A comprehensive atlas of perineuronal net distribution and colocalization with parvalbumin in the adult mouse brain.

Perineuronal nets (PNNs) surround specific neurons in the brain and are involved in various forms of plasticity and clinical conditions. However, our understanding of the PNN role in these phenomena is limited by the lack of highly quantitative maps of PNN distribution and association with specific cell types. Here, we present a comprehensive atlas of Wisteria floribunda agglutinin (WFA)-positive PNNs and colocalization with parvalbumin (PV) cells for over 600 regions of the adult mouse brain. Data analysis shows that PV expression is a good predictor of PNN aggregation. In the cortex, PNNs are dramatically enriched in layer 4 of all primary sensory areas in correlation with thalamocortical input density, and their distribution mirrors intracortical connectivity patterns. Gene expression analysis identifies many PNN-correlated genes. Strikingly, PNN-anticorrelated transcripts are enriched in synaptic plasticity genes, generalizing PNNs' role as circuit stability factors.

Selective Disruption of Perineuronal Nets in Mice Lacking Crtl1 is Sufficient to Make Fear Memories Susceptible to Erasure.

The ability to store, retrieve, and extinguish memories of adverse experiences is an essential skill for animals' survival. The cellular and molecular factors that underlie such processes are only partially known. Using chondroitinase ABC treatment targeting chondroitin sulfate proteoglycans (CSPGs), previous studies showed that the maturation of the extracellular matrix makes fear memory resistant to deletion. Mice lacking the cartilage link protein Crtl1 (Crtl1-KO mice) display normal CSPG levels but impaired CSPG condensation in perineuronal nets (PNNs). Thus, we asked whether the presence of PNNs in the adult brain is responsible for the appearance of persistent fear memories by investigating fear extinction in Crtl1-KO mice. We found that mutant mice displayed fear memory erasure after an extinction protocol as revealed by analysis of freezing and pupil dynamics. Fear memory erasure did not depend on passive loss of retention; moreover, we demonstrated that, after extinction training, conditioned Crtl1-KO mice display no neural activation in the amygdala (Zif268 staining) in comparison to control animals. Taken together, our findings suggest that the aggregation of CSPGs into PNNs regulates the boundaries of the critical period for fear extinction.

Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency.

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.

Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency.

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.

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.

Behavioral impulsivity is associated with pupillary alterations and hyperactivity in CDKL5 mutant mice.

Cyclin-dependent kinase-like 5 (Cdkl5) deficiency disorder (CDD) is a severe neurodevelopmental condition caused by mutations in the X-linked Cdkl5 gene. CDD is characterized by early-onset seizures in the first month of life, intellectual disability, motor and social impairment. No effective treatment is currently available and medical management is only symptomatic and supportive. Recently, mouse models of Cdkl5 disorder have demonstrated that mice lacking Cdkl5 exhibit autism-like phenotypes, hyperactivity and dysregulations of the arousal system, suggesting the possibility to use these features as translational biomarkers. In this study, we tested Cdkl5 male and female mutant mice in an appetitive operant conditioning chamber to assess cognitive and motor abilities, and performed pupillometry to assess the integrity of the arousal system. Then, we evaluated the performance of artificial intelligence models to classify the genotype of the animals from the behavioral and physiological phenotype. The behavioral results show that CDD mice display impulsivity, together with low levels of cognitive flexibility and perseverative behaviors. We assessed arousal levels by simultaneously recording pupil size and locomotor activity. Pupillometry reveals in CDD mice a smaller pupil size and an impaired response to unexpected stimuli associated with hyperlocomotion, demonstrating a global defect in arousal modulation. Finally, machine learning reveals that both behavioral and pupillometry parameters can be considered good predictors of CDD. Since early diagnosis is essential to evaluate treatment outcomes and pupillary measures can be performed easily, we proposed the monitoring of pupil size as a promising biomarker for CDD.

MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex.

Visual cortical circuits show profound plasticity during early life and are later stabilized by molecular "brakes" limiting excessive rewiring beyond a critical period. The mechanisms coordinating the expression of these factors during the transition from development to adulthood remain unknown. We found that miR-29a expression in the visual cortex dramatically increases with age, but it is not experience-dependent. Precocious high levels of miR-29a blocked ocular dominance plasticity and caused an early appearance of perineuronal nets. Conversely, inhibition of miR-29a in adult mice using LNA antagomirs activated ocular dominance plasticity, reduced perineuronal nets, and restored their juvenile chemical composition. Activated adult plasticity had the typical functional and proteomic signature of critical period plasticity. Transcriptomic and proteomic studies indicated that miR-29a manipulation regulates the expression of plasticity brakes in specific cortical circuits. These data indicate that miR-29a is a regulator of the plasticity brakes promoting age-dependent stabilization of visual cortical connections.

Cyclocreatine treatment ameliorates the cognitive, autistic and epileptic phenotype in a mouse model of Creatine Transporter Deficiency.

Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr.

Novel translational phenotypes and biomarkers for creatine transporter deficiency.

Creatine transporter deficiency is a metabolic disorder characterized by intellectual disability, autistic-like behaviour and epilepsy. There is currently no cure for creatine transporter deficiency, and reliable biomarkers of translational value for monitoring disease progression and response to therapeutics are sorely lacking. Here, we found that mice lacking functional creatine transporter display a significant alteration of neural oscillations in the EEG and a severe epileptic phenotype that are recapitulated in patients with creatine transporter deficiency. In-depth examination of knockout mice for creatine transporter also revealed that a decrease in EEG theta power is predictive of the manifestation of spontaneous seizures, a frequency that is similarly affected in patients compared to healthy controls. In addition, knockout mice have a highly specific increase in haemodynamic responses in the cerebral cortex following sensory stimuli. Principal component and Random Forest analyses highlighted that these functional variables exhibit a high performance in discriminating between pathological and healthy phenotype. Overall, our findings identify novel, translational and non-invasive biomarkers for the analysis of brain function in creatine transporter deficiency, providing a very reliable protocol to longitudinally monitor the efficacy of potential therapeutic strategies in preclinical, and possibly clinical, studies.

Site-specific abnormalities in the visual system of a mouse model of CDKL5 deficiency disorder.

CDKL5 deficiency disorder (CDD) is a neurodevelopmental disorder characterized by a severe global developmental delay and early-onset seizures. Notably, patients show distinctive visual abnormalities often clinically diagnosed as cortical visual impairment. However, the involvement of cerebral cortical dysfunctions in the origin of the symptoms is poorly understood. CDD mouse models also display visual deficits, and cortical visual responses can be used as a robust biomarker in CDKL5 mutant mice. A deeper understanding of the circuits underlying the described visual deficits is essential for directing preclinical research and translational approaches. Here, we addressed this question in two ways: first, we performed an in-depth morphological analysis of the visual pathway, from the retina to the primary visual cortex (V1), of CDKL5 null mice. We found that the lack of CDKL5 produced no alteration in the organization of retinal circuits. Conversely, CDKL5 mutants showed reduced density and altered morphology of spines and decreased excitatory synapse marker PSD95 in the dorsal lateral geniculate nucleus and in V1. An increase in the inhibitory marker VGAT was selectively present in V1. Second, using a conditional CDKL5 knockout model, we showed that selective cortical deletion of CDKL5 from excitatory cells is sufficient to produce abnormalities of visual cortical responses, demonstrating that the normal function of cortical circuits is dependent on CDKL5. Intriguingly, these deficits were associated with morphological alterations of V1 excitatory and inhibitory synaptic contacts. In summary, this work proposes cortical circuit structure and function as a critically important target for studying CDD.

A Nervous System-Specific Model of Creatine Transporter Deficiency Recapitulates the Cognitive Endophenotype of the Disease: a Longitudinal Study.

Mutations in creatine (Cr) transporter (CrT) gene lead to cerebral creatine deficiency syndrome-1 (CTD), an orphan neurodevelopmental disorder presenting with brain Cr deficiency, intellectual disability, seizures, movement and autistic-like behavioral disturbances, language and speech impairment. We have recently generated a murine model of CTD obtained by ubiquitous deletion of 5-7 exons in the CrT gene. These mice showed a marked Cr depletion, associated to early and progressive cognitive impairment, and autistic-like defects, thus resembling the key features of human CTD. Given the importance of extraneural dysfunctions in neurodevelopmental disorders, here we analyzed the specific role of neural Cr in the CTD phenotype. We induced the conditional deletion of Slc6a8 gene in neuronal and glial cells by crossing CrT floxed mice with the Nestin::Cre recombinase Tg (Nes-cre) 1Kln mouse. We report that nervous system-specific Cr depletion leads to a progressive cognitive regression starting in the adult age. No autistic-like features, including repetitive and stereotyped movements, routines and rituals, are present in this model. These results indicate that Cr depletion in the nervous system is a pivotal cause of the CTD pathological phenotype, in particular with regard to the cognitive domain, but extraneural actors also play a role.

Mir-132/212 is required for maturation of binocular matching of orientation preference and depth perception.

MicroRNAs (miRNAs) are known to mediate post-transcriptional gene regulation, but their role in postnatal brain development is still poorly explored. We show that the expression of many miRNAs is dramatically regulated during functional maturation of the mouse visual cortex with miR-132/212 family being one of the top upregulated miRNAs. Age-downregulated transcripts are significantly enriched in miR-132/miR-212 putative targets and in genes upregulated in miR-132/212 null mice. At a functional level, miR-132/212 deletion affects development of receptive fields of cortical neurons determining a specific impairment of binocular matching of orientation preference, but leaving orientation and direction selectivity unaltered. This deficit is associated with reduced depth perception in the visual cliff test. Deletion of miR-132/212 from forebrain excitatory neurons replicates the binocular matching deficits. Thus, miR-132/212 family shapes the age-dependent transcriptome of the visual cortex during a specific developmental window resulting in maturation of binocular cortical cells and depth perception.

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice.

CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60-80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder.

A mouse model for creatine transporter deficiency reveals early onset cognitive impairment and neuropathology associated with brain aging.

Mutations in the creatine (Cr) transporter (CrT) gene lead to cerebral creatine deficiency syndrome-1 (CCDS1), an X-linked metabolic disorder characterized by cerebral Cr deficiency causing intellectual disability, seizures, movement and autistic-like behavioural disturbances, language and speech impairment. Since no data are available about the neural and molecular underpinnings of this disease, we performed a longitudinal analysis of behavioural and pathological alterations associated with CrT deficiency in a CCDS1 mouse model. We found precocious cognitive and autistic-like defects, mimicking the early key features of human CCDS1. Moreover, mutant mice displayed a progressive impairment of short and long-term declarative memory denoting an early brain aging. Pathological examination showed a prominent loss of GABAergic synapses, marked activation of microglia, reduction of hippocampal neurogenesis and the accumulation of autofluorescent lipofuscin. Our data suggest that brain Cr depletion causes both early intellectual disability and late progressive cognitive decline, and identify novel targets to design intervention strategies aimed at overcoming brain CCDS1 alterations.

Dendritic Spine Instability in a Mouse Model of CDKL5 Disorder Is Rescued by Insulin-like Growth Factor 1.

BACKGROUND: CDKL5 (cyclin-dependent kinase-like 5) is mutated in many severe neurodevelopmental disorders, including atypical Rett syndrome. CDKL5 was shown to interact with synaptic proteins, but an in vivo analysis of the role of CDKL5 in dendritic spine dynamics and synaptic molecular organization is still lacking. METHODS: In vivo two-photon microscopy of the somatosensory cortex of Cdkl5(-/y) mice was applied to monitor structural dynamics of dendritic spines. Synaptic function and plasticity were measured using electrophysiological recordings of excitatory postsynaptic currents and long-term potentiation in brain slices and assessing the expression of synaptic postsynaptic density protein 95 (PSD-95). Finally, we studied the impact of insulin-like growth factor 1 (IGF-1) treatment on CDKL5 null mice to restore the synaptic deficits. RESULTS: Adult mutant mice showed a significant reduction in spine density and PSD-95-positive synaptic puncta, a reduction of persistent spines, and impaired long-term potentiation. In juvenile mutants, short-term spine elimination, but not formation, was dramatically increased. Exogenous administration of IGF-1 rescued defective rpS6 phosphorylation, spine density, and PSD-95 expression. Endogenous cortical IGF-1 levels were unaffected by CDKL5 deletion. CONCLUSIONS: These data demonstrate that dendritic spine stabilization is strongly regulated by CDKL5. Moreover, our data suggest that IGF-1 treatment could be a promising candidate for clinical trials in CDKL5 patients.

A novel mouse model of creatine transporter deficiency.

Mutations in the creatine (Cr) transporter (CrT) gene lead to cerebral creatine deficiency syndrome-1 (CCDS1), an X-linked metabolic disorder characterized by cerebral Cr deficiency causing intellectual disability, seizures, movement  and behavioral disturbances, language and speech impairment ( OMIM #300352). CCDS1 is still an untreatable pathology that can be very invalidating for patients and caregivers. Only two murine models of CCDS1, one of which is an ubiquitous knockout mouse, are currently available to study the possible mechanisms underlying the pathologic phenotype of CCDS1 and to develop therapeutic strategies. Given the importance of validating phenotypes and efficacy of promising treatments in more than one mouse model we have generated a new murine model of CCDS1 obtained by ubiquitous deletion of 5-7 exons in the Slc6a8 gene. We showed a remarkable Cr depletion in the murine brain tissues and cognitive defects, thus resembling the key features of human CCDS1. These results confirm that CCDS1 can be well modeled in mice. This CrT (-/y) murine model will provide a new tool for increasing the relevance of preclinical studies to the human disease.

Experience-dependent expression of miR-132 regulates ocular dominance plasticity.

miR-132 is a CREB-induced microRNA that is involved in dendritic spine plasticity. We found that visual experience regulated histone post-translational modifications at a CRE locus that is important for miR-212 and miR-132 cluster transcription, and regulated miR-132 expression in the visual cortex of juvenile mice. Monocular deprivation reduced miR-132 expression in the cortex contralateral to the deprived eye. Counteracting this miR-132 reduction with an infusion of chemically modified miR-132 mimic oligonucleotides completely blocked ocular dominance plasticity.

Food restriction enhances visual cortex plasticity in adulthood.

Neural circuits display a heightened sensitivity to external stimuli during well-established windows in early postnatal life. After the end of these critical periods, brain plasticity dramatically wanes. The visual system is one of the paradigmatic models for studying experience-dependent plasticity. Here we show that food restriction can be used as a strategy to restore plasticity in the adult visual cortex of rats. A short period of food restriction in adulthood is able both to reinstate ocular dominance plasticity and promote recovery from amblyopia. These effects are accompanied by a reduction of intracortical inhibition without modulation of brain-derived neurotrophic factor expression or extracellular matrix structure. Our results suggest that food restriction could be investigated as a potential way of modulating plasticity.

ERK pathway activation bidirectionally affects visual recognition memory and synaptic plasticity in the perirhinal cortex.

ERK 1,2 pathway mediates experience-dependent gene transcription in neurons and several studies have identified its pivotal role in experience-dependent synaptic plasticity and in forms of long term memory involving hippocampus, amygdala, or striatum. The perirhinal cortex (PRHC) plays an essential role in familiarity-based object recognition memory. It is still unknown whether ERK activation in PRHC is necessary for recognition memory consolidation. Most important, it is unknown whether by modulating the gain of the ERK pathway it is possible to bidirectionally affect visual recognition memory and PRHC synaptic plasticity. We have first pharmacologically blocked ERK activation in the PRHC of adult mice and found that this was sufficient to impair long term recognition memory in a familiarity-based task, the object recognition task (ORT). We have then tested performance in the ORT in Ras-GRF1 knock-out (KO) mice, which exhibit a reduced activation of ERK by neuronal activity, and in ERK1 KO mice, which have an increased activation of ERK2 and exhibit enhanced striatal plasticity and striatal mediated memory. We found that Ras-GRF1 KO mice have normal short term memory but display a long term memory deficit; memory reconsolidation is also impaired. On the contrary, ERK1 KO mice exhibit a better performance than WT mice at 72 h retention interval, suggesting a longer lasting recognition memory. In parallel with behavioral data, LTD was strongly reduced and LTP was significantly smaller in PRHC slices from Ras-GRF1 KO than in WT mice while enhanced LTP and LTD were found in PRHC slices from ERK1 KO mice.

The short-time structural plasticity of dendritic spines is altered in a model of Rett syndrome.

The maturation of excitatory transmission comes about through a developmental period in which dendritic spines are highly motile and their number, form and size are rapidly changing. Surprisingly, although these processes are crucial for the formation of cortical circuitry, little is known about possible alterations of these processes in brain disease. By means of acute in vivo 2-photon imaging we show that the dynamic properties of dendritic spines of layer V cortical neurons are deeply affected in a mouse model of Rett syndrome (RTT) at a time around P25 when the neuronal phenotype of the disease is still mild. Then, we show that 24h after a subcutaneous injection of IGF-1 spine dynamics is restored. Our study demonstrates that spine dynamics in RTT mice is severely impaired early during development and suggest that treatments for RTT should be started very early in order to reestablish a normal period of spine plasticity.