Aβ can trigger AD.

(A) Proteolytic processing of APP by β-secretase and γ-secretase leads to the generation of Aβ protein. Red asterisks: mutations that cause familial AD; green asterisk: a protective mutation. Insert: typical amyloid plaques and neurofibrillary tangles of AD pathology. (B) One way to depict the amyloid cascade. Individual steps in the cascade may evoke distinct microglial responses. Aβ, amyloid β-protein; AD, Alzheimer disease; APP, amyloid precursor protein.</p

Apparent reasons for “failure” in some previous anti-amyloid clinical trials.

Apparent reasons for “failure” in some previous anti-amyloid clinical trials.</p

Pathology of early-onset and late-onset AD.

Similar Aβ protein and tau pathology in a tissue sample from a patient with “sporadic” late-onset AD and an individual with a mutation in PS1, encoding presenilin-1, which causes early-onset familial AD. Shown is the gyrus parahippocampalis immunostained with the antibodies 4G8, which labels Aβ, and AT8, which labels ptau. Figure kindly provided by Dr. Thomas Arzberger (Center for Neuropathology and Prion Research, Ludwig-Maximilians-Universität, Munich). Aβ, amyloid β-protein; AD, Alzheimer disease; ptau, phospho-tau.</p

Relationship of plaque lowering and less cognitive decline across anti-Aβ antibody trials.

Graphical relationship between amyloid-lowering effect (abscissa) of various anti-Aβ antibodies (“compounds”) in individual completed therapeutic trials and their effects on clinical outcome on the CDR-SB test (ordinate). The trend line moving toward the lower left corner across these trials signifies the relationship. (Obtained from reference [74]). Aβ, amyloid β-protein; CDR-SB, Clinical Dementia Rating-Sum of Boxes; PET, positron emission tomography.</p

No SpMN axonopathy in Grna and Grnb single and double KOs.

<p><b>A</b>: Schematic illustration of a zebrafish embryo at 28hpf (lateral view) and a detail of the region above the end of the yolk extension imaged for the analysis of SpMN axons (lateral view). <b>B:</b> In Grna and Grnb single and double KOs the SpMN axons show no extended branching. Whole-mount immunofluorescence staining of 28hpf embryos with znp1 antibody. The 5 SpMN axons above the end of the yolk extension are shown. Images taken by spinning disk confocal microscopy. Anterior to the left. Lateral view. Orthogonal projections. Scale bar: 100μm. <b>C-E</b>: Quantification of the SpMN axon length in homozygous and heterozygous Grna and Grnb single and double KOs and wildtype siblings. The SpMN axon length of the 5 SpMN axons (1–5) above the end of the yolk extension is measured from the exit point of the spinal cord to the tip of the growth cone. <b>C</b>: Homozygous and heterozygous Grna KOs and wildtype siblings. n = 30. <b>D</b>: Homozygous and heterozygous Grnb KOs and wildtype siblings. n = 30. <b>E</b>: Homozygous and heterozygous Grna and Grnb KOs and wildtype siblings. n = 25. S.E.M. Two-way ANOVA. Bonferroni post-test. All non-significant (n.s.).</p

Generation of Grna and Grnb KOs using ZFNs.

<p><b>A</b>: Schematic illustration of human GRN and zebrafish Granulins. Human GRN has 7 ½ granulin domains, while 12 granulin domains are found in Grna, 9 in Grnb, and 1 ½ in Grn1 and Grn2. Grey: signal peptide. Black numbers: amino acids. Darker colour and white letters/numbers: granulin domains. <b>B-C:</b> Localisation of ZFN target sequences in <i>grna</i> and <i>grnb</i> and predicted protein sequence of selected alleles. The genomic structure of <i>grna</i> and <i>grnb</i> is depicted. ZFNs targeting <i>grna</i> and <i>grnb</i> are located in the first and fourth coding exon, respectively. ZFN-induced genomic lesions in <i>grna</i> can be detected with the restriction enzyme (RE) Eco91I and in <i>grnb</i> with the RE XcmI. Grey boxes: untranslated region (UTR). Coloured boxes: coding region. Light blue: ZFN binding sites in <i>grna</i>. Light red: ZFN binding sites in <i>grnb</i>. Green lines: binding sites of the RE. Dashed green line: cut site of the RE. Protein sequences of wildtype (wt) <i>grna</i> and 4 <i>grna</i> mutation alleles as well as wt <i>grnb</i> and 3 <i>grnb</i> mutation alleles are shown. *: Stop. <b>D-E</b>: Grna and Grnb protein is lost in all mutants. <b>D</b>: Grna signal is lost in all adult kidney samples from grna^−/− mutants, whereas a signal is present in wt. A Calnexin blot serves as a loading control. <b>E</b>: The Grnb signal observed in wt is lost in all 1.5dpf samples from <i>grnb</i>^−/− mutants. Injection of <i>grnb</i> mRNA leads to an increase in signal. The loading control α-tubulin is present in all samples.</p

No disease-related biochemical alterations in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants.

<p><b>A-C</b>: <i>ctsd</i> mRNA expression and Ctsd protein levels in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants. <b>A</b>: <i>ctsd</i> mRNA expression at 5dpf and in 7mpf as well as 22mpf brain samples derived from <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants compared to wildtype controls. 5dpf: p = 1.0. 7mpf: p = 0.4. 22mpf: p = 0.7. <b>B</b>: Western blots showing Ctsd and α-tubulin in 5dpf whole lysis samples as well as 7mpf and 22mpf brain samples from <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype controls. <b>C</b>: Quantification of Ctsd from the Western blots shown in B normalized to α-tubulin. 5dpf: n = 4. 7mpf and 22mpf: n = 3. 5dpf: p = 0.8857. 7mpf: p = 1.0. 22mpf: p = 0.7. <b>D</b>: <i>stat3</i> mRNA levels in 5dpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and 7mpf as well as 22mpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutant brains compared to wildtype control samples. 5dpf: p = 0.1. 7mpf: p = 0.4. 22mpf: p = 0.4. <b>E</b>: <i>flnca</i> mRNA levels in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} larvae at 5dpf and 7mpf as well as 22mpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} brain samples in comparison to wildtype samples. 5dpf: p = 0.4. 7mpf: p = 0.7. 22mpf: p = 0.7. <b>F</b>: <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} larvae of 5dpf and 7mpf and 22mpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} brain samples are analysed for <i>flncb</i> mRNA expression levels and compared to wildtype. 5dpf: p = 0.1. 7mpf: p = 0.4. 22mpf: p = 0.4. A,D-E: Normalized to <i>actb1</i> and <i>tbp</i>. qPCR. 5dpf: n = 4. 7mpf and 22mpf: n = 3. A,C-E: S.E.M. Mann-Whitney test (two-tailed).</p

The number of MPCs is equal.

<p><b>A</b>: Schematic illustration of a zebrafish embryo at 24hpf (lateral view) and a detail of the region above the end of the yolk extension imaged for the analysis of the MPCs (lateral view). The four somites, which were considered for the quantification of MPCs are marked with red lines. <b>B</b>: Immunofluorescence staining with Pax7 at 24hpf in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype embryos. In each image two xanthophores are exemplarily marked with a red x. Images taken by spinning disk confocal microscopy. Anterior to the left. Lateral view. Orthogonal projections. Scale bar: 50μm. <b>C</b>: Quantification of Pax7-positive cells in the 4 somites (1–4) above the end of the yolk extension in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype embryos. Only Pax7-positive cells at the surface of the somites were counted. n = 15. S.E.M. Mann-Whitney test (two-tailed). All n.s. S1: p = 0.0854. S2: p = 0.7977. S3: p = 0.3489. S4: p = 0.2337.</p

No microgliosis and neurodegeneration in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants.

<p><b>A</b>: Schematic illustration of a zebrafish larvae at 3dpf (lateral view) and a detail of the region (red line), dorsal view, imaged for the analysis of neutral red positive particles (B). The dashed red line marks the area that was imaged in the time lapse recordings of microglia (C). <b>B</b>: The number of neutral red positive particle in the region illustrated in A (Z-stack) is unchanged in wildtype and <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants. n = 15. S.E.M. Mann-Whitney test (two-tailed). p = 0.2884. <b>C-E</b>: Microglia in Tg(<i>apoeb</i>:lynEGFP) <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype larvae at 3dpf are indistinguishable. <b>C:</b> Still images of the time lapse recordings in the optic tectum recorded by spinning disk confocal microscopy. Two microglia cells marked in each genotype by a white and yellow arrow. Dorsal view. Anterior to the left. n = 3. Scale bar: 50μm. Recording time: 60min. 1frame/min. <b>D:</b> The distance microglia move within one hour in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype larvae. Quantification of n = 3x5 randomly selected microglia from the time lapse recordings shown in C. S.E.M. Mann-Whitney test (two-tailed). p = 0.0671. <b>E:</b> Processes in the <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype larvae persist for same durations. Quantification of n = 3x5 randomly selected processes from the time lapse recordings shown in C. S.E.M. Mann-Whitney test (two-tailed). p = 0.8296. <b>F:</b> Schematic illustration of a zebrafish larvae at 5dpf (lateral view) and a detail of the region, dorsal view, imaged for the analysis of acridine orange (AO) positive cells. <b>G</b>: The number of acridine orange positive cells in the region illustrated in C (Z-stack) is unchanged in wildtype and <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants. n = 15. S.E.M. Mann-Whitney test (two-tailed). p = 0.69.</p

<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants swim like wildtype.

<p><b>A</b>: The swim path of wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is shown. PTZ treatment was used as a positive control. 5dpf. Movements < 2mm/s: black lines. Movements 2–6mm/s: green lines. Movements > 6mm/s: red lines. Recording time: 5min. <b>B</b>: The total distance moved within 5min in wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is shown. Wt-<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>}: p = 0.2386. Wt-DMSO: p = 0.0534. Wt-PTZ: ***p = 0.0002. DMSO-PTZ: **p = 0.004. <b>C</b>: A graph of the mean velocity of wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is shown. Time frame: 5min. Wt-<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>}: p = 0.5657. Wt-DMSO: p = 0.8081. Wt-PTZ: *p = 0.0137. DMSO-PTZ: **p = 0.0014. <b>D</b>: Percentage of time spent for movements with a velocity above 2mm/s in wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is plotted. Time frame: 5min. Wt-<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>}: p = 0.2585. Wt-DMSO: p = 0.0668. Wt-PTZ: **p = 0.0016. DMSO-PTZ: **p = 0.0037. <b>B-D:</b> n = 18. S.E.M. Mann-Whitney test (two-tailed).</p