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Clinically Early-Stage Cspα Mutation Carrier Exhibits Remarkable Terminal Stage Neuronal Pathology with Minimal Evidence of Synaptic Loss Bruno A

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Clinically Early-Stage Cspα Mutation Carrier Exhibits Remarkable Terminal Stage Neuronal Pathology with Minimal Evidence of Synaptic Loss Bruno A Benitez et al. Acta Neuropathologica Communications (2015) 3:73 DOI 10.1186/s40478-015-0256-5 CASE REPORT Open Access Clinically early-stage CSPα mutation carrier exhibits remarkable terminal stage neuronal pathology with minimal evidence of synaptic loss Bruno A. Benitez1* , Nigel J. Cairns2,3,6, Robert E. Schmidt3, John C. Morris2, Joanne B. Norton4, Carlos Cruchaga4,6 and Mark S. Sands1,5,6 Abstract Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (AD-ANCL) is a multisystem disease caused by mutations in the DNAJC5 gene. DNAJC5 encodes Cysteine String Protein-alpha (CSPα), a putative synaptic protein. AD-ANCL has been traditionally considered a lysosomal storage disease based on the intracellular accumulation of ceroid material. Here, we report for the first time the pathological findings of a patient in a clinically early stage of disease, which exhibits the typical neuronal intracellular ceroid accumulation and incipient neuroinflammation but no signs of brain atrophy, neurodegeneration or massive synaptic loss. Interestingly, we found minimal or no apparent reductions in CSPα or synaptophysin in the neuropil. In contrast, brain homogenates from terminal AD-ANCL patients exhibit significant reductions in SNARE-complex forming presynaptic protein levels, including a significant reduction in CSPα and SNAP-25. Frozen samples for the biochemical analyses of synaptic proteins were not available for the early stage AD-ANLC patient. These results suggest that the degeneration seen in the patients with AD-ANCL reported here might be a consequence of both the early effects of CSPα mutations at the cellular soma, most likely lysosome function, and subsequent neuronal loss and synaptic dysfunction. Keywords: Neuronal ceroid lipofuscinosis, Cysteine string protein alpha, Synaptic loss, Neurodegenerative disease, Lysosome DNAJC5 Background lysosomal proteins. The function of some of these pro- The neuronal ceroid lipofuscinoses (NCLs) are the most teins is largely unknown and their relationship to lyso- common group of inherited neurodegenerative diseases some function is unclear [3]. in children [1]. NCLs are a genetically, clinically and Adult-onset NCLs (ANCL) represent up to 10 % of pathologically heterogeneous group of disorders, unified NCL cases [4]. ANCL patients exhibit the typical auto- by the presence of intracellular accumulation of auto- fluorescent granular osmiophilic deposits (GROD) in fluorescent ceroid-lipofuscin aggregates (a complex mix- neurons and skin cells [4, 5]. In recent years, the pro- ture of proteins, lipids and metals) [1, 2]. The age at gress in sequencing technology has supported the identi- onset of clinical symptoms and differences in ultrastruc- fication of a plethora of novel disease-causing genes in tural features of the storage material underlie the noso- multiple families with ANCL-like clinical features and logical spectrum of NCLs [1, 2]. NCLs are traditionally GROD-type storage material. This indicates that the considered lysosomal storage diseases (LSD), even genetic architecture of ANCL is more varied and com- though several of the mutant proteins are not primary plex than previously thought [3]. Most NCLs have a recessive pattern of inheritance; one * Correspondence: [email protected] exception is the autosomal dominant, adult onset neur- 1 Department of Medicine, Washington University School of Medicine, St. onal ceroid lipofuscinosis (AD-ANCL) (MIM #162350). Louis, MO 63110, USA Full list of author information is available at the end of the article AD-ANCL patients are asymptomatic until around the © 2015 Benitez et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Benitez et al. Acta Neuropathologica Communications (2015) 3:73 Page 2 of 10 third decade [5]. The first symptom is usually new-onset Materials and methods seizures (tonic-clonic type) followed by rapid cognitive Mutation analysis decline, motor impairment and finally, death in the ab- DNAJC5-Exon4 (Forward, 5'-ggaaggcagtatccccacctggaac-3'; sence of visual impairments [5]. The primary clinical and Reverse, 5'-cggcacagtgtcagtgccctcc-3') was amplified on pathological features of AD-ANCL include progressive Applied Biosystems (Applied Biosystems, Carlsbad, and widespread neurodegenerative processes resulting in California, USA) 96-Well GeneAmp® PCR System remarkable cortical and cerebellar atrophy [5]. Nearly all 9700 Thermal Cyclers using a touchdown protocol. of our current understanding of AD-ANCL is based on PCR products were amplified under the same conditions analysis of postmortem tissue from terminal cases. To our (25-μlvolumecontaining10×PfuUltra™ HF reaction knowledge, there is no report characterizing both early buffer (Stratagene, La Jolla, California, USA), 5× Betaine and late pathology events of AD-ANCL cases. (Sigma-aldrich, St Louis, USA), 100 μMeachdNTP, AD-ANCL is caused by a heterozygous point mutation 200 nM each primer, 0.4 PfuUltra™ High-Fidelity DNA (p.L115R) and an in-frame codon deletion (p.L116Δ)in Polymerase (Promega); PCR profile: 94 °C followed by the DnaJ (Hsp40) homolog, subfamily C, member 5 34 cycles of 45 s at 94 °C, 45 s at 62°, and 1 min at 72 °C). (DNAJC5) gene [6–8]. The DNAJC5 gene encodes CSPα. PCR product purification was completed with Exosap-IT CSPα is a cytosolic and membrane-bound co-chaperone (USB Corporation). Sequencing was performed both in with a putative neuroprotective function [9]. The func- forward and reverse direction using BigDye® Terminator tion of CSPα in the central nervous system is primarily v3.1 Cycle Sequencing Kit (ABI) on an ABI 3130 sequen- deduced from data collected from CSPα-deficient mice cer. Sequence traces were analyzed using Sequencher and flies, which exhibit massive age-dependent neurode- (v4.7, Gene Codes Corp, Ann Arbor, Michigan, USA). generation affecting mainly active neurons and photore- ceptors [9, 10]. CSPα is widely considered a member of Neuropathological Methods the presynaptic machinery responsible for proteostasis After death, the consent for brain removal was obtained and synapse maintenance [11–13]. Defective synaptic from next-of-kin. Briefly, the left hemibrain was pre- soluble NSF attachment protein receptor (SNARE)- served in 10 % phosphate-buffered formalin for neuro- complex assembly, due mostly but not exclusively to the pathologic assessment; the right hemibrain was snap reduction in soluble NSF attachment protein-25 (SNAP- frozen and stored at −80 °C for biochemical studies [18]. 25) levels, appears to be responsible for the neurodegen- Unfortunately, no flash frozen tissue was obtained from eration seen in CSPα-deficient mice [10, 11, 14, 15]. the early stage AD-ANCL patient. Tissue blocks were There are approximately 20 additional proteins that taken from the frontal, temporal, parietal, and occipital demonstrate altered expression patterns in the absence lobes, amygdala, hippocampus, basal ganglia, thalamus, of CSPα in mice [13] that can also be part of its patho- brainstem, cerebellum, and spinal cord when available. genic cascade. Following formic acid (95 %) pretreatment for 5 min, The histological hallmark of AD-ANCL is the intra- histology was undertaken and included hematoxylin and cellular accumulation of autofluorescent storage ma- eosin and a modified Bielschowsky silver impregnation. terial (AFSM) [4, 5]. Although there are similarities in Immunohistochemistry was performed using the follow- the neurodegenerative process between AD-ANCL ing primary antibodies: glial fibrillary acidic protein patients and CSPα-deficient mice, no ceroid accumu- (GFAP, 1:2,000; Dako, Glostrup, Denmark), ionized lation has been reported in CSPα-deficient mice or calcium-binding adapter molecule 1 (Iba1, 1:2,000; flies [12, 16]. Currently, it is unknown how mutations Wako Chemicals USA Inc., Richmond, VA), synaptophy- in DNAJC5/CSPα result in the formation of AFSM. A sin (1:1,000; Abcam, Cambridge, MA) and CSPα/ dominant negative effect was initially proposed as the DNAJC5 (AB1576, 1:1,000, Millipore, Temecula, CA). pathogenic mechanism for AD-ANCL-causing muta- tions in CSPα based on both its inheritance pattern and a reduction in neuropil CSPα staining in terminal Densitometry AD-ANCL cases [7, 12, 17]. Densitometry of synaptophysin and CSPα/DNAJC5 im- In this report, we describe the clinical and patho- munoreactivity was assessed in the cortical ribbon of the logical features of a patient in a clinically early stage of middle frontal gyrus, an area available in all mutation the disease with all the pathological cellular changes carriers and non-carriers, and included the gyral crest found in terminal AD-ANCL cases but with no apparent and sulcal depth. The intensity of immunoreactivity reduction in CSPα or synaptophysin in the neuropil. In (luminance measured as arbitrary values) was determined contrast, terminal AD-ANCL patients exhibit significant following immunohistochemistry of all cases treated in a reductions of presynaptic protein levels, including a single batch using Densita
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