Tau reduction with artificial microRNAs modulates neuronal physiology and improves tauopathy phenotypes in mice.

Abnormal tau accumulation is the hallmark of several neurodegenerative diseases, named tauopathies. Strategies aimed at reducing tau in the brain are promising therapeutic interventions, yet more precise therapies would require targeting specific nuclei and neuronal subpopulations affected by disease while avoiding global reduction of physiological tau. Here, we developed artificial microRNAs directed against the human MAPT mRNA to dwindle tau protein by engaging the endogenous RNA interference pathway. In human differentiated neurons in culture, microRNA-mediated tau reduction diminished neuronal firing without affecting neuronal morphology or impairing axonal transport. In the htau mouse model of tauopathy, we locally expressed artificial microRNAs in the prefrontal cortex (PFC), an area particularly vulnerable to initiating tau pathology in this model. Tau knockdown prevented the accumulation of insoluble and hyperphosphorylated tau, modulated firing activity of putative pyramidal neurons, and improved glucose uptake in the PFC. Moreover, such tau reduction prevented cognitive decline in aged htau mice. Our results suggest target engagement of designed tau-microRNAs to effectively reduce tau pathology, providing a proof of concept for a potential therapeutic approach based on local tau knockdown to rescue tauopathy-related phenotypes.

Oxidative stress associated with spatial memory impairment and social olfactory deterioration in female mice reveals premature aging aroused by perinatal protein malnutrition.

Early-life adversity, like perinatal protein malnutrition, increases the vulnerability to develop long-term alterations in brain structures and function. This study aimed to determine whether perinatal protein malnutrition predisposes to premature aging in a murine model and to assess the cellular and molecular mechanisms involved. To this end, mouse dams were fed either with a normal (NP, casein 20%) or a low-protein diet (LP, casein 8%) during gestation and lactation. Female offspring were evaluated at 2, 7 and 12 months of age. Positron emission tomography analysis showed alterations in the hippocampal CA3 region and the accessory olfactory bulb of LP mice during aging. Protein malnutrition impaired spatial memory, coinciding with higher levels of reactive oxygen species in the hippocampus and sirt7 upregulation. Protein malnutrition also led to higher senescence-associated ß-galactosidase activity and p21 expression. LP-12-month-old mice showed a higher number of newborn neurons that did not complete the maturation process. The social-odor discrimination in LP mice was impaired along life. In the olfactory bulb of LP mice, the senescence marker p21 was upregulated, coinciding with a downregulation of Sirt2 and Sirt7. Also, LP-12-month-old mice showed a downregulation of catalase and glutathione peroxidase, and LP-2-month-old mice showed a higher number of newborn neurons in the subventricular zone, which then returned to normal values. Our results show that perinatal protein malnutrition causes long-term impairment in cognitive and olfactory skills through an accelerated senescence phenotype accompanied by an increase in oxidative stress and altered sirtuin expression in the hippocampus and olfactory bulb.

Systemic Ghrelin Treatment Induces Rapid, Transient, and Asymmetric Changes in the Metabolic Activity of the Mouse Brain.

INTRODUCTION: Ghrelin regulates a variety of functions by acting in the brain. The targets of ghrelin in the mouse brain have been mainly mapped using immunolabeling against c-Fos, a transcription factor used as a marker of cellular activation, but such analysis has several limitations. Here, we used positron emission tomography in mice to investigate the brain areas responsive to ghrelin. METHODS: We analyzed in male mice the brain areas responsive to systemically injected ghrelin using positron emission tomography imaging of 18F-fluoro-2-deoxyglucose (18F-FDG) uptake, an indicator of metabolic rate. Additionally, we studied if systemic administration of fluorescent ghrelin or native ghrelin displays symmetric accessibility or induction of c-Fos, respectively, in the brain of male mice. RESULTS: Ghrelin increased 18F-FDG uptake in few specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain at 0-10-min posttreatment. At the 10-20 and 20-30 min posttreatment, ghrelin induced mixed changes in 18F-FDG uptake in specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain, as well as in areas of the olfactory areas, hippocampal and retrohippocampal regions, hypothalamus, pons, medulla, and even the cerebellum. Ghrelin-induced changes in 18F-FDG uptake were transient and asymmetric. Systemically administrated fluorescent-ghrelin-labeled midline brain areas known to contain fenestrated capillaries and the hypothalamic arcuate nucleus, where a symmetric labeling was observed. Ghrelin treatment also induced a symmetric increased c-Fos labeling in the arcuate nucleus. DISCUSSION/CONCLUSION: Systemically injected ghrelin transiently and asymmetrically affects the metabolic activity of the brain of male mice in a wide range of areas, in a food intake-independent manner. The neurobiological bases of such asymmetry seem to be independent of the accessibility of ghrelin into the brain.

SMaRT modulation of tau isoforms rescues cognitive and motor impairments in a preclinical model of tauopathy.

Tau is a microtubule-associated protein predominantly expressed in neurons, which participates in microtubule polymerization and axonal transport. Abnormal tau metabolism leads to neurodegenerative diseases named tauopathies, such as Alzheimer's disease and frontotemporal dementia. The alternative splicing of exon 10 (E10) in the primary transcript produces tau protein isoforms with three (3R) or four (4R) microtubule binding repeats, which are found in equal amounts in the normal adult human brain. Several tauopathies are associated with abnormal E10 alternative splicing, leading to an imbalance between 3R and 4R isoforms, which underlies disease. Correction of such imbalance represents a potential disease-modifying therapy for those tauopathies. We have previously optimized a trans-splicing RNA reprogramming strategy to modulate the 3R:4R tau content in a mouse model of tauopathy related to tau mis-splicing (htau mice), and showed that local modulation of E10 inclusion in the prefrontal cortex prevents cognitive decline, neuronal firing impairments and hyperphosphorylated tau accumulation. Furthermore, local shifting of 3R-4R tau into the striatum of htau mice prevented motor coordination deficits. However, a major bottleneck of our previous work is that local splicing regulation was performed in young mice, before the onset of pathological phenotypes. Here we tested whether regulation of tau E10 splicing could rescue tau pathology phenotypes in htau mice, after the onset of cognitive and motor impairments, comparable to early stages of human tauopathies. To determine phenotypic time course and affected brain nuclei, we assessed htau mice using behavioural tests and microPET FDG imaging over time, similarly to diagnosis methods used in patients. Based on these analyses, we performed local delivery of pre-trans splicing molecules to regulate E10 inclusion either into the medial prefrontal cortex (mPFC) or the striatum at 6-month-old once behavioral phenotypes and metabolic changes were detected. Tau isoforms modulation into the mPFC restored cognitive performance in mice that previously showed mild to severe memory impairment while motor coordination deficit was rescued after striatal injection of trans-splicing molecules. Our data suggest that tau regulation could recover pathological phenotypes early after phenotypic onset, raising promising perspectives for the use of RNA based therapies in tauopathies related to MAPT abnormal splicing.

Impaired social cognition caused by perinatal protein malnutrition evokes neurodevelopmental disorder symptoms and is intergenerationally transmitted.

Nutritional inadequacy before birth and during postnatal life can seriously interfere with brain development and lead to persistent deficits in learning and behavior. In this work, we asked if protein malnutrition affects domains of social cognition and if these phenotypes can be transmitted to the next generation. Female mice were fed with a normal or hypoproteic diet during pregnancy and lactation. After weaning, offspring were fed with a standard chow. Social interaction, social recognition memory, and dominance were evaluated in both sexes of F1 offspring and in the subsequent F2 generation. Glucose metabolism in the whole brain was analyzed through preclinical positron emission tomography. Genome-wide transcriptional analysis was performed in the medial prefrontal cortex followed by gene-ontology enrichment analysis. Compared with control animals, malnourished mice exhibited a deficit in social motivation and recognition memory and displayed a dominant phenotype. These altered behaviors, except for dominance, were transmitted to the next generation. Positron emission tomography analysis revealed lower glucose metabolism in the medial prefrontal cortex of F1 malnourished offspring. This brain region showed genome-wide transcriptional dysregulation, including 21 transcripts that overlapped with autism-associated genes. Our study cannot exclude that the lower maternal care provided by mothers exposed to a low-protein diet caused an additional impact on social cognition. Our results showed that maternal protein malnutrition dysregulates gene expression in the medial prefrontal cortex, promoting altered offspring behavior that was intergenerationally transmitted. These results support the hypothesis that early nutritional deficiency represents a risk factor for the emergence of symptoms associated with neurodevelopmental disorders.

Correlation between 99mTc-TRODAT-1 SPECT and 18F-FDOPA PET in patients with Parkinson's disease: a pilot study.

OBJECTIVE: To determine whether technetium-99m-labeled tropane derivative single-photon emission computed tomography (99mTc-TRODAT-1 SPECT) provides results comparable to those of the less widely available, less accessible tool fluorine-18-labeled fluorodopa positron-emission tomography (18F-FDOPA PET) in the setting of a movement disorders clinic. MATERIALS AND METHODS: In this prospective pilot study, eight subjects with a clinical diagnosis of Parkinson's disease were randomly selected from among patients under treatment at a movement disorders clinic and submitted to 99mTc-TRODAT-1 SPECT and 18F-FDOPA PET. The results were read by two experienced observers, and a semiquantitative analysis was performed. RESULTS: The visual and semiquantitative analyses were concordant for all studies, showing that radiotracer uptake in the contralateral striatum on the most affected side was lower when 99mTc-TRODAT-1 SPECT was employed. The semiquantitative analysis demonstrated a significant correlation between 18F-FDOPA PET and 99mTc-TRODAT-1 SPECT (r = 0.73; p < 0.01). CONCLUSION: It appears that 99mTc-TRODAT-1 SPECT is a valid option for the study of dopaminergic function in a clinical setting.

OBJETIVO: Determinar se a 99mTc-TRODAT-1 SPECT fornece resultados comparáveis aos da 18F-FDOPA PET, ferramenta menos acessível e menos amplamente disponível, no contexto de uma clínica de distúrbios do movimento. MATERIAIS E MÉTODOS: Neste estudo prospectivo, oito indivíduos com diagnóstico clínico de doença de Parkinson foram selecionados aleatoriamente entre pacientes em tratamento em uma clínica de distúrbios do movimento e submetidos a 99mTc-TRODAT-1 SPECT e 18F-FDOPA PET. Os resultados foram lidos por dois observadores experientes e uma análise semiquantitativa foi realizada. RESULTADOS: As análises visual e semiquantitativa foram concordantes para todos os estudos, mostrando que a captação do radiotraçador no estriado contralateral do lado mais afetado foi menor quando a 99mTc-TRODAT-1 SPECT foi empregada. A análise semiquantitativa demonstrou uma correlação significativa entre 18F-FDOPA PET e 99mTc-TRODAT-1 SPECT (r = 0,73; p < 0,01). CONCLUSÃO: A 99mTc-TRODAT-1 SPECT parece ser uma opção válida para o estudo da função dopaminérgica em um ambiente clínico.

Correlation between 99mTc-TRODAT-1 SPECT and 18F-FDOPA PET in patients with Parkinson's disease: a pilot study / Correlação entre 99mTc-TRODAT-1 SPECT e 18F-FDOPA PET em pacientes com doença de Parkinson: um estudo piloto

Abstract Objective: To determine whether technetium-99m-labeled tropane derivative single-photon emission computed tomography (99mTc-TRODAT-1 SPECT) provides results comparable to those of the less widely available, less accessible tool fluorine-18-labeled fluorodopa positron-emission tomography (18F-FDOPA PET) in the setting of a movement disorders clinic. Materials and Methods: In this prospective pilot study, eight subjects with a clinical diagnosis of Parkinson's disease were randomly selected from among patients under treatment at a movement disorders clinic and submitted to 99mTc-TRODAT-1 SPECT and 18F-FDOPA PET. The results were read by two experienced observers, and a semiquantitative analysis was performed. Results: The visual and semiquantitative analyses were concordant for all studies, showing that radiotracer uptake in the contralateral striatum on the most affected side was lower when 99mTc-TRODAT-1 SPECT was employed. The semiquantitative analysis demonstrated a significant correlation between 18F-FDOPA PET and 99mTc-TRODAT-1 SPECT (r = 0.73; p < 0.01). Conclusion: It appears that 99mTc-TRODAT-1 SPECT is a valid option for the study of dopaminergic function in a clinical setting.

Resumo Objetivo: Determinar se a 99mTc-TRODAT-1 SPECT fornece resultados comparáveis aos da 18F-FDOPA PET, ferramenta menos acessível e menos amplamente disponível, no contexto de uma clínica de distúrbios do movimento. Materiais e Métodos: Neste estudo prospectivo, oito indivíduos com diagnóstico clínico de doença de Parkinson foram selecionados aleatoriamente entre pacientes em tratamento em uma clínica de distúrbios do movimento e submetidos a 99mTc-TRODAT-1 SPECT e 18F-FDOPA PET. Os resultados foram lidos por dois observadores experientes e uma análise semiquantitativa foi realizada. Resultados: As análises visual e semiquantitativa foram concordantes para todos os estudos, mostrando que a captação do radiotraçador no estriado contralateral do lado mais afetado foi menor quando a 99mTc-TRODAT-1 SPECT foi empregada. A análise semiquantitativa demonstrou uma correlação significativa entre 18F-FDOPA PET e 99mTc-TRODAT-1 SPECT (r = 0,73; p < 0,01). Conclusão: A 99mTc-TRODAT-1 SPECT parece ser uma opção válida para o estudo da função dopaminérgica em um ambiente clínico.

Neurobiological substrates underlying corpus callosum hypoconnectivity and brain metabolic patterns in the valproic acid rat model of autism spectrum disorder.

Atypical connectivity between brain regions and altered structure of the corpus callosum (CC) in imaging studies supports the long-distance hypoconnectivity hypothesis proposed for autism spectrum disorder (ASD). The aim of this study was to unveil the CC ultrastructural and cellular changes employing the valproic acid (VPA) rat model of ASD. Male Wistar rats were exposed to VPA (450 mg/kg i.p.) or saline (control) during gestation (embryonic day 10.5), and maturation, exploration, and social behavior were subsequently tested. Myelin content, ultrastructure, and oligodendroglial lineage were studied in the CC at post-natal days 15 (infant) and 36 (juvenile). As a functional outcome, brain metabolic activity was determined by positron emission tomography. Concomitantly with behavioral deficits in juvenile VPA rats, the CC showed reduced myelin basic protein, conserved total number of axons, reduced percentage of myelinated axons, and aberrant and less compact arrangements of myelin sheath ultrastructure. Mature oligodendrocytes decreased and oligodendrocyte precursors increased in the absence of astrogliosis or microgliosis. In medial prefrontal and somatosensory cortices of juvenile VPA rats, myelin ultrastructure and oligodendroglial lineage were preserved. VPA animals exhibited global brain hypometabolism and local hypermetabolism in brain regions relevant for ASD. In turn, the CC of infant VPA rats showed reduced myelin content but preserved oligodendroglial lineage. Our findings indicate that CC hypomyelination is established during infancy and prior to oligodendroglial pattern alterations, which suggests that axon-oligodendroglia communication could be compromised in VPA animals. Thus, CC hypomyelination may underlie white matter alterations and contribute to atypical patterns of connectivity and metabolism found in ASD.

Tau mis-splicing correlates with motor impairments and striatal dysfunction in a model of tauopathy.

Tauopathies are neurodegenerative diseases caused by the abnormal metabolism of the microtubule associated protein tau (MAPT), which is highly expressed in neurons and critically involved in microtubule dynamics. In the adult human brain, the alternative splicing of exon 10 in MAPT pre-mRNA produces equal amounts of protein isoforms with either three (3R) or four (4R) microtubule binding domains. Imbalance in the 3R:4R tau ratio is associated with primary tauopathies that develop atypical parkinsonism, such as progressive supranuclear palsy and corticobasal degeneration. Yet, the development of effective therapies for those pathologies is an unmet goal. Here we report motor coordination impairments in the htau mouse model of tauopathy which harbour abnormal 3R:4R tau isoforms content, and in contrast to TauKO mice, are unresponsive to l-DOPA. Preclinical-PET imaging, array tomography and electrophysiological analyses indicated the dorsal striatum as the candidate structure mediating such phenotypes. Indeed, local modulation of tau isoforms by RNA trans-splicing in the striata of adult htau mice, prevented motor coordination deficits and restored basal neuronal firing. Together, these results suggest that abnormal striatal tau isoform content might lead to parkinsonian-like phenotypes and demonstrate a proof of concept that modulation of tau mis-splicing is a plausible disease-modifying therapy for some primary tauopathies.

Impaired brain glucose metabolism and presynaptic dopaminergic functioning in a mouse model of schizophrenia.

BACKGROUND: Schizophrenia is a disease diagnosed by visible signs and symptoms from late adolescence to early adulthood. The etiology of this disease remains unknown. An objective diagnostic approach is required. Here, we used a mouse model that shows schizophrenia-like phenotypes to study brain glucose metabolism and presynaptic dopaminergic functioning by positron emission tomography (PET) and immunohistochemistry. PET scannings were performed on mice after the administration of [18F]-FDG or [18F]-F-DOPA. Glucose metabolism was evaluated in basal conditions and after the induction of a hyperdopaminergic state. RESULTS: Mutant animals show reduced glucose metabolism in prefrontal cortex, amygdala, and nucleus reuniens under the hyperdopaminergic state. They also show reduced [18F]-F-DOPA uptake in prefrontal cortex, substantia nigra reticulata, raphe nucleus, and ventral striatum but increased [18F]-F-DOPA uptake in dorsal striatum. Mutant animals also show reduced tyrosine hydroxylase expression on midbrain neurons. CONCLUSIONS: Dopamine D2 mutant animals show reduced glucose metabolism and impaired presynaptic dopaminergic functioning, in line with reports from human studies. This mouse line may be a valuable model of schizophrenia, useful to test novel tracers for PET scanning diagnostic.

The lateral neocortex is critical for contextual fear memory reconsolidation.

Memories are a product of the concerted activity of many brain areas. Deregulation of consolidation and reprocessing of mnemonic traces that encode fearful experiences might result in fear-related psychopathologies. Here, we assessed how pre-established memories change with experience, particularly the labilization/reconsolidation of memory, using the whole-brain analysis technique of positron emission tomography in male mice. We found differences in glucose consumption in the lateral neocortex, hippocampus and amygdala in mice that underwent labilization/reconsolidation processes compared to animals that did not reactivate a fear memory. We used chemogenetics to obtain insight into the role of cortical areas in these phases of memory and found that the lateral neocortex is necessary for fear memory reconsolidation. Inhibition of lateral neocortex during reconsolidation altered glucose consumption levels in the amygdala. Using an optogenetic/neuronal recording-based strategy we observed that the lateral neocortex is functionally connected with the amygdala, which, along with retrograde labeling using fluorophore-conjugated cholera toxin subunit B, support a monosynaptic connection between these areas and poses this connection as a hot-spot in the circuits involved in reactivation of fear memories.

Quantification methods comparison in brain 18F-FDOPA PET.

18F-FDOPA PET is one of the most widely used molecular imaging techniques to assess presynaptic dopaminergic activity. A variety of analytical methods have been developed to quantify 18F-FDOPA PET images and in most, the striatal-to-occipital ratio (SOR) is used as a quantitative parameter. A manual strategy is typically used for quantification purposes, which can have some caveats, being time-consuming and having some inter-rater variability. In the present study we aimed to test whether automated quantification methods can provide an efficient alternative to manual quantification to overcome its limitations and compare each method's capacity to discriminate between normal and abnormal subjects. 18F-FDOPA PET images of 60 subjects were analyzed and quantified with one manual and two automated methods. SUVRs were obtained for caudate and putamen nucleus in both cases. We were able to reach the same level of discrimination with manual and automated strategies, and a threshold for normal/abnormal discrimination could be obtained. We believe automated strategies for VOI quantification can help molecular imaging physicians in the process of interpretation of studies, making the process faster, yet reliable and objective.

Sociability deficits after prenatal exposure to valproic acid are rescued by early social enrichment.

Background: Autism spectrum disorder (ASD) is characterized by impaired social interactions and repetitive patterns of behavior. Symptoms appear in early life and persist throughout adulthood. Early social stimulation can help reverse some of the symptoms, but the biological mechanisms of these therapies are unknown. By analyzing the effects of early social stimulation on ASD-related behavior in the mouse, we aimed to identify brain structures that contribute to these behaviors. Methods: We injected pregnant mice with 600-mg/kg valproic acid (VPA) or saline (SAL) at gestational day 12.5 and evaluated the effect of weaning their offspring in cages containing only VPA animals, only SAL animals, or mixed. We analyzed juvenile play at PD21 and performed a battery of behavioral tests in adulthood. We then used preclinical PET imaging for an unbiased analysis of the whole brain of these mice and studied the function of the piriform cortex by c-Fos immunoreactivity and HPLC. Results: Compared to control animals, VPA-exposed animals play less as juveniles and exhibit a lower frequency of social interaction in adulthood when reared with other VPA mice. In addition, these animals were less likely to investigate social odors in the habituation/dishabituation olfactory test. However, when VPA animals were weaned with control animals, these behavioral alterations were not observed. Interestingly, repetitive behaviors and depression-related behaviors were not affected by social enrichment. We also found that VPA animals present high levels of glucose metabolism bilaterally in the piriform cortex (Pir), a region known to be involved in social behaviors. Moreover, we found alterations in the somatosensory, motor, and insular cortices. Remarkably, these effects were mostly reversed after social stimulation. To evaluate if changes in glucose metabolism in the Pir correlated with changes in neuronal activity, we measured c-Fos immunoreactivity in the Pir and found it increased in animals prenatally exposed to VPA. We further found increased dopamine turnover in the Pir. Both alterations were largely reversed by social enrichment. Conclusions: We show that early social enrichment can specifically rescue social deficits in a mouse model of ASD. Our results identified the Pir as a structure affected by VPA-exposure and social enrichment, suggesting that it could be a key component of the social brain circuitry.

The neural substrate of gesture recognition.

Previous studies have linked action recognition with a particular pool of neurons located in the ventral premotor cortex, the posterior parietal cortex and the superior temporal sulcus (the mirror neuron system). However, it is still unclear if transitive and intransitive gestures share the same neural substrates during action-recognition processes. In the present study, we used event-related functional magnetic resonance imaging (fMRI) to assess the cortical areas active during recognition of pantomimed transitive actions, intransitive gestures, and meaningless control actions. Perception of all types of gestures engaged the right pre-supplementary motor area (pre-SMA), and bilaterally in the posterior superior temporal cortex, the posterior parietal cortex, occipitotemporal regions and visual cortices. Activation of the posterior superior temporal sulcus/superior temporal gyrus region was found in both hemispheres during recognition of transitive and intransitive gestures, and in the right hemisphere during the control condition; the middle temporal gyrus showed activation in the left hemisphere when subjects recognized transitive and intransitive gestures; activation of the left inferior parietal lobe and intraparietal sulcus (IPS) was mainly observed in the left hemisphere during recognition of the three conditions. The most striking finding was the greater activation of the left inferior frontal gyrus (IFG) during recognition of intransitive actions. Results show that a similar neural substrate, albeit, with a distinct engagement underlies the cognitive processing of transitive and intransitive gestures recognition. These findings suggest that selective disruptions in these circuits may lead to distinct clinical deficits.