Spectrum of Ocular Manifestations in CLN2-Associated Batten (Jansky-Bielschowsky) Disease Correlate with Advancing Age and Deteriorating Neurological Function

<div><p>Background</p><p>Late infantile neuronal ceroid lipofuscinosis (LINCL), one form of Batten’s disease is a progressive neurodegenerative disorder resulting from a <i>CLN2</i> gene mutation. The spectrum of ophthalmic manifestations of LINCL and the relationship with neurological function has not been previously described.</p> <p>Methods</p><p>Patients underwent ophthalmic evaluations, including anterior segment and dilated exams, optical coherence tomography, fluorescein and indocyanine green angiography. Patients were also assessed with the LINCL Neurological Severity Scale. Ophthalmic findings were categorized into one of five severity scores, and the association of the extent of ocular disease with neurological function was assessed.</p> <p>Results</p><p>Fifty eyes of 25 patients were included. The mean age at the time of exam was 4.9 years (range 2.5 to 8.1). The mean ophthalmic severity score was 2.6 (range 1 to 5). The mean neurological severity score was 6.1 (range 2 to 11). Significantly more severe ophthalmic manifestations were observed among older patients (p<0.005) and patients with more severe neurological findings (p<0.03). A direct correlation was found between the Ophthalmic Severity Scale and the Weill Cornell Neurological Scale (p<0.002). A direct association was also found between age and the ophthalmic manifestations (p<0.0002), with older children having more severe ophthalmic manifestations.</p> <p>Conclusions</p><p>Ophthalmic manifestations of LINCL correlate closely with the degree of neurological function and the age of the patient. The newly established LINCL Ophthalmic Scale may serve as an objective marker of LINCL severity and disease progression, and may be valuable in the evaluation of novel therapeutic strategies for LINCL, including gene therapy.</p> </div

Correlation of Weill Cornell LINCL Ophthalmic Severity Scale and Age.

<p>A direct association was found between age and the Weill Cornell LINCL Ophthalmic Severity Scale (Kendall’s tau =3.79, p<0.0002), with older patients having more severe ophthalmic manifestations (Kruskal-Wallis Test: X² = 8.2, p<0.005).</p

Weill Cornell LINCL Ophthalmic Severity Score 2.

<p><b>A</b>. Dilated fundus photograph of Patient 10 reveals subtle pigmentary changes in the fovea (arrow). The optic nerve and vessels appearing normal. <b>B</b>. Late-phase FA and C. ICGA of Patient 17 shows a faint area of central hyper-fluorescence (arrow) surrounded by hypo-fluorescence. <b>D</b>. SD-OCT of patient 10 demonstrates normal retinal architecture outside the fovea (arrows). <b>E</b>. Enlargement of the fovea and para-foveal regions of the same OCT exposes outer retinal abnormalities including the disruption of the ellipsoid hyper-reflective band (*). The external limiting membrane, however, appears intact. FA – fluorescein angiogram, ICGA - indocyanine green angiogram, SD-OCT – spectral domain optical coherence tomography.</p

Association of Weill Cornell LINCL Ophthalmic Severity Scale and Weill Cornell LINCL Neurological Score.

<p>A direct correlation was found between this new Weill Cornell LINCL Ophthalmic Severity Scale and the previously validated neurological scale (Kendall’s tau = -0.476, p<0.002). More severe ophthalmic manifestations were observed in patients with more severe neurological findings (Kruskal-Wallis Test: X² = 4.8, p<0.03).</p

Weill Cornell LINCL Ophthalmic Severity Score 1.

<p><b>A</b>. Dilated fundus photograph of patient 14 showing normal appearing optic nerve, vessels and fovea. <b>B</b>. Mid-phase FA and <b>C</b>. ICGA of patient 20 appear normal. <b>D</b>. SD-OCT of the same patient demonstrates normal retinal architecture without disruption of the outer retina. FA – fluorescein angiogram, ICGA - indocyanine green angiogram, SD-OCT – spectral domain optical coherence tomography.</p

Weill Cornell LINCL Ophthalmic Severity Score 4.

<p><b>A</b>. Dilated fundus photograph of patient 9 shows a “bull’s eye” maculopathy with concentric rings of pigmentary changes emanating from the fovea (arrow). Some optic nerve pallor with attenuation of retinal vessels is also noted. <b>B</b>. Late phase FA of the right eye of patient 9 showing the “bull’s eye” maculopathy. <b>C</b>. Late phase FA of the left eye of patient 8 demonstrates a similar pattern within the macula. <b>D</b>. Late phase ICGA of the right eye of patient 9 correlates with the FA. <b>E</b>. Late phase ICGA of the left eye of patient 8. <b>F</b>. SD-OCT of patient 8 demonstrates considerable outer retinal abnormalities (*), including outer retinal atrophy and buildup of hyper-reflective material at the level of the retinal pigment epithelium. This outer retinal disruption typically extends less than 2 disc diameters (*), with normal appearing retinal architecture beyond the central fovea (arrows). <b>G</b>. Interestingly, the hyper-reflective material in some patients (including patient 16 shown here), appears to preferentially accumulate in the immediate parafoveal region (arrows). The late-phase FA (<b>B</b> and <b>D</b>) and ICGA (<b>C</b> and <b>E</b>) images show the hyper- and hypo-fluorescence areas (arrows) corresponding to the pigmentary changes on the color photograph (A) and the outer retinal abnormalities noted on the SD-OCT (<b>F</b> and <b>G</b>). FA – fluorescein angiogram, ICGA - indocyanine green angiogram, SD-OCT – spectral domain optical coherence tomography.</p

PINK1 constructs and their expressions.

<p>A) A schematic depiction of PINK1 constructs. Full length wild type, L347P-, or E417G- PINK1 tagged with Flag, V5, or GFP are indicated. Several truncated PINK1 tagged with Flag or V5 are also depicted. M stands for mitochondrial targeting sequence. B) Confirmation of the expression of the above constructs in HEK 293 cells. HEK293 cells were transfected by various PINK1 constructs, and their lysates were analyzed by Western blots with Flag antibody (Left panel), V5 antibody (the middle panel) or GFP antibody (the right Panel). Lane 1–10 are lysates from cells transfected by plasmids with the same numbering as shown in A). The lysates from the cells transfected with the empty cloning vector without PINK1 insert were used as controls (labeled as C). The results demonstrated that the expression of all constructs yielded recombinant PINK1 proteins with expected molecular weights.</p

Proteasome function is impaired by mutant or loss of PINK1.

<p>Proteasome activity was measured from SH-SY5Y cells expressing mutant PINK1 (A), and PC12 cells expressing siRNA against PINK1 (B, C, D). A) Fluorescence of fluorogenic proteasome substrate Suc-LLVY-AMC (Calbiochem) is positively correlated with proteasome function. No statistically significant changes were detected in proteasome activity between control SH-SY5Y cells and the cells expressing wild type PINK1 (n = 8, p = 0.484, paired student t test). There was a statistically significant decrease of proteasome activity in the SH-SY5Y cells expressing L347P-PINK1 (23% reduction, n = 7, p = 0.018, paired student t test) or in SH-SY5Y cells expressing E417G-PINK1 (19.4% reduction, n = 8, p = 0.012, paired student t test) compared to cells expressing wild type PINK1. MG132, a proteasome inhibitor, was used as a negative control. The bottom panel is a Western analysis of the above samples with the 20S α subunit Ab for normalization. B) Proteasome activity was measured in 20 µg of cell lysate isolated from wild type control PC12 cells (open diamond) or SiPINK1-4 PC12 cell line (filled circle) for 60 min after 30 min incubation. Wild type PC12 cells lysate treated with MG132 (filled triangle) was used as a negative control. The result revealed that the kinetic of proteasome activity monitored over 60 min was markedly decreased in the cells with reduced PINK1. The bottom panel is a Western analysis of the above samples with the 20S α subunit antibody for normalization. C) Histographic presentation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004597#pone-0004597-g007" target="_blank">Figure 7B</a>. The reduction of PINK1 by siRNA impairs the proteasome activity (31.8% reduction, n = 8, p = 0.01, ANOVA). Experiments were repeated with SiPINK1-2 PC12 cell line, and consistent results were obtained (data not shown). D) PINK1 mediated proteasome activity deficit confirmed by another independent method in the HeLa cells. Compared to control (CFP-de transfection), siRNA against PINK1 (siPINK1) knocked down PINK1 and led to a sigfinicant inhibition of CFP degradation (p = 0.0001, ANOVA) to an extent similar to direct proteasome inhibition by MG132 (p = 0.0014, ANOVA). A scrambled siRNA (siSCR) had no effect (p = 0.876, ANOVA). The RNAi sequences are: GAGAGGUCCAAGCAACUA TT and CCUGGUCGACUACCCUGAU TT.</p

Proteasome function is impaired by reduction of ATP.

<p>Proteasome function is ATP dependent. Fluorescent CFP was fused to degron, a signaling peptide that directs its protein to proteasome for degradation. An increase of fluorescence (open circle) indicates a reduction of proteasome function. ATP production was inhibited by 2-deoxyglucose (2DG), and ATP content was measured with the ATP Assay Kit (Calbiochem) for luminescence (filled circle). A) Increasing dosages of 2DG caused a decrease in ATP production (filled circle) and enhanced proteasome inhibition (open circle). B) Compared to non-treated cells in DMEM, there is a significant proteasome inhibition by 6 mM 2DG (p = 0.0004, ANOVA) and the proteasome inhibitor MG132 (p = 0.0001, ANOVA).</p

Dimerization of PINK1 via the kinase domain.

<p>In all experiments, PINK1 with different tags were co-transfected in pairs into HEK293 cells. A tag was used for immunoprecipitation (IP), and Ab against another tag was used for Western blots (WB). Results were confirmed in COS and HeLa cells (data not shown). A) Wild type PINK1 form dimers. Left lanes in all three panels: lysate from cells without transfection as negative controls; right lanes in all panels: lysate from cells co-transfected with a pair of PINK1 constructs. In the left and middle panels, PINK1-Flag and PINK1-V5 were co-transfected. Anti-Flag Ab could co-IP PINK1-V5 (left panel) and vice versa (middle panel), indicating that PINK1-Flag and PINK1-V5 form a dimer. PINK1 dimerization is confirmed when PINK1-GFP and PINK1-V5 were co-transfected, anti-GFP Ab could co-IP PINK1-V5 (right panel). B) Wild type and mutant PINK1 form homo-dimers via the kinase domain. Lysates from cells expressing PINK1-V5 and PINK1_1-245-Flag (lane 1), PINK1-Flag and PINK1_1-509-V5 (lane 2), or PINK1-Flag and PINK1_1-525-V5 were isolated (lane 3), and subject to Western analysis with V5 antibody (middle panel, input control) or Flag antibody (bottom panel, input control) to confirm the expression of expected tagged recombinant protein. These lysates were then immunoprecipitated with mouse anti-V5 Ab, and subjected to Western analyses with rabbit anti-Flag Ab (upper panel; Co-IP). PINK1_1-245-Flag abolished dimerization (lane 1), whereas PINK1_1-525-V5 and PINK1_1-509-V5 could dimerize normally (lanes 2 and 3). Thus amino acid residues 246–509 are necessary for dimerization. L347P and E417G mutations did not disrupt the PINK1-PINK1 interaction (lane 5 and 6). C) Mutant PINK1 can also form hetero-dimers with wild type PINK. Lysates from cells expressing 1) PINK1-V5, 2) PINK1-V5 and PINK1-Flag, 3) PINK1-V5 and PINK1-L347P-Flag, 4) PINK1-V5 and PINK1-E417G-Flag were isolated and immunoprecipitated with rabbit anti-Flag Ab. The IP and Co-IP fractions were then subjected to Western analyses with mouse anti-V5 Ab (upper panel, Co-IP) or mouse anti-Flag Ab (lower panel, control IP). Wild type PINK1 form dimers (lane 2), and the disease-causing PINK1 mutations did not affect the dimerization (lanes 3, 4).</p