, a protein of the postsynaptic density-membrane associated guanylate kinase family. Association of ADAM10 with synapse-associated protein 97 governs enzyme trafficking and activity at synapses. Interfering with the ADAM10/synapse-associated protein 97 complex for 2 weeks by means of a cell-permeable peptide strategy is sufficient to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimers disease. After 2 weeks of treatment, we detected progressive Alzheimers disease-like neuropathology, with an increase of β-amyloid aggregate production and of tau hyperphosphorylation, and a selective alteration of N-methyl-d-aspartic acid receptor subunit composition in the postsynaptic compartment of mouse brain. Behavioural and electrophysiological deficits were also induced by peptide treatment.

The γ-secretase modulator CHF5074 restores memory and hippocampal synaptic plasticity in plaque-free Tg2576 mice.

Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimers disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

S 15535, a benzodioxopiperazine acting as presynaptic agonist and postsynaptic 5‐HT1A receptor antagonist, prevents the impairment of spatial learning caused by intrahippocampal scopolamine

The effect of S 15535 (4‐benzodioxan‐5‐yl)1‐(indan‐2‐yl)piperazine), an agonist at presynaptic and antagonist at postsynaptic 5‐HT1A receptors, on the impairment of spatial learning caused by intrahippocampal scopolamine in a two‐platform spatial discrimination task was studied. Scopolamine (4.0 μg μl−1), injected bilaterally into the CA1 region of the dorsal hippocampus 10 min before each training session, impaired choice accuracy with no effect on choice latency and errors of omission. Administered subcutaneously 30 min before each training session, S 15535 1.0 (but not 0.3) mg kg−1 did not modify choice accuracy but prevented its impairment by intrahippocampal scopolamine. WAY 100635, a 5‐HT1A receptor antagonist, injected into the dorsal raphe at 1.0 μg 0.5 μl−1 5 min before scopolamine, had no effect on choice accuracy and latency or errors of omission and did not modify the effect of scopolamine but completely antagonized the effect of S 15535 (1.0 mg kg−1) on scopolamine‐induced impairment of choice accuracy. The results confirm a previous report ( Carli et al., 1998 ) that stimulation of presynaptic 5‐HT1A receptors in the dorsal raphe counteracts the deficit caused by intrahippocampal scopolamine, probably by facilitating the transfer of facilitatory information from the entorhinal cortex to the hippocampus. Drugs that stimulate action on presynaptic 5‐HT1A receptors, such as S 15535 and other partial 5‐HT1A receptors agonists, may be useful in the symptomatic treatment of human memory disturbances associated with loss of cholinergic innervation to the hippocampus.