This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of the Lewy bodies and Lewy neurites found in Parkinson's disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addition of preformed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds the recruitment of endogenous α-syn into aggregates characterized by detergent insolubility and hyperphosphorylation. Aggregate formation follows a lag phase of 2-3 d, followed by formation in axons by days 4-7, spread to somatodendritic compartments by days 7-10 and neuron death ~14 d after PFF addition. Here we provide methods and highlight the crucial steps for PFF formation, PFF addition to cultured hippocampal neurons and confirmation of aggregate formation. Neurons derived from various brain regions from nontransgenic and genetically engineered mice and rats can be used, allowing interrogation of the effect of specific genes on aggregate formation.

Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates.

We have shown that it is possible to transmit bovine spongiform encephalopathy (BSE) to a sheep by transfusion with whole blood taken from another sheep during the symptom-free phase of an experimental BSE infection. BSE and variant Creutzfeldt-Jakob disease (vCJD) In human beings are caused by the same infectious agent, and the sheep-BSE experimental model has a similar pathogenesis to that of human vCJD. Although UK blood transfusions are leucodepleted--a possible protective measure against any risk from blood transmission--this report suggests that blood donated by symptom-free vCJD-infected human beings may represent a risk of spread of vCJD infection among the human population of the UK.

Transmission of TSEs by blood transfusion in sheep

The era of iatrogenic Creutzfeldt-Jakob disease (CJD) has nearly closed; only occasional cases with exceptionally long incubation periods are still appearing. The principal sources of these outbreaks are contaminated growth hormone (226 cases) and dura mater grafts (228 cases) derived from human cadavers with undiagnosed CJD infections; a small number of additional cases are caused by neurosurgical instrument contamination, corneal grafts, gonadotrophic hormone, and secondary infection with variant CJD transmitted by transfusion of blood products. No new sources of disease have been identified, and current practices, which combine improved recognition of potentially infected persons with new disinfection methods for fragile surgical instruments and biological products, should continue to minimize the risk for iatrogenic disease until a blood screening test for the detection of preclinical infection is validated for human use.

/pdf/iatrogenic-creutzfeldt-jakob-disease-final-assessment-1j9fw9vvb6.pdf

Iatrogenic Creutzfeldt-Jakob Disease, Final Assessment

Quality transitivity and traceability system of herbal medicine products based on quality markers

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 103ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.

/pdf/highly-efficient-prion-transmission-by-blood-transfusion-1lvh3xf0r4.pdf

Highly Efficient Prion Transmission by Blood Transfusion

Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" albumin solutions.

In vitro infectivity assay for prion titration for application to the evaluation of the prion removal capacity of biological products manufacturing processes.

Decontamination of prions in a plasma product manufacturing environment.

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Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" albumin solutions.

In vitro infectivity assay for prion titration for application to the evaluation of the prion removal capacity of biological products manufacturing processes.

Decontamination of prions in a plasma product manufacturing environment.