Antisense oligonucleotides targeting the miR-29b binding site in the GRN mRNA increase progranulin translation.

Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3' UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3' UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.

FTD-associated behavioural and transcriptomic abnormalities in 'humanized' progranulin-deficient mice: A novel model for progranulin-associated FTD.

Frontotemporal dementia (FTD) is an early onset dementia characterized by neuropathology and behavioural changes. A common genetic cause of FTD is haploinsufficiency of the gene progranulin (GRN). Mouse models of progranulin deficiency have provided insight into progranulin neurobiology, but the description of phenotypes with preclinical relevance has been limited in the currently available heterozygous progranulin-null mice. The identification of robust and reproducible FTD-associated behavioural, neuropathological, and biochemical phenotypes in progranulin deficient mice is a critical step in the preclinical development of therapies for FTD. In this work, we report the generation of a novel, 'humanized' mouse model of progranulin deficiency that expresses a single, targeted copy of human GRN in the absence of mouse progranulin. We also report the in-depth, longitudinal characterization of humanized progranulin-deficient mice and heterozygous progranulin-null mice over 18 months. Our analysis yielded several novel progranulin-dependent physiological and behavioural phenotypes, including increased marble burying, open field hyperactivity, and thalamic microgliosis in both models. RNAseq analysis of cortical tissue revealed an overlapping profile of transcriptomic dysfunction. Further transcriptomic analysis offers new insights into progranulin neurobiology. In sum, we have identified several consistent phenotypes in two independent mouse models of progranulin deficiency that are expected to be useful endpoints in the development of therapies for progranulin-deficient FTD. Furthermore, the presence of the human progranulin gene in the humanized progranulin-deficient mice will expedite the development of clinically translatable gene therapy strategies.

AAV5-miHTT-mediated huntingtin lowering improves brain health in a Huntington's disease mouse model.

Huntingtin (HTT)-lowering therapies show great promise in treating Huntington's disease. We have developed a microRNA targeting human HTT that is delivered in an adeno-associated serotype 5 viral vector (AAV5-miHTT), and here use animal behaviour, MRI, non-invasive proton magnetic resonance spectroscopy and striatal RNA sequencing as outcome measures in preclinical mouse studies of AAV5-miHTT. The effects of AAV5-miHTT treatment were evaluated in homozygous Q175FDN mice, a mouse model of Huntington's disease with severe neuropathological and behavioural phenotypes. Homozygous mice were used instead of the more commonly used heterozygous strain, which exhibit milder phenotypes. Three-month-old homozygous Q175FDN mice, which had developed acute phenotypes by the time of treatment, were injected bilaterally into the striatum with either formulation buffer (phosphate-buffered saline + 5% sucrose), low dose (5.2 × 109 genome copies/mouse) or high dose (1.3 × 1011 genome copies/mouse) AAV5-miHTT. Wild-type mice injected with formulation buffer served as controls. Behavioural assessments of cognition, T1-weighted structural MRI and striatal proton magnetic resonance spectroscopy were performed 3 months after injection, and shortly afterwards the animals were sacrificed to collect brain tissue for protein and RNA analysis. Motor coordination was assessed at 1-month intervals beginning at 2 months of age until sacrifice. Dose-dependent changes in AAV5 vector DNA level, miHTT expression and mutant HTT were observed in striatum and cortex of AAV5-miHTT-treated Huntington's disease model mice. This pattern of microRNA expression and mutant HTT lowering rescued weight loss in homozygous Q175FDN mice but did not affect motor or cognitive phenotypes. MRI volumetric analysis detected atrophy in four brain regions in homozygous Q175FDN mice, and treatment with high dose AAV5-miHTT rescued this effect in the hippocampus. Like previous magnetic resonance spectroscopy studies in Huntington's disease patients, decreased total N-acetyl aspartate and increased myo-inositol levels were found in the striatum of homozygous Q175FDN mice. These neurochemical findings were partially reversed with AAV5-miHTT treatment. Striatal transcriptional analysis using RNA sequencing revealed mutant HTT-induced changes that were partially reversed by HTT lowering with AAV5-miHTT. Striatal proton magnetic resonance spectroscopy analysis suggests a restoration of neuronal function, and striatal RNA sequencing analysis shows a reversal of transcriptional dysregulation following AAV5-miHTT in a homozygous Huntington's disease mouse model with severe pathology. The results of this study support the use of magnetic resonance spectroscopy in HTT-lowering clinical trials and strengthen the therapeutic potential of AAV5-miHTT in reversing severe striatal dysfunction in Huntington's disease.

Intracerebroventricular Administration of AAV9-PHP.B SYN1-EmGFP Induces Widespread Transgene Expression in the Mouse and Monkey Central Nervous System.

Viral vectors made from adeno-associated virus (AAV) have emerged as preferred tools in basic and translational neuroscience research to introduce or modify genetic material in cells of interest. The use of viral vectors is particularly attractive in nontransgenic species, such as nonhuman primates. Injection of AAV solutions into the cerebrospinal fluid is an effective method to achieve a broad distribution of a transgene in the central nervous system. In this study, we conducted injections of AAV9-PHP.B, a recently described AAV capsid mutant, in the lateral ventricle of mice and rhesus macaques. To enhance the expression of the transgene (the tag protein emerald green fluorescent protein [EmGFP]), we used a gene promoter that confers high neuron-specific expression of the transgene, the human synapsin 1 (SYN1) promoter. The efficacy of the viral vector was first tested in mice. Our results show that intracerebroventricular injections of AAV9-PHP.B SYN1-EmGFP-woodchuck hepatitis virus posttranscriptional regulatory element resulted in neuronal EmGFP expression throughout the mice and monkey brains. We have provided a thorough characterization of the brain regions expressing EmGFP in both species. EmGFP was observed in neuronal cell bodies over the whole cerebral cortex and in the cerebellum, as well as in some subcortical regions, including the striatum and hippocampus. We also observed densely labeled neuropil in areas known to receive projections from these regions. Double fluorescence studies demonstrated that EmGFP was expressed by several types of neurons throughout the mouse and monkey brain. Our results demonstrate that a single injection in the lateral ventricle is an efficient method to obtain transgene expression in many cortical and subcortical regions, obviating the need of multiple intraparenchymal injections to cover large brain areas. The use of intraventricular injections of AAV9-PHP.B SYN1-EmGFP could provide a powerful approach to transduce widespread areas of the brain and may contribute to further development of methods to genetically target-specific populations of neurons.

Human progranulin-expressing mice as a novel tool for the development of progranulin-modulating therapeutics.

The granulin protein (also known as, and hereafter referred to as, progranulin) is a secreted glycoprotein that contributes to overall brain health. Heterozygous loss-of-function mutations in the gene encoding the progranulin protein (Granulin Precursor, GRN) are a common cause of familial frontotemporal dementia (FTD). Gene therapy approaches that aim to increase progranulin expression from a single wild-type allele, an area of active investigation for the potential treatment of GRN-dependent FTD, will benefit from the availability of a mouse model that expresses a genomic copy of the human GRN gene. Here we report the development and characterization of a novel mouse model that expresses the entire human GRN gene in its native genomic context as a single copy inserted into a defined locus (Hprt) in the mouse genome. We show that human and mouse progranulin are expressed in a similar tissue-specific pattern, suggesting that the two genes are regulated by similar mechanisms. Human progranulin rescues a phenotype characteristic of progranulin-null mice, the exaggerated and early deposition of the aging pigment lipofuscin in the brain, indicating that the two proteins are functionally similar. Longitudinal behavioural and neuropathological analyses revealed no significant differences between wild-type and human progranulin-overexpressing mice up to 18 months of age, providing evidence that long-term increase of progranulin levels is well tolerated in mice. Finally, we demonstrate that human progranulin expression can be increased in the brain using an antisense oligonucleotide that inhibits a known GRN-regulating micro-RNA, demonstrating that the transgene is responsive to potential gene therapy drugs. Human progranulin-expressing mice represent a novel and valuable tool to expedite the development of progranulin-modulating therapeutics.

Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles.

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Isolating cells from adult murine brain for validation of cell-type specific cre-mediated deletion.

BACKGROUND: TheCre/loxP system allows for the temporal and spatial investigation of the expression of a single gene in the nervous system. Current methods of validating conditional knock-out mouse models rely on heterogeneous brain tissue or primary culture. These methods may assess the extent of genetic knockdown in the brain but do not provide age-appropriate, cell-type specific information. NEW METHOD: We isolated specific cell types from adult murine brain using FACS to assess cell type-specific gene expression in conditional mouse models. RESULTS: We identified robust but incomplete genetic knockdown in microglia isolated from two separate microglia-specific knockout models. COMPARISONWITH EXISTING METHODS(S): Genetic knockdown in isolated adult microglia differed significantly from cultured primary microglia. CONCLUSIONS: Differences observed in primary cultured microglia compared to isolated adult microglia suggest that current methods used to validate microglia-specific gene deletion over-estimate deletion efficiency. Assessment of gene expression in isolated adult microglia provides a more accurate assessment of Cre-mediated gene deletion.

Mutant huntingtin expression in microglia is neither required nor sufficient to cause the Huntington's disease-like phenotype in BACHD mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in the HTT gene and is characterized by early and selective striatal neurodegeneration. The huntingtin (HTT) protein is ubiquitously expressed in many tissues and the cellular pathogenesis of the disease is not fully understood. Immune cell dysfunction due to mutant HTT (mHTT) expression and aberrant immune system activation in HD patients suggests that inflammatory processes may contribute to HD pathogenesis. Here we used the BACHD mouse model of HD, which carries a conditional transgene expressing full-length human mHTT, to selectively deplete mHTT expression in myeloid lineage cells, including microglia, and evaluated the effects on HD-related behavior and neuropathology. In the converse experiment, we depleted mHTT expression in the majority of cells in the brain but specifically excluding microglia and again evaluated behavior and neuropathology. In mice with myeloid-specific mHTT-depletion, we observed no significant rescue of any behavioral or neuropathological outcome measures, while neural-specific knockout mice showed significant rescue of body weight, rotarod performance and striatal volume. We conclude that mHTT expression in microglia, though clearly affecting specific aspects of microglia function, does not alter disease pathogenesis in the BACHD mouse model. This may have implications for current or future therapeutic trials testing immune-modulating drugs in HD patients.

Selective depletion of microglial progranulin in mice is not sufficient to cause neuronal ceroid lipofuscinosis or neuroinflammation.

BACKGROUND: Progranulin deficiency due to heterozygous null mutations in the GRN gene are a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations are thought to be a rare cause of neuronal ceroid lipofuscinosis (NCL). Aged progranulin-knockout (Grn-null) mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. In the brain, progranulin is predominantly expressed in neurons and microglia, and previously, we demonstrated that neuronal-specific depletion of progranulin does not recapitulate the neuropathological phenotype of Grn-null mice. In this study, we evaluated whether selective depletion of progranulin expression in myeloid-lineage cells, including microglia, causes NCL-like neuropathology or neuroinflammation in mice. METHODS: We generated mice with progranulin depleted in myeloid-lineage cells by crossing mice homozygous for a floxed progranulin allele to mice expressing Cre recombinase under control of the LyzM promotor (Lyz-cKO). RESULTS: Progranulin expression was reduced by approximately 50-70% in isolated microglia compared to WT levels. Lyz-cKO mice aged to 12 months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for many of these measures in Grn-null animals. To evaluate the functional effect of reduced progranulin expression in isolated microglia, primary cultures were stimulated with controlled standard endotoxin and cytokine release was measured. While Grn-null microglia display a hyper-inflammatory phenotype, Lyz-cKO and WT microglia secreted similar levels of inflammatory cytokines. CONCLUSION: We conclude that progranulin expression from either microglia or neurons is sufficient to prevent the development of NCL-like neuropathology in mice. Furthermore, microglia that are deficient for progranulin expression but isolated from a progranulin-rich environment have a normal inflammatory profile. Our results suggest that progranulin acts, at least partly, in a non-cell autonomous manner in the brain.

Conditional loss of progranulin in neurons is not sufficient to cause neuronal ceroid lipofuscinosis-like neuropathology in mice.

Progranulin deficiency due to heterozygous null mutations in the GRN gene is a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations cause neuronal ceroid lipofuscinosis (NCL). Aged progranulin-knockout mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. Progranulin is a secreted glycoprotein expressed in both neurons and microglia, but not astrocytes, in the brain. We generated conditional progranulin-knockout mice that lack progranulin in nestin-expressing cells (Nes-cKO mice), which include most neurons as well as astrocytes. We confirmed near complete knockout of progranulin in neurons in Nes-cKO mice, while microglial progranulin levels remained similar to that of wild-type animals. Overall brain progranulin levels were reduced by about 50% in Nes-cKO, and no Grn was detected in primary Nes-cKO neurons. Nes-cKO mice aged to 12months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for most of these measures in Grn-null animals. We conclude that neuron-specific loss of progranulin is not sufficient to cause similar neuropathological changes to those seen in constitutive Grn-null animals. Our results suggest that increased lipofuscinosis and gliosis in Grn-null animals are not caused by intrinsic progranulin deficiency in neurons, and that microglia-derived progranulin may be sufficient to maintain neuronal health and homeostasis in the brain.

Progranulin in neurodegenerative disease.

Loss-of-function mutations in the progranulin gene are a common cause of familial frontotemporal dementia (FTD). The purpose of this review is to summarize the role of progranulin in health and disease, because the field is now poised to begin examining therapeutics that alter endogenous progranulin levels. We first review the clinical and neuropathological phenotype of FTD patients carrying mutations in the progranulin gene, which suggests that progranulin-mediated neurodegeneration is multifactorial and influenced by other genetic and/or environmental factors. We then examine evidence for the role of progranulin in the brain with a focus on mouse model systems. A better understanding of the complexity of progranulin biology in the brain will help guide the development of progranulin-modulating therapies for neurodegenerative disease.

Progranulin promotes activation of microglia/macrophage after pilocarpine-induced status epilepticus.

Progranulin (PGRN) haploinsufficiency accounts for up to 10% of frontotemporal lobe dementia. PGRN has also been implicated in neuroinflammation in acute and chronic neurological disorders. Here we report that both protein and mRNA levels of cortical and hippocampal PGRN are significantly enhanced following pilocarpine-induced status epilepticus. We also identify intense PGRN immunoreactivity that colocalizes with CD11b in seizure-induced animals, suggesting that PGRN elevation occurs primarily in activated microglia and macrophages. To test the role of PGRN in activation of microglia/macrophages, we apply recombinant PGRN protein directly into the hippocampal formation, and observe no change in the number of CD11b(+) microglia/macrophages in the dentate gyrus. However, with pilocarpine-induced status epilepticus, PGRN application significantly increases the number of CD11b(+) microglia/macrophages in the dentate gyrus, without affecting the extent of hilar cell death. In addition, the number of CD11b(+) microglia/macrophages induced by status epilepticus is not significantly different between PGRN knockout mice and wildtype. Our findings suggest that status epilepticus induces PGRN expression, and that PGRN potentiates but is not required for seizure-induced microglia/macrophage activation.

Sensitivity to neurotoxic stress is not increased in progranulin-deficient mice.

Loss-of-function mutations in the progranulin (GRN) gene are a common cause of autosomal dominant frontotemporal lobar degeneration, a fatal and progressive neurodegenerative disorder common in people less than 65 years of age. In the brain, progranulin is expressed in multiple regions at varying levels, and has been hypothesized to play a neuroprotective or neurotrophic role. Four neurotoxic agents were injected in vivo into constitutive progranulin knockout (Grn(-/-)) mice and their wild-type (Grn(+/+)) counterparts to assess neuronal sensitivity to toxic stress. Administration of 3-nitropropionic acid, quinolinic acid, kainic acid, and pilocarpine induced robust and measurable neuronal cell death in affected brain regions, but no differential cell death was observed between Grn(+/+) and Grn(-/-) mice. Thus, constitutive progranulin knockout mice do not have increased sensitivity to neuronal cell death induced by the acute chemical models of neuronal injury used in this study.

Synaptic dysfunction in progranulin-deficient mice.

Progranulin haploinsufficiency is a common cause of familial frontotemporal dementia (FTD), but the role of progranulin in the brain is poorly understood. To investigate the role of murine progranulin (Grn) in the CNS in vivo, we generated mice targeted at the progranulin locus (Grn) using a gene-trap vector. Constitutive progranulin knockout mice (GrnKO) show moderate abnormalities in anxiety-related behaviors, social interactions, motor coordination, and novel object recognition at 8months of age, many of which differ between males and females. Analysis of synaptic transmission in 10-12 month old GrnKO male mice indicates altered synaptic connectivity and impaired synaptic plasticity. Additionally, apical dendrites in pyramidal cells in the CA1 region of the hippocampus in GrnKO males display an altered morphology and have significantly decreased spine density compared to wild-type (WT) mice. The observed changes in behavior, synaptic transmission, and neuronal morphology in GrnKO mice occur prior to neuropathological abnormalities, most of which are apparent at 18 but not at 8 months of age. We conclude that progranulin deficiency leads to reduced synaptic connectivity and impaired plasticity, which may contribute to FTD pathology in human patients.

Progranulin expression in the developing and adult murine brain.

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative condition characterized by focal degeneration of the frontal and temporal lobes of the brain. Autosomal dominantly inherited mutations of the progranulin gene (GRN) have been identified as the cause of a subset of cases of familial FTLD. In order to better understand the function of progranulin in the central nervous system (CNS), we have assessed the spatiotemporal expression pattern of both the murine progranulin gene (Grn) and the protein (Grn) by using transgenic knock-in mice expressing a reporter gene from the Grn locus and by immunohistochemistry, respectively. We compared Grn expression with a panel of established markers for distinct neuronal developmental stages and specific cell lineages at time points ranging from embryonic day 13.5 through to the mature adult. We find that Grn is expressed in both neurons and microglia within the CNS, but that it shows a different developmental expression pattern in each cell type. Grn expression in neurons increases as the cells mature, whereas expression in microglia varies with the cells' state of activation, being specifically upregulated in microglia in response to excitotoxic injury. Our results suggest that progranulin plays distinct roles in neurons and microglia, both of which likely contribute to overall neuronal health and function.