Sequencing the serotonergic neuron translatome reveals a new role for Fkbp5 in stress.

Serotonin is a key mediator of stress, anxiety, and depression, and novel therapeutic targets within serotonin neurons are needed to combat these disorders. To determine how stress alters the translational profile of serotonin neurons, we sequenced ribosome-associated RNA from these neurons after repeated stress in male and female mice. We identified numerous sex- and stress-regulated genes. In particular, Fkbp5 mRNA, which codes for the glucocorticoid receptor co-chaperone protein FKBP51, was consistently upregulated in male and female mice following stress. Pretreatment with a selective FKBP51 inhibitor into the dorsal raphe prior to repeated forced swim stress decreased resulting stress-induced anhedonia. Our results support previous findings linking FKBP51 to stress-related disorders and provide the first evidence suggesting that FKBP51 function may be an important regulatory node integrating circulating stress hormones and serotonergic regulation of stress responses.

Resistance, vulnerability and resilience: A review of the cognitive cerebellum in aging and neurodegenerative diseases.

In the context of neurodegeneration and aging, the cerebellum is an enigma. Genetic markers of cellular aging in cerebellum accumulate more slowly than in the rest of the brain, and it generates unknown factors that may slow or even reverse neurodegenerative pathology in animal models of Alzheimer's Disease (AD). Cerebellum shows increased activity in early AD and Parkinson's disease (PD), suggesting a compensatory function that may mitigate early symptoms of neurodegenerative pathophysiology. Perhaps most notably, different parts of the brain accumulate neuropathological markers of AD in a recognized progression and generally, cerebellum is the last brain region to do so. Taken together, these data suggest that cerebellum may be resistant to certain neurodegenerative mechanisms. On the other hand, in some contexts of accelerated neurodegeneration, such as that seen in chronic traumatic encephalopathy (CTE) following repeated traumatic brain injury (TBI), the cerebellum appears to be one of the most susceptible brain regions to injury and one of the first to exhibit signs of pathology. Cerebellar pathology in neurodegenerative disorders is strongly associated with cognitive dysfunction. In neurodegenerative or neurological disorders associated with cerebellar pathology, such as spinocerebellar ataxia, cerebellar cortical atrophy, and essential tremor, rates of cognitive dysfunction, dementia and neuropsychiatric symptoms increase. When the cerebellum shows AD pathology, such as in familial AD, it is associated with earlier onset and greater severity of disease. These data suggest that when neurodegenerative processes are active in the cerebellum, it may contribute to pathological behavioral outcomes. The cerebellum is well known for comparing internal representations of information with observed outcomes and providing real-time feedback to cortical regions, a critical function that is disturbed in neuropsychiatric disorders such as intellectual disability, schizophrenia, dementia, and autism, and required for cognitive domains such as working memory. While cerebellum has reciprocal connections with non-motor brain regions and likely plays a role in complex, goal-directed behaviors, it has proven difficult to establish what it does mechanistically to modulate these behaviors. Due to this lack of understanding, it's not surprising to see the cerebellum reflexively dismissed or even ignored in basic and translational neuropsychiatric literature. The overarching goals of this review are to answer the following questions from primary literature: When the cerebellum is affected by pathology, is it associated with decreased cognitive function? When it is intact, does it play a compensatory or protective role in maintaining cognitive function? Are there theoretical frameworks for understanding the role of cerebellum in cognition, and perhaps, illnesses characterized by cognitive dysfunction? Understanding the role of the cognitive cerebellum in neurodegenerative diseases has the potential to offer insight into origins of cognitive deficits in other neuropsychiatric disorders, which are often underappreciated, poorly understood, and not often treated.

AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress.

NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration.

Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism.

Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery. IMPORTANCE: Adeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism.

Intravitreal ranibizumab therapy for retinal capillary hemangioblastoma related to von Hippel-Lindau disease.

PURPOSE: To evaluate the effect of intravitreal ranibizumab on retinal capillary hemangioblastomas (RCHs) associated with von Hippel-Lindau (VHL) disease that are not amenable or responsive to standard therapy. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Five patients with VHL-associated RCH with exudative changes and visual loss. METHODS: Monthly intravitreal injections of ranibizumab (0.5 mg) were given over a course of 6 months for a total of 7 injections, with additional injections considered until week 52. The final study visit was designated as 8 weeks after the final study injection. MAIN OUTCOME MEASURES: The primary outcome was the change in best-corrected visual acuity (BCVA) of >/=15 letters at the final visit compared with baseline. Secondary outcomes included change in lesion size, exudation as assessed clinically and by fluorescein angiography, change in retinal thickness as evaluated by optical coherence tomography, and adverse event assessments. RESULTS: Patients received an average of 10.0+/-3.1 injections over an average period of 47+/-14 weeks, including follow-up. Mean change in BCVA was a decrease of 9+/-20 letters, with 1 patient gaining >/=15 letters, and 2 patients losing >/=15 letters. Changes in both lesion size and exudation were variable. CONCLUSIONS: Intravitreal ranibizumab, delivered as monotherapy every 4 weeks, had minimal beneficial effects on most VHL-related RCHs. Possible treatment efficacy was demonstrated in the patient with the smallest lesion with less exudation. Future prospective studies are needed to determine the potential role of an antiangiogenic agent, possibly in combination with other therapies for the treatment of such advanced ocular tumors associated with VHL.

Perivascular mural cells of the mouse choroid demonstrate morphological diversity that is correlated to vasoregulatory function.

OBJECTIVE: Perivascular mural cells of the choroid have been implicated in physiological functioning as well as in retinal disease pathogenesis. However details regarding their form and function are not well understood. We aim to characterize choroidal mural cells in the adult mouse choroid in terms of their distribution and morphology, and correlate these to their contractile behavior. METHODS: Sclerochoroidal flat-mounted explants were prepared from albino transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives the expression of green fluorescent protein (GFP). α-SMA-expressing smooth muscle cells and pericytes in the living choroid were thereby rendered fluorescent and imaged with confocal microscopy and live-cell imaging in situ. RESULTS: Choroidal perivascular mural cells demonstrate significant diversity in terms of their distribution and morphology at different levels of the vasculature. They range from densely-packed circumferentially-oriented cells that provide complete vascular coverage in primary arteries to widely-spaced stellate-shaped cells that are distributed sparsely over terminal arterioles. Mural cells at each level are immunopositive for contractile proteins α-SMA and desmin and demonstrate vasoconstrictory contractile movements in response to endothelin-1 and the calcium ionophore, A23187, and vasodilation in response to the calcium chelator, BAPTA. The prominence of vasoregulatory contractile responses varies with mural cell morphology and density, and is greater in vessels with dense coverage of mural cells with circumferential cellular morphologies. In the choriocapillaris, pericytes demonstrate a sparse, horizontal distribution and are selectively distributed only to the scleral surface of the choriocapillaris. CONCLUSIONS: Diversity and regional specialization of perivascular mural cells may subserve varying requirements for vasoregulation in the choroid. The model of the α-SMA-GFP transgenic albino mouse provides a useful and intact system for the morphological and functional study of choroidal mural cells.

Microglial morphology and dynamic behavior is regulated by ionotropic glutamatergic and GABAergic neurotransmission.

PURPOSE: Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or "resting" conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates "resting" microglial morphology and behavior. METHODS: We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia. RESULTS: Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels. CONCLUSIONS: Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of "resting" microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity.

Methods to promote Notch signaling at the biomaterial interface and evaluation in a rafted organ culture model.

The Notch signaling pathway is a promising target for controlling cell fate choices at the biomaterial-tissue interface. Building on our previous work in developing Notch-signaling biomaterials, we evaluated various immobilization schemes for Notch ligands, and their effect on human foreskin keratinocytes. A peptide sequence derived from the Jagged-1 DSL-region and immobilized to poly(2-hydroxyethyl methacrylate) (polyHEMA) showed no bioactivity in relation to the Notch-CSL pathway. The full-length Jagged-1 protein immobilized directly to the polyHEMA surface showed activity in signaling the Notch-CSL pathway. However, an indirect affinity immobilization approach yielded a stronger signal. Human keratinocytes plated on bound Jagged-1 showed upregulated involucrin, keratin 10, and loricrin protein expression, with this expression being cell density-dependent. Utilizing a human foreskin rafted organ culture model as a bridge between in vitro and in vivo studies, Jagged-1-modified or control polyHEMA rods were implanted in human foreskin and cultured at the air-medium interface. Keratinocyte proliferation was suppressed and intermediate-stage differentiation promoted in Jagged-1-modified rods compared with control rods. Thus, Notch-signaling biomaterials provide a robust approach to control keratinocyte differentiation and may find application to other progenitor and stem cells.

Regulation of dynamic behavior of retinal microglia by CX3CR1 signaling.

PURPOSE: Microglia in the central nervous system display a marked structural dynamism in their processes in the resting state. This dynamic behavior, which may play a constitutive surveying role in the uninjured neural parenchyma, is also highly responsive to tissue injury. The role of CX3CR1, a chemokine receptor expressed in microglia, in regulating microglia morphology and dynamic behavior in the resting state and after laser-induced focal injury was examined. METHODS: Time-lapse confocal imaging of retinal explants was used to evaluate the dynamic behavior of retinal microglia labeled with green fluorescent protein (GFP). Transgenic mice in which CX3CR1 signaling was ablated (CX3CR1(GFP/GFP)/CX3CR1(-/-)) and preserved (CX3CR1(+/GFP)/CX3CR1(+/-)) were used. RESULTS: Retinal microglial density, distribution, cellular morphology, and overall retinal tissue anatomy were not altered in young CX3CR1(-/-) animals. In the absence of CX3CR1, retinal microglia continued to exhibit dynamic motility in their processes. However, rates of process movement were significantly decreased, both under resting conditions and in response to tissue injury. In addition, microglia migration occurring in response to focal laser injury was also significantly slowed in microglia lacking CX3CR1. CONCLUSIONS: CX3CR1 signaling in retinal microglia, though not absolutely required for the presence of microglial dynamism, plays a role in potentiating the rate of retinal microglial process dynamism and cellular migration. CX3CL1 signaling from retinal neurons and endothelial cells likely modulates dynamic microglia behavior so as to influence the level of microglial surveillance under basal conditions and the rate of dynamic behavior in response to tissue injury.

Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy.

PURPOSE: Retinal microglia have been implicated in the pathogenesis of various retinal diseases, but their basic function and cellular phenotype remain incompletely understood. Here, the authors used a novel ex vivo retinal imaging preparation to examine the behavioral phenotype of living retinal microglia in intact tissue and in response to injury. METHODS: Fluorescence-labeled microglia in retinal explants from CX3CR1(+/GFP) transgenic mice were observed using time-lapse confocal imaging. High spatial and temporal resolution imaging parameters were used to follow dynamic microglial behavior in real time. RESULTS: Under normal conditions, resting retinal microglia are not static in structure but instead exhibit extensive structural dynamism in their cellular processes. Process movements are highly random in direction but are balanced to maintain overall cellular symmetry and arbor size. At rest, however, these exuberant process movements do not result in overt cellular migration. After focal laser injury, microglial processes increase significantly in their motility and direct themselves toward the injury site. Microglia rapidly transition their morphologies from symmetric to polarized toward the laser lesion. Microglia also transition from a fixed to a migratory phenotype, translocating through tissue while retaining their ramified morphology. CONCLUSIONS: Retinal microglia normally occupying uninjured tissue display a continuous, dynamic behavior that suggests functions of tissue surveillance and intercellular communication. Microglial behavior is highly regulated by, and immediately responsive to, focal tissue injury and may constitute a therapeutic cellular response to focal laser photocoagulation. Ex vivo live imaging in the retina is an experimental approach well suited to the study of dynamic aspects of microglial physiology.