Awilda M. Rosario

IL-10 Alters Immunoproteostasis in APP Mice, Increasing Plaque Burden and Worsening Cognitive Behavior

Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimers disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1)-mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10-expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely because of direct interaction with aggregated Aβ in the IL-10-expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain.

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

Conformational templating of α-synuclein aggregates in neuronal-glial cultures

BackgroundGenetic studies have established a causative role for α-synuclein (αS) in Parkinson’s disease (PD), and the presence of αS aggregates in the form of Lewy body (LB) and Lewy neurite (LN) protein inclusions are defining pathological features of PD. Recent data has established that extracellular αS aggregates can induce intracellular αS pathologies supporting the hypothesis that αS pathology can spread via a “prion-like” self-templating mechanism.ResultsHere we investigated the potential for conformational templating of αS intracellular aggregates by seeding using recombinant wild-type and PD-linked mutant (A53T and E46K) αS in primary mixed neuronal-glial cultures. We find that wild-type and A53T αS fibrils predominantly seed flame-like inclusions in both neurons and astrocytes of mixed primary cultures; whereas the structurally distinct E46K fibrils seed punctate, rounded inclusions. Notably, these differences in seeded inclusion formation in these cultures reflect differences in inclusion pathology seen in transgenic mice expressing the A53T or E46K αS mutants. We further show that the inclusion morphology is dictated primarily by the seed applied rather than the form of αS expressed. We also provide initial evidence that αS inclusion pathology can be passaged in primary astrocyte cultures.ConclusionThese studies establish for the first time that αS aggregation in cultured cells can occur by a morphological self-templating mechanism.

Widespread and Efficient Transduction of Spinal Cord and Brain Following Neonatal AAV Injection and Potential Disease Modifying Effect in ALS Mice

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.

Re-Opening the Critical Window for Estrogen Therapy

A decline in estradiol (E2)-mediated cognitive benefits denotes a critical window for the therapeutic effects of E2, but the mechanism for closing of the critical window is unknown. We hypothesized that upregulating the expression of estrogen receptor α (ERα) or estrogen receptor β (ERβ) in the hippocampus of aged animals would restore the therapeutic potential of E2 treatments and rejuvenate E2-induced hippocampal plasticity. Female rats (15 months) were ovariectomized, and, 14 weeks later, adeno-associated viral vectors were used to express ERα, ERβ, or green fluorescent protein (GFP) in the CA1 region of the dorsal hippocampus. Animals were subsequently treated for 5 weeks with cyclic injections of 17β-estradiol-3-benzoate (EB, 10 μg) or oil vehicle. Spatial memory was examined 48 h after EB/oil treatment. EB treatment in the GFP (GFP + EB) and ERβ (ERβ + EB) groups failed to improve episodic spatial memory relative to oil-treated animals, indicating closing of the critical window. Expression of ERβ failed to improve cognition and was associated with a modest learning impairment. Cognitive benefits were specific to animals expressing ERα that received EB treatment (ERα + EB), such that memory was improved relative to ERα + oil and GFP + EB. Similarly, ERα + EB animals exhibited enhanced NMDAR-mediated synaptic transmission compared with the ERα + oil and GFP + EB groups. This is the first demonstration that the window for E2-mediated benefits on cognition and hippocampal E2 responsiveness can be reinstated by increased expression of ERα. SIGNIFICANCE STATEMENT Estradiol is neuroprotective, promotes synaptic plasticity in the hippocampus, and protects against cognitive decline associated with aging and neurodegenerative diseases. However, animal models and clinical studies indicate a critical window for the therapeutic treatment such that the beneficial effects are lost with advanced age and/or with extended hormone deprivation. We used gene therapy to upregulate expression of the estrogen receptors ERα and ERβ and demonstrate that the window for estradiols beneficial effects on memory and hippocampal synaptic function can be reinstated by enhancing the expression of ERα. Our findings suggest that the activity of ERα controls the therapeutic window by regulating synaptic plasticity mechanisms involved in memory.

Microglia-specific targeting by novel capsid-modified AAV6 vectors

Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1–9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter–driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies.

Complex Relationships between Substrate Sequence and Sensitivity to Alterations in γ-Secretase Processivity Induced by γ-Secretase Modulators

γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-β (Aβ) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer’s disease modify the profile of Aβ isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aβ42 and in some circumstances Aβ43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aβ42, respectively. As GSMs lower the level of production of pathogenic forms of long Aβ isoforms, they are of great interest as potential Alzheimer’s disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.

A human monoclonal IgG that binds aβ assemblies and diverse amyloids exhibits anti-amyloid activities in vitro and in vivo.

Alzheimers disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aβ and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aβ and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aβ in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aβ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

Increased brain hemopexin levels improve outcomes after intracerebral hemorrhage

Following intracerebral hemorrhage (ICH), extracellular heme precipitates secondary brain injury, which results in irreversible brain damage and enduring neurological deficits. Hemopexin (Hpx) is an endogenous protein responsible for scavenging heme, thereby modulating its intrinsic proxidant/proinflammatory properties. Although Hpx is present in the brain, the endogenous levels are insufficient to combat the massive heme overload following ICH. We hypothesized that increasing brain Hpx levels would improve ICH outcomes. Unique recombinant adeno-associated viral vectors were designed to specifically overexpress Hpx within the mouse brain. Western blotting, ELISA, and immunohistochemistry of brain homogenates/sections, CSF, and serum were performed. As compared to controls, Hpx mice have increased Hpx protein levels in all three types of biospecimens evaluated, which results in 45.6 ± 6.9% smaller lesions and improved functional recovery after ICH (n=14–19/group, p < 0.05). Local mechanistic analyses show significantly less tissue injury, trends toward smaller hematoma volumes, unchanged heme oxygenase 1 and iron levels, and significantly increased microgliosis and decreased astrogliosis and lipid peroxidation. Peripheral levels of heme-related markers indicate a positive modulation of iron-binding capacity. These findings reveal that high local Hpx levels improve ICH outcomes, likely through both central and peripheral clearance mechanisms, and establish the potential for therapeutically administering clinical-grade Hpx for ICH.

Amyloid β peptides overexpression in retinal pigment epithelial cells via AAV-mediated gene transfer mimics AMD-like pathology in mice

Age-related macular degeneration (AMD) is a progressive retinal neurodegenerative disorder characterized by extracellular deposits known as drusen. A major constituent of drusen deposits are Alzheimer disease-associated amyloid β (Aβ) peptides. To understand the etiology of Aβ proteostasis in AMD, we delivered recombinant adeno-associated virus (AAV) encoding Aβ42 and Aβ40 peptides fused to BRI2 protein by intraocular injection in C57BL/6J mice. Endogenous protease cleavage of such constructs leads to production of secreted Aβ42 and Aβ40 respectively. We demonstrate that overexpression of secreted Aβ40 or Aβ42 resulted in dramatic induction of drusen-like deposits by 2 months’ post-injection. These drusen-like deposits were immunopositive for Aβ and complement proteins but did not stain for conventional amyloid dyes, such as Thioflavin S. Both injected cohorts showed gliosis and degenerative changes, though ERG responses were minimally affected. Intriguingly, simultaneous overexpression of BRI-Aβ40 or BRI-Aβ42 together resulted in dose-dependent and cumulative changes reminiscent of AMD type pathology - drusen-like deposits, severe reduction in ERG responses, photoreceptor cell loss and gliosis. Here, we have established a physiological model of Aβ containing deposits in wild-type mice that recapitulates major retinal pathophysiological features of AMD and will be instrumental in mechanistic understanding and development of therapeutic strategies against AMD.