Gianfranco Puoti

Sporadic human prion diseases: molecular insights and diagnosis

Human prion diseases can be sporadic, inherited, or acquired by infection. Distinct clinical and pathological characteristics separate sporadic diseases into three phenotypes: Creutzfeldt-Jakob disease (CJD), fatal insomnia, and variably protease-sensitive prionopathy. CJD accounts for more than 90% of all cases of sporadic prion disease; it is commonly categorised into five subtypes that can be distinguished according to leading clinical signs, histological lesions, and molecular traits of the pathogenic prion protein. Three subtypes affect prominently cognitive functions whereas the other two impair cerebellar motor activities. An accurate and timely diagnosis depends on careful clinical examination and early performance and interpretation of diagnostic tests, including electroencephalography, quantitative assessment of the surrogate markers 14-3-3, tau, and of the prion protein in the CSF, and neuroimaging. The reliability of CSF tests is improved when these tests are interpreted alongside neuroimaging data.

Creutzfeldt-Jakob disease: Carnoy's fixative improves the immunohistochemistry of the proteinase K-resistant prion protein.

The neuropathological diagnosis of Creutzfeldt‐Jakob disease relies on the immunohistochemical demonstration of the proteinase‐K resistant form of the prion protein (PrPres) in the brain tissue. The antigenicity of PrPres is strongly reduced by the formalin solution widely used to fix the tissue, thus the PrPres immunoreactivity is inconsistently detectable in formalin‐fixed tissue. A better PrPres immunostaining can be obtained by using Carnoys fixing solution, which is composed of ethanol, chloroform and acetic acid (6:3:1). PrPres can easily be extracted from Carnoys‐fixed, paraplast‐embedded tissue. Accordingly, Carnoys‐fixed tissue can prior to immunolabeling be subjected to proteinase K and guanidine thio‐cyanate, which respectively eliminate the normal cellular form of prion protein and promote protein denaturation. In comparison with the best protocols for formalin‐fixed tissue (i.e. ‐ hydrolytic autoclaving or autoclaving in distilled water followed by formic acid and guanidine thiocyanate), PrPres immunostaining carried out on sections cut from Carnoys‐fixed, paraplast‐embedded tissue blocks and subjected to proteinase K and guanidine thiocyanate, proved more successful to detect and map both diffuse and focal PrPres immunoreactivity, and to correlate the immunoreactivity pattern with MV polymorphism at PRNP codon 129 and PrPres banding and glycosyla‐tion pattern revealed by Western blot.

Hereditary cerebral hemorrhage with amyloidosis associated with the E693K mutation of APP.

OBJECTIVE To report the clinical, genetic, neuroimaging, and neuropathologic studies of patients with the hereditary cerebral hemorrhage with amyloidosis linked to the APP E693K mutation. DESIGN Case series. Clinical details and laboratory results were collected by direct evaluation and previous medical records. DNA analysis was carried out in several affected subjects and healthy individuals. Neuropathologic examination was performed in 2 subjects. SETTING Southern Lombardy, Italy. Patients Individuals with and without amyloidosis in 4 unrelated Italian families (N = 37). Main Outcome Measure Genotype-phenotype relationship. RESULTS The affected individuals presented with recurrent headache and multiple strokes, followed by epilepsy and cognitive decline in most of them. The disease was inherited with an autosomal dominant trait and segregated with the APP E693K mutation. Neuroimaging demonstrated small to large hematomas, subarachnoid bleeding, scars with hemosiderin deposits, small infarcts, and leukoaraiosis. Amyloid-beta immunoreactivity was detected in the wall of leptomeningeal and parenchymal vessels and in the neuropil, whereas phosphorylated tau, neurofibrillary changes, and neuritic plaques were absent. CONCLUSIONS These findings expand the number of APP mutations linked to hereditary cerebral hemorrhage with amyloidosis, reinforcing the link between this phenotype and codon 693 of APP.

Sporadic Creutzfeldt-Jakob disease: the extent of microglia activation is dependent on the biochemical type of PrPSc.

In prion-related encephalopathies, microglial activation occurs early and is dependent on accumulation of disease-specific forms of the prion protein (PrPSc) and may play a role in nerve cell death. Previously, we found that different types of PrPSc (i.e. type 1 and type 2) coexisted in approximately 25% of patients with sporadic Creutzfeldt-Jakob disease (CJD); and a close relationship was detected between PrPSc type, the pattern of PrP immunoreactivity, and extent of spongiform degeneration. To investigate whether microglial reaction is related to the biochemical type and deposition pattern of PrPSc, we carried out a neuropathologic and biochemical study on 26 patients with sporadic CJD, including all possible genotypes at codon 129 of the prion protein gene. By quantitative analysis, we demonstrated that strong microglial activation was associated with type 1 PrPSc and diffuse PrP immunoreactivity, whereas type 2 PrPSc and focal PrP deposits were accompanied by mild microglia reaction. These findings support the view that the phenotypic heterogeneity of sporadic CJD is largely determined by the physicochemical properties of distinct PrPSc conformers.

The ε isoform of 14-3-3 protein is a component of the prion protein amyloid deposits of Gerstmann-Sträussler-Scheinker disease

The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic events, such as transduction pathway modulation, cell cycle control, and apoptosis. Seven isoforms have been identified that are abundant in the brain, preferentially localized in neurons. Remarkable increases in 14-3-3 are seen in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease (CJD), and it has been found in pathologic inclusions of several neurodegenerative diseases. Moreover, the &zgr; isoform has been detected in prion protein (PrP) amyloid deposits of CJD patients. To further investigate the cerebral distribution of 14-3-3 in prion-related encephalopathies, we carried out an immunohistochemical and biochemical analysis of brain tissue from patients with Gerstmann-Sträussler-Scheinker disease (GSS) and sporadic, familial and acquired forms of CJD, using specific antibodies against the seven 14-3-3 isoforms. The study showed a strong immunoreactivity of PrP amyloid plaques of GSS patients for the 14-3-3 &egr; isoform, but not for the other isoforms. The &egr; isoform of 14-3-3 was not found in PrP deposits of CJD. These results indicate that the &egr; isoform of 14-3-3 is a component of PrP amyloid deposits of GSS and suggest that this is the sole 14-3-3 isoform specifically involved in the neuropathologic changes associated with this disorder.

A novel insertional mutation in the prion protein gene: clinical and bio-molecular findings

A young man, presenting with early onset of personality and behavioural changes followed by slowly progressive cognitive impairment associated with marked bi-parietal cerebral atrophy, was found to carry a novel seven extra-repeat insertional mutation in the prion protein gene (PRNP). In vitro, the mutated recombinant prion protein (PrP) showed biochemical properties that were consistent with pathological PrP variants. Our results further underline the heterogeneity of neurological pictures associated with insertional mutations of PRNP, indicating the diagnostic difficulties of sporadic cases with early-onset atypical dementia.

Rehabilitation of gesture imitation: a case study with fMRI.

Acquired disorders of gesture imitation are amenable to treatment, but with poor generalisation toward gestures not included in the training program. We investigated the neural basis of this item-specific recovery in a patient with a slowly progressive posterior cortical atrophy, by means of an fMRI study comparing imitation of rehabilitated and not-rehabilitated gestures. Results suggested that in our patient gesture imitation recruited the mirror system and additional areas relevant to gesture analysis and preparation. Imitation of rehabilitated gestures activated the mirror neuron system, and also left dorsolateral prefrontal cortex and putamen, and the right anterior temporal cortex. This suggests that item-specific recovery was based on interaction of circuitry of imitation with neural systems involved in emotional and motivational processing.

Heidenhain variant in two patients with inherited V210I Creutzfeldt-Jakob disease.

Objective: To report two members of the same family carrying the valine to isoleucine point mutation of the prion protein gene (PRNP) and presenting with visual symptoms as initial manifestation as in the “Heidenhain variant” of sporadic Creutzfeldt–Jakob disease (CJD). Methods: Patients underwent neurological examination, electroencephalogram (EEG), brain magnetic resonance images (MRI) and cerebrospinal fluid (CSF) analysis including the Real Time Quaking Induced Conversion (RT-QuIC) test. Disease-specific mutations and polymorphism at codon 129 of the PRNP gene were also studied. Results: Isolated visual symptoms characterized disease onset of both patients followed by progressive neurological signs, dementia and death in 3 (proband) and 9 (his aunt) months. RT-QuIC analysis of CSF samples of both patients revealed the presence of the pathological prion protein and DNA analysis the V210I point mutation of the PRNP and methionine homozygosity at the polymorphic codon 129. Conclusions: This report suggests to consider the diagnosis of V210I genetic CJD in patients presenting with the Heidenhain form of CJD and highlights the importance of genetic testing in all patients with isolated visual manifestations at onset followed by progressive neurological signs and dementia.

New-Onset Refractory Status Epilepticus Mimicking Herpes Virus Encephalitis

New-onset refractory status epilepticus (NORSE) is a recently defined clinical entity that describes patients who present with status epilepticus of unclear etiology that is highly refractory to therapy. Magnetic resonance imaging (MRI) of NORSE usually discloses no specific abnormalities except for an occasional mild T2/FLAIR hyperintense signal of the mesial temporal lobe. Here, we report a peculiar case of NORSE in which brain MRI showed massive alteration of both temporal lobes, with features strongly supporting the diagnosis of herpes virus encephalitis, but lacking any laboratory evidence of viral infection in the blood and cerebrospinal fluid. It showed also striking signal alterations in the thalamus, which got worse in the course of the disease. This report emphasizes the possibility that seizure activity alone plays a critical role in both determining the disease and whether it will be self-sustaining.

Coexistence of cavernous hemangioma and other vascular malformations of the orbit. A report of three cases.

Coexistence of orbital cavernous hemangioma and other vascular malformations is unusual and few cases have been reported. We describe the clinical and radiological features of three cases of orbital cavernous hemangiomas associated with other vascular malformations, selected reviewing a series of 181 cases of cavernous hemangiomas. All patients were males (age ranging from 43 to 67 years) without vascular systemic disorders and/or a clinical syndrome. They experienced slow progressive exophthalmos. One of them developed acute pulsatile proptosis (case 2), while another experienced slow progressive diplopia (case 3). In one case vascular lesions were bilateral (case 3) and in two patients two different lesions coexisted in the same orbit (cases 1 and 2). All patients underwent surgical excision, which was partial in two cases. Two patients had cavernous hemangiomas in association with a venous malformation (a varix in case 1 and a lymphangioma in case 2), while in the other ones (case 3) cavernous hemangioma was associated with a low-flow arteriovenous malformation. No patient denied visual impairment postoperatively. Few cases of orbital cavernous hemangiomas coexisting with other vascular malformations have been reported in the literature. This entity seems to be an association of different variants of orbital vascular malformations, presenting with a wide spectrum of clinical forms and probably with the same pathogenesis.