The role of progranulin in the brain

Frontotemporal lobar degeneration (FTLD) is a devastating, late-onset neurodegenerative disorder that causes profound behavioral abnormalities, language impairment, and alterations in personality in affected patients. As many as 40% of cases have a family history, and of these approximately 25% are due to loss-of-function mutations in the progranulin (GRN) gene. Progranulin-mediated FTLD, inherited in an autosomal dominant manner, is caused by haploinsufficiency. The purpose of this thesis was to characterize a novel mouse model of progranulin deficiency in order to study the role of progranulin in the brain and to understand how progranulin deficiency leads to neurodegeneration. The expression pattern of progranulin in the murine brain was examined using a lacZ reporter transgenic mouse line and immunohistochemistry. Progranulin is expressed at varying levels in all regions of the brain examined. Expression is strongest in the thalamus and hippocampus, moderate in the cortex, and low in the striatum and cerebellum. Double immunofluorescent labeling identified progranulin expression in mature neurons and microglia, the resident immune cells of the brain. Subsequently, a mouse model of progranulin deficiency was generated and characterized for abnormalities in behavior and neuropathology. The tests and outcome measures used were designed to reflect the behavioral and neuropathological changes observed in FTLD patients carrying GRN mutations. Heterozygous loss of progranulin produced no measurable alterations in behavior or pathology. Homozygous loss of progranulin in mice caused subtle behavioral abnormalities, intense microgliosis and astrogliosis throughout the brain, and exaggerated deposition of the aging pigment lipofuscin. Strain-dependent differences in the penetrance and expressivity of these phenotypes was observed, indicating that progranulin-dependent neurodegenerative phenotypes are modified by genetic background. In conclusion, progranulin-deficient mice develop neuropathological phenotypes that indicate increased neuronal stress and a decline in overall brain health that progresses with age. Although dramatic behavioral changes and overt neuronal loss similar to what is seen FTLD patients is not apparent in progranulin-deficient mice, the presence of robust, age-dependent neurodegenerative abnormalities makes progranulin knockout mice a useful model for certain aspects of progranulin-dependent FTLD.Medicine, Faculty ofMedical Genetics, Department ofGraduat

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.Medicine, Faculty ofOther UBCMedical Genetics, Department ofMedicine, Department ofNeurology, Division ofReviewedFacult

Additional file 1: Figure S1. of Selective depletion of microglial progranulin in mice is not sufficient to cause neuronal ceroid lipofuscinosis or neuroinflammation

Progranulin mRNA, intracellular, and secreted protein levels correlate in primary microglia cultures. (A) Progranulin mRNA levels were reduced by approximately 50% in Het cultures compared to WT cultures and not detectable in GrnKO cultures. N = 2–3 wells/per genotype. (B) Progranulin protein quantified by ELISA on cell lysate and normalized to total protein per well shows a corresponding decrease of about 50% in Het cultures compared to WT cultures and negligible levels in GrnKO cultures. N = 6 wells/genotype. (C) Secreted progranulin detected by ELISA on conditioned media again shows progranulin reduced to approximately 50% in Het cultures compared to WT cultures and not detectable in GrnKO cultures. N = 6 wells/genotype. (PDF 90 kb

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

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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 δ1-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

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