Ephraim F. Trakhtenberg

Soluble Adenylyl Cyclase Activity Is Necessary for Retinal Ganglion Cell Survival and Axon Growth

cAMP is a critical second messenger mediating activity-dependent neuronal survival and neurite growth. We investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in CNS retinal ganglion cells (RGCs). We found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated sAC inhibition dramatically decreased RGC survival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knock-out mice or after specifically inhibiting tmACs. These data identify a novel sAC-mediated cAMP signaling pathway regulating RGC survival and axon growth, and suggest new neuroprotective or regenerative strategies based on sAC modulation.

Regulation of Intrinsic Axon Growth Ability at Retinal Ganglion Cell Growth Cones

PURPOSE Mammalian central nervous system neurons fail to regenerate after injury or disease, in part due to a progressive loss in intrinsic axon growth ability after birth. Whether lost axon growth ability is due to limited growth resources or to changes in the axonal growth cone is unknown. METHODS Static and time-lapse images of purified retinal ganglion cells (RGCs) were analyzed for axon growth rate and growth cone morphology and dynamics without treatment and after manipulating Kruppel-like transcription factor (KLF) expression or applying mechanical tension. RESULTS Retinal ganglion cells undergo a developmental switch in growth cone dynamics that mirrors the decline in postnatal axon growth rates, with increased filopodial adhesion and decreased lamellar protrusion area in postnatal axonal growth cones. Moreover, expressing growth-suppressive KLF4 or growth-enhancing KLF6 transcription factors elicits similar changes in postnatal growth cones that correlate with axon growth rates. Postnatal RGC axon growth rate is not limited by an inability to achieve axon growth rates similar to embryonic RGCs; indeed, postnatal axons support elongation rates up to 100-fold faster than postnatal axonal growth rates. Rather, the intrinsic capacity for rapid axon growth is due to both growth cone pausing and retraction, as well as to a slightly decreased ability to achieve rapid instantaneous rates of forward progression. Finally, we observed that RGC axon and dendrite growth are regulated independently in vitro. CONCLUSIONS Together, these data support the hypothesis that intrinsic axon growth rate is regulated by an axon-specific growth program that differentially regulates growth cone motility.

Regulating Set-β's Subcellular Localization Toggles Its Function between Inhibiting and Promoting Axon Growth and Regeneration

The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-β are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is disrupted in Alzheimers disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-β was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed, whereas Set-β localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimers disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-β in primary neurons, raising the hypothesis that nuclear Set-β may preferentially regulate gene expression whereas Set-β at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-β to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation.

β1 integrin-focal adhesion kinase (FAK) signaling modulates retinal ganglion cell (RGC) survival.

Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin’s major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs’ survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.

The role of serotonin in axon and dendrite growth.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays multiple roles in the enteric, peripheral, and central nervous systems (CNS). Although its most prominent biological function is as a signal transmission messenger from pre- to postsynaptic neurons, other roles such as shaping brain development and regulating neurite growth have also been described. Here, we review the less well-studied role of 5-HT as a modulator of neurite growth. 5-HT has been shown to regulate neurite growth in multiple systems and species, including in the mammalian CNS. 5-HT predominantly appears to suppress neurite growth, but depending on the model system and 5-HT receptor subtype, in rare cases, it may promote neurite outgrowth and elongation. Failure of axon regeneration in the adult mammalian CNS is a major problem in multiple diseases, and understanding how 5-HT receptors signal opposing effects on neurite growth may lead to novel neuroregenerative therapies, by targeting either 5-HT receptors or their downstream signaling pathways.

The N-terminal Set-β Protein Isoform Induces Neuronal Death

Background: Set-β protein can suppress or promote neuronal recovery. We investigated Set-β isoforms to understand its different roles in neurons. Results: Neurons express several Set-β transcripts, Set-β proteins localize to different compartments, and the N-terminal Set-β induces neuronal death. Conclusion: N-terminal Set-β induces neuronal death. Other isoforms could play different roles. Significance: Different Set-β isoforms could underlie its diverse roles in neurons. Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death.

Zinc chelation and Klf9 knockdown cooperatively promote axon regeneration after optic nerve injury

ABSTRACT The inability of axons to regenerate over long‐distances in the central nervous system (CNS) limits the recovery of sensory, motor, and cognitive functions after various CNS injuries and diseases. Although pre‐clinical studies have identified a number of manipulations that stimulate some degree of axon growth after CNS damage, the extent of recovery remains quite limited, emphasizing the need for improved therapies. Here, we used traumatic injury to the mouse optic nerve as a model system to test the effects of combining several treatments that have recently been found to promote axon regeneration without the risks associated with manipulating known tumor suppressors or oncogenes. The treatments tested here include TPEN, a chelator of mobile (free) zinc (Zn2+); shRNA against the axon growth‐suppressing transcription factor Klf9; and the atypical growth factor oncomodulin combined with a cAMP analog. Whereas some combinatorial treatments produced only marginally stronger effects than the individual treatments alone, co‐treatment with TPEN and Klf9 knockdown had a substantially stronger effect on axon regeneration than either one alone. This combination also promoted a high level of cell survival at longer time points. Thus, Zn2+ chelation in combination with Klf9 suppression holds therapeutic potential for promoting axon regeneration after optic nerve injury, and may also be effective for treating other CNS injuries and diseases. HighlightsThe effects of combining treatments that promote axon regeneration were tested.Traumatic injury to the mouse optic nerve was used as a model system.Zinc chelator TPEN and knockdown of Klf9 had the strongest effect on axon regeneration.TPEN and knockdown of Klf9 increased retinal ganglion cell survival by 7‐fold.

Cell types differ in global coordination of splicing and proportion of highly expressed genes.

Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules.

Serotonin receptor 2C regulates neurite growth and is necessary for normal retinal processing of visual information

Serotonin (5HT) is present in a subpopulation of amacrine cells, which form synapses with retinal ganglion cells (RGCs), but little is known about the physiological role of retinal serotonergic circuitry. We found that the 5HT receptor 2C (5HTR2C) is upregulated in RGCs after birth. Amacrine cells generate 5HT and about half of RGCs respond to 5HTR2C agonism with calcium elevation. We found that there are on average 83 5HT+ amacrine cells randomly distributed across the adult mouse retina, all negative for choline acetyltransferase and 90% positive for tyrosine hydroxylase. We also investigated whether 5HTR2C and 5HTR5A affect RGC neurite growth. We found that both suppress neurite growth, and that RGCs from the 5HTR2C knockout (KO) mice grow longer neurites. Furthermore, 5HTR2C is subject to post‐transcriptional editing, and we found that only the edited isoforms suppressive effect on neurite growth could be reversed by a 5HTR2C inverse agonist. Next, we investigated the physiological role of 5HTR2C in the retina, and found that 5HTR2C KO mice showed increased amplitude on pattern electroretinogram. Finally, RGC transcriptional profiling and pathways analysis suggested partial developmental compensation for 5HTR2C absence. Taken together, our findings demonstrate that 5HTR2C regulates neurite growth and RGC activity and is necessary for normal amplitude of RGC response to physiologic stimuli, and raise the hypothesis that these functions are modulated by a subset of 5HT+/ChAT‐/TH+ amacrine cells as part of retinal serotonergic circuitry.

Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes

Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes.Retinal ganglion cells (RGCs) are diverse in cellular function and physiology. This study demonstrates additional RGC heterogeneity using single cell transcriptomic analyses to classify 40 classes of RGCs in early postnatal mice before eye opening.