NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Altered Functionality, Morphology, and Vesicular Glutamate Transporter Expression of Cortical Motor Neurons from a Presymptomatic Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a lethal disorder characterized by the gradual degeneration of motor neurons in the cerebrospinal axis. Whether upper motor neuron hyperexcitability, which is a feature of ALS, provokes dysfunction of glutamate metabolism and degeneration of lower motor neurons via an anterograde process is undetermined. To examine whether early changes in upper motor neuron activity occur in association with glutamatergic alterations, we performed whole-cell patch-clamp recordings to analyze excitatory properties of Layer V cortical motor neurons and excitatory postsynaptic currents (EPSCs) in presymptomatic G93A mice modeling familial ALS (fALS). We found that G93A Layer V pyramidal neurons exhibited altered EPSC frequency and rheobase values indicative of their hyperexcitability status. Biocytin loading of these hyperexcitable neurons revealed an expansion of their basal dendrite arborization. Moreover, we detected increased expression levels of the vesicular glutamate transporter 2 in cortical Layer V of G93A mice. Altogether our data show that functional and structural neuronal alterations associate with abnormal glutamatergic activity in motor cortex of presymptomatic G93A mice. These abnormalities, expected to enhance glutamate release and to favor its accumulation in the motor cortex, provide strong support for the view that upper motor neurons are involved early on in the pathogenesis of ALS.

Postsynaptic alteration of NR2A subunit and defective autophosphorylation of alphaCaMKII at threonine-286 contribute to abnormal plasticity and morphology of upper motor neurons in presymptomatic SOD1G93A mice, a murine model for amyotrophic lateral sclerosis.

Although amyotrophic lateral sclerosis (ALS) has long been considered as a lower motor neuron (MN) disease, degeneration of upper MNs arising from a combination of mechanisms including insufficient growth factor signaling and enhanced extracellular glutamate levels is now well documented. The observation that these mechanisms are altered in presymptomatic superoxide dismutase (SOD1) mice, an ALS mouse model, suggests that defective primary motor cortex (M1) synaptic activity might precede the onset of motor disturbances. To examine this point, we assessed the composition of AMPAR and NMDAR subunits and of the alphaCa²(+)/calmodulin-dependent kinase autophosphorylation at threonine-286 in the triton insoluble fraction from the M1 in postnatal P80-P85 SOD1(G93A) and wild-type mice. We show that presymptomatic SOD1(G93A) exhibit a selective decrease of NR2A subunit expression and of the alphaCa²(+)/calmodulin-dependent kinase autophosphorylation at threonine-286 in the triton insoluble fraction of upper MNs synapses. These molecular alterations are associated with synaptic plasticity defects, and a reduction in upper MN dendritic outgrowth revealing that abnormal neuronal connectivity in the M1 region precedes the onset of motor symptoms. We suggest that the progressive disruption of M1 corticocortical connections resulting from the SOD1(G93A) mutation might extend to adjacent regions and promote development of cognitive/dementia alterations frequently associated with ALS.

Offline ventral subiculum-ventral striatum serial communication is required for spatial memory consolidation.

The hippocampal formation is considered essential for spatial navigation. In particular, subicular projections have been suggested to carry spatial information from the hippocampus to the ventral striatum. However, possible cross-structural communication between these two brain regions in memory formation has thus far been unknown. By selectively silencing the subiculum-ventral striatum pathway we found that its activity after learning is crucial for spatial memory consolidation and learning-induced plasticity. These results provide new insight into the neural circuits underlying memory consolidation and establish a critical role for off-line cross-regional communication between hippocampus and ventral striatum to promote the storage of complex information.

Region-specific changes in the microanatomy of single dendritic spines over time might account for selective memory alterations in ageing hAPPsweTg2576 mice, a mouse model for Alzheimer disease.

Tg2576 mice over-expressing human mutant APP (hAPPswe) show progressive impairments in hippocampal plasticity and episodic memory while fronto-striatal plasticity and procedural memory remain intact. Here we examine the status of synaptic connectivity in the hippocampus and the dorsolateral striatum (DLS) of 3- and 15-month-old Tg2576 and wild-type mice through the analysis of single dendritic spines microanatomy. We found that, in each region, all mice showed a global reduction in the size of spines as a function of age. Ageing mutants, however, exhibited smaller spines with shorter necks on CA1 pyramidal neurons but larger spines with longer necks on DLS spiny neurons compared to their age-matched wild-type controls. Our findings indicate that hippocampal and DLS dendritic spines in hAPPswe mutants undergo a different pattern of morphological changes over time and point to minor alterations in the microanatomy of DLS spines as a compensatory mechanism maintaining procedural abilities in the ageing mutants.

AD-Related N-Terminal Truncated Tau Is Sufficient to Recapitulate In Vivo the Early Perturbations of Human Neuropathology: Implications for Immunotherapy.

The NH2tau 26-44 aa (i.e., NH2htau) is the minimal biologically active moiety of longer 20-22-kDa NH2-truncated form of human tau-a neurotoxic fragment mapping between 26 and 230 amino acids of full-length protein (htau40)-which is detectable in presynaptic terminals and peripheral CSF from patients suffering from AD and other non-AD neurodegenerative diseases. Nevertheless, whether its exogenous administration in healthy nontransgenic mice is able to elicit a neuropathological phenotype resembling human tauopathies has not been yet investigated. We explored the in vivo effects evoked by subchronic intracerebroventricular (i.c.v.) infusion of NH2htau or its reverse counterpart into two lines of young (2-month-old) wild-type mice (C57BL/6 and B6SJL). Six days after its accumulation into hippocampal parenchyma, significant impairment in memory/learning performance was detected in NH2htau-treated group in association with reduced synaptic connectivity and neuroinflammatory response. Compromised short-term plasticity in paired-pulse facilitation paradigm (PPF) was detected in the CA3/CA1 synapses from NH2htau-impaired animals along with downregulation in calcineurin (CaN)-stimulated pCREB/c-Fos pathway(s). Importantly, these behavioral, synaptotoxic, and neuropathological effects were independent from the genetic background, occurred prior to frank neuronal loss, and were specific because no alterations were detected in the control group infused with its reverse counterpart. Finally, a 2.0-kDa peptide which biochemically and immunologically resembles the injected NH2htau was endogenously detected in vivo, being present in hippocampal synaptosomal preparations from AD subjects. Given that the identification of the neurotoxic tau species is mandatory to develop a more effective tau-based immunological approach, our evidence can have important translational implications for cure of human tauopathies.

Progressive cognitive decline in a transgenic mouse model of Alzheimer's disease overexpressing mutant hAPPswe.

The possibility of detecting progressive changes in cognitive function reflecting the spatio-temporal pattern of beta-amyloid peptide (Abeta) deposition was investigated in Tg2576 mice overexpressing the human mutant amyloid precursor protein (hAPP). Here, we show that at 7 months of age, Tg2576 mice exhibited a selective deficit in hippocampus-based operations including a defective habituation of object exploration, a lack of reactivity to spatial novelty and a disruption of allothetic orientation in a cross-shaped maze. At 14 months of age, Tg2576 mice displayed a more extended pattern of behavioral abnormalities, because they failed to react to object novelty and exclusively relied on motor-based orientation in the cross-shaped maze. However, an impaired reactivity to spatial and object novelty possibly reflecting age-related attention deficits also emerged in aged wild-type mice. These findings further underline that early cognitive markers of AD can be detected in Tg2576 mice before Abeta deposition occurs and suggest that as in humans, cognitive deterioration progressively evolves from an initial hippocampal syndrome to global dementia because of the combined effect of the neuropathology and aging.

Altered long-term corticostriatal synaptic plasticity in transgenic mice overexpressing human CU/ZN superoxide dismutase (GLY(93)-->ALA) mutation.

Apart from the extensive loss of motor neurons, degeneration of midbrain dopaminergic cells has been described in both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Mice overexpressing the mutant human Cu/Zn superoxide dismutase (SOD1) show an ALS-like phenotype in that they show a progressive death of motor neurons accompanied by degeneration of dopaminergic cells. To describe the functional alterations specifically associated with this dopaminergic dysfunction, we have investigated the corticostriatal synaptic plasticity in mice overexpressing the human SOD1 (SOD1+) and the mutated (Gly(93)-->Ala) form (G93A+) of the same enzyme. We show that repetitive stimulation of the corticostriatal pathway generates long-term depression (LTD) in SOD1+ mice and in control (G93A-/SOD1-) animals, whereas in G93A+ mice the same stimulation generates an N-methyl-D-aspartic acid receptor-dependent long-term potentiation. No significant alterations were found in the intrinsic membrane properties of striatal medium spiny neurons and basal corticostriatal synaptic transmission of G93A+ mice. Bath perfusion of dopamine or the D(2) dopamine receptor agonist quinpirole restored LTD in G93A+ mice. Consistent with these in vitro results, habituation of locomotor activity and striatal-dependent active avoidance learning were impaired in G93A+ mice. Thus, degeneration of dopaminergic neurons in the substantia nigra of G93A+ mice causes substantial modifications in striatal synaptic plasticity and related behaviors, and may be a cellular substrate of the extrapyramidal motor and cognitive disorders observed in familial and sporadic ALS.

Parallel modifications of spatial memory performances, exploration patterns, and hippocampal theta rhythms in fornix-damaged rats: reversal by oxotremorine.

Learning scores and degrees of divergence of the exploratory patterns (EP) displayed during the acquisition stage of a radial eight-arm maze task were examined in fornix-damaged and sham-operated rats injected either with oxotremorine (0.1 mg/kg) or saline. Modifications of hippocampal rhythmic slow activity (theta) recorded in each condition were analyzed in CA1 and dentate gyrus. Dorsal fornix sections reduced choice accuracy but also induced the adoption of weakly divergent EP. Oxotremorine in animals with lesions reinstates both learning scores and degree of divergence of EP at the levels respectively observed in saline sham-operated animals. Finally, oxotremorine in sham-operated animals did not significantly improve choice accuracy but strongly modified the EP. Preoperatively, theta rhythms indicated a decrease of frequency after oxotremorine administration. Postoperatively, they showed an increase of frequency in animals with lesions that were reinstated at the preoperative level by oxotremorine.

Ibotenic lesions of the nucleus accumbens promote reactivity to spatial novelty in nonreactive DBA mice: implications for neural mechanisms subserving spatial information encoding.

The role of the nucleus accumbens (NA) in forming spatial representations was investigated in C57BL/6 (C57) and DBA/2 (DBA) inbred mice. One week before testing, bilateral excitotoxic lesions were performed in the NA using ibotenic acid. Testing consisted of placing mice in an arena containing 5 objects at a fixed location and, after habituation to the object configuration, examining their reactivity to the displacement (spatial novelty) or the substitution (object novelty) of some of these objects. C57 mice reacted to spatial novelty and DBA mice did not. Both strains, however, reacted to object novelty. The lesion had no effect on C57 mice's performance, but in the DBA mice, it promoted a clear reaction to spatial novelty that was absent in control animals. Radial maze performance also was improved in DBA with NA lesions. Results suggest the NA as a possible site for modulating spatially mediated behaviors in poor-performing subjects.

Open field behaviours and spatial learning performance in C57BL/6 mice: early stage effects of chronic GM1 ganglioside administration.

One month intact C57BL/6 mice were treated with GM1 ganglioside for 3 consecutive weeks. At 2 months of age, treated and control mice were observed in the open-field situation and tested for spatial learning in a radial eight-arm maze. The results showed that, in the open-field, treated mice displayed less freezing but more rearing behavior than control animals. In the radial maze, GM1-treated mice made more correct path choices before the first error within each trial than control mice. However, this improvement was limited to the first stage of training. These results suggest an early stimulating effect of the GM1 ganglioside treatment which could facilitate adaptive reactions to new situations.

Involvement of glutamatergic and dopaminergic systems in the reactivity of mice to spatial and non-spatial change.

Injections of glutamatergic NMDA as well as dopaminergic antagonists produce selective place- but not cue-learning deficits in associative spatial tasks. The present work was aimed at examining if the blockade of NMDA and dopaminergic receptors interferes with the encoding of spatial information in a non-associative task specifically designed for rodents. CD1 mice injected with MK-801 (0.1 and 0.25 mg/kg), haloperidol (0.04 and 0.08 mg/kg), a combination of the lower doses of each drug (haloperidol: 0.04 mg/kg and MK-801: 0.1 mg/kg) or saline were placed in an open field containing five objects and their reactivity to the displacement (spatial change) or the substitution (non-spatial change) of some of these objects was examined. The results show that saline-injected mice reacted to spatial as to non-spatial change by increasing the time spent exploring the displaced objects or the substituted one. Both doses of MK-801 prevented mice from detecting spatial change but did not affect their reactivity to the novel object. Both doses of haloperidol abolished the reactivity of mice to spatial change but the higher dose of the drug also altered the reaction to non-spatial change. Taken together, the present results indicate that the blockade of dopaminergic or glutamatergic NMDA receptors abolishes the detection of spatial novelty. The well-documented impairing effects of haloperidol and MK-801 on spatial learning may, therefore, be the consequence of a drug-induced inability in forming and/or updating spatial representations. The effects of haloperidol was, however, less specific than that of MK-801, since haloperidol always modified activity together with the response to spatial change and, at the higher dose, abolished the detection of both spatial and non-spatial change. Finally, haloperidol pretreatment was found to enhance the effect of MK-801 thus suggesting a possible interaction between the two systems in modulating these behavioral responses.

N-methyl-D-aspartate receptors in the nucleus accumbens are involved in detection of spatial novelty in mice.

The aim of this study was to investigate the role played by intra-accumbens N-methyl-D-aspartate (NMDA) receptors in spatial information encoding. For this purpose, the effect of local administration of both competitive (AP-5) and non-competitive (MK-801) NMDA antagonists was assessed in a task designed to estimate the ability of rodents to encode spatial relationships between discrete stimuli. The task consists of placing mice in an open field containing five objects and, after three sessions of habituation, examining their reactivity to object displacement (spatial novelty) and object substitution (object novelty). The results show that both doses of MK-801 (0.15 and 0.3 microg/side) induced a selective impairment in the capability of mice to detect spatial novelty. A similar effect was obtained by injecting the low dose of the competitive antagonist AP-5 (0.1 microg/side), whereas the high dose (0.15 microg/side) abolished detection of both spatial and object novelty. Taken together, these results show that intra-accumbens injections of low doses of competitive and non-competitive NMDA antagonists can produce selective deficits in processing spatial information resembling those observed after hippocampal damage. Moreover, the fact that pharmacological treatments spare memory processes involved in habituation suggests that NMDA antagonists may interfere with the formation of spatial representations rather than producing memory deficits per se.

Contextual-dependent effects of nucleus accumbens lesions on spatial learning in mice.

The effect of nucleus accumbens lesions on radial maze performance of C57BL/6 and DBA/2 mice was assessed under distinct extra-maze cuing conditions. Among sham-lesioned mice, C57BL/6 performed better under rich than poor cuing conditions whereas DBA performed in the same fashion under both conditions. In C57BL/6, a disruptive effect of lesions was found only in mice tested under rich cuing. Conversely, in DBA/2, the lesions improved performance under poor cuing and disrupted performance under rich cuing. In that strain, a possible lesion-induced enhancement of attention to background stimuli improving performance under poor cuing but producing interference under rich cuing is suggested. In general, the lesions effect seemed to depend on the strain predisposition to implement configural or cue-based responding.

Selective effects of hippocampal and frontal cortex lesions on a spatial learning problem in two inbred strains of mice.

The effects of dorsal hippocampal and medial frontal lesions of the cortex on a spatial learning problem were studied in two inbred strains of mice (C57BL/6 and DBA/2) which present both neuroanatomical differences of such structures and various patterns of spontaneous exploration. The results showed that hippocampal lesions produced impairments of the learning performance in each strain of mouse, but the temporal distribution of the errors over the experiment was found to be strain dependent. On the other hand, medial frontal cortex lesions selectively affected the learning performances since the acquisition process of only the C57BL/6 lesioned mice differed significantly from the other groups. The effects of these lesions are discussed in terms of genetically associated differences of brain structures and functions. It is suggested that investigations of such differences can provide an experimental model for the study of functional and structural recovery.

Spatial learning and memory, maze running strategies and cholinergic mechanisms in two inbred strains of mice.

The acquisition process of the radial maze task was studied in two inbred strains of mice, C57BL/6 and DBA/2. A quantitative and qualitative evaluation of performance was performed and the pretest level of activity was measured. The results showed a significant correlation between activity and performance since the highly active C57BL/6 mice exhibited better performance of the radial maze task than the less active DBA/2 mice. Moreover, for correct trials, strain-dependent maze-running strategies were observed: while both strains displayed about the same percentage of clockwise and spatial strategies, it was observed that among the spatial strategies C57BL/6 used a larger number of different correct solutions. Subsequently, the effect of scopolamine administration on working memory processes was assessed in sequential and discrete trials. A different reactivity of each strain to anti-cholinergic treatment was found in discrete trials since only DBA/2 mice were impaired. The effect of scopolamine is discussed in relation to the different models of information processing involved in learning and memorizing the experimental rule.

Spatial learning in two inbred strains of mice: genotype-dependent effect of amygdaloid and hippocampal lesions.

Spatial learning performance and maze-running strategies were estimated in two inbred strains of mice, C57BL/6 and DBA/2, submitted to an 8-arm radial maze task. Subsequently the genotype-dependent effect of hippocampus and amygdala on the mastering of this task was examined as a function of the different acquisition model provided by each strain. The results firstly show that unoperated C57BL/6 mice reach a higher level of performance and develop a stronger preference for adjacent arms - 45 degrees angle - turns than unoperated DBA/2 mice. In the high learner C57BL/6 strain, both hippocampal and amygdaloid lesions impair performance and modify maze-running strategies. With practice, however, the difference between amygdala-lesioned mice and controls disappears while that between hippocampus-lesioned mice and controls persists. Conversely, in the low learner DBA/2 strain, hippocampal lesions have a negative effect on a single parameter of performance, while amygdaloid lesions only affect maze-running strategies. Taken together, these results confirm the specific control exerted by the hippocampus on spatial learning. Moreover, they suggest that the amygdala can parallel the role of the hippocampus as far as the baseline level of performance of the strain considered is high.

The amygdala mediates the impairing effect of the selective kappa-opioid receptor agonist U-50,488 on memory in CD1 mice.

The effect of the selective kappa-opioid receptor agonist U-50,488 on passive avoidance behaviour was studied in CD1 mice bearing lesions of the amygdaloid complex. The results have shown that post-trial injections of U-50,488 in unoperated mice as well as lesions of the amygdaloid complex in untreated mice decreased retention performances. No further impairment was observed in lesioned mice treated with U-50,488, indicating that amygdala is involved in the mediation of the effects of kappa-opioid receptor agonists on memory. The possibility that kappa-opioid receptors could modulate the memory retention of CD1 mice by influencing their emotional state is discussed.

Enhancement by oxiracetam of passive avoidance improvement induced by the presynaptic muscarinic antagonist secoverine in mice.

Post-trial administration of secoverine (0.5, 1 and 2.5 mg/kg), a presynaptic muscarinic blocker, improved retention in mice tested in a passive avoidance task. The nootropic drug oxiracetam (50 mg/kg), given before both acquisition and retention trial, had no effect alone, but significantly enhanced secoverine improving effects. Performance improvements were also found in combining ineffective doses of the two compounds. Even if not necessarily through a direct and specific action, oxiracetam might activate cholinergic function. Thus, performance improvements resulting from the combination of oxiracetam and secoverine may be due to a simultaneous activation of different cholinergic mechanisms.

Clonidine reverses spatial learning deficits and reinstates theta frequencies in rats with partial fornix section.

Rats received knife-cuts to the dorsal fornix or sham-operations. Half of the animals from each group were injected with clonidine (0.01 mg/kg) and the others with saline before each daily trail of a 10-trial radial 8-arm maze task. The number of choices before the first repetition and the run time were used as performance indices. Lesioned rats were significantly impaired in the acquisition of this task. Clonidine-treated rats, lesioned or not, had an acquisition profile indistinguishable from that of sham-operated saline-injected rats, in spite of their increased run time. When tested one week after the last learning trial in a no-drug condition, lesioned rats treated with clonidine throughout learning maintained a high level of performance during the 5-day retraining phase. A parallel analysis of theta rhythms recorded in an independent group of rats placed in equivalent treatment and/or lesion conditions was then performed. Preoperatively, clonidine injections decreased theta frequency during both alert immobility and movement. Partial fornix lesions produced an increase in theta frequency. Finally, clonidine in fornix-damaged rats decreased theta frequency, thus reinstating the postoperative values at a level statistically no different from that recorded preoperatively. The role of clonidine in restoring the function of the septo-hippocampal input in partially fornix-damaged rats through a noradrenergic modulation of hippocampal acetylcholine release is discussed.