Activity-mediated secretion of progranulin-containing granules

Progranulin (PGRN) is a multi-functional, secreted growth factor expressed in a variety of tissues throughout the body. In the central nervous system (CNS), PGRN is expressed in microglia as well as in a number of neuronal populations and has been shown to promote neuronal survival, enhance neurite outgrowth and regulate inflammation and development. Mutations in the progranulin (GRN) gene have been identified as a major cause of autosomal dominant frontotemporal dementia (FTD) with tau-negative inclusions. The majority of GRN mutations result in the production of a null allele and reduced PGRN expression. However, the normal functions of PGRN in the CNS remain poorly understood. Our study examines the secretion characteristics of PGRN in neurons. To study the secretion of PGRN from axons and dendrites, we have fused a pH-sensitive optical reporter of exocytosis, superecliptic pHluorin, to PGRN (PGRN-SEP). We demonstrate that activity enhances the secretion of PGRN from axons and dendrites with different temporal profiles of secretion. We show, using calcium blockers and calcium-free media, that activity-mediated secretion of PGRN requires Ca²⁺ entry via voltage-gated calcium channels (VGCC). We postulate that activity-dependent secretion of PGRN may enhance the formation and maturation of synapses as treatment of hippocampal neurons with recombinant PGRN results in an increase in synapse density.Medicine, Faculty ofGraduat

Dose and Radioadaptive Response Analysis of Micronucleus Induction in Mouse Bone Marrow

Enhanced cellular DNA repair efficiency and suppression of genomic instability have been proposed as mechanisms underlying radio-adaptive responses following low-dose radiation exposures. We previously showed that low-dose γ irradiation does not generate radio-adaptation by lowering radiation-induced cytogenetic damage in mouse spleen. Since radiation may exert tissue-specific effects, we extended these results here by examining the effects of γ radiation on cytogenetic damage and proliferative index in bone marrow erythrocytes of C57BL/6 and BALB/c mice. In C57BL/6 mice, the induction of micronuclei in polychromatic erythrocytes (MN-PCE) was observed at radiation doses of 100 mGy and greater, and suppression of erythroblast maturation occurred at doses of >500 mGy. A linear dose–response relationship for MN-PCE frequencies in C57BL/6 mice was established for radiation doses between 100 mGy and 1 Gy, with departure from linearity at doses of >1 Gy. BALB/c mice exhibited increased MN-PCE frequencies above baseline following a 20 mGy radiation exposure but did not exhibit radio-sensitivity relative to C57BL/6 mice following 2 Gy exposure. Radio-adaptation of bone marrow erythrocytes was not observed in either strain of mice exposed to low-dose priming γ irradiation (single doses of 20 mGy or 100 mGy or multiple 20 mGy doses) administered at various times prior to acute 2 Gy irradiation, confirming the lack of radio-adaptive response for induction of cytogenetic damage or suppression or erythrocyte proliferation/maturation in bone marrow of these mouse strains

Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

<div><p>Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.</p></div

Shortening the gap to 7 nucleotides and evaluation at higher doses.

<p>(A) Replacing PS-nucleotides with RNase H resistant chemical modifications and shortening the gap from 9 to 7 nucleotides. The top 4 candidates are shown. Primary Hu97/18 neurons were treated with ASO at 1–10000 nM for 6 days. (B) Western blot and quantitation of HTT protein levels. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (C) Western blots showing full length and cleaved spectrin. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 8–14 per data point at 0–1000 nM and n = 4–6 at 1250–10,000 nM. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p

ASO screening pipeline.

<p>(A) HD-SNPs in the <i>HTT</i> gene: blue = HD-SNPs, pink = previous human fibroblasts screen, grey = Hu97/18 screen; green Rs numbers = SNPs identified as the most RNase-H-active sites (B) ASO development pipeline: The number of targeted SNPs and ASOs tested are shown above and below the column bars, respectively. 50 SNPs are enriched on HD alleles and ASOs targeting 24 of these were previously screened for mHTT mRNA silencing. ASOs targeting 10 SNPs were screened in primary Hu97/18 neurons for HTT protein suppression and tolerability. Then, ASOs with modifications to the wings targeting 4 of these SNPs were screened. Microwalk SAR and 7-base gap SAR was done for oligos targeting SNP Rs7685686. Lastly, higher ASO concentrations and longer treatment durations were tested.</p

Spectrin cleavage after extended treatment duration.

<p>Primary Hu97/18 neurons were treated with ASO at 1–1000 nM for 6, 10, or 15 days. Western blots showing full length spectrin and cleaved spectrin (120 kDa) after 6 (black), 10 (green), and 15 (blue) days of treatment. Spectrin fragment is normalized to calnexin and then to the untreated sample. HTT membranes were reprobed for spectrin. Representative images are shown. Data are presented as mean ± SD with n = 6–12 per data point. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p

Microwalk of the SNP position within the gap.

<p>(A, B) Diagram of microwalk ASOs and HTT mRNA silencing in primary human HD fibroblasts. (A) Starting from A3, we moved one cEt modification to the 5′ wing (ekkk-9-ke) and moved the SNP site from position 4 to 14 (B) Similarly, we moved one cEt modification to the 3′ wing (ek-9-kkke) and moved the SNP site from position 2 to 12. mHTT and wtHTT mRNA were normalized to total RNA and then to the untreated sample. n = 2 per data point. A subset of ASOs from preliminary fibroblast screen marked by #, were evaluated in primary Hu97/18 neurons at 4–1000 nM for 6 days. (C) Western blots of HTT protein and quantitations. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (D) Western blots showing full length and cleaved spectrin. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 6–10 per data point. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p

ASO screen at 4 SNPs using two different cEt motifs.

<p>(A) ASOs with two different cEt-modified wing motifs (ekek-9-keke and ekk-9-kke) were compared to the parent MOE oligos (5e-9-5e). Primary Hu97/18 neurons were treated with ASO at 1–1000 nM for 6 days. (B) HTT Western blot and quantitations. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (C) Western blots showing full length and cleaved spectrin. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 6–8 per data point. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p