Upregulation of astroglial connexin 30 impairs hippocampal synaptic activity and recognition memory.

Astrocytes crucially contribute to synaptic physiology and information processing. One of their key characteristics is to express high levels of connexins (Cxs), the gap junction-forming protein. Among them, Cx30 displays specific properties since it is postnatally expressed and dynamically upregulated by neuronal activity and modulates cognitive processes by shaping synaptic and network activities, as recently shown in knockout mice. However, it remains unknown whether local and selective upregulation of Cx30 in postnatal astrocytes within a physiological range modulates neuronal activities in the hippocampus. We here show in mice that, whereas Cx30 upregulation increases the connectivity of astroglial networks, it decreases spontaneous and evoked synaptic transmission. This effect results from a reduced neuronal excitability and translates into an alteration in the induction of synaptic plasticity and an in vivo impairment in learning processes. Altogether, these results suggest that astroglial networks have a physiologically optimized size to appropriately regulate neuronal functions.

Publisher Correction: Progenitors from the central nervous system drive neurogenesis in cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Fast calcium transients in dendritic spines driven by extreme statistics.

[This corrects the article DOI: 10.1371/journal.pbio.2006202.].

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns.

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Neuronal A2A receptor exacerbates synapse loss and memory deficits in APP/PS1 mice.

Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

Progenitors from the central nervous system drive neurogenesis in cancer.

Autonomic nerve fibres in the tumour microenvironment regulate cancer initiation and dissemination, but how nerves emerge in tumours is currently unknown. Here we show that neural progenitors from the central nervous system that express doublecortin (DCX+) infiltrate prostate tumours and metastases, in which they initiate neurogenesis. In mouse models of prostate cancer, oscillations of DCX+ neural progenitors in the subventricular zone-a neurogenic area of the central nervous system-are associated with disruption of the blood-brain barrier, and with the egress of DCX+ cells into the circulation. These cells then infiltrate and reside in the tumour, and can generate new adrenergic neurons. Selective genetic depletion of DCX+ cells inhibits the early phases of tumour development in our mouse models of prostate cancer, whereas transplantation of DCX+ neural progenitors promotes tumour growth and metastasis. In humans, the density of DCX+ neural progenitors is strongly associated with the aggressiveness and recurrence of prostate adenocarcinoma. These results reveal a unique crosstalk between the central nervous system and prostate tumours, and indicate neural targets for the treatment of cancer.

Reactive astrocytes promote proteostasis in Huntington's disease through the JAK2-STAT3 pathway.

Huntington's disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntington's disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntington's disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models. Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntington's disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntington's disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons. Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntington's disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes.

Development of an AAV-based model of tauopathy targeting retinal ganglion cells and the mouse visual pathway to study the role of microglia in Tau pathology.

Tauopathy is a typical feature of Alzheimer's disease of major importance because it strongly correlates with the severity of cognitive deficits experienced by patients. During the pathology, it follows a characteristic spatiotemporal course which takes its origin in the transentorhinal cortex, and then gradually invades the entire forebrain. To study the mechanisms of tauopathy, and test new therapeutic strategies, it is necessary to set-up relevant and versatile in vivo models allowing to recapitulate tauopathy. With this in mind, we have developed a model of tauopathy by overexpression of the human wild-type Tau protein in retinal ganglion cells in mice (RGCs). This overexpression led to the presence of hyperphosphorylated forms of the protein in the transduced cells as well as to their progressive degeneration. The application of this model to mice deficient in TREM2 (Triggering Receptor Expressed on Myeloid cells-2, an important genetic risk factor for AD) as well as to 15-month-old mice showed that microglia actively participate in the degeneration of RGCs. Surprisingly, although we were able to detect the transgenic Tau protein up to the terminal arborization of RGCs at the level of the superior colliculi, spreading of the transgenic Tau protein to post-synaptic neurons was detected only in aged animals. This suggests that there may be neuron-intrinsic- or microenvironment mediators facilitating this spreading that appear with aging.

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

PKCδ-positive GABAergic neurons in the central amygdala exhibit tissue-type plasminogen activator: role in the control of anxiety.

Tissue plasminogen activator (tPA) is a serine protease expressed in several brain regions and reported to be involved in the control of emotional and cognitive functions. Nevertheless, little is known about the structure-function relationships of these tPA-dependent behaviors. Here, by using a new model of constitutive tPA-deficient mice (tPAnull), we first show that tPA controls locomotor activity, spatial cognition and anxiety. To investigate the brain structures involved in these tPA-dependent behavioral phenotypes, we next generated tPAflox mice allowing conditional tPA deletion (cKO) following stereotaxic injections of adeno-associated virus driving Cre-recombinase expression (AAV-Cre-GFP). We demonstrate that tPA removal in the dentate gyrus of the hippocampus induces hyperactivity and partial spatial memory deficits. Moreover, the deletion of tPA in the central nucleus of the amygdala, but not in the basolateral nucleus, induces hyperactivity and reduced anxiety-like level. Importantly, we prove that these behaviors depend on the tPA present in the adult brain and not on neurodevelopmental disorders. Also, interestingly, our data show that tPA from Protein kinase-C delta-positive (PKCδ) GABAergic interneurons of the lateral/ capsular part of adult mouse central amygdala controls emotional functions through neuronal activation of the medial central amygdala. Together, our study brings new data about the critical central role of tPA in behavioral modulations in adult mice.

Fast calcium transients in dendritic spines driven by extreme statistics.

Fast calcium transients (<10 ms) remain difficult to analyse in cellular microdomains, yet they can modulate key cellular events such as trafficking, local ATP production by endoplasmic reticulum-mitochondria complex (ER-mitochondria complex), or spontaneous activity in astrocytes. In dendritic spines receiving synaptic inputs, we show here that in the presence of a spine apparatus (SA), which is an extension of the smooth ER, a calcium-induced calcium release (CICR) is triggered at the base of the spine by the fastest calcium ions arriving at a Ryanodyne receptor (RyR). The mechanism relies on the asymmetric distributions of RyRs and sarco/ER calcium-ATPase (SERCA) pumps that we predict using a computational model and further confirm experimentally in culture and slice hippocampal neurons. The present mechanism for which the statistics of the fastest particles arriving at a small target, followed by an amplification, is likely to be generic in molecular transduction across cellular microcompartments, such as thin neuronal processes, astrocytes, endfeets, or protrusions.

Neuronal tau species transfer to astrocytes and induce their loss according to tau aggregation state.

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocytes in the hippocampus, whereas the accumulation of tau aggregates does not affect their survival. Thus, astrocytes are not mere bystanders of neuronal pathology. Our results strongly suggest that tau pathology in astrocytes may significantly contribute to clinical symptoms.

Time-Course of Alterations in the Endocannabinoid System after Viral-Mediated Overexpression of α-Synuclein in the Rat Brain.

Since the discovery of α-synuclein as the major component in Lewy bodies, research into this protein in the context of Parkinson's disease pathology has been exponential. Cannabinoids are being investigated as potential therapies for Parkinson's disease from numerous aspects, but still little is known about the links between the cannabinoid system and the pathogenic α-synuclein protein; understanding these links will be necessary if cannabinoid therapies are to reach the clinic in the future. Therefore, the aim of this study was to investigate the time-course of alterations in components of the endocannabinoid system after viral-mediated α-synuclein overexpression in the rat brain. Rats were given unilateral intranigral injections of AAV-GFP or AAV-α-synuclein and sacrificed 4, 8 and 12 weeks later for qRT-PCR and liquid chromatography-mass spectrometry analyses of the endocannabinoid system, in addition to histological visualization of α-synuclein expression along the nigrostriatal pathway. As anticipated, intranigral delivery of AAV-α-synuclein induced widespread overexpression of human α-synuclein in the nigrostriatal pathway, both at the mRNA level and the protein level. However, despite this profound α-synuclein overexpression, we detected no differences in CB1 or CB2 receptor expression in the nigrostriatal pathway; however, interestingly, there was a reduction in the expression of neuroinflammatory markers. Furthermore, there was a reduction in the levels of the endocannabinoid 2-AG and the related lipid immune mediator OEA at week 12 post-surgery, indicating that α-synuclein overexpression triggers dysregulation of the endocannabinoid system. Although this research does show that the endocannabinoid system is impacted by α-synuclein, further research is necessary to more comprehensively understand the link between the cannabinoid system and the α-synuclein aspect of Parkinson's disease pathology in order for cannabinoid-based therapies to be feasible for the treatment of this disease in the coming years.

THY-Tau22 mouse model accumulates more tauopathy at late stage of the disease in response to microglia deactivation through TREM2 deficiency.

The role played by microglia has taken the center of the stage in the etiology of Alzheimer's disease (AD). Several genome-wide association studies carried out on large cohorts of patients have indeed revealed a large number of genetic susceptibility factors corresponding to genes involved in neuroinflammation and expressed specifically by microglia in the brain. Among these genes TREM2, a cell surface receptor expressed by microglia, arouses strong interest because its R47H variant confers a risk of developing AD comparable to the ε4 allele of the APOE gene. Since this discovery, a growing number of studies have therefore examined the role played by TREM2 in the evolution of amyloid plaques and neurofibrillary tangles, the two brain lesions characteristic of AD. Many studies report conflicting results, reflecting the complex nature of microglial activation in AD. Here, we investigated the impact of TREM2 deficiency in the THY-Tau22 transgenic line, a well-characterized model of tauopathy. Our study reports an increase in the severity of tauopathy lesions in mice deficient in TREM2 occurring at an advanced stage of the pathology. This exacerbation of pathology was associated with a reduction in microglial activation indicated by typical morphological features and altered expression of specific markers. However, it was not accompanied by any further changes in memory performance. Our longitudinal study confirms that a defect in microglial TREM2 signaling leads to an increase in neuronal tauopathy occurring only at late stages of the disease.

Connexin 30 controls astroglial polarization during postnatal brain development.

Astrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates in situ the extension and ramification of astroglial processes. However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an in vitro wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/ß1 integrin/Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the ß1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. In vivo, connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development.

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo.

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson's disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) "cell-autonomous". Here, we explored whether ΔLRRK2G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2G2019S or its "dead" kinase form, ΔLRRK2DK, and human α-syn with the A53T mutation (AAV-α-synA53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-synA53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-synA53T with AAV-ΔLRRK2G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-synA53T alone or with AAV-ΔLRRK2DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2G2019 alone, through cell-autonomous mechanisms.

The intellectual disability protein Oligophrenin-1 controls astrocyte morphology and migration.

Astrocytes are involved in several aspects of neuronal development and properties which are altered in intellectual disability (ID). Oligophrenin-1 is a RhoGAP protein implicated in actin cytoskeleton regulation, and whose mutations are associated with X-linked ID. Oligophrenin-1 is expressed in neurons, where its functions have been widely reported at the synapse, as well as in glial cells. However, its roles in astrocytes are still largely unexplored. Using in vitro and in vivo models of oligophrenin1 disruption in astrocytes, we found that oligophrenin1 regulates at the molecular level the RhoA/ROCK/MLC2 pathway in astroglial cells. We also showed at the cellular level that oligophrenin1 modulates astrocyte morphology and migration both in vitro and in vivo, and is involved in glial scar formation. Altogether, these data suggest that oligophrenin1 deficiency alters not only neuronal but also astrocytic functions, which might contribute to the development of ID.

The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo.

The G2019S substitution in the kinase domain of LRRK2 (LRRK2G2019S) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2WT or LV-ΔLRRK2G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2WT and ΔLRRK2G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.

Potentiating tangle formation reduces acute toxicity of soluble tau species in the rat.

Tauopathies are neurodegenerative diseases characterized by the aggregation of tau protein. These pathologies exhibit a wide variety of clinical and anatomo-pathological presentations, which may result from different pathological mechanisms. Although tau inclusions are a common feature in all these diseases, recent evidence instead implicates small oligomeric aggregates as drivers of tau-induced toxicity. Hence in vivo model systems displaying either soluble or fibrillary forms of wild-type or mutant tau are needed to better identify their respective pathological pathways. Here we used adeno-associated viruses to mediate gene transfer of human tau to the rat brain to develop models of pure tauopathies. Two different constructs were used, each giving rise to a specific phenotype developing in less than 3 months. First, hTAUWT overexpression led to a strong hyperphosphorylation of the protein, which was associated with neurotoxicity in the absence of any significant aggregation. In sharp contrast, its co-expression with the pro-aggregation peptide TauRD-ΔK280 in the hTAUProAggr group strongly promoted its aggregation into Gallyas-positive neurofibrillary tangles, while preserving neuronal survival. Our results support the hypothesis that soluble tau species are key players of tau-induced neurodegeneration.

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin.

The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration.