Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness

Physiologic maturation is both extrinsically and intrinsically regulated in progenitor-derived neurons

During development, newly-differentiated neurons undergo several morphological and physiological changes to become functional, mature neurons. Physiologic maturation of neuronal cells derived from isolated stem or progenitor cells may provide insight into maturation in vivo but is not well studied. As a step towards understanding how neuronal maturation is regulated, we studied the developmental switch of response to the neurotransmitter GABA, from excitatory depolarization to inhibitory hyperpolarization. We compared acutely isolated retinal ganglion cells (RGCs) at various developmental stages and RGCs differentiated in vitro from embryonic retinal progenitors for the effects of aging and, independently, of retinal environment age on their GABA receptor (GABA R) responses, elicited by muscimol. We found that neurons generated in vitro from progenitors exhibited depolarizing, immature GABA responses, like those of early postnatal RGCs. As progenitor-derived neurons aged from 1 to 3 weeks, their GABA responses matured. Interestingly, signals secreted by the early postnatal retina suppressed acquisition of mature GABA responses. This suppression was not associated with changes in expression of GABA R or of the chloride co-transporter KCC2, but rather with inhibition of KCC2 dimerization in differentiating neurons. Taken together, these data indicate GABA response maturation depends on release of inhibition by developmentally regulated diffusible signals from the retina

Sox2 regulates astrocytic and vascular development in the retina

Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis

Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease. cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth and for the survival of retinal ganglion cells following optic nerve injury