PrPres deposition in the retina is a common finding of sporadic, familial and iatrogenic Creutzfeldt-Jakob diseases (CJD)

Text Creutzfeldt-Jakob disease (CJD) is clinically characterized by progressive dementia and neuropathologically characterized by deposits of a protease-resistant isoform of the prion protein (PrP) in the central nervous system. PrP deposits in the neural retina were identified in the outer and inner plexiform layers (OPL and IPL) in a limited number of sporadic CreutzfeldtJakob diseases (sCJD) and two variant CJDs [1, 2]. However, the presence of PrP in the neural retina remains unknown in other types of CJDs. Therefore, we analyzed 16 prion cases from our brain bank, including sporadic, familial, and iatrogenic CJDs by using retinal sections [3]. At the time of autopsy, full permission was obtained from each patient’s next-of-kin. The posterior portion of the eye ball was removed with a scalpel, leaving the cornea and lens for funereal purposes. The following cases were available: nine cases of sCJD (MM1), two cases of sCJD (MM1 + 2, MM1 > 2), one case of sCJD (MM2), three cases of familial CJD (fCJD) (two of V180I and one of M232R), and one case of iatrogenic CJD (cadaveric dura mater graft, dCJD). We classified sCJDs based on the Parchi’s methodology [4, 5]. We also used four autopsy-confirmed neurological cases as controls (Table 1). For immunohistochemical studies to detect PrP, formalin-fixed and formic acid-treated sections of the retina were immunolabeled with monoclonal antibodies specific to prion proteins 3F4 (109–112) (1:200, Biolegend, USA) and 12F10 (142–160) (1200, Bertin Bioreagent, France). The retinal sections were processed by using a Ventana Discovery automated immunostainer. Evaluations of 3F4 and 12F10-immunoreactive deposits (PrP-irs) of the outer and inner plexiform layers were performed by using both antibodies in a semiquantitative manner: 0 = none, 1 = positive and scattered, 2 = positive (Table 1). PrP-irs staining was weak or focal in the outer and inner nuclear layers (ONL and INL), as well as in the ganglion cell and nerve fiber layers (GCL and NFL); thus, we calculated the frequency of cases with PrP-irs staining, separated on the basis of each anatomical region of the neural retina. In all CJD cases, 3F4 and 12F10-irs were consistently and clearly observed in the OPL and IPL of the neural retina (Fig. 1a). In our series, 12F10-irs staining was stronger than 3F4-irs. PrP-irs staining exhibited granular and fine synaptic patterns in the OPL and IPL, respectively. Although PrP-irs staining was always present in both OPL and IPL, PrP-irs staining was stronger in sCJD (MM2), fCJD, and dCJD cases than in sCJD (MM1) cases (Fig. 1a). In some instances, fine-dot PrP-irs staining was observed in the INL, ONL, GCL and NFL. More consistent findings were observed in sCJD (MM2), fCJD, and dCJD cases (Fig. 1b). No PrP-irs staining was present in the photoreceptor cell layer. In addition, there was no amyloid-beta (4G8), p-tau (AT8), p-synuclein, or TDP43-irs staining in the retina. No PrP-irs staining was observed in retinas from control cases. There was no clear PrP-irs staining in the optic nerves. Clinical characteristics, such as age at onset, duration, gender, and initial presentation were not associated with the presence or absence of PrP-irs in the retina. Our methodology analyzing the posterior portion of the eye * Correspondence: msktakaobrb@gmail.com Department of Neurology, Saitama International Medical Center, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298, Japan Department of Neurology and Brain Bank, Mihara Memorial Hospital, 366 Ohtemachi, Isesaki, Gunma 372-0006, Japan Full list of author information is available at the end of the article

(1:200, Biolegend, USA) and 12F10 (142-160) (1200, Bertin Bioreagent, France). The retinal sections were processed by using a Ventana Discovery automated immunostainer. Evaluations of 3F4 and 12F10-immunoreactive deposits (PrP res -irs) of the outer and inner plexiform layers were performed by using both antibodies in a semiquantitative manner: 0 = none, 1 = positive and scattered, 2 = positive (Table 1). PrP res -irs staining was weak or focal in the outer and inner nuclear layers (ONL and INL), as well as in the ganglion cell and nerve fiber layers (GCL and NFL); thus, we calculated the frequency of cases with PrP res -irs staining, separated on the basis of each anatomical region of the neural retina.
In all CJD cases, 3F4 and 12F10-irs were consistently and clearly observed in the OPL and IPL of the neural retina (Fig. 1a). In our series, 12F10-irs staining was stronger than 3F4-irs. PrP res -irs staining exhibited granular and fine synaptic patterns in the OPL and IPL, respectively. Although PrP res -irs staining was always present in both OPL and IPL, PrP res -irs staining was stronger in sCJD (MM2), fCJD, and dCJD cases than in sCJD (MM1) cases (Fig. 1a).
In some instances, fine-dot PrP res -irs staining was observed in the INL, ONL, GCL and NFL. More consistent findings were observed in sCJD (MM2), fCJD, and dCJD cases (Fig. 1b). No PrP res -irs staining was present in the photoreceptor cell layer. In addition, there was no amyloid-beta (4G8), p-tau (AT8), p-synuclein, or TDP-43-irs staining in the retina. No PrP res -irs staining was observed in retinas from control cases. There was no clear PrP res -irs staining in the optic nerves.
Clinical characteristics, such as age at onset, duration, gender, and initial presentation were not associated with the presence or absence of PrP res -irs in the retina. Our methodology analyzing the posterior portion of the eye ball accurately reflected the pathologic condition of the neural retina in prion diseases. Indeed, a previous study showed that PrP res -irs were not prominent in the anterior portion of the neural retina [2].
Our study is the first to describe PrP res -irs within the retina in a series of cases of sCJD (MM1), sCJD (MM2, MM1 + 2), fCJD, and dCJD. Although we did not quantitatively evaluate the amount of PrP res in each case, PrP res -irs in the OPL and IPL may be more prominent in fCJD and dCJD cases. In addition, PrP res -irs were occasionally observed within layers of neural retina other than the OPL and IPL.
Protease-sensitive normal cellular PrP was identified in the neural retina of healthy controls [1]. Because no 3F4-irs and 12F10-irs were present in control cases, we suspect that 3F4-irs and 12F10-irs in the retina of the present cases reflect PrP res accumulation. Head et al. performed a detailed analysis of PrP res in the neural retina [1,2]; they found that PrP res -irs were present in the OPL (granular pattern) and IPL (synaptic pattern) in one case of sCJD (MM1) and two cases of variant CJD. PrP res was reported as less detectable in the neural retina of sCJD (MM1) [2]. Another study reported the presence of PrP res in the neural retina in vCJD, but not in sCJD, by Western blotting analysis [6]. In Gerstmann-Sträussler-Scheinker disease (F198S), PrP deposits were found in the inner portion of the OPL [7]. We suspect that the type of prion disease is associated with the pattern and severity of PrP res -irs in the neural retina. There was no clear association between the clinical duration and PrP res -irs in the neural retina.
The present study has some limitations. The sample sizes were small, except for cases of sCJD (MM1). This study was able to describe PrP res staining in the OPL and IPL; thus, PrP res must be analyzed in other layers of the retina with another methodology, such as laser micro-dissection or biochemical analysis, because PrP res -irs staining in other layers was very weak. In the future, specific eye examinations may become a potential biomarker for the clinical diagnosis of prion diseases, similar to potential clinical diagnosis of AD by detection of amyloid-beta deposits in the retina [8]. In terms of infection protection, we need to understand PrP res accumulation in the neural retina is common findings even in atypical clinical form of sCJD (MM2, MM1 + 2) as well as fCJD and dCJD.  Availability of data and materials All data generated or analyzed during this study are included in this published article.
Authors' contributions MT: conceptualization, methodology, autopsy, investigation (neuropathological analysis), and writing of the manuscript; HK and BM: conceptualization of clinical study; TK: Genetic and biochemical analysis. All authors read and approved the final maniscript.
Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the intuitional review board of Mihara Memorial Hospital (084-02). We obtained written informed consent from the deceased relatives for autopsy and further neuropathological analysis, and all subjects were registered with our brain bank for future research. The brain bank was approved by the Ethics Committee of Mihara Memorial Hospital for neuropathological analysis (072-01, 078-01, 085-01).

Consent for publication
We obtained written informed consent from the deceased relatives for publication.

Competing interests
The authors declare that they have no competing interests.

Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.