Single-cell multi-omics defines the cell-type specific impact of splicing factor mutations on human hematopoietic differentiation

The formation and remodeling of synaptic contacts require the precise distribution and trafficking of proteins to specialized compartments. This dynamic trafficking of synaptic proteins is partly controlled by S-palmitoylation, which is the most common form of post-translational lipid modification in the brain. Notably, several studies have shown that synaptic proteins can be differentially palmitoylated in response to synaptic activity. However, it is unclear how changes in synaptic activity alters protein palmitoylation. Here we show that increasing synaptic activity in primary rat hippocampal cultures using a well-established chemical LTP (cLTP) paradigm leads to post-translational modifications in ZDHHC enzymes which in turn impact enzyme stability, protein interaction and function. Notably, we observe the same post-translational modifications following the acquisition of contextual fear memory in vivo. In contrast to that observed for palmitoylating enzymes, we observed no activity-induced changes in the activity of thioesterases nor post-translational modifications of the thioesterase, ABHD17. These findings suggest that the differential palmitoylation of synaptic proteins following synaptic stimulation is mediated through post-translational modifications of ZDHHC enzymes that in turn regulate enzyme stability and function, and not through changes in depalmitoylating thioesterases.

Dynamic regulation of palmitoyltransferases by synaptic activity

Mammalian spinal cord injury (SCI) occurs by a primary mechanical trauma that is exacerbated by a secondary cellular insult in the form of neuroinflammation, cell death, and reactive gliosis. The lack of functional recovery may be attributed to the inability of mammals to replace lost neurons. In zebrafish (Danio rerio), the secondary insult is attenuated and allows for the development of a spinal microenvironment that fosters regeneration. Radial glia lining the zebrafish central canal undergo injury-induced proliferation and neuronal differentiation, yet the molecular mechanisms that underlie these processes remain elusive. Previous reports have identified roles for purinergic P2Y2 and P2X7 receptors in mammalian neural progenitor cell proliferation and neuronal differentiation, respectively. Given that the purinergic system is evolutionarily conserved among vertebrates, we hypothesize that radial glia proliferation and neurogenesis following zebrafish SCI may be mediated by P2X7 and P2Y2 signaling. Preliminary immunohistochemical data suggests that radial glia express P2X7 and P2Y2 receptors. Preliminary Western blotting data shows significant upregulation of P2Y2 receptor expression at 1 day following zebrafish SCI, as well as significant downregulation of P2X7 receptor expression at 7 days. This contrasts the more gradual increase in P2Y2 receptor expression as well as the general increase in P2X7 receptor expression following mammalian SCI. Specific blockers for P2X7 and P2Y2 will be used to ascertain the roles of these receptors in the zebrafish SCI response. This work is well suited to advance our current understanding of successful central nervous system regeneration and elucidate the signaling pathways involved.

Adaptive purinergic P2X7 and P2Y2 signaling following spinal cord injury in Danio rerio

Subiculum pyramidal neurons form the main output of the hippocampus, and are classically viewed as giving rise to memory by branched structures that receive proximal and distal input. Here, we identified a sparse pyramidal cell subpopulation within the subiculum that diverges from classical pyramidal neuron characteristics across connectivity, morphology, and intrinsic activity. Via transcriptomics, we identified a sparse excitatory neuron population that occupies the deepest layer of the subiculum, and generated a new transgenic mouse line that allows cre-mediated cell-type-specific access of these deep cells. Combining this transgenic line with cell-type-specific viral tracing tools, we discovered that this deep cell population forms dedicated projections to the anterior thalamic nuclei, a key brain region in spatial working memory. Moreover, these deep cells lack radial oblique dendrites typical of classical subiculum pyramidal cells. Motivated by this unique local and long-range structure of these cells, we investigated potential differences in intrinsic function via whole cell patch-clamp recordings. Recording results revealed that these deep cells exhibited an increased input resistance compared to classical pyramidal cells in the subiculum. Altogether, our data provides evidence for a previously unknown subiculum pyramidal cell subtype that exhibits unique structural and intrinsic characteristics, which could indicate a potentially specialized role in spatial memory. Future work will be integral to identifying the functional correlates of this atypical population.

Atypical subiculum pyramidal neurons form dedicated projections and exhibit morphological and electrophysiological differences

Synaptic plasticity, characterized by long-term potentiation(LTP) and long-term depression (LTD), is an important physiological phenomenon that is thought to underlie the mechanism of learning and memory formation. Dysregulation in synaptic plasticity is associated with various neurological disorders. Successful treatments to enhance learning have been hard to target to the lack of understanding in the mechanisms underlying synaptic plasticity. Synaptopodin (SP), a novel actin-associated protein, was found to be an important regulator of synaptic plasticity and learning. SP is found in a subset of dendritic spines, namely the larger, functionally stronger synapses. It is associated with an intracellular organelle the spine apparatus which is responsible for calcium release from internal calcium stores and local protein synthesis. Previous research demonstrated that SP knock-out (SPKO) mice have impaired NMDA- dependent LTP and spatial learning ability but no change in NMDA-dependent LTD. mGluR-LTD is another form of LTD in the hippocampus and its expression mechanisms are much less understood than NMDA-LTD. mGluR-LTD requires IP3R- dependent calcium signaling and protein synthesis-dependent pathway. Its dysregulation has been associated with learning deficit in many neurological diseases, in particular, Fragile X syndrome, the most prevalent neurodevelopmental disorder. Using SPKO mice, we found a reduction in mGluR-LTD compared to wildtype mice. We report that mGluR-LTD - through activation of mGluR1 but not mGluR5 - drives selective loss of the larger more stable excitatory synapses distinguished by the lack of SP. We also observed a signifcant reduction of IP3R in SPKO spines. These findings identify spines that harbor SP as the cellular locus of mGluR-LTD.

Synaptopodin, a novel actin-associated protein, plays important role in mGluR-LTD

Latrophyllins belong to the family of adhesion G protein-coupled receptors, capable of binding to actin-associated scaffold proteins. They are expressed in various tissues, suggesting that they might participate in biological processes that are ubiquitous. Here we focus on actin cytoskeleton dynamics to explore the role of latrophilins in mammalian cells. Overexpression of each latrophilin isoform comparably increased cell volume while modifying the net profile of F-actin-dependent cell structures, as evaluated by confocal microscopy analysis. Latrophilin deletion mutants evidenced that direct coupling to the intracellular machinery was a requirement for modulating cell structures. The association between latrophilins and the actin cytoskeleton was detected by co-immunoprecipitation assays and corroborated with immunocytochemistry analysis. Consistent with the destabilization of F-actin structures, latrophilin isoforms constitutively induced a prominent increase in the activity of actin depolymerizing factor, cofilin, evaluated by western blot assays. Intercellular adhesion events stabilized by heterophilic Teneurin-4 trans-interactions disrupted latrophilin colocalization with F-actin and led to an isoform-specific rescue of cell extensions. Therefore, we found that constitutive signaling of latrophyllins and their activation by ligand directly involves the actin cytoskeleton machinery

Study of the mechanisms involved in the remodeling of the actin cytoskeleton induced by Latrophyllins 1,2 and 3

Aging is associated with cognitive decline and memory loss in humans. In rats, age-associated impairment of spatial and olfactory learning has been observed. We investigated how neuronal excitability changes during aging both in pyramidal neurons of the piriform cortex (PC) and in the CA1 layer of the hippocampus, two key areas involved in these behaviors. In young (<5 months old) and aging Sprague-Dawley rats (>18 months old), we used whole-cell patch clamp recording to measure intrinsic electrophysiological properties of pyramidal neurons in each region. To preserve the health of slices, rats were intracardially perfused with cold and bubbled (95% O2, 5% CO2) artificial cerebrospinal fluid prior to dissection and slicing. Sagittal slices were incubated in N-methyl-D-glucamine (NMDG)-based recovery solution for 10 minutes, and then transferred to a HEPES buffered holding solution for full recovery. In whole-cell configuration, cells were injected with depolarizing currents to generate a current-voltage (I-V) relationship, where somatic and action potential (AP) properties were measured. In addition, current was injected to characterize slow afterhyperpolarization (AHP). In both regions, AP threshold was significantly higher for aged rats, with larger fast AHP. Aged hippocampal neurons also displayed a reduced excitability, along with higher input resistance and a significantly larger slow AHP. These differences in intrinsic neuronal function may contribute to underlying age-related mechanisms of reduced synaptic plasticity and overall cognitive decline.

The effects of aging on intrinsic properties of pyramidal neurons in the piriform cortex and hippocampus

Despite demonstrations that a number of different types of sensory stimuli can evoke calcium responses in glia, a comprehensive understanding of the mechanisms underlying the generation of these events in intact animals remains limited. To thoroughly investigate the signalling pathways mediating the recruitment of glial activation during visual stimulation, we generated Xenopus laevis tadpoles which express GCaMP6s in all cells of the brain and used in vivo two-photon microscopy to simultaneously image both neurons and glia in the developing retinotectal system of intact animals. Using a systematic combination of visual stimulation and pharmacological manipulation during live imaging, we were able to demonstrate that radial astrocytes in the optic tectum are highly responsive to visual stimulation, and that visually-evoked responses in these glia continue to occur following broad blockade of all glutamate receptors and the subsequent silencing of post- but not pre-synaptic neuronal activity. We observed that visually-evoked responses in radial astrocytes are silenced following the additional blockade of either glutamate transporters or sodium-calcium exchangers (NCX) and that the blockade of NCX alone is sufficient to prevent radial astrocytes from responding to visual stimulation, suggesting NCX may play a pivotal role in glia during early development of the visual system.

Visually-evoked calcium transients in glia are mediated by sodium-calcium exchangers in the developing retinotectal system

Theta rhythms (4-12 Hz) are crucial for hippocampal network coordination and functions, including memory and anxiety. Lesion and pharmacological studies have shown that the median raphe nucleus (MR) exerts a strong modulation over theta rhythms. However, the MR is composed of mixed neuronal populations, including serotonergic neurons (5-HT) and glutamatergic neurons expressing the vesicular glutamate transporter type 3 (VGLUT3). In this study, we aim to evaluate the contribution of these two largely distinct median raphe neuronal populations to theta rhythm properties. We used optogenetics to activate either 5HT or VGLUT3 neurons in the median raphe (MR) of freely behaving mice. To do so, we injected CRE-dependent AAV vectors in the MR of either SERT-CRE or VGLUT3-CRE mice, to selectively transfect 5-HT or VGLUT3 MR neurons with the excitatory opsin ChETAET/TC. An optic fiber implanted above the MR allowed light delivery and activation of 5-HT or VGLUT3 neurons, while simultaneously recording theta rhythms in the hippocampus during REM sleep and awake locomotion. Our results show that 5HT and VGLUT3 neurons participate in the modulation of hippocampal theta frequency and power. These results suggest a potential role for MR 5-HT and VGLUT3 neurons in the modulation of hippocampal functions such as memory and anxiety.

3Hippocampal theta rhythm modulation by the glutamatergic and serotonergic raphe

BACKGROUND AND AIM: Microglia are the principal immune cells of the brain. The consensus today is that once activated microglia/macrophages can acquire a wide repertoire of profiles ranging from the classical pro-inflammatory to alternative and more protective phenotypes. Over the past decade, we and others have shown that optimal and timely activation of microglial cells and innate immunity is instrumental in the control of the inflammation-induced damage to CNS. To add to complexity, increasing evidence suggests a marked sexual dimorphism in the processes associated with microglial activation following different types of brain injuries. At present, the in vivo molecular mechanisms involved in the control of microglia and/or macrophage immune profiles remain elusive METHODS: To decipher the molecular mechanisms underlying microglial activation in vivo, we created a transgenic model in which Flag/EGFP was fused to the N-terminus of the large subunit ribosomal protein L10a and expressed under the transcriptional control of a myeloid specific gene promoter Thus, a simple high affinity immunoprecipitation assay from brain homogenates allows a pull down of the transcriptome and/or proteome from the targeted cell type. By isolating both ribosome-attached mRNAs and peptides, we obtained a snapshot of the dynamic translational state of microglial ribosomes with mRNAs as input and newly synthesized peptides as output. Using this strategy, we identified mRNA and protein signatures associated with microglial activation. RESULTS: By using in vivo model-system for analysis of dynamic translational state of microglia ribosomes with mRNAs as input and newly synthesized peptides as an output, we discovered a marked dissociation of microglia mRNA and protein networks following innate immune challenge. We found that the highly up-regulated and polysome- associated mRNAs are not translated resulting in two distinct and rather divergent microglia molecular signatures: i) a highly specialized immune and pro-inflammatory mRNA signature and ii) a more immunomodulatory and homeostatic protein signature. As molecular mechanism, we discovered a selective 3'UTR-mediated translational suppression of highly expressed immune mRNAs. Furthermore, we identified a novel and previously unknown role for RNA binding protein Serine/Arginine-Rich Splicing Factor 3 SRSF3 as a master suppressor/regulator of innate immune genes translation in activated microglia. CONCLUSIONS: Our recent work suggests involvement of SRSF3 -mediated mechanisms in the modulation of immune profiles of activated microglia in different type of brain injuries and/or pathologies. Better understanding of SRFS3- mediated immunomodulation may open new avenues for therapeutic modulation of innate immune response following brain injuries.

Microglial cells after brain injuries: sexually dimorphic and lost in translation

Microglia are the resident immune cells in the central nervous system (CNS). Originally thought to be primarily responsible for disposing of cellular debris and responding to neural insults, emerging research now shows that microglia are highly dynamic cells involved in a variety of neural processes, including development. The hypothalamus is a brain region critical for maintaining homeostatic processes such as energy balance, thirst, food intake, reproduction, and circadian rhythms. Given that microglia colonize the embryonic brain alongside key steps of hypothalamic development and that the loss of microglia causes obesity in newborn pups, consistent with a defect in neuroendocrine signaling, we explored how microglia influence these nearby hypothalamic progenitors. We found that embryonic microglia directly and indirectly interact with hypothalamic radial glial cells by both touching and clipping of their projections and also by releasing signaling cues, respectively. These interactions were increased following maternal stress, suggesting that fetal microglia might be sensors to the external environment. Combined, these data demonstrate that microglia can influence overall development of this important brain region, which might contribute to diseases later in life.

Embryonic hypothalamic microglia: a sensor for maternal stressors and potential mediator of neurodevelopmental diseases

Recent optogenetic technologies have demonstrated not only light-dependent control of neuronal activity but also the regulation of target molecules by light, suggesting their potential applications to study cellular functions at the molecular level in the brain. Here, we introduce our optogenetic approaches for the non-invasive manipulation of intracellular cAMP processes with light in target neurons of the brain. cAMP is a ubiquitous secondary messenger for intracellular signalling pathways, mediating sensory transduction and neuromodulation. However, cAMP dynamics and related spatiotemporal functions remain elusive in the brain due to limitations of spatiotemporal specificity of pharmacological and genetic approaches to acutely perturb cAMP function. We have optimized and further established genetically encoded light-sensitive cAMP metabolic enzymes to enhance or suppress cAMP levels in living neurons. By expressing the light-sensitive cAMP metabolic enzymes with cAMP fluorescence indicators, we successfully monitored and manipulated cAMP levels of living hippocampal pyramidal neurons in murine hippocampal brain slices with two-photon laser microscopy, validating our optogenetic approaches in the target hippocampal neurons. Furthermore, we incorporated these optogenetic techniques directly in the target hippocampal neurons of freely behaving mice with brain-implanted fiber optic LED devices for light-dependent manipulation of cAMP in the intact brain. We will discuss in vivo optogenetic approaches in combination with murine hippocampus-dependent behavioural tests.

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Single-cell multi-omics defines the cell-type specific impact of splicing factor mutations on human hematopoietic differentiation

Dynamic regulation of palmitoyltransferases by synaptic activity

Adaptive purinergic P2X7 and P2Y2 signaling following spinal cord injury in Danio rerio

Atypical subiculum pyramidal neurons form dedicated projections and exhibit morphological and electrophysiological differences

Synaptopodin, a novel actin-associated protein, plays important role in mGluR-LTD

Study of the mechanisms involved in the remodeling of the actin cytoskeleton induced by Latrophyllins 1,2 and 3

The effects of aging on intrinsic properties of pyramidal neurons in the piriform cortex and hippocampus

Visually-evoked calcium transients in glia are mediated by sodium-calcium exchangers in the developing retinotectal system

3Hippocampal theta rhythm modulation by the glutamatergic and serotonergic raphe

Microglial cells after brain injuries: sexually dimorphic and lost in translation

Embryonic hypothalamic microglia: a sensor for maternal stressors and potential mediator of neurodevelopmental diseases

A toolbox for the optogenetic control of cAMP in vivo

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