Handbook of Clinical Neurology, Vol. 153 (3rd series) Human Prion Diseases M. Pocchiari and J. Manson, Editors https://doi.org/10.1016/B978-0-444-63945-5.00010-6 Copyright © 2018 Elsevier B.V. All rights reserved

Chapter 10 Variably protease-sensitive prionopathy

SILVIO NOTARI1, BRIAN S. APPLEBY1,2,3,4, AND PIERLUIGI GAMBETTI1* 1Department of Pathology, Case Western Reserve University, Cleveland, OH, United States 2National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, United States 3Department of Neurology, Case Western Reserve University, Cleveland, OH, United States 4Department of Psychiatry, Case Western Reserve University, Cleveland, OH, United States

Abstract

Variably protease-sensitive prionopathy (VPSPr), originally identified in 2008, was further characterized and renamed in 2010. Thirty-seven cases of VPSPr have been reported to date, consistent with estimated prevalence of 0.7–1.7% of all sporadic prion diseases. The lack of gene mutations establishes VPSPr as a sporadic form of human prion diseases, along with sporadic Creutzfeldt–Jakob disease (sCJD) and spo- radic fatal insomnia. Like sCJD, VPSPr affects patients harboring any of the three genotypes, MM, MV, and VVat the prion protein (PrP) gene polymorphic codon 129, with VPSPr VVaccounting for 65% of all VPSPr cases. Distinguishing clinical features include a median 2-year duration and presentation with psy- chiatric signs, speech/language impairment, or cognitive decline. Neuropathology comprises moderate spongiform degeneration, PrP amyloid miniplaques, and a target-like or plaque-like PrP deposition. The abnormal PrP associated with VPSPr typically forms an electrophoretic profile of five to seven bands (according to the antibody) presenting variable protease resistance depending on the 129 genotype. The familial prion disease associated with the V180I PrP gene mutation which harbors an abnormal PrP with similar electrophoretic profile might serve as a model for VPSPr. Transmission to animals has definitively established VPSPr as a prion disease. Because of its recent identification, rarity, and the elusiveness of its abnormal PrP, VPSPr remains largely understudied.

INTRODUCTION immunohistochemical techniques. However, they were negative following biochemical analysis by gel electro- Variably protease-sensitive prionopathy (VPSPr) was TSE phoresis and immunoblotting of PrP , a test that is typ- originally described in 2008 based on a study of 11 US ically performed following treatment with proteinase subjects (Gambetti et al., 2008). The identification of this TSE K (PK) given the common PrP protease resistance rare and clinically rather indistinct novel condition, in prion diseases. Enhanced analyses demonstrated the and its prompt independent validation that followed, TSE presence of a peculiar form of PrP that was particu- was made possible by the existence of many national larly sensitive to PK digestion and displayed a distinctive prion surveillance centers worldwide dedicated to the multiple-band profile upon immunoblotting. examination and tissue banking of prion diseases. Genetic analysis established that all subjects were During regular examination of acquired cases at the homozygotes for the codon valine (V) at position 129 US National Prion Disease Pathology Surveillance of the prion protein (PrP) gene, the site of a common Center (NPDPSC), it was noted that in rare but recurring and nonpathogenic methionine polymorphism, M129V cases, brain tissues tested positive for the presence of (Parchi et al., 1999; Gambetti et al., 2003, 2011). More- abnormal, disease-related prion protein (PrPTSE)by over, no mutation was found in and adjacent to the coding

*Correspondence to: Pierluigi Gambetti, Department of Pathology, Case Western Reserve University, Cleveland, OH, USA. E-mail: [email protected] 176 S. NOTARI ET AL. region of the PrP gene where genetic mutations reside in estimated that VPSPr accounts for one death per 4 years inherited prion diseases, thus defining VPSPr as a spo- in the 60 million UK population. Adding together the radic prion disease (Gambetti et al., 2003, 2008; Kong unpublished cases stored by the US and UK prion sur- et al., 2004). A subsequent study (Zou et al., 2010) veillance centers brings the total number of known reporting 15 additional cases from the United States VPSPr cases worldwide to at least 77. However, it is and Italy established that all three M129V genotypes, likely that VPSPr is underreported, given that in most MM, MV, and VV, not just VV, were affected in VPSPr. cases it is initially misdiagnosed as one of the non- This property makes VPSPr the only sporadic human Alzheimer dementias, conditions in which autopsies prion disease that affects the three 129 genotypes besides are not regularly performed (Puoti et al., 2012). sporadic Creutzfeldt–Jakob disease (sCJD) (Chapter 9). This second study also revealed that PrPTSE resistance to DEFINITION PK treatment varied according to the 129 genotype. This finding led to the amendment of the disease label VPSPr is a novel sporadic prion disease, which may from the original protease-sensitive prionopathy (PSPr) account for almost 2% of all sporadic prion diseases. – in the first 2008 publication to the current VPSPr Similar to sporadic Creutzfeldt Jakob disease (sCJD), (Gambetti et al., 2008; Zou et al., 2010). VPSPr affects individuals harboring each of the three Two additional VPSPr features emerged. First, VPSPr genotypes at codon 129 of the PrP gene, thus segregating prevalence in the three 129 genotypes was found to be VPSPr into three subtypes: MM, MV, and VV. The MM 11%, MV 24%, VV 65% (Table 10.1), which is almost M129V genotype slightly affects disease phenotype TSE the opposite of that associated with sCJD (MM 68%, MV and PrP features. Overall, mean age at onset is 16%, VV 16%) (Collins et al., 2006), indicating that the 70 and duration 2 years. Clinical presentation often role played by the 129 genotype is different in the two dis- includes one or more of three sets of signs: psychiatric eases.Second, severalVPSPrpatients haveafamily history abnormalities, speech/language impairment, and cogni- of cognitive impairment, which raises the possibility of a tive decline (Table 10.1). Histopathologic features genetic component in VPSPr. Unfortunately, no genetic include moderate cortical and subcortical spongiform or neuropathologic examination has been performed on degeneration (SD) and miniplaques in the cerebellum, cognitively impaired relatives of VPSPr patients. while PrP immunostaining often reveals a distinctive target-like granular pattern. Depending on the antibody used, PrPTSE forms a five- or seven-band ladder-like EPIDEMIOLOGY electrophoretic profile with increasing protease resis- To date, 37 cases of VPSPr have been published. They tance from VV to MM subtypes. include 25 from the United States (Gambetti et al., 2008; Zou et al., 2010; Cannon et al., 2014), five from CLINICAL FEATURES the United Kingdom (Head et al., 2009, 2010, 2013); Demographics two each from Italy and Spain (Giaccone et al., 2007; Rodríguez-Martínez et al., 2010, 2012; Zou et al., VPSPr is unique given its departure from the clinical 2010), and one each from Austria, Germany, and The aspects typically observed in other human prion diseases. Netherlands (Krebs et al., 2007; Jansen et al., 2010; The clinical diagnosis of VPSPr is difficult because of its Assar et al., 2015). One of the US cases was diagnosed nonspecific clinical presentation and noncontributory incidentally at autopsy of a 93-year-old neurologically diagnostic tests that are typically suggestive of prion asymptomatic individual (Ghoshal et al., 2015). As for disease. the M129V polymorphic genotype, the 37 cases include The polymorphism at codon 129 of the PrP gene 24 VV homozygotes, nine MV heterozygotes, and four affects many aspects of this prion disease, including sus- MM homozygotes. In addition, the US NPDPSC lists a ceptibility to the disease, age at onset, illness duration, total of 57 cases of VPSPr (inclusive of the 25 published) clinical symptoms, and diagnostic test results from a total of 3279 sporadic prion disease cases, which (Table 10.2). Contrary to sCJD, the majority of VPSPr would estimate the VPSPr prevalence at 1.7% among all cases are homozygous for valine at codon 129 (65%), sporadic prion diseases (US National Prion Disease and the minority are homozygous for methionine Pathology Surveillance Center, 2017). Thirteen VPSPR (11%) (Table 10.2). VPSPr is typically a mid- to late-life cases are stored by the National CJD Surveillance and illness with a mean age at onset of 70 years (range 48–87 Research Unit in the United Kingdom, which by the years) (Puoti et al., 2012). The presence of valine at same calculation would result in a 0.7% prevalence of codon 129 seemingly has a dose–effect relationship VPSPr there (UK National CJD Research and toward a younger age at onset (mean age at onset: Surveillance Unit, 2017). Head et al. (2013) have VV ¼ 67, MV ¼ 74, MM ¼ 78 years) (Table 10.2). Table 10.1 Classification of variably protease-sensitive prionopathy

Clinical feature Histopathology SDa PrP immunostaining patterna

129 Onset Course subtype N (years) (months) Type of presentationb C. cort BG Cereb C. cortex Cereb

MMc 366 56 Parkinsonian, ataxic, cognitive ++ ++ + Granular, plaque-like of various sizes Granular, small (55–78) (41–78) plaque-like MV 9 73 37 Psychiatric, parkinsonian, +++ +++ Fine granular with rarer larger granule or Amyloid (65–81) (7–72) cognitive behavioral, aphasic plaque-like miniplaques, VVd 24 64 30 Psychiatric, ataxic, cognitive, As above but forming target-like plaque-like, (48–77) (10–78) behavioral, spastic gate, gaze structures granular palsy aTypical spongiform degeneration (SD) severity, rated relatively to the three genotypes, and typical immunostaining patterns. bOften clinical presentation included two types coexisting or in rapid succession. cMM group includes four cases, but the fourth was asymptomatic. dIncludes one case treated with doxycycline for 4 years. BG, basal ganglia; C. cort, cerebral cortex; Cereb, cerebellum. Table 10.2 Clinical characteristics of variable protease-sensitive prionopathy (VPSPr)

PRNP codon 129 polymorphism

Met-Met Met-Val Val-Val All genotypes

Mean age at onset 78 (64–87) 74 (65–81) 67 (48–77) 70 (48–87) (years, range) Duration 41 (10–73) 34 (7–91) 18 (10–60) 24 (7–91) (months, range) Family history of 33% 0% 60% 42% dementia Clinical presentation Psychiatric symptoms, aphasia, Psychiatric symptoms, Psychiatric symptoms, cognitive Psychiatric symptoms, speech/language parkinsonism, ataxia cognitive impairment impairment, aphasia impairment, cognitive impairment Advanced clinical Cognitive impairment, aphasia, Ataxia, parkinsonism, ataxia Ataxia, parkinsonism, myoclonus Ataxia, parkinsonism, myoclonus symptoms myoclonus EEG suggestive of prion 50% 25% 0% 9% disease CSF (14-3-3 and tau) 50% 0% 37% 21% sensitivity Brain MRI suggestive of 0% 0% 5% 3% prion disease

CSF, cerebrospinal fluid; EEG, electroencephalogram; MRI, magnetic resonance imaging. VARIABLY PROTEASE-SENSITIVE PRIONOPATHY 179 An important difference between VPSPr and other Table 10.3 sporadic prion diseases is its prolonged illness duration, Proposed clinical criteria for variable protease-sensitive which likely affects clinical ascertainment of cases. The prionopathy duration of VPSPr is best measured in years as opposed to months, with a mean disease duration of 2 years (Puoti A. Symptoms (both 1 and 2) et al., 2012). Cases with durations over 6 and 7 years 1. Cognitive impairment have been described in the literature (Appleby et al., 2. Two or more of the following: (a) Psychiatric symptoms 2009; Head et al., 2013). The codon 129 polymorphism (b) Parkinsonism or aphasia affects disease duration, with the presence of valine (c) Ataxia or myoclonus resulting in a shorter duration, which is the opposite of B. Duration < 8 years what is observed in sCJD (Table 10.2)(Parchi et al., C. Lack of alternative etiology or phenotype divergence from 1999; Puoti et al., 2012). Because of the insidious onset other neurodegenerative atypical dementias and slower progression of clinical symptoms compared to sCJD, it is likely more difficult to accurately determine the time of disease onset in VPSPr cases. tests used to diagnose prion disease are often unhelpful in Clinical course the diagnosis of VPSPr. Hence, the ability to successfully diagnose VPSPr relies on the clinical phenotype and The clinical presentation and course in VPSPr can best be exclusion of other etiologies. described as an atypical dementia and differ from the phenotype observed in most cases of sCJD. One or more of a triad of symptoms characterizes the clinical presen- Electroencephalogram (EEG) tation of VPSPr: psychiatric symptoms, speech/language Periodic sharp-wave complexes (PSWCs) on EEG are impairment, and cognitive decline (Puoti et al., 2012). suggestive of prion disease and are part of the World Most VV and MV cases present with psychiatric symp- Health Organization’s diagnostic criteria for sCJD toms, cognitive decline, and/or aphasia. Cases homozy- (World Health Organization, 1998). The presence of gous for methionine have initial symptoms more similar PSWCs is rare in VPSPr, with an estimated overall sen- to classic sCJD with psychiatric symptoms, aphasia, par- sitivity of 9% (Puoti et al., 2012). PSWCs have been kinsonism, and ataxia. Psychiatric symptoms often detected in 25% of MV and 50% of MM cases. No include disinhibition, euphoria, impulsivity, and apathy reported VV cases have demonstrated PSWCs. Typi- (Zou et al., 2010). Speech and language impairments cally, EEGs are normal or demonstrate generalized slow- include anomic or semantic aphasia with or without dys- ing (Gambetti et al., 2008). In some cases, EEGs are arthria. Cognitive impairment is most often associated initially normal and then progress to generalized slowing with frontal lobe dysfunction (e.g., dysexecutive later in the disease course (Head et al., 2009; Rodríguez- syndrome). Martínez et al., 2012). Whereas the initial presentation of VPSPr is mostly characterized by cortical impairments, subcortical and cerebellar symptoms arise later in the disease course. Cerebrospinal fluid Cognitive decline is observed in all cases at some point The increase of certain cerebrospinal fluid (CSF) pro- in the disease (Puoti et al., 2012). Parkinsonism, ataxia, teins can be suggestive of prion disease. Rise of 14-3-3 and myoclonus often develop later in the disease course. in CSF with a clinical duration less than 2 years is part Additionally, certain codon 129 polymorphisms are of the World Health Organization diagnostic criteria marked by a paucity of specific symptoms. For example, for sCJD (World Health Organization, 1998). Unfortu- aphasia is rare in MV cases and psychiatric symptoms are nately, CSF 14-3-3 protein is typically negative or rare in MM cases (Zou et al., 2010). Clinical criteria weakly positive (e.g., nondiagnostic) in VPSPr including symptoms are proposed in Table 10.3. (Gambetti et al., 2008; Head et al., 2009; Jansen et al., 2010; Rodríguez-Martínez et al., 2012; Assar et al., CLINICAL TESTS 2015). However, a study detected positive CSF 14-3-3 protein in 50% of all UK VPSPr cases (Head et al., Diagnostic workup 2013). When CSF 14-3-3 is combined with tau, diagnos- Many cases of sCJD do not have a classic CJD clinical tic sensitivity is 21% in all VPSPr cases with differing phenotype, but are often ascertained during the diagnos- sensitivities by codon 129 genotype (Puoti et al., tic workup because of a test that is suggestive of prion 2012). No MV cases demonstrated diagnostic 14-3- disease (Appleby et al., 2014). Unfortunately, the typical 3/tau levels, whereas 50% of MM and 37% of VV cases 180 S. NOTARI ET AL. had CSF results suggestive of prion disease. In the UK may initially be diagnosed as having a mood disorder series, CSF S100b was positive in 75% of cases (Head (Gambetti et al., 2008; Assar et al., 2015). et al., 2013), though this is not a diagnostic test that is As diagnosis of VPSPr currently stands, it is very dif- routinely used in the diagnosis of prion disease. Two ficult to discern the above atypical dementias from VV cases of VPSPr were reported to have elevated VPSPr in the initial stages of the disease. In fact, diagno- CSF protein levels (175 mg/dL and 126 mg/dL), the sig- sis of prion disease is often not considered until later in nificance of which is unknown (Gambetti et al., 2008). the disease course (Gambetti et al., 2008). One item that CSF from one case of VPSPr was tested with real-time may signal a diagnosis of VPSPr, as opposed to one of the quaking-induced conversion (RT-QuIC) and found to above atypical dementias, is a divergence from the be negative (Lattanzio et al., 2017). However, using the expected clinical course of a previously diagnosed atyp- second generation RT-QuIC (IQ-CSF) 3 out of 3 cases ical dementia. For example, ataxia would not be expected of VPSPr were positive (Franceschini et al., 2017). in any of the above conditions and should prompt the consideration of VPSPr as the etiology. Additionally, a Neuroimaging faster than expected disease progression may also be sug- gestive of VPSPr. Aside from disease-specific modalities Brain magnetic resonance imaging (MRI) is a sensitive (e.g., amyloid imaging), neuroimaging may be mislead- and specific diagnostic tool for sCJD. Hyperintensity ing and lead to an initial diagnosis of frontotemporal on diffusion-weighted imaging (DWI) is typically seen lobar degeneration due to frontal atrophy or asymmetric in the basal ganglia and/or in a gyral pattern of the cortex hypoperfusion on SPECT. in sCJD (Zerr et al., 2009); however; these brain MRI findings suggestive of prion disease are typically not pre- sent in VPSPr. Only two cases to date have demonstrated NEUROPATHOLOGY brain MRI findings suggestive of prion disease, both of The common histopathologic feature to all codon 129 which were VV at codon 129 (Zou et al., 2010; Assar genotypic subtypes of VPSPr is a moderate degree of et al., 2015). Brain MRIs of VPSPr cases typically dem- SD, which similarly affects cerebral neocortex, entorhi- onstrate diffuse cortical atrophy, with some cases show- nal cortex, basal ganglia, and thalamus, and to a lesser ing preferential atrophy of the frontal and/or parietal degree the molecular layer of the cerebellum, while typ- lobes and the cerebellum (Gambetti et al., 2008; ically the hippocampus is spared (Fig. 10.1)(Jansen Rodríguez-Martínez et al., 2012; Head et al., 2013). et al., 2010; Zou et al., 2010; Head et al., 2013). The Overall, diagnostic sensitivity of brain MRI is 3% SD brain distribution is similar to, but less severe than, (Puoti et al., 2012). Single-photon emission computed that of sCJDMM1 (sCJD associated with PrP MM geno- tomography (SPECT) has been performed in three cases type and PrPTSE type 1), the most common subtype of of VPSPr. All cases demonstrated hypoperfusion, which sCJD. However, the SD vacuoles of VPSPr differ in size was global in one case and asymmetric in two (Gambetti since they are on average 50% larger than those of et al., 2008; Rodríguez-Martínez et al., 2012). sCJDMM1 (9 3 vs. 6 1) and spread over a larger DIFFERENTIAL DIAGNOSIS range of sizes (Parchi et al., 1999; Zou et al., 2010); how- ever they are smaller and not as closely clustered or con- Because the clinical features of VPSPr resemble atypical fluent as the vacuoles in the sCJDMM2 subtype (Parchi dementias, these cases are most often initially misdiag- et al., 1999; Zou et al., 2010). nosed as non-Alzheimer disease dementias. The most A distinctive feature is the presence of very small common clinical misdiagnoses of VPSPr include prion plaques in the molecular layer of the cerebellum normal-pressure hydrocephalus,dementiawithLewy bod- and, rarely, the hippocampal region that are often irreg- ies, and frontotemporal lobar degeneration (Gambetti ular in shape and may comprise several cores et al., 2011). One can certainly understand how VPSPr (Fig. 10.2)(Gambetti et al., 2008; Zou et al., 2010; may be initially misdiagnosed as an atypical dementia. Head et al., 2013; Cannon et al., 2014). Immunoelectron The prominent symptoms of aphasia, parkinsonism, and microscopic examination of cerebellar and hippocampal frontal lobe syndrome are suggestive of several frontotem- miniplaques demonstrated poorly demarcated structures poral lobar degeneration subtypes, including frontotem- containing clusters of PrP-labeled fibrils embedded in an poral dementia, primary progressive aphasia, semantic amorphous matrix. These features have been interpreted dementia, progressive supranuclearpalsy,and corticobasal as consistent with poorly formed or immature amyloid syndrome. The prominent parkinsonian symptoms with plaques (Gambetti et al., 2008; Cannon et al., 2014). In cognitive impairment may also be suggestive of dementia VV cases, thioflavin S and FSB (bis-styrylbenzenes) with Lewy bodies or Parkinson disease dementia. Because staining procedures confirmed the presence of amyloid of the prominence of psychiatric symptoms, some cases in many cerebellar miniplaques, while the cerebral VARIABLY PROTEASE-SENSITIVE PRIONOPATHY 181

Fig. 10.1. Spongiform degeneration and prion protein (PrP) immunohistochemical pattern in the three genotypes of variably protease-sensitive prionopathy (VPSPr). Top row: mild to moderate spongiform degeneration in the three genotypes comprised of approximately 60% larger vacuoles than sporadic CJDMM1, the most common subtype of sporadic Creutzfeldt–Jakob disease. Middle and lower rows: PrP immunohistochemistry: fine granular deposits sometimes distributed in a tigroid pattern in VPSPr- 129VV (VV, middle row); at higher magnification (lowest row) occasionally finer granules surround bigger ones in a target-like formation. Similar granular immunostaining in VPSPr-129MV but with less noticeable tigroid and target-like patterns (MV, mid- dle and lowest rows). Plaque-like immunostaining in a fine granular background in VPSPr-129MM. Antibody to PrP 3F4. (Reproduced from Zou WQ, Puoti G, Xiao X, et al. (2010) Variably protease-sensitive prionopathy: a new sporadic disease of the prion protein. Ann Neurol 68: 162–172, with permission from John Wiley.) neocortex remained seemingly negative (Flaherty et al., surrounding a larger central one. A peculiar selective 2007; Puoti et al., unpublished data). immune staining of the hippocampal molecular layer PrP immunostaining generally codistributes with SD has also been noted in VV cases (Gambetti et al., and typically shows coarse granules in a background of a 2008). In the cerebellum the pattern consists of fine granular or “synaptic” pattern, often shaped as small rounded deposits, which often show the tinctorial target-like formations with rounded clusters of granules characteristics of real plaques and correspond to the 182 S. NOTARI ET AL.

Fig. 10.2. Histopathology and prion protein (PrP) immunohistochemistry of the cerebellar molecular layer. (A) Miniplaques often with multiple cores in VPSPr-129VV and -129MV. (B) Miniplaques immunostain intensely as round, compact, and sharply bor- dered structures in the 129VV and 129MV VPSPr genotypes. To date cerebellar miniplaques have not been reported in VPSPr- 129MM. In this condition PrP immunostaining of cerebellar molecular layer demonstrates more loose, plaque-like structures rather than plaque (B, MM). Antibody to PrP 3F4. (Reproduced from Zou WQ, Puoti G, Xiao X, et al. (2010) Variably protease-sensitive prionopathy: a new sporadic disease of the prion protein. Ann Neurol 68: 162–172, with permission from John Wiley.) miniplaques described above in the cerebellar 2010; Head et al., 2013). Similarly, presence of PrP amy- molecular layer. Other cerebellar regions are not immu- loid miniplaques, while well documented in VVand MV nostained. Miniplaques and plaque-like deposits are also subsets, has not been reported in MM homozygotes (Zou seen in the dorsal parts of the midbrain (Gambetti et al., 2010; Head et al., 2013). Combined, these findings et al., 2008). seem to outline a trend running from the 129VV to the Separating neuropathologic features of the individual 129MM subset toward the formation of PrPTSE that subtypes of VPSPr according to the 129 genotype is chal- shows an increasing propensity to aggregate and less lenging because the variations are not substantial and the to form amyloid. This trend would be inversely related MM and MV subtype cohorts are still relatively small. to codon V dosage. Overall, VV and MV cases seem to share more similar- Another reported but little explored issue is the occur- ities between them than with the MM subtype. While rence of co-pathologies in VPSPr. The majority of cases all subtypes share similar SD distribution profiles and belonging to the three 129 genotypes harbor tau pathol- similar vacuole sizes, the severity appears to be compa- ogy preferentially in the lower medial temporal cortex, rably higher in the VV and MV subsets than in the MM especially entorhinal and transitional cortices and hippo- (Fig. 10.1)(Zou et al., 2010). The immunostaining pat- campal gyrus, which has been rated Braak stage 2 or terns in the cerebral cortex of the VV subset most often higher, while the neocortex is minimally involved. Ab consist of the aforementioned target-like formations. plaques may also co-occur (Braak and Braak, 1995; This pattern seems to become less well organized and Giaccone et al., 2007; Head et al., 2010; Rodríguez- detectable in MV heterozygotes and is virtually lost in Martínez et al., 2012). Ballooned neurons have been MM homozygotes, where the immunostaining pattern reported in two cases and pretangle formations have been is primarily plaque-like (Fig. 10.1)(Giaccone et al., observed in pigmented nuclei of one case (Giaccone 2007; Rodríguez-Martínez et al., 2010; Zou et al., et al., 2007; Rodriguez-Martinez et al., 2010; Cannon VARIABLY PROTEASE-SENSITIVE PRIONOPATHY 183 et al., 2014). Five cases harbored comorbidities of anchorless counterpart of the above two fragments, and VPSPR and neurodegenerative diseases that included the 7 kDa is a fragment that lacks a large portion of both diffuse Lewy body disease (Head et al., 2010; Assar N-and C-termini and is unglycosylated (Fig. 10.3)(Zou et al., 2015), argyrophilic grain disease (Giaccone et al., 2010; Pirisinu et al., 2013). These conclusions are et al., 2007; Rodríguez-Martínez et al., 2010), and amyo- consistent with the result of enzymatic deglycosylation, trophic lateral sclerosis (Cannon et al., 2014). which, as expected, reduces the original five fragments to three: the 20-kDa unglycosylated fragment, its DISEASE-ASSOCIATED PRION PROTEIN 17-kDa anchorless counterpart, and the 7-kDa internal fragment (Fig. 10.3)(Zou et al., 2010). The PrPTSE associated with VPSPr displays a panoply of Antibodies to the PrP C-terminus demonstrate the peculiar features (Gambetti et al., 2008; Zou et al., 2010; presence of two additional bands: one with an electro- Puoti et al., 2012). Following appropriate immunoblot phoretic mobility of 12–13 kDa that matches the procedures, PK-resistant PrPTSE (resPrPTSE)associated CTF12/13, two C-terminal fragments described in all with VPSPr forms a distinctive ladder profile that com- sCJD subtypes (Zou et al., 2003), and one migrating at prises five or seven fragments (depending on the use of 7 kDa. The latter, despite the similar molecular weight, antibodies to the PrP N-terminus or to the C-terminus) must be quite different from the 7-kDa fragment detected (Fig. 10.3). The resPrPTSE fragments originate exclusively by the N-terminal antibody and rather may be an addi- from the monoglycosylated and unglycosylated isoforms, tional C-terminal fragment extensively truncated at the which results in the lack of diglycosylated isoform in the N-terminus. resPrPTSE immunoblot profile even though this glycoform The peculiar lack of the PrPTSE diglycosylated form is present in the PK-untreated total PrP preparations has been shown to result from the presence of only the (Gambetti et al., 2008; Zou et al., 2010). isoform monoglycosylated at residue 197 among the The five PrPTSE immunoblot bands demonstrated by resPrPTSE fragments, while neither the isoform monogly- N-terminus antibodies comprise fragments of 26, 23, 20, cosylated at residue 181 nor the 181 and 197 diglycosy- 17, and 7 kDa (Fig. 10.3)(Zou et al., 2010). Based on lated isoform is converted to resPrPTSE (Xiao molecular weight and experiments with hydrofluoric et al., 2013). acid to assess the presence of the glycolipid anchor, The N- and C-termini of the 7-kDa fragment have been the five resPrPTSE fragments have been tentatively characterized by epitope mapping (Pirisinu et al., 2013). identified (Notari et al., 2008; Dagdanova et al., 2010; The N-terminus appears ragged with alternative termini Notari et al., unpublished data). Fragments 26 and at residues 97, 99, and 103, while the C-terminus has been 20 kDa originate from the monoglycosylated and ungly- identified at residue 152. This amino acid sequence would cosylated isoforms, following N-terminus truncation by predict a relative mass of 5–6 kDa. No similar data are PK treatment; the 23- and 17-kDa fragments are the available for the N-termini of the other fragments.

Fig. 10.3. Immunoblot of proteinase K-treated prion protein (resPrPTSE) from VPSPr-129MV and sCJDVV2 subtype demon- strated by an N-terminus antibody and diagrammatic representation of all bands detected by antibodies to N- and C-terminus. (A) The immunoblot compares the five-band ladder profile formed by resPrPTSE associated with VPSPr with the three-band profile of a sporadic Creutzfeldt–Jakob disease subtype. Antibody 1E4. (B) In black: four fragments detected by antibodies to the N-terminus; In light gray: the fragment recognized by antibodies to C-terminus, the so-called CTF-12/13 (Zou et al., 2003); in deep gray: the two different fragments as for N- and C-terminus but comigrating 7-kDa fragments, one detected by antibodies to the N-terminus and the other by antibodies to C-terminus. Diglyc: diglycosylated isoform; Monoglyc: monoglycosylated; M.anchor ̶: presumed monoglycosylated anchorless; U.anchor ̶: presumed unglycosylated anchorless; Unglyc: unglycosylated. 184 S. NOTARI ET AL. VV MM MV PK (µg/ml) 0 5 10 25 50 1000 5 10 25 50 100 0 5 10 25 50 100 kDa 37 kDa 29 23 20 17

7

Fig. 10.4. Proteinase K (PK) titration of disease-associated prion protein (PrPTSE) from VPSPr associated with the 129VV, 129MM, or 129MV genotype. Brain homogenates from 129VV, 129MM, and 129MV genotypes were treated with PK at various concentrations between 0 and 100 mg/mL and probed with 1E4. Overall, the PK resistance of the 7-kDa fragment is similarly high in all three VPSPr 129 genotypes. The other four fragments of the ladder (arrows) are definitely less PK-resistant in VPSPr-129VV than in the other two VPSPr genotypes. Besides the 7 kDa, three fragments (23, 20, and 17 kDa) in VPSPr-129MM and two (23 and 17 kDa) in -129MV are highly resistant to PK digestion. (Reproduced from Zou WQ, Puoti G, Xiao X, et al. (2010) Variably protease-sensitive prionopathy: a new sporadic disease of the prion protein. Ann Neurol 68: 162–172, with permission from John Wiley.)

Two other distinctive features of VPSPr PrPTSE are sedimentation in sucrose gradients and fractionation by amount and sensitivity to protease digestion. Analyses gel filtration, both of which separate PrPTSE aggregates of the representation of total PrPTSE (comprising both according to size (Rhodes et al., 2009), have shown that PK-sensitive and resistant components) carried out in VPSPr PrPTSE forms far fewer large aggregates than the VPSPr-VV have shown that total PrPTSE accounts for PrPTSE of sCJDVV1 (30% vs. 58% of total PrPTSE), approximately 3.5% of PrP preparations, including nor- with few aggregates exceeding 2000 kDa. Similar data mal or cellular PrP (PrPC) and PrPTSE, about 16 times less have been obtained by Saverioni and colleagues than in the sCJDMM1 subtype (Gambetti et al., 2008). (2013) in a study comparing VPSPr with other sCJD. Furthermore, according to this study, 24% of the total However, VPSPr data differ much less from those of PrPTSE is PK-resistant, compared to nearly 90% in the Gerstmann–Str€aussler–Scheinker (GSS) subtype carry- sCJDMM1 subtype. These data explain the low amount ing the A117V PrP gene mutation (GSSA117V) of resPrPTSE in VPSPr (Gambetti et al., 2008). (Gambetti et al., 2008). Moreover, the banding pattern A compounding issue is that antibody 3F4, which is formed on immunoblot by PrPTSE collected from the widely used to probe human PrPTSE species, does not fractions that contain large aggregates differed in VPSPr react well with some fragments of VPSPr-associated from that of sCJDVV1 (the sCJD subtype used in this PrPTSE, particularly in the VV subtype (Gambetti comparative study) and, to a lesser extent, from that of et al., 2008). As for the PK sensitivity of PrPTSE,itis GSSA117V. highest in the VV subtype and lower in subtypes MV Second,PrPTSE conformationalstabilityhasbeentested and MM (Fig. 10.4). In fact, PK titration experiments with the conformational stability and solubility assay, showed that VPSPr MM and MV have a low PK sensi- which derives stability data by measuring the amount of tivity even compared with those of sCJD subtypes the denaturing agent guanidine hydrochloride needed to TSE (Saverioni et al., 2013). However, PK sensitivity also solubilize 50% of native total PrP (GdnHCL1/2) varies among the five resPrPTSE fragments recognized (Pirisinuetal.,2013).The conformational stability and sol- by antibodies to the N-terminus. The 7-kDa fragment ubility assay has demonstrated that PrPTSE conformational is the most PK-resistant across the three subtypes; it is stability is higher in VPSPr VV (GdnHCL1/2 2.18) than the only highly PK-resistant fragment in VPSPr VV in the VPSPr MV and MM subtypes that have similar while two more fragments, 23 kDa and 17 kDa, show GdnHCL1/2 values (1.63 and 1.67, respectively) significant and similar PK resistance in the MV and (Pirisinu et al., 2013). Similar results have been obtained MM subtypes (Fig. 10.4)(Zou et al., 2010). Notably, with the comparable conformational stability assay all these three fragments are anchorless. (Peretz et al., 2001; Cali et al., 2009;Calietal., Several tests performed to assess the conformational unpublished data). characteristics of VPSPr total PrPTSE have underlined The properties of the resPrPTSE associated with divergences with PrPTSE species associated with sCJD VPSPr invite two considerations. First, the lack of the subtypes (Gambetti et al., 2008; Pirisinu et al., 2013; 181 glycoform in VPSPr resPrPTSE is likely due to the Saverioni et al., 2013; Cali et al., unpublished data). First, incompetence of the normal 181 glycoform to adopt VARIABLY PROTEASE-SENSITIVE PRIONOPATHY 185 the resPrPTSE conformation characteristics of VPSPr inoculated four lines of Tg mice expressing PrPC with and, therefore, to acquire PK resistance. Alternatively, 129 residues M or Vat levels ranging from one- to eight- it has been suggested that the 181 glycan is anomalous fold normal human brain levels on a murine PrPC null and hinders the conversion of the 181 glycoform to background. Brain homogenates (BH) from 12 VPSPr resPrPTSE (Nishina et al., 2006; Xiao et al., 2013). Sec- subjects that comprised six VV, four MV, and two MM ond, the presence of both anchor-linked and anchorless subtypes were used as inocula. Mice of the same lines conformers of the mono- and unglycosylated isoforms served as negative and positive controls. None of the (fragments 26 and 23, as well as 20 and 17 kDa, respec- 112 mice challenged with VPSPr BH developed clinical tively) exemplifies the remarkable heterogeneity of signs. 54% of the mice challenged with matching (i.e., PrPTSE in VPSPr, which must reflect multiple confor- having the same M or V genotype) BH from VPSPr mations. As a result of this heterogeneity, a fraction of VVor MM, showed histologic lesions that immunoreacted these two isoforms is PK-resistant at the C-terminus with PrP antibodies and 34% had positive immunoblots, while the other is not and becomes anchorless. Further- further confirming the presence of PrPTSE (Fig. 10.5). more, the size of the C-terminus region that remains There appeared to be no significant variation in attack rate PK-sensitive in the two anchorless fragments differs between high (8) and intermediate (3) PrP expression from that of the 7-kDa fragment. Based on the relative mice. Five of six VPSPr VV BH and one of two MM BH mass and detectability with the antibody to the C- inoculated into 129 matching mice transmitted the disease. terminus, the PK-sensitive C-terminal region of the The non-transmissible MM BH was from a clinically 23- and 17-kDa anchorless fragments must be small once asymptomatic subject diagnosed at autopsy. the mass reduction due to loss of the anchor is taken into No transmission occurred when the 129 genotype was account. In contrast, according to epitope mapping data, mismatched (i.e., BH VV was inoculated to 129M or the 7-kDa fragment is generated by PK digestion of MM into 129V mice). Surprisingly, there was also no nearly 80 C-terminus residues. transmission when BH MV was inoculated to 129V or Finally, the original report of VPSPr also described 129M mice. These findings clearly point to an amino the limited presence of the three-band resPrPTSE isoform acid sequence barrier introduced by the M129V with a resPrPTSE type 1 electrophoretic mobility that is polymorphism. typically associated with subtypes of sCJD (Gambetti Histologic lesions of two types were observed. The et al., 2008). Significant to minimal amounts of first featured poorly structured and often clustered pla- three-band resPrPTSE were detected in putamen, thala- ques, most often located at the border of the hippocam- mus, and substantia nigra of three out of eight cases pus and the overlying white matter, and occasionally in tested (Gambetti et al., 2008). This finding indicates that periventricular regions (Fig. 10.5). Plaques appeared to the three-band resPrPTSE characteristic of sCJD may be more prevalent in mice expressing PrPC 8 fold than occasionally occur in subcortical regions of VPSPr sub- 3 fold and to be more compact and better formed follow- jects. More recently, Peden et al. (2014) and Rodríguez- ing inoculation with BH from VPSPr VV than MM Martínez et al. (2012) and their colleagues have reported (Fig. 10.5). The lesion of the second type consisted of the presence of sCJD resPrPTSE in the cerebellum of three focal SD located in the lacunosum-moleculare layer of cases of VPSPr VV. In the latter report, sCJD resPrPTSE the hippocampal formation (Fig. 10.5). Both plaques was type 2, but the PrP immunostaining pattern of the and SD areas were intensely reactive with PrP antibodies. cerebellum was apparently consistent with VPSPr rather Widespread PrP deposition was not detected in any of than sCJDVV2, as would be expected by the presence of the mice challenged with any VPSPr BH. Immunoblot resPrPTSE type 2 in a subject harboring the VV genotype. demonstrated small amounts of resPrPTSE forming a All of these features, but especially the number and ladder resembling (Fig. 10.6), but apparently more heterogeneity of the resPrPTSE fragments that imply dis- PK-resistant than that associated with VPSPr, which tinct conformations, currently establish resPrPTSE asso- was exclusively observed in the 8 PrP expression ciated with VPSPr as a unique resPrPTSE isoform or 129V Tg mice. Deglycosylation reduced the bands to strain among sporadic human prion diseases. three that matched the electrophoretic mobility of the corresponding VPSPr bands (Fig. 10.6). No PrPTSE EXPERIMENTAL TRANSMISSION AND was recovered in negative controls while all sCJD sub- IN VITRO AMPLIFICATION types transmitted to positive controls with efficiency comparable to that reported in other studies. Surpris- Experimental transmission ingly, none of the positive mice transmitted the disease VPSPr has been experimentally transmitted to transgenic on second passage. (Tg) mice in two concurrent studies (Diack et al., 2014; Very similar results were obtained by Diack and Notari et al., 2014a). Notari and colleagues (2014a) coworkers (2014), following inoculation of BH from 186 S. NOTARI ET AL.

Fig. 10.5. Immunohistochemistry and histopathology brain lesions in Tg mice inoculated with variably protease-sensitive priono- pathy (VPSPr) extracts. (A) Clusters of prion plaques typically observed at the border of hippocampus and overlying white matter (Tg(HuPrP129V)8); box marks area shown in (B). (B) At higher magnification, plaques appear often confluent or fragmented and are intensely prion protein (PrP)-immunoreactive; at hematoxylin and eosin stain, plaques show an amorphous core lacking the spiny appearance of typical kuru plaques (inset). (C) In (Tg(HuPrP129M)2) mice inoculated with VPSPr-129MM, PrP aggre- gates generally appear looser and form fewer well-bounded plaques. (D–F) Second type of lesion consisting of PrP granular deposits in the lacunosum-moleculare layer of the hippocampal formation (D and E) that codistribute with spongiform degener- ation (F) (box in D marks area shown in E). Antibody 3F4. (Reproduced from Notari S, Xiao X, Espinosa JC, et al. (2014a) Transmission characteristics of variably protease-sensitive prionopathy. Emerg Infect Dis 20: 2006–2014.)

Fig. 10.6. Immunoblot of abnormal prion protein (PrPTSE) recovered from brains of variably protease-sensitive prionopathy (VPSPr)-inoculated Tg mice and controls. (A) Extract from brains of VPSPr-inoculated mice (VPSPr+), treated with increasing amounts of proteinase K (PK) show PK-resistant PrPTSE (resPrPTSE) fragments with a ladder-like electrophoretic profile even at high concentrations of PK (50 mg/mL). Nonspecific bands are seen in negative control mice (VPSPr–). The banding pattern in VPSPr+ roughly recapitulates that of resPrPTSE from the VPSPr inoculum (VPSPr Inoc). (B) VPSPr+ extracts treated with PK (25 mg/mL) and PNGase F show three resPrPTSE bands migrating at 20 kDa, 17 kDa, and 7 kDa that replicate those of sim- ilarly treated VPSPr inoculum (VPSPr Inoc). No bands can be detected in the negative Tg mice (VPSPr–). All preparations were run on the same gel, but unnecessary lanes were removed. Antibody 1E4. (Reproduced from Notari S, Xiao X, Espinosa JC, et al. (2014a) Transmission characteristics of variably protease-sensitive prionopathy. Emerg Infect Dis 20: 2006–2014.) VARIABLY PROTEASE-SENSITIVE PRIONOPATHY 187 two subjects with VPSPr-VVand one with VPSPr-MV to cyclic amplification (PMCA) by two groups. Successful, Tg mice that were produced by gene targeting and albeit weak, amplification of VPSPr-VV PrPTSE has been expressed human PrP MM, MV and VV at physiologic reported by Peden et al. (2014), using two kinds of sub- levels. Transmission succeeded only with VPSPr-VV. strate: “human PrP expressing Tg mice” and human Challenge of Tg mice with BH mismatched for the 129 brain. The stronger amplification was obtained with Tg residue also seems to support the presence of a primary mice as substrate. Additionally, in this study, a PrPTSE structure barrier, as in the study of Notari and colleagues species, described as similar to the sCJD type 2 and found (2014a), although perhaps not as strict, since VPSPr VV in the cerebellum of two VV cases, was positively ampli- was transmitted to 129M Tg mice, albeit to only one of fied with PMCA. Notari and coworkers, in a preliminary 15 inoculated mice. Histologic lesions and patterns of study (Gambetti et al., 2012; Notari et al., 2014b), suc- PrP deposition were virtually identical, although lesions ceeded in amplifying VPSPr PrPTSE using human PrPC apparently were less frequent, presumably due to the from different Tg mice as substrate. The highest ampli- lower PrP expression levels of the mice used in this study. fication was obtained using PrPC 129V from Tg152 mice Plaques were shown to contain amyloid by thioflavin expressing four to eight times normal levels of PrPC as Sstaining.NoPrPTSE was demonstrated by immunoblot substrate (Telling et al., 1995). With Tg mice expressing analyses. No data were reported on second passage. PrP129M or 129V at the same lower level (2)(Kong Both studies prompt several considerations. VPSPr et al., 2005 and unpublished data), amplification was MV failed to transmit and at present remains the only sensitive to the M129V polymorphism and occurred only VPSPr subtype with no evidence of transmission. The when 129 residues matched; however these mice sup- lack of clinical signs in PrPTSE-positive mice is likely ported a less robust amplification. Following PK treat- due to the focality of the lesions and their location in rel- ment, the amplified resPrPTSE reproduced the same atively silent brain regions. Prion plaques similar in type electrophoretic ladder-like profile as the seed and location to those described in these two studies have (Gambetti et al., 2012; Notari et al., 2014b). been observed previously in Tg and wild-type mice chal- Peden et al. (2014) also demonstrated the capacity of lenged with human BH or in vitro preparations that share VPSPr to convert recPrP to its misfolded form by real- the property of harboring plaques or seeding plaque for- time quaking-induced conversion (RT-QuIC), another mation (Piccardo et al., 2007; Asante et al., 2013; amplification technique. Pirisinu et al., 2016). The failure to transmit the disease These studies definitively establish the propensity of upon second passage is puzzling but not unprecedented VPSPr to replicate in vitro. (Wadsworth et al., 2004; Piccardo et al., 2007, 2013). TSE Based on the low amount of resPrP in VPSPr, which ANIMAL MODELS indicates a low conversion efficiency, it has been pro- posed that due to limited amplification and propagation It has recently been found that resPrPTSE associated with of the seed at first passage, along with the relative short in a familial form of CJD harboring the V180I PrP gene lifespan of the mouse, the actual resPrPTSE concentration mutation (fCJDV180I) displays an electrophoretic profile in the whole brain of the inoculated mouse is lower than and antibody immunoreactivity that mimic those of that of the inoculum (Notari et al., 2014a). Furthermore, VPSPr (Fig. 10.7)(Xiao et al., 2013). The electrophoretic the failure of the second passage also suggests that there profile, when probed with N-terminus antibodies, is no significant adaptation of the seed to the host during includes five bands that co-migrate with those of VPSPr, the first passage in VPSPr transmission despite the long although show some difference in the ratios. Remark- incubation. Future studies of VPSPr transmission to ably, as in VPSPr, both monoglycosylated at residue more receptive hosts, such as the bank vole (Nonno 181 and diglycosylated isoforms fail to convert to et al., 2012), as well as experiments of PrPTSE rescue resPrPTSE; thus, of the three glycoforms only the 197 from brains of mice used in these two studies may clarify monoglycosylated is converted even though total PrP this issue. from fCJDV180I contains both monoglycosylated and Nonetheless, both these studies definitively establish diglycosylated isoforms like VPSPr. The virtually iden- that VPSPr is a transmissible prion disease and further tical mobility of the five bands supports the notion that highlight the unique properties of VPSPr PrPTSE among the resPrPTSE fragments have common properties in sporadic prion diseases. the two diseases, and therefore anchorless fragments may also be present in fCJDV180I. These striking similar- ities indicate that the resPrPTSE isoforms associated with In vitro amplification VPSPr and fCJDV180I share similar conformations in The propensity of VPSPr PrPTSE to convert PrPC to its spite of the different etiologies of the two diseases misfolded form was analyzed with protein misfolding (genetic in fCJDV180, sporadic in VPSPr). Because it is 188 S. NOTARI ET AL. the original cohort of 11 VPSPr VV patients (Gambetti et al., 2008). Two additional VV patients had a family history of a condition similar to amyotrophic lateral scle- rosis (or amyotrophic lateral sclerosis was a comorbidity (Jansen et al., 2010; Cannon et al., 2014). Since no muta- tion has been detected in the coding region of the PrP gene in any of VPSPr-affected subjects, the presence of a variation involving other regions of the PrP gene or other parts of the genome needs to be investigated to assess the role of a genetic component in VPSPr. VPSPr-associated PrPTSE also has unique features, including the inability to acquire resistance to proteases by the PrP glycosylated at residue 181, regardless of whether it is the mono- or diglycosylated isoform, and by part of the C-terminus. Furthermore, data from exper- imental transmission also indicate that, compared to most Fig. 10.7. Immunoblot of proteinase K-resistant prion protein subtypes of sCJD, conversion of the host PrPC and prop- from variably protease-sensitive prionopathy (VPSPr) and agation of newly formed PrPTSE are slow and very limited. V180I brain extracts. The electrophoretic mobility of the indi- These features need to be clarified to begin understanding vidual bands is very similar, consistent with similar primary the pathogenetic mechanisms that underlie VPSPr. structure. Representation of at least two bands, monoglycosy- lated and unglycosylated anchorless (arrow and 17 kDa), is noticeably different. Antibody1E4. REFERENCES Appleby BS,ApplebyKK,Crain BJetal. (2009).Characteristics – a genetic disease, fCJDV180I would lend itself better to of established and proposed sporadic Creutzfeldt Jakob disease variants. Arch Neurol 66: 208–215. the study of pathogenetic mechanisms and might be a Appleby BS, Rincon-Beardsley TD, Appleby KK et al. (2014). valid model to shed light on VPSPr. 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