Stabilizing Immature Dendritic Spines in the Auditory Cortex: A Key Mechanism for mTORC1-Mediated Enhancement of Long-Term Fear Memories.

Mammalian target of rapamycin (mTOR) pathway has emerged as a key molecular mechanism underlying memory processes. Although mTOR inhibition is known to block memory processes, it remains elusive whether and how an enhancement of mTOR signaling may improve memory processes. Here we found in male mice that the administration of VO-OHpic, an inhibitor of the phosphatase and tensin homolog (PTEN) that negatively modulates AKT-mTOR pathway, enhanced auditory fear memory for days and weeks, while it left short-term memory unchanged. Memory enhancement was associated with a long-lasting increase in immature-type dendritic spines of pyramidal neurons into the auditory cortex. The persistence of spine remodeling over time arose by the interplay between PTEN inhibition and memory processes, as VO-OHpic induced only a transient immature spine growth in the somatosensory cortex, a region not involved in long-term auditory memory. Both the potentiation of fear memories and increase in immature spines were hampered by rapamycin, a selective inhibitor of mTORC1. These data revealed that memory can be potentiated over time by the administration of a selective PTEN inhibitor. In addition to disclosing new information on the cellular mechanisms underlying long-term memory maintenance, our study provides new insights on the molecular processes that aid enhancing memories over time.SIGNIFICANCE STATEMENT The neuronal mechanisms that may help improve the maintenance of long-term memories are still elusive. The inhibition of mammalian-target of rapamycin (mTOR) signaling shows that this pathway plays a crucial role in synaptic plasticity and memory formation. However, whether its activation may strengthen long-term memory storage is unclear. We assessed the consequences of positive modulation of AKT-mTOR pathway obtained by VO-OHpic administration, a phosphatase and tensin homolog inhibitor, on memory retention and underlying synaptic modifications. We found that mTOR activation greatly enhanced memory maintenance for weeks by producing a long-lasting increase of immature-type dendritic spines in pyramidal neurons of the auditory cortex. These results offer new insights on the cellular and molecular mechanisms that can aid enhancing memories over time.

mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a devastating rare neurodevelopmental disease without a cure, caused by mutations of the serine/threonine kinase CDKL5 highly expressed in the forebrain. CDD is characterized by early-onset seizures, severe intellectual disabilities, autistic-like traits, sensorimotor and cortical visual impairments (CVI). The lack of an effective therapeutic strategy for CDD urgently demands the identification of novel druggable targets potentially relevant for CDD pathophysiology. To this aim, we studied Class I metabotropic glutamate receptors 5 (mGluR5) because of their important role in the neuropathological signs produced by the lack of CDKL5 in-vivo, such as defective synaptogenesis, dendritic spines formation/maturation, synaptic transmission and plasticity. Importantly, mGluR5 function strictly depends on the correct expression of the postsynaptic protein Homer1bc that we previously found atypical in the cerebral cortex of Cdkl5-/y mice. In this study, we reveal that CDKL5 loss tampers with (i) the binding strength of Homer1bc-mGluR5 complexes, (ii) the synaptic localization of mGluR5 and (iii) the mGluR5-mediated enhancement of NMDA-induced neuronal responses. Importantly, we showed that the stimulation of mGluR5 activity by administering in mice specific positive-allosteric-modulators (PAMs), i.e., 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) or RO6807794, corrected the synaptic, functional and behavioral defects shown by Cdkl5-/y mice. Notably, in the visual cortex of 2 CDD patients we found changes in synaptic organization that recapitulate those of mutant CDKL5 mice, including the reduced expression of mGluR5, suggesting that these receptors represent a promising therapeutic target for CDD.

p140Cap regulates the composition and localization of the NMDAR complex in synaptic lipid rafts.

The N-Methyl-D-aspartate receptors (NMDAR) are key players in both physiological and pathological synaptic plasticity because of their involvement in many aspects of neuronal transmission as well as learning and memory. The contribution in these events of different types of GluN2A-interacting proteins is still unclear. The p140Cap scaffold protein acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders and regulates synaptic functions like the stabilization of mature dendritic spine, memory consolidation, long-term potentiation, and depression. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap knockout male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons and p140Cap expression in knockout neurons can rescue GluN2A and PSD95 colocalization. p140Cap is crucial in the recruitment of GluN2A-containing NMDARs and, consequently, in regulating NMDARs intrinsic properties. p140Cap is associated to synaptic lipid-raft (LR) and to soluble postsynaptic membranes and GluN2A and PSD95 are less recruited into synaptic LR of p140Cap knockout male mice. g-STED microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in LR in an activity-dependent fashion. In the synaptic compartment p140Cap influences the association between GluN2A and PSD95 and modulates GluN2A enrichment into LR. Overall, such increase in these membrane domains rich in signalling molecules results in improved signal transduction efficiency.SIGNIFICANT STATEMENTHere we originally show that the adaptor protein p140Cap directly binds the GluN2A subunit of NMDAR and modulates the GluN2A-associated molecular network. Moreover, we show for the first time that p140Cap also associates to synaptic lipid rafts and controls the selective recruitment of GluN2A and PSD95 to this specific compartment. Finally, g-STED microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in lipid rafts in an activity-dependent fashion. Overall, our findings provide the molecular and functional dissection of p140Cap as a new active member of a highly dynamic synaptic network involved in memory consolidation, LTP and LTD that are known to be altered in neurological and psychiatric disorders.

Age-Related Cognitive and Motor Decline in a Mouse Model of CDKL5 Deficiency Disorder is Associated with Increased Neuronal Senescence and Death.

CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene. Children affected by CDD display a clinical phenotype characterized by early-onset epilepsy, intellectual disability, motor impairment, and autistic-like features. Although the clinical aspects associated with CDKL5 mutations are well described in children, adults with CDD are still under-characterized. Similarly, most animal research has been carried out on young adult Cdkl5 knockout (KO) mice only. Since age represents a risk factor for the worsening of symptoms in many neurodevelopmental disorders, understanding age differences in the development of behavioral deficits is crucial in order to optimize the impact of therapeutic interventions. Here, we compared young adult Cdkl5 KO mice with middle-aged Cdkl5 KO mice, at a behavioral, neuroanatomical, and molecular level. We found an age-dependent decline in motor, cognitive, and social behaviors in Cdkl5 KO mice, as well as in breathing and sleep patterns. The behavioral decline in older Cdkl5 KO mice was not associated with a worsening of neuroanatomical alterations, such as decreased dendritic arborization or spine density, but was paralleled by decreased neuronal survival in different brain regions such as the hippocampus, cortex, and basal ganglia. Interestingly, we found increased ß-galactosidase activity and DNA repair protein levels, γH2AX and XRCC5, in the brains of older Cdkl5 KO mice, which suggests that an absence of Cdkl5 accelerates neuronal senescence/death by triggering irreparable DNA damage. In summary, this work provides evidence that CDKL5 may play a fundamental role in neuronal survival during brain aging and suggests a possible worsening with age of the clinical picture in CDD patients.

Long-term effect of neonatal inhibition of APP gamma-secretase on hippocampal development in the Ts65Dn mouse model of Down syndrome.

Neurogenesis impairment is considered a major determinant of the intellectual disability that characterizes Down syndrome (DS), a genetic condition caused by triplication of chromosome 21. Previous evidence obtained in the Ts65Dn mouse model of DS showed that the triplicated gene APP (amyloid precursor protein) is critically involved in neurogenesis alterations. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain) resulting from APP cleavage by gamma-secretase increase the transcription of Ptch1, a Sonic Hedgehog (Shh) receptor that keeps the mitogenic Shh pathway repressed. Previous evidence showed that neonatal treatment with ELND006, an inhibitor of gamma-secretase, reinstates the Shh pathway and fully restores neurogenesis in Ts65Dn pups. In the framework of potential therapies for DS, it is extremely important to establish whether the positive effects of early intervention are retained after treatment cessation. Therefore, the goal of the current study was to establish whether early treatment with ELND006 leaves an enduring trace in the brain of Ts65Dn mice. Ts65Dn and euploid pups were treated with ELND006 in the postnatal period P3-P15 and the outcome of treatment was examined at ~one month after treatment cessation. We found that in treated Ts65Dn mice the pool of proliferating cells in the hippocampal dentate gyrus (DG) and total number of granule neurons were still restored as was the number of pre- and postsynaptic terminals in the stratum lucidum of CA3, the site of termination of the mossy fibers from the DG. Accordingly, patch-clamp recording from field CA3 showed functional normalization of the input to CA3. Unlike in field CA3, the number of pre- and postsynaptic terminals in the DG of treated Ts65Dn mice was no longer fully restored. The finding that many of the positive effects of neonatal treatment were retained after treatment cessation provides proof of principle demonstration of the efficacy of early inhibition of gamma-secretase for the improvement of brain development in DS.

Two distinct types of depolarizing afterpotentials are differentially expressed in stellate and pyramidal-like neurons of entorhinal-cortex layer II.

Two types of principal neurons, stellate cells and pyramidal-like cells, are found in medial entorhinal-cortex (mEC) layer II, and are believed to represent two distinct channels of information processing and transmission in the entorhinal cortex-hippocampus network. In this study, we found that depolarizing afterpotentials (DAPs) that follow single action potentials (APs) evoked from various levels of holding membrane voltage (Vh ) show distinct properties in the two cells types. In both, an evident DAP followed the AP at near-threshold Vh levels, and was accompanied by an enhancement of excitability and spike-timing precision. This DAP was sensitive to voltage-gated Na(+)-channel block with TTx, but not to partial removal of extracellular Ca(2+). Application of 5-µM anandamide, which inhibited the resurgent and persistent Na(+) -current components in a relatively selective way, significantly reduced the amplitude of this particular DAP while exerting poor effects on the foregoing AP. In the presence of background hyperpolarization, DAPs showed an opposite behavior in the two cell types, as in stellate cells they became even more prominent, whereas in pyramidal-like cells their amplitude was markedly reduced. The DAP observed in stellate cells under this condition was strongly inhibited by partial extracellular-Ca(2+) removal, and was sensitive to the low-voltage-activated Ca(2+)-channel blocker, NNC55-0396. This Ca(2+) dependence was not observed in the residual DAP evoked in pyramidal-like cells from likewise negative Vh levels. These results demonstrate that two distinct mechanism of DAP generation operate in mEC layer-II neurons, one Na(+)-dependent and active at near-threshold Vh levels in both stellate and-pyramidal-like cells, the other Ca(2+)-dependent and only expressed by stellate cells in the presence of background membrane hyperpolarization.