Silvia Middei

Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease

Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimers disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimers disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3–dependent mechanism that drives synaptic failure and contributes to cognitive dysfunction in Alzheimers disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages.

Phosphodiesterase type IV inhibition prevents sequestration of CREB binding protein, protects striatal parvalbumin interneurons and rescues motor deficits in the R6/2 mouse model of Huntington's disease

The phosphodiesterase type IV inhibitor rolipram increases cAMP response element‐binding protein (CREB) phosphorylation and exerts neuroprotective effects in both the quinolinic acid rat model of Huntington’s disease ( DeMarch et al., 2007 ) and the R6/2 mouse including sparing of striatal neurons, prevention of neuronal intranuclear inclusion formation and attenuation of microglial reaction ( DeMarch et al., 2008 ). In this study, we sought to determine if rolipram has a beneficial role in the altered distribution of CREB binding protein in striatal spiny neurons and in the motor impairments shown by R6/2 mutants. Moreover, we investigated whether rolipram treatment altered the degeneration of parvalbuminergic interneurons typical of Huntington’s disease ( Fusco et al., 1999 ). Transgenic mice and their wild‐type controls from a stable colony maintained in our laboratory were treated with rolipram (1.5 mg/kg) or saline daily starting from 4 weeks of age. The cellular distribution of CREB binding protein in striatal spiny neurons was assessed by immunofluorescence, whereas parvalbuminergic neuron degeneration was evaluated by cell counts of immunohistochemically labeled tissue. Motor coordination and motor activity were also examined. We found that rolipram was effective in preventing CREB binding protein sequestration into striatal neuronal intranuclear inclusions, sparing parvalbuminergic interneurons of R6/2 mice, and rescuing their motor coordination and motor activity deficits. Our findings demonstrate the possibility of reversing pharmacologically the behavioral and neuropathological abnormalities of symptomatic R6/2 mice and underline the potential therapeutic value of phosphodiesterase type IV inhibitors in Huntington’s disease.

Synaptic Adaptations of CA1 Pyramidal Neurons Induced by a Highly Effective Combinational Antidepressant Therapy

BACKGROUND Antidepressants (AD) need to be chronically administered (weeks to months) to provide beneficial effects. Evidence suggests that combined administration of inhibitors of monoamine reuptake and phosphodiesterase type 4 allows a highly effective therapeutic action. Also, this coadministration more rapidly boosts the cyclic adenosine monophosphate (cAMP) pathway, which is normally activated during chronic treatment of single compounds. Little is known, however, about how this augmentation therapy affects the core mechanism of glutamatergic plasticity. We therefore investigated how in vivo combinational subchronic rolipram and imipramine (scRI) treatment affects depressive behavior, cAMP-dependent transcription, and glutamatergic transmission in the hippocampus, a region critically implicated in depression. METHODS Antidepressant properties of scRI were investigated through the forced swim test. Changes in cAMP-dependent transcription and synaptic transmission of CA1 pyramidal cells were explored with green fluorescent protein, enzyme-linked immunosorbent assay, electrophysiology recordings, and Golgi-Cox staining. RESULTS We demonstrate that scRI displays robust antidepressant properties compared with single-drug treatments and increases hippocampal c-Fos expression and brain-derived neurotrophic factor protein levels. These effects are accompanied by a specific increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors in already existing synapses. Finally, both acute and subchronic treatments led to enhancement of long-term potentiation but differently affected spine density and morphology, with scRI administration specifically resulting in a large increase in stubby spines. CONCLUSIONS We conclude that scRI is highly effective in providing antidepressive effects, including the hippocampal transcriptional alterations normally observed with longer single-drug treatments. Furthermore, we identified scRI-induced modifications in glutamatergic transmission that probably underlie the beneficial action of this combinational therapy.

Learning discloses abnormal structural and functional plasticity at hippocampal synapses in the APP23 mouse model of Alzheimer's disease

B6-Tg/Thy1APP23Sdz (APP23) mutant mice exhibit neurohistological hallmarks of Alzheimers disease but show intact basal hippocampal neurotransmission and synaptic plasticity. Here, we examine whether spatial learning differently modifies the structural and electrophysiological properties of hippocampal synapses in APP23 and wild-type mice. While no genotypic difference was found in the pseudotrained mice, training elicited a stronger increase in spine density and a more rapid decay of long-term potentiation (LTP) in APP23 mutants. Thus, learning discloses mutation-related abnormalities regarding dendritic spine formation and LTP persistence, thereby suggesting that although unaltered in naïve synapses, plasticity becomes defective at the time it comes into play.

CREB is necessary for synaptic maintenance and learning-induced changes of the AMPA receptor GluA1 subunit.

The transcription factor cAMP response element binding protein (CREB) is a key protein implicated in memory, synaptic plasticity and structural plasticity in mammals. Whether CREB regulates the synaptic incorporation of hippocampal glutamatergic receptors under basal and learning‐induced conditions remains, however, mostly unknown. Using double‐transgenic mice conditionally expressing a dominant negative form of CREB (CREBS133A, mCREB), we analyzed how chronic loss of CREB function in adult hippocampal glutamatergic neurons impacts the levels of the AMPA and NMDA receptors subunits within the post‐synaptic densities (PSD). In basal (naïve) conditions, we report that inhibition of CREB function was associated with a specific reduction of the AMPAR subunit GluA1 and a proportional increase in its Ser845 phosphorylated form within the PSD. These molecular alterations correlated with a reduction in AMPA receptors mEPSC frequency, with a decrease in long‐term potentiation (LTP), and with an increase in long‐term depression (LTD). The basal levels other major synaptic proteins (GluA2/3, GluN1, GluN2A, and PSD95) within the PSD were not affected by CREB inhibition. Blocking CREB function also impaired contextual fear conditioning (CFC) and selectively blocked the CFC‐driven enhancement of GluA1 and its Ser845 phosphorylated form within the PSD, molecular changes normally observed in wild‐type mice. CFC‐driven enhancement of other synaptic proteins (GluA2/3, GluN1, GluN2A, and PSD95) within the PSD was not significantly perturbed by the loss of CREB function. These findings provide the first evidence that, in vivo, CREB is necessary for the specific maintenance of the GluA1 subunit within the PSD of hippocampal neurons in basal conditions and for its trafficking within the PSD during the occurrence of learning.

Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals

D-aspartate (D-Asp) is an atypical amino acid, which is especially abundant in the developing mammalian brain, and can bind to and activate N-methyl-D-Aspartate receptors (NMDARs). In line with its pharmacological features, we find that mice chronically treated with D-Asp show enhanced NMDAR-mediated miniature excitatory postsynaptic currents and basal cerebral blood volume in fronto-hippocampal areas. In addition, we show that both chronic administration of D-Asp and deletion of the gene coding for the catabolic enzyme D-aspartate oxidase (DDO) trigger plastic modifications of neuronal cytoarchitecture in the prefrontal cortex and CA1 subfield of the hippocampus and promote a cytochalasin D-sensitive form of synaptic plasticity in adult mouse brains. To translate these findings in humans and consistent with the experiments using Ddo gene targeting in animals, we performed a hierarchical stepwise translational genetic approach. Specifically, we investigated the association of variation in the gene coding for DDO with complex human prefrontal phenotypes. We demonstrate that genetic variation predicting reduced expression of DDO in postmortem human prefrontal cortex is mapped on greater prefrontal gray matter and activity during working memory as measured with MRI. In conclusion our results identify novel NMDAR-dependent effects of D-Asp on plasticity and physiology in rodents, which also map to prefrontal phenotypes in humans.

The strain-specific involvement of nucleus accumbens in latent inhibition might depend on differences in processing configural- and cue-based information between C57BL/6 and DBA mice.

Latent inhibition (LI) consists of decreased associative strength between an elemental stimulus (CS: tone) paired with an unconditioned stimulus (US: footshock) following non-reinforced pre-exposure to the tone. In view of the differences shown by C57BL/6 (C57) and DBA/2 (DBA) mice in processing elemental vs. configural stimuli, the present experiments were designed (1) to assess whether these differences were likely to interfere with the capability of each strain to show LI, and (2) to verify the extent to which lesions of the nucleus accumbens, which have been reported to enhance attention towards contextual stimuli under certain conditions, might interfere with the development of LI. C57 and DBA mice with Nacc or sham lesions were given two periods (4 or 7 days) of pre-exposure to a CS (tone) then subjected to two CS-US pairings given on a single day. On the day after, freezing to the tone was examined in each group. Results show that, following the shorter period of pre-exposure, LI developed in sham-lesioned DBA but did not in sham-lesioned C57. Nacc lesions, however, were found (1) to block LI in DBA but (2) to promote LI in C57. After the longer period of pre-exposure LI was observed in both strain and lesion conditions. In general, these results confirm that strain differences in processing the tone as a single elemental cue (DBA) or, alternatively, as a part of a contextual configural stimulus (C57) can interfere with the development of LI. In addition, they indicate that Nacc lesions, that are susceptible to increase attention to the background, might modify the salience of the tone and produce opposite effect on LI according to the strain specialisation to show elemental or configural responding.

CREB selectively controls learning-induced structural remodeling of neurons

The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons remains elusive. Using transgenic mice conditionally expressing a dominant-negative CREB (CREBS133A: mCREB) mutant, we found that inhibiting CREB function does not alter spine density, spine morphology, and levels of polymerized actin in naive CA1 neurons. CREB inhibition, however, impaired contextual fear conditioning and produced a learning-induced collapse of spines associated with a blockade of learning-dependent increase in actin polymerization. Blocking mCREB expression with doxycycline rescued memory and restored a normal pattern of learning-induced spines, demonstrating that CREB controls structural adaptations of neurons selectively involved in memory formation.

Enhanced procedural learning following beta-amyloid protein (1-42) infusion in the rat.

Wistar rats receiving intracerebroventricular infusion of the &bgr;-amyloid protein (A&bgr;1-42) or of the inactive fragment (A&bgr;1-42) were subjected to the cross-maze task. According to the standard protocol, rats were released from the south arm and trained to collect food at the end of the east arm. After a 5-day training period, they were given a probe trial during which they were released from the north arm and allowed to choose either the east arm (place learning) or the west arm (response learning). Control rats showed predominant place learning whereas all rats receiving (A&bgr;1-42) showed response learning. These data indicate that exposure to (A&bgr;1-42) does not only impair cognitive responding but elicits strong procedural (motor-based) responding.

Reversible inactivation of hippocampus and dorsolateral striatum in C57BL/6 and DBA/2 inbred mice failed to show interaction between memory systems in these genotypes

C57BL/6 and DBA/2 mice with cannulae inserted bilaterally in the dorsal hippocampus or the dorsolateral striatum were released from the south arm of a cross maze and trained to find food in the east arm. Probe trials on which mice were released from the north arm were given following short or prolonged training. Prior to the probe trials, mice received intra-hippocampal or intra-striatal injections of lidocaine or saline. Results show that saline-injected C57BL/6 were fundamentally place learners whereas saline-injected DBA/2 mice did not engage any predominant system. Inactivating the hippocampus or the dorsolateral striatum in C56BL/6 mice disrupted place learning without promoting response learning. Inactivating the same brain sites in DBA/2 mice did not affect their behaviour. Thus, contrary to that observed in rats, disrupting the neural substrate of one memory system can abolish learning in that system but does not promote the use of another system in these genotypes.