• CREUTZFELDT-JAKOB DISEASE
• NEUROPATHOLOGY
• HISTOPATHOLOGY
• POST MORTEM
• PRION

Variably protease-sensitive prionopathy (VPSPr) is a recently described neurodegenerative disorder characterised by the presence of spongiform encephalopathy and an unusual immunostaining and immunoblotting pattern for the disease-associated prion protein (PrP^Sc).1 This links VPSPr to human prion diseases, which are uniformly fatal disorders. The clinical symptoms and the longer duration of illness make VPSPr distinct from sporadic or idiopathic Creutzfeldt-Jakob disease (sCJD).1 Doxycycline treatment has been evaluated in patients with prion disease, however, there is little evidence that it can reverse the clinical symptoms or reduce the underlying disease progression once established.2 We present a patient with VPSPr who received doxycycline and survived for an extended period of time in an akinetic and mute state.

Neuropathological examination was performed using published methods and various anti-PrP antibodies.3 Frozen tissues from selected brain regions were available for biochemical analysis. Tissues were analysed for the presence of protease-resistant PrP (PrP^res) as previously described (see online supplementary material).4

In May 2007, a registered psychiatrist suspected an organic affective disorder in a 54-year-old Austrian woman. Two months earlier, medical work-up for presumed weight loss of 16 kg within the past 18 months had been unremarkable. In June 2007, the patient was admitted to a clinic that specialised in disorders of the nervous system. Her family history was negative for neurodegenerative diseases and there was no evidence of exposure to toxins. She reported depressed mood and short-term memory problems, and difficulties with balance, walking, driving and cooking. On neuropsychological examination she was oriented to time, place, person and situation, Mini-Mental State Examination score was 22/30, clock drawing test score was 3/9. She had word-finding difficulties, ideational apraxia, acalculia and visuoconstructive deficits. She displayed affective incontinence with crying fits. She had gait ataxia, and extensor plantar responses were observed with increased tone in arms and legs. Baseline MRI of the brain revealed symmetric increased signal in the basal ganglia in T2-weighted images and, less marked, cortical ribbon hyperintensity with widespread restricted diffusion in the cortex (figure 1A, B). EEG studies showed generalised slowing only; Western blot assay for the 14-3-3 protein in the cerebrospinal fluid was weakly positive. Accordingly, this case fulfilled surveillance criteria for probable sCJD. Analysis of the PRNP gene excluded any mutation and revealed VV homozygosity at codon 129. Following the guidelines of an ongoing therapeutic trial,2 the patient was treated with 100 mg doxycycline per day. Until December 2008, she was examined five times (see online supplementary material). By November 2007 she developed extrapyramidal signs and dystonic postures and frequent falls. Repeated EEG still showed generalised slowing. There was no myoclonus. From July 2008 onwards she was in a state of akinetic mutism. She was repeatedly treated with antibiotics for aspiration pneumonia and urinary tract infections. In April 2011 levetiracetame was started due to new-onset focal epilepsy. Doxycycline was stopped after 4 years. Diffusion restriction was less pronounced 3.5 years after baseline imaging (see online supplementary material). CT scan showed atrophy 4.5 years after baseline MRI imaging (see online supplementary material). The patient died in May 2013, 6 years after recognition of the initial symptoms and after nearly 5 years in an akinetic and mute state.

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Figure 1

Cranial MRI, neuropathology and immunoblotting observations. T2-weighted images showing bilateral hyperintense signal in the basal ganglia (A). Diffusion weighted images showing restricted diffusion in the basal ganglia as well as in widespread areas of the cortex (B). H&E staining revealing spongiform change in the frontal cortex (C), more prominent reactive astrogliosis in the caudate nucleus (D) and mild vacuolation in the molecular layer of the cerebellum (E). Granular and synaptic PrP immunoreactivity appearing in loose clusters is shown in the parietal cortex (F and enlarged in G). Granular PrP deposits in the thalamus (H). Prominent PrP immunoreactivity in the cerebellum (I and enlarged in J). Prominent α-synuclein immunoreactive Lewy-related pathology in the substantia nigra (K). Western blot analysis (L–N) of frontal cortex (L lanes 2,4,6,8 and M, N lane 3) 10% w/v brain homogenate from this case, loaded as 5 μL without proteinase K digestion (L lane 2), or as 0.5 μL (L lane 6), 5 μL (L lane 4), or 50 μL collected by centrifugation (L lane 8) after proteinase K digestion. Molecular mass standards (L and M, N, lanes 1) and ∼5 μL of 10% w/v proteinase K digested frontal cortex from sporadic Creutzfeldt-Jakob disease (CJD) MM1 subtype (L lanes 3 and 7, M, N lanes 2 and 10), sporadic CJD VV2A subtype (L lane 10, M, N lane 8) and variant CJD (L lane 5 and 9, M, N lane 9) are shown for reference. In (M, N) 50 μL of samples of 10% w/v brain homogenates of frontal cortex (lane 3) occipital cortex (lane 4), basal ganglia (lane 5), white matter from frontal lobe (lane 6) and cerebellum (lane 7) were proteinase K digested and collected by centrifugation before Western blot analysis. Western blot is shown after short (M) and long (N) exposure times. The prion protein antibody used was 3F4. Bar in (A) represents 150 μm for A–C, and G; 200 μm for D; 500 μm for H; and 50 μm for F and I–O.

Histopathological features comprised spongiform change with intermediate-sized vacuoles, mild to moderate reactive gliosis in cortical and subcortical areas (figure 1C–E) and moderate neuronal loss and presence of eosinophilic cytoplasmic Lewy bodies in the brainstem pigmented nuclei. Immunostaining for PrP showed a diffuse synaptic pattern with loose clusters of granular deposits in the cortex (figure 1F, G). In the thalamus and cerebellar cortex mostly the granular-like and microplaque-like PrP immunoreactivity was seen (figure 1H–J). Immunostaining for phospho-τ (AT8) revealed small neuritic profiles. Immunostaining for α-synuclein showed Lewy bodies, thin and thick neurites, and dots (figure 1K), corresponding to Braak stage 4 of Lewy-related pathology or transitional/limbic type according to the McKeith criteria of dementia with Lewy bodies (for details on neuropathological alterations see online supplementary file).

Western blot analysis of a specimen of frontal cortex from this case showed abundant PrP prior to limited digestion with proteinase K (figure 1L). Following digestion with 50 μg/mL proteinase K, two faint bands were seen: one between 20 and 30 kDa and the other in the low molecular mass region. Analysis of a greater volume of brain homogenate (with PrP^res collected by centrifugation) showed a single strong clear band of approximately 8 kDa (figure 1L, M). This approximately 8 kDa band was present in occipital cortex, basal ganglia, cerebellum in addition to frontal cortex, but was not detectable in a specimen of white matter from the cerebrum (figure 1M, N). In the cerebellum the approximately 8 kDa band was accompanied by the presence of two bands with similar electrophoretic mobilities to the bottom and middle bands of the type 2A PrP^res profile that is found in some forms of CJD. A very faint band of approximately 17 kDa was also seen, giving the appearance of a ladder of PrP^res fragments (figure 1M, N).

The neuropathological and biochemical features of this case are consistent with VPSPr.1 ,4 The clinical course was biphasic; within 14 months the symptoms had progressed to akinetic mutism, following which the patient survived for 5 years. Although the early psychiatric symptoms were similar to those described in VPSPr cases,1 the clinical phenotype was compatible also with sCJD, including the striatal and cortical increased signals detected in baseline MRI, and the positive cerebrospinal fluid 14-3-3 test,5 which have been reported only in a single VPSPr case with VV homozygosity at codon 129.1 While prolonged survival is not unusual in VPSPr cases,1 in none of the reported VPSPr cases has there been such a protracted state of akinetic mutism reported. In contrast to the present case, in sCJD long-standing akinetic mutism is associated with severe pathology in the cortex and white matter, referred to as the panencephalopathic form of CJD. Although the long survival associated with less prominent histopathological alterations could suggest that doxycycline might have reduced the neurotoxic effect of disease-associated PrP, this observation merits further confirmation.