SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration.

Reactive astrogliosis is a common pathological hallmark of CNS injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. These SrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains of Srf knockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases, SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce ß-amyloid plaques in mouse models of Parkinson's and Alzheimer's disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

Region-specific heterogeneity in neuronal nuclear morphology in young, aged and in Alzheimer's disease mouse brains.

Neurons in the mammalian brain exhibit enormous structural and functional diversity across different brain regions. Compared to our understanding of the morphological diversity of neurons, very little is known about the heterogeneity of neuronal nuclear morphology and how nuclear size changes in aging and diseased brains. Here, we report that the neuronal cell nucleus displays differences in area, perimeter, and circularity across different anatomical regions in the mouse brain. The pyramidal neurons of the hippocampal CA3 region exhibited the largest area whereas the striatal neuronal nuclei were the smallest. These nuclear size parameters also exhibited dichotomous changes with age across brain regions-while the neocortical and striatal neurons showed a decrease in nuclear area and perimeter, the CA3 neurons showed an increase with age. The nucleus of parvalbumin- and calbindin-positive interneurons had comparable morphological features but exhibited differences between brain regions. In the context of activity-dependent transcription in response to a novel environment, there was a decrease in nuclear size and circularity in c-Fos expressing neurons in the somatosensory cortex and hippocampal CA1 and CA3. In an APP/PS1 mutant mouse model of Alzheimer's disease (AD), the neuronal nuclear morphology varies with plaque size and with increasing distance from the plaque. The neuronal nuclear morphology in the immediate vicinity of the plaque was independent of the plaque size and the morphology tends to change away from the plaque. These changes in the neuronal nuclear size and shape at different ages and in AD may be attributed to changes in transcriptional activity. This study provides a detailed report on the differences that exist between neurons in nuclear morphology and can serve as a basis for future studies.

Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG.

Synapse associated protein-97/Human Disk Large (SAP97/hDLG) is a conserved, alternatively spliced, modular, scaffolding protein critical in regulating the molecular organization of cell-cell junctions in vertebrates. We confirm that the molecular determinants of first order phase transition of SAP97/hDLG is controlled by morpho-functional changes in its nanoscale organization. Furthermore, the nanoscale molecular signatures of these signalling islands and phase transitions are altered in response to changes in cytosolic Ca2+. Additionally, exchange kinetics of alternatively spliced isoforms of the intrinsically disordered region in SAP97/hDLG C-terminus shows differential sensitivities to Ca2+ bound Calmodulin, affirming that the molecular signatures of local phase transitions of SAP97/hDLG depends on their nanoscale heterogeneity and compositionality of isoforms.

Automated morphometric analysis with SMorph software reveals plasticity induced by antidepressant therapy in hippocampal astrocytes.

Nervous system development and plasticity involve changes in cellular morphology, making morphological analysis a valuable exercise in the study of nervous system development, function and disease. Morphological analysis is a time-consuming exercise requiring meticulous manual tracing of cellular contours and extensions. We have developed a software tool, called SMorph, to rapidly analyze the morphology of cells of the nervous system. SMorph performs completely automated Sholl analysis. It extracts 23 morphometric features based on cell images and Sholl analysis parameters, followed by principal component analysis (PCA). SMorph was tested on neurons, astrocytes and microglia and reveals subtle changes in cell morphology. Using SMorph, we found that chronic 21-day treatment with the antidepressant desipramine results in a significant structural remodeling in hippocampal astrocytes in mice. Given the proposed involvement of astroglial structural changes and atrophy in major depression in humans, our results reveal a novel kind of structural plasticity induced by chronic antidepressant administration.

Vinculin-mediated axon growth requires interaction with actin but not talin in mouse neocortical neurons.

The actin-binding protein vinculin is a major constituent of focal adhesion, but its role in neuronal development is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using functional mutants, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had an inhibitory effect. Interestingly, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching, and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobility of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.

Quantifying molecular aggregation by super resolution microscopy within an excitatory synapse from mouse hippocampal neurons.

Super-resolution microscopy (SRM) has been widely adopted to probe molecular distribution at excitatory synapses. We present an SRM paradigm to evaluate the nanoscale organization heterogeneity between neuronal subcompartments. Using mouse hippocampal neurons, we describe the identification of the morphological characteristics of nanodomains within functional zones of a single excitatory synapse. This information can be used to correlate structure and function at molecular resolution in single synapses. The protocol can be applied to immunocytochemical/histochemical samples across different imaging paradigms. For complete details on the use and execution of this protocol, please refer to Kedia et al. (2021).

Alteration in synaptic nanoscale organization dictates amyloidogenic processing in Alzheimer's disease.

Despite intuitive insights into differential proteolysis of amyloid precursor protein (APP), the stochasticity behind local product formation through amyloidogenic pathway at individual synapses remain unclear. Here, we show that the major components of amyloidogenic machinery namely, APP and secretases are discretely organized into nanodomains of high local concentration compared to their immediate environment in functional zones of the synapse. Additionally, with the aid of multiple models of Alzheimer's disease (AD), we confirm that this discrete nanoscale chemical map of amyloidogenic machinery is altered at excitatory synapses. Furthermore, we provide realistic models of amyloidogenic processing in unitary vesicles originating from the endocytic zone of excitatory synapses. Thus, we show how an alteration in the stochasticity of synaptic nanoscale organization contributes to the dynamic range of C-terminal fragments ß (CTFß) production, defining the heterogeneity of amyloidogenic processing at individual synapses, leading to long-term synaptic deficits as seen in AD.

SRF Is Required for Maintenance of Astrocytes in Non-Reactive State in the Mammalian Brain.

Astrocytes play several critical roles in the normal functioning of the mammalian brain, including ion homeostasis, synapse formation, and synaptic plasticity. Following injury and infection or in the setting of neurodegeneration, astrocytes become hypertrophic and reactive, a process termed astrogliosis. Although acute reactive gliosis is beneficial in limiting further tissue damage, chronic gliosis becomes detrimental for neuronal recovery and regeneration. Several extracellular factors have been identified that generate reactive astrocytes; however, very little is known about the cell-autonomous transcriptional mechanisms that regulate the maintenance of astrocytes in the normal non-reactive state. Here, we show that conditional deletion of the stimulus-dependent transcription factor, serum response factor (SRF) in astrocytes (SrfGFAPCKO) results in astrogliosis marked by hypertrophic morphology and increased expression of GFAP, vimentin, and nestin. These reactive astrocytes were not restricted to any specific brain region and were seen in both white and gray matter in the entire brain. This astrogliosis persisted throughout adulthood concomitant with microglial activation. Importantly, the Srf mutant mouse brain did not exhibit any cell death or blood brain barrier (BBB) deficits suggesting that apoptosis and leaky BBB are not the causes for the reactive phenotype. The mutant astrocytes expressed more A2 reactive astrocyte marker genes and the SrfGFAPCKO mice exhibited normal neuronal numbers indicating that SRF-deficient gliosis astrocytes are not neurotoxic. Together, our findings suggest that SRF plays a critical role in astrocytes to maintain them in a non-reactive state.

Shh-Mediated Increase in ß-Catenin Levels Maintains Cerebellar Granule Neuron Progenitors in Proliferation.

Cerebellar granule neuron progenitors (CGNPs) give rise to the cerebellar granule neurons in the developing cerebellum. Generation of large number of these neurons is made possible by the high proliferation rate of CGNPs in the external granule layer (EGL) in the dorsal cerebellum. Here, we show that upregulation of ß-catenin can maintain murine CGNPs in a state of proliferation. Further, we show that ß-catenin mRNA and protein levels can be regulated by the mitogen Sonic hedgehog (Shh). Shh signaling led to an increase in the level of the transcription factor N-myc. N-myc was found to bind the ß-catenin promoter, and the increase in ß-catenin mRNA and protein levels could be prevented by blocking N-myc upregulation downstream of Shh signaling. Furthermore, blocking Wingless-type MMTV integration site (Wnt) signaling by Wnt signaling pathway inhibitor Dickkopf 1 (Dkk-1) in the presence of Shh did not prevent the upregulation of ß-catenin. We propose that in culture, Shh signaling regulates ß-catenin expression through N-myc and results in increased CGNP proliferation.

Centrosome Inheritance Does Not Regulate Cell Fate in Granule Neuron Progenitors of the Developing Cerebellum.

An inherent asymmetry exists between the two centrosomes of a dividing cell. One centrosome is structurally more mature (mother centrosome) than the other (daughter centrosome). Post division, one daughter cell inherits the mother centrosome while the other daughter cell inherits the daughter centrosome. Remarkably, the kind of centrosome inherited is associated with cell fate in several developmental contexts such as in radial glial progenitors in the developing mouse cortex, Drosophila neuroblast divisions and in Drosophila male germline stem cells. However, the role of centrosome inheritance in granule neuron progenitors in the developing cerebellum has not been investigated. Here, we show that mother and daughter centrosomes do exist in these progenitors, and the amount of pericentriolar material (PCM) each centrosome possesses is different. However, we failed to observe any correlation between the fate adopted by the daughter cell and the nature of centrosome it inherited.

Genetic rescue of functional senescence in synaptic and behavioral plasticity.

STUDY OBJECTIVES: Aging has been linked with decreased neural plasticity and memory formation in humans and in laboratory model species such as the fruit fly, Drosophila melanogaster. Here, we examine plastic responses following social experience in Drosophila as a high-throughput method to identify interventions that prevent these impairments. PATIENTS OR PARTICIPANTS: Wild-type and transgenic Drosophila melanogaster. DESIGN AND INTERVENTIONS: Young (5-day old) or aged (20-day old) adult female Drosophila were housed in socially enriched (n = 35-40) or isolated environments, then assayed for changes in sleep and for structural markers of synaptic terminal growth in the ventral lateral neurons (LNVs) of the circadian clock. MEASUREMENTS AND RESULTS: When young flies are housed in a socially enriched environment, they exhibit synaptic elaboration within a component of the circadian circuitry, the LNVs, which is followed by increased sleep. Aged flies, however, no longer exhibit either of these plastic changes. Because of the tight correlation between neural plasticity and ensuing increases in sleep, we use sleep after enrichment as a high-throughput marker for neural plasticity to identify interventions that prolong youthful plasticity in aged flies. To validate this strategy, we find three independent genetic manipulations that delay age-related losses in plasticity: (1) elevation of dopaminergic signaling, (2) over-expression of the transcription factor blistered (bs) in the LNVs, and (3) reduction of the Imd immune signaling pathway. These findings provide proof-of-principle evidence that measuring changes in sleep in flies after social enrichment may provide a highly scalable assay for the study of age-related deficits in synaptic plasticity. CONCLUSIONS: These studies demonstrate that Drosophila provides a promising model for the study of age-related loss of neural plasticity and begin to identify genes that might be manipulated to delay the onset of functional senescence.

SRF phosphorylation by glycogen synthase kinase-3 promotes axon growth in hippocampal neurons.

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

L450W and Q455K Col8a2 knock-in mouse models of Fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy.

PURPOSE: We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two α2 collagen VIII (Col8a2) knock-in mouse models and human FECD tissues. METHODS: In vivo corneal endothelial cell (CEC) counts and morphology were analyzed by clinical confocal microscopy. Ultrastructural analysis of CECs was performed by transmission electron microscopy. Real-time PCR and Western blotting were performed using total RNA, and protein extracted from mouse CECs and human CECs obtained from FECD and autopsy patients. RESULTS: Both Col8a2 mouse models exhibited hallmarks of FECD; however, the Col8a2(L450W/L450W) mice exhibited a milder phenotype compared to the Col8a2(Q455K/Q455K) mice. Both models exhibited upregulation of the unfolded protein response (UPR) as evidenced by dilated rough endoplasmic reticulum (RER), and upregulation of UPR-associated genes and proteins. Real-time PCR of Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) CECs at 40 weeks revealed a 2.1-fold (P < 0.05) and a 5.2-fold (P < 0.01) upregulation of the autophagy marker Dram1, respectively. Real-time PCR of human FECD endothelium revealed a 10.4-fold upregulation of DRAM1 (P < 0.0001) compared to autopsy controls. CONCLUSIONS: The Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) mouse models of FECD showed distinct endothelial cell phenotypes. Dram1 was associated with activation of the UPR and increased autophagy. Overexpression of this gene in mouse and human FECD endothelial cells suggested a role for altered autophagy in this disease.

Serum response factor controls transcriptional network regulating epidermal function and hair follicle morphogenesis.

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality. Mice lacking SRF in epidermis displayed morphogenetic defects, including an eye-open-at-birth phenotype and lack of whiskers. SRF-null skin exhibited abnormal morphology, hyperplasia, aberrant expression of differentiation markers and transcriptional regulators, anomalous actin organization, enhanced inflammation, and retarded hair follicle (HF) development. Transcriptional profiling experiments uncovered profound molecular changes in SRF-null E17.5 epidermis and revealed that many previously identified SRF target CArG box-containing genes were markedly upregulated in SRF-null epidermis, indicating that SRF may function to repress transcription of a subset of its target genes in epidermis. Remarkably, when transplanted onto nude mice, engrafted SRF-null skin lacked hair but displayed normal epidermal architecture with proper expression of differentiation markers, suggesting that although keratinocyte SRF is essential for HF development, a cross-talk between SRF-null keratinocytes and the surrounding microenvironment is likely responsible for the barrier-deficient mutant epidermal phenotype.

A critical cell-intrinsic role for serum response factor in glial specification in the CNS.

Astrocytes and oligodendrocytes play crucial roles in nearly every facet of nervous system development and function, including neuronal migration, synaptogenesis, synaptic plasticity, and myelination. Previous studies have widely characterized the signaling pathways important for astrocyte differentiation and unveiled a number of transcription factors that guide oligodendrocyte differentiation in the CNS. However, the identities of the transcription factors critical for astrocyte specification in the brain remain unknown. Here we show that deletion of the stimulus-dependent transcription factor, serum response factor (SRF), in neural precursor cells (NPCs) (Srf-Nestin-cKO) results in nearly 60% loss in astrocytes and 50% loss in oligodendrocyte precursors at birth. Cultured SRF-deficient NPCs exhibited normal growth rate and capacity to self-renew. However, SRF-deficient NPCs generated fewer astrocytes and oligodendrocytes in response to several lineage-specific differentiation factors. These deficits in glial differentiation were rescued by ectopic expression of wild-type SRF in SRF-deficient NPCs. Interestingly, ectopic expression of a constitutively active SRF (SRF-VP16) in NPCs augmented astrocyte differentiation in the presence of pro-astrocytic factors. However, SRF-VP16 expression in NPCs had an inhibitory effect on oligodendrocyte differentiation. In contrast, mice carrying conditional deletion of SRF in developing forebrain neurons (Srf-NEX-cKO) did not exhibit any deficits in astrocytes in the brain. Together, our observations suggest that SRF plays a critical cell-autonomous role in NPCs to regulate astrocyte and oligodendrocyte specification in vivo and in vitro.

Activation of intracellular metabotropic glutamate receptor 5 in striatal neurons leads to up-regulation of genes associated with sustained synaptic transmission including Arc/Arg3.1 protein.

The G-protein coupled receptor, metabotropic glutamate receptor 5 (mGluR5), is expressed on both cell surface and intracellular membranes in striatal neurons. Using pharmacological tools to differentiate membrane responses, we previously demonstrated that cell surface mGluR5 triggers rapid, transient cytoplasmic Ca(2+) rises, resulting in c-Jun N-terminal kinase, Ca(2+)/calmodulin-dependent protein kinase, and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein (CREB) phosphorylation, whereas stimulation of intracellular mGluR5 induces long, sustained Ca(2+) responses leading to the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Elk-1 (Jong, Y. J., Kumar, V., and O'Malley, K. L. (2009) J. Biol. Chem. 284, 35827-35838). Using pharmacological, genetic, and bioinformatics approaches, the current findings show that both receptor populations up-regulate many immediate early genes involved in growth and differentiation. Activation of intracellular mGluR5 also up-regulates genes involved in synaptic plasticity including activity-regulated cytoskeletal-associated protein (Arc/Arg3.1). Mechanistically, intracellular mGluR5-mediated Arc induction is dependent upon extracellular and intracellular Ca(2+) and ERK1/2 as well as calmodulin-dependent kinases as known chelators, inhibitors, and a dominant negative Ca(2+)/calmodulin-dependent protein kinase II construct block Arc increases. Moreover, intracellular mGluR5-induced Arc expression requires the serum response transcription factor (SRF) as wild type but not SRF-deficient neurons show this response. Finally, increased Arc levels due to high K(+) depolarization is significantly reduced in response to a permeable but not an impermeable mGluR5 antagonist. Taken together, these data highlight the importance of intracellular mGluR5 in the cascade of events associated with sustained synaptic transmission.

Serum response factor is required for cortical axon growth but is dispensable for neurogenesis and neocortical lamination.

Previous studies have shown that neuron-specific deletion of serum response factor (SRF) results in deficits in tangential cell migration, guidance-dependent circuit assembly, activity-dependent gene expression, and synaptic plasticity in the hippocampus. Furthermore, SRF deletion in mouse embryonic stem cells causes cell death in vitro. However, the requirement of SRF for early neuronal development including neural stem cell homeostasis, neurogenesis, and axonal innervations remains unknown. Here, we report that SRF is critical for development of major axonal tracts in the forebrain. Conditional mutant mice lacking SRF in neural progenitor cells (Srf-Nestin-cKO) exhibit striking deficits in cortical axonal projections including corticostriatal, corticospinal, and corticothalamic tracts, and they show a variable loss of the corpus callosum. Neurogenesis and interneuron specification occur normally in the absence of SRF and the deficits in axonal projections were not due to a decrease or loss in cell numbers. Radial migration of neurons and neocortical lamination were also not affected. No aberrant cell death was observed during development, whereas there was an increase in the number of proliferative cells in the ventricular zone from embryonic day 14 to day 18. Similar axonal tract deficits were also observed in mutant mice lacking SRF in the developing excitatory neurons of neocortex and hippocampus (Srf-NEX-cKO). Together, these findings suggest distinct roles for SRF during neuronal development; SRF is specifically required in a cell-autonomous manner for axonal tract development but is dispensable for cell survival, neurogenesis, neocortical lamination, and neuronal differentiation.

Use-dependent plasticity in clock neurons regulates sleep need in Drosophila.

Sleep is important for memory consolidation and is responsive to waking experience. Clock circuitry is uniquely positioned to coordinate interactions between processes underlying memory and sleep need. Flies increase sleep both after exposure to an enriched social environment and after protocols that induce long-term memory. We found that flies mutant for rutabaga, period, and blistered were deficient for experience-dependent increases in sleep. Rescue of each of these genes within the ventral lateral neurons (LNVs) restores increased sleep after social enrichment. Social experiences that induce increased sleep were associated with an increase in the number of synaptic terminals in the LNV projections into the medulla. The number of synaptic terminals was reduced during sleep and this decline was prevented by sleep deprivation.