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Purpose: Complement C1q, the initiating molecule of the classical complement cascade, is involved in synapse elimination and neuronal loss in neurodegenerative diseases including glaucoma. Here we report an evaluation of the safety, tolerability, and ocular pharmacokinetics (PK) and pharmacodynamics of intravitreal (IVT) injections of ANX007, an anti-C1q monoclonal antibody fragment that blocks activation of the classical complement cascade. Design: An open-label, single-dose-escalation phase Ia study followed by a double-masked, randomized, sham-controlled, repeat-injection phase Ib study. Participants: A total of 26 patients with primary open-angle glaucoma. Methods: Nine patients with primary open-angle glaucoma (mean Humphrey visual field deviation between -3 and -18 decibels [dB]) were enrolled in phase Ia and received single doses of ANX007 (1.0 mg, n = 3; 2.5 mg, n = 3; or 5.0 mg, n = 3). Seventeen patients (mean Humphrey visual field deviation between -3 and -24 dB) were enrolled in phase Ib and randomized to 2 monthly doses of ANX007 (sham, n = 6; 2.5 mg ANX007, n = 6; or 5 mg ANX007, n = 5). Main Outcome Measures: Safety and tolerability (including laboratory evaluation of urinalysis, complete blood count, and serum chemistries), ANX007 PK, target engagement, and immunogenicity. Results: The mean age overall was 70 years in phase Ia and 68 years in phase Ib. In both studies, no serious adverse events were observed, no non-ocular treatment-emergent adverse events (TEAEs) attributable to study drug were reported, and ocular TEAEs were mild. Intraocular pressure returned to normal levels for all patients within 45 minutes of IVT injection. No clinically significant deviations in laboratory results were observed. In the phase Ib study, C1q in the aqueous humor was reduced to undetectable levels in both the 2.5 mg and 5 mg cohorts 4 weeks after the first ANX007 dose. Conclusions: In these studies, single and repeat IVT ANX007 injections were well tolerated and demonstrated full target engagement 4 weeks after dosing with both low and high doses, supporting monthly or less-frequent dosing. Further investigation in neurodegenerative ocular diseases is warranted. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.
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Purpose: C1q and the classical complement cascade are key regulators of synaptic pruning, and their aberrant activation has been implicated in neurodegenerative ophthalmic diseases including geographic atrophy and glaucoma. The antigen-binding fragment antibody ANX007 specifically recognizes globular head groups of C1q to block substrate binding and functionally inhibit classical complement cascade activation. ANX007 was assessed in nonclinical studies of biodistribution and C1q target engagement in the eye following intravitreal (IVT) administration in cynomolgus monkeys. Methods: Female juvenile cynomolgus monkeys (n = 12) received a single bilateral dose of 1 or 5 mg ANX007/eye, with vitreous and non-perfused tissue samples collected approximately 4 weeks later. In a separate study, male (n = 6/5) and female (n = 6/5) animals received repeat bilateral dosing of 1, 2.5, or 5 mg ANX007/eye on days 1 and 29, with aqueous and vitreous collections on day 44 or day 59. Tissues from the 5 mg/eye repeat-dose group were perfused, and retina, choroid, and optic nerve samples were collected approximately 2 and 4 weeks post-last dose. Results: Following a single dose of ANX007, vitreous levels of free drug were measurable through 4 weeks at both the 1 and 5 mg dose levels, with approximately 3-day half-life. With repeat dose of 5 mg/eye, free-ANX007 was measurable 4 weeks post-last dose in perfused retina and choroid and up to approximately 2 weeks post-last dose in optic nerve. There was a strong correlation between C1q target engagement and free drug levels in aqueous and vitreous humors and retinal tissue. Conclusions: Following IVT administration, ANX007 distributes to sites within the retina that are relevant to neurodegenerative ophthalmic disease with clear evidence of C1q target engagement. Based on its mechanism of action inhibiting C1q and its downstream activity, ANX007 is predicted to mitigate tissue damage driven by classical complement activation in the retina. These data support further clinical evaluation of ANX007.
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Retina , Cuerpo Vítreo , Animales , Masculino , Femenino , Macaca fascicularis , Distribución Tisular , Retina/metabolismo , Cuerpo Vítreo/metabolismo , Fragmentos Fab de InmunoglobulinasRESUMEN
Although traumatic brain injury (TBI) acutely disrupts the cortex, most TBI-related disabilities reflect secondary injuries that accrue over time. The thalamus is a likely site of secondary damage because of its reciprocal connections with the cortex. Using a mouse model of mild TBI (mTBI), we found a chronic increase in C1q expression specifically in the corticothalamic system. Increased C1q expression colocalized with neuron loss and chronic inflammation and correlated with disruption in sleep spindles and emergence of epileptic activities. Blocking C1q counteracted these outcomes, suggesting that C1q is a disease modifier in mTBI. Single-nucleus RNA sequencing demonstrated that microglia are a source of thalamic C1q. The corticothalamic circuit could thus be a new target for treating TBI-related disabilities.
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Lesiones Encefálicas/complicaciones , Complemento C1q/fisiología , Fases del Sueño , Trastornos del Sueño-Vigilia/etiología , Trastornos del Sueño-Vigilia/fisiopatología , Tálamo/fisiopatología , Animales , Lesiones Encefálicas/fisiopatología , Complemento C1q/genética , Modelos Animales de Enfermedad , Epilepsia/fisiopatología , Ratones , Microglía/metabolismo , Tálamo/metabolismoRESUMEN
Traumatic brain injury (TBI) is a leading cause of long-term neurological disability. Currently there is no effective pharmacological treatment for patients suffering from the long-lasting symptoms of TBI. We recently discovered that colony stimulating factor 1 (CSF1), an essential regulator of macrophage homeostasis, is neuroprotective and reduces neuroinflammation in two models of neurological disease in mice. Here we used a mouse model of repetitive mild TBI (mTBI) to examine whether CSF1 would attenuate cognitive deficits and improve pathological outcomes in two paradigms. In the acute paradigm, a single bolus treatment of CSF1 administered 24 h after injury significantly reduces memory impairment and astrocyte reactivity assessed 3 months later. In the chronic paradigm, the mice were tested 3 months after mTBI when they showed cognitive deficits. The mice were then randomly assigned to receive CSF1 or PBS (as control) treatment. After one month of treatment, the PBS-treated mice remained cognitively impaired, but the CSF1-treated showed significant improvements in cognitive function. RNA-seq and Ingenuity Pathway Analysis reveals CSF1 treatment alters cognition- and memory-related transcriptomic changes and pathways. The results of this study show that acute as well as delayed CSF1 treatment attenuate chronically impaired cognitive functions and improve pathological outcomes long after mTBI. The wide therapeutic time window of CSF1, together with the fact that CSF1 is approved for human use in clinical trials, strongly supports the potential clinical usefulness of this treatment in patients with mTBI.
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Conmoción Encefálica , Lesiones Traumáticas del Encéfalo , Trastornos del Conocimiento , Disfunción Cognitiva , Animales , Conmoción Encefálica/complicaciones , Conmoción Encefálica/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Disfunción Cognitiva/tratamiento farmacológico , Modelos Animales de Enfermedad , Humanos , Factor Estimulante de Colonias de Macrófagos , RatonesRESUMEN
Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.
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Envejecimiento/genética , Envejecimiento/fisiología , Regulación de la Expresión Génica , Especificidad de Órganos/genética , Animales , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/genética , Femenino , Cadenas J de Inmunoglobulina/genética , Cadenas J de Inmunoglobulina/metabolismo , Masculino , Ratones , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , RNA-Seq , Análisis de la Célula Individual , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de Tiempo , TranscriptomaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call 'lipid-droplet-accumulating microglia' (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.
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Envejecimiento/patología , Encéfalo/patología , Lípidos , Microglía/patología , Animales , Humanos , Inflamación/patología , RatonesRESUMEN
An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.
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Envejecimiento/sangre , Encéfalo/metabolismo , Células-Madre Neurales/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adolescente , Adulto , Anciano , Envejecimiento/inmunología , Envejecimiento/metabolismo , Animales , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Eliminación de Gen , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Microglía/metabolismo , Células-Madre Neurales/citología , Molécula 1 de Adhesión Celular Vascular/sangre , Molécula 1 de Adhesión Celular Vascular/genética , Adulto JovenRESUMEN
Brain aging and neurodegeneration are associated with prominent microglial reactivity and activation of innate immune response pathways, commonly referred to as neuroinflammation. One such pathway, the type I interferon response, recognizes viral or mitochondrial DNA in the cytoplasm via activation of the recently discovered cyclic dinucleotide synthetase cGAS and the cyclic dinucleotide receptor STING. Here we show that the FDA-approved antiviral drug ganciclovir (GCV) induces a type I interferon response independent of its canonical thymidine kinase target. Inhibition of components of the STING pathway, including STING, IRF3, Tbk1, extracellular IFNß, and the Jak-Stat pathway resulted in reduced activity of GCV and its derivatives. Importantly, functional STING was necessary for GCV to inhibit inflammation in cultured myeloid cells and in a mouse model of multiple sclerosis. Collectively, our findings uncover an unexpected new activity of GCV and identify the STING pathway as a regulator of microglial reactivity and neuroinflammation.
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Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Microglía/metabolismo , Animales , Animales Recién Nacidos , Antivirales/uso terapéutico , Células Cultivadas , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Femenino , Adyuvante de Freund/toxicidad , Ganciclovir/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Monocitos/efectos de los fármacos , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/inmunología , Toxina del Pertussis/toxicidad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Although central nervous system-resident microglia are believed to be ineffective at phagocytosing and clearing amyloid-ß (Aß), a major pathological hallmark of Alzheimer's disease (AD), it has been suggested that peripheral myeloid cells constitute a heterogeneous cell population with greater Aß-clearing capabilities. Here, we demonstrate that the conditional ablation of resident microglia in CD11b-HSVTK (TK) mice is followed by a rapid repopulation of the brain by peripherally derived myeloid cells. We used this system to directly assess the ability of peripheral macrophages to reduce Aß plaque pathology and therefore depleted and replaced the pool of resident microglia with peripherally derived myeloid cells in Aß-carrying APPPS1 mice crossed to TK mice (APPPS1;TK). Despite a nearly complete exchange of resident microglia with peripheral myeloid cells, there was no significant change in Aß burden or APP processing in APPPS1;TK mice. Importantly, however, newly recruited peripheral myeloid cells failed to cluster around Aß deposits. Even additional anti-Aß antibody treatment aimed at engaging myeloid cells with amyloid plaques neither directed peripherally derived myeloid cells to amyloid plaques nor altered Aß burden. These data demonstrate that mere recruitment of peripheral myeloid cells to the brain is insufficient in substantially clearing Aß burden and suggest that specific additional triggers appear to be required to exploit the full potential of myeloid cell-based therapies for AD.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Células Mieloides/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Movimiento Celular , Modelos Animales de Enfermedad , Ganciclovir/farmacología , Masculino , Ratones , Microglía/fisiología , VacunaciónRESUMEN
Aberrant microglial responses contribute to neuroinflammation in many neurodegenerative diseases, but no current therapies target pathogenic microglia. We discovered unexpectedly that the antiviral drug ganciclovir (GCV) inhibits the proliferation of microglia in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (MS), as well as in kainic acid-induced excitotoxicity. In EAE, GCV largely prevented infiltration of T lymphocytes into the central nervous system (CNS) and drastically reduced disease incidence and severity when delivered before the onset of disease. In contrast, GCV treatment had minimal effects on peripheral leukocyte distribution in EAE and did not inhibit generation of antibodies after immunization with ovalbumin. Additionally, a radiolabeled analogue of penciclovir, [(18)F]FHBG, which is similar in structure to GCV, was retained in areas of CNS inflammation in EAE, but not in naive control mice, consistent with the observed therapeutic effects. Our experiments suggest GCV may have beneficial effects in the CNS beyond its antiviral properties.
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Antivirales/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Ganciclovir/farmacología , Microglía/efectos de los fármacos , Animales , Antivirales/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Proliferación Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Ganciclovir/farmacocinética , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/patología , Ovalbúmina/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patologíaRESUMEN
Prion diseases are associated with accumulation of the amyloid form of the prion protein, but the mechanisms of toxicity are unknown. Amyloid toxicity is also associated with fungal prions. In Podospora anserina, the simultaneous presence of [Het-s] prion and its allelic protein HET-S causes cell death in a self-/nonself-discrimination process. Here, using the prion form of a fragment of HET-s ([PrD(157)(+)]), we show that [Het-s]-HET-S toxicity can be faithfully recapitulated in yeast. Overexpression of Hsp40 chaperone, Sis1, rescues this toxicity by curing cells of [PrD(157)(+)]. We find no evidence for toxic [PrD(157)(+)] conformers in the presence of HET-S. Instead, [PrD(157)(+)] appears to seed HET-S to accumulate at the cell periphery and to form aggregates distinct from visible [PrD(157)(+)] aggregates. Furthermore, HET-S mutants that cause HET-S to be sequestered into [PrD(157)(+)] prion aggregates are not toxic. The localization of HET-S at the cell periphery and its association with cell death was also observed in the native host Podospora anserina. Thus, upon interaction with [Het-s], HET-S localizes to the cell periphery, and this relocalization, rather than the formation of mixed HET-s/HET-S aggregates, is associated with toxicity.
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Amiloide/metabolismo , Proteínas Fúngicas/análisis , Proteínas Fúngicas/metabolismo , Podospora/citología , Priones/metabolismo , Amiloide/análisis , Amiloide/genética , Proteínas Fúngicas/genética , Expresión Génica , Proteínas del Choque Térmico HSP40/genética , Mutación , Podospora/genética , Podospora/metabolismo , Priones/análisis , Priones/genética , Multimerización de Proteína , Estructura Terciaria de Proteína , Regulación hacia ArribaRESUMEN
Most scoring assays for yeast prions are dependent on specific genetic markers and constructs that differ for each prion. Here we describe a simple colour assay for the [URE3] prion that works in the 74D-964 strain frequently used to score the [PSI(+)] prion. Although this assay can only be used to score for [URE3] in the [psi(-)] version of the strain, it makes it easier to examine the effects of host mutations or environmental changes on [URE3] or [PSI(+)] using a colour assay in the identical genetic background.
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Colorimetría/métodos , Glutatión Peroxidasa/química , Factores de Terminación de Péptidos/genética , Priones/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/química , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Mutación , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Various proteins, like the infectious yeast prions and the noninfectious human Huntingtin protein (with expanded polyQ), depend on a Gln or Asn (QN)-rich region for amyloid formation. Other prions, e.g., mammalian PrP and the [Het-s] prion of Podospora anserina, although still able to form infectious amyloid aggregates, do not have QN-rich regions. Furthermore, [Het-s] and yeast prions appear to differ dramatically in their amyloid conformation. Despite these differences, a fusion of the Het-s prion domain to GFP (Het-sPrD-GFP) can propagate in yeast as a prion called [Het-s](y). We analyzed the properties of two divergent prions in yeast: [Het-s](y) and the native yeast prion [PSI(+)] (prion form of translational termination factor Sup35). Curiously, the induced appearance and transmission of [PSI(+)] and [Het-s](y) aggregates is remarkably similar. Overexpression of tagged prion protein (Sup35-GFP or Het-sPrD-GFP) in nonprion cells gives rise to peripheral, and later internal, ring/mesh-like aggregates. The cells with these ring-like aggregates give rise to daughters with one (perivacuolar) or two (perivacuolar and juxtanuclear) dot-like aggregates per cell. These line, ring, mesh, and dot aggregates are not really the transmissible prion species and should only be regarded as phenotypic markers of the presence of the prions. Both [PSI(+)] and [Het-s](y) first appear in daughters as numerous tiny dot-like aggregates, and both require the endocytic protein, Sla2, for ring formation, but not propagation.
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Proteínas Fúngicas/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/genética , Actinas/metabolismo , Sitios de Unión , División Celular , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Microscopía Fluorescente , Mutación , Factores de Terminación de Péptidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transformación Genética , Vacuolas/metabolismoRESUMEN
Several cellular chaperones have been shown to affect the propagation of the yeast prions [PSI(+)], [PIN(+)] and [URE3]. Ssa1 and Ssa2 are Hsp70 family chaperones that generally cause pro-[PSI(+)] effects, since dominant-negative mutants of Ssa1 or Ssa2 cure [PSI(+)], and overexpression of Ssa1 enhances de novo [PSI(+)] appearance and prevents curing by excess Hsp104. In contrast, Ssa1 was shown to have anti-[URE3] effects, since overexpression of Ssa1 cures [URE3]. Here we show that excess Ssa1 or Ssa2 can also cure [PSI(+)]. This curing is enhanced in the presence of [PIN(+)]. During curing, Sup35-GFP fluorescent aggregates get bigger and fewer in number, which leads to their being diluted out during cell division, a phenotype that was also observed during the curing of [PSI(+)] by certain variants of [PIN(+)]. The sizes of the detergent-resistant [PSI(+)] prion oligomers increase during [PSI(+)] curing by excess Ssa1. Excess Ssa1 likewise leads to an increase in oligomer sizes of low, medium and very high [PIN(+)] variants. While these phenotypes are also caused by inhibition of Hsp104 or Sis1, the overexpression of Ssa1 did not cause any change in Hsp104 or Sis1 levels.