Creutzfeldt-Jakob’s disease or Prion Disease or Mad Cow disease It belongs to a group of neurodegenerative diseases called Transmissible Spongiform Encephalopathy (TSE). The infectious agents responsible for TSE are Prions, PrP c converted into PrP sc. They are generally hard to eliminate, and are extremely resistant to: Heat Radiation Disinfection Protein digestion (degradation)

Creutzfeldt-Jakob’s Disease (Mad cow disease or Prion disease)

Severe Brain Atrophy in CDJ’s patient

KURU: laughing death -Papua New Guinea (1957) KURU and prion’s disease: similarities in the symptoms and in cerebellar ataxia

Models for conformational conversion of PrP c into PrP sc

Tissues in which PrP sc accumulates !!! vCJD is of oral origin, and PrP sc could take years before converting PrP c Role of oral mucosas in first accumulating then spreading PrP sc

Symptoms in Creutzfeldt-Jakob’s Disease -It could take years (decades) before a carrier of prion disease will become fully symptomatic. -Symptoms are characterized by cognitive decline, which may be fulminant and progress to akinetic mutism within few weeks. -Cerebellar signs are evident (balance and coordination dysfunction -ataxia, changes in gait, rigid posture, and seizures). mood swings depression anxiety memory lapses social withdrawal clumsiness or lack of coordination insomnia

As the disease rapidly progresses, patients with all forms of CJD generally experience: * visual deterioration and eventual blindness * dementia * involuntary muscle contractions * muscle paralysis * slurred speech * difficulty swallowing * incontinence * coma

Cow affected by Bovine Spongiform Encephalitis

Diagnosis: -Electroencephalography -MRI “Probable” CJD is based on the clinical symptoms. -Correct and definite diagnosis only post-mortem, performing immunohistochemical analysis of brain sections. -Biopsy of the tonsils and, in 30% of the cases, of skeletal muscles can confirm CDJ. The target of the analysis is the presence of the scrapie, protease-K resistant form of Prion Protein (PrP sc ). This form is transported in the hematopoietic system, tends to accumulate in the lymphatic system, is enriched in the B-lymphocytes, and is present also in the mucosa olfactoria.

ControlCJD Absence of Protease-K-digested PrP in CJD used for diagnosis Prion protein

Spongiform (intracellular vacuolation) change in the cortical gray matter of the brain, characteristic of TSEs and prions aggregates Walker et al.,

*Neuronal death *Neuronal apoptosis *Astrogliosis (as a cause or a consequence of inflammation) *Protein misfolding and aggregation *Precipitation of aggregates (proteinaceous material) both at an intracellular and extracellular level (amyloid plaques) Features of TSEs and CJD

Deposition of fibrillar proteinacious material in Creutzfeldt-Jakob’s disease (prion disease) Prion disease: Alteration in the prion protein lead to both intracellular and extracellular accumulation of amyloid aggregates, plaques, similar to those characteristic of AD, and positive to prion protein staining. Probably, replication and accumulation of the protease insensitive PrP sc results in fibril formation and plaque deposition. Alzheimer’s Creutzfeldt-Jakob’s Aguzzi A, Haass C. Science Oct 31;302(5646): Review.

Epidemiology of Creutzfeldt-Jakob’s disease (CJD) CJD is, among the Transmissible Spongiform Encephalopathies, the most diffuse one. CJD can be classified as Sporadic sCJD: etiology not known, caused by both exogenous and endogenous factors, represents 85% of all the cases of CJD. In the United States, there are approximately 200 sporadic CJD cases per year. Familial fCJD: caused by mutations in the gene for PrP (prion protein). 15% of CJD cases are inherited. Iatrogenic iCJD: caused by the spreading of the infectious agent due to contaminated surgical tools, to the transplantation of tissues, or to the administration of pituitary hormones from deceased patients affected by the disease. 1% of CJD cases. Variant vCJD: caused by the transmission of Bovine Spongiform Encephalopathy (BSE) prion to humans (aka Mad Cow disease).

Incidence of BSE reported worldwide

Incidence of vCJD reported worldwide

The prion protein: functional domains and mutations causing inherited prion’s diseases

OR: not required for PrPc function, it might influence the change of conformation in PrPsc, as OR KO mice do not propagate the disease. Protects from apoptosis. CC1: probably involved in protein internalization/trafficking. CC1 KO mice are viable and could develop the disease. CC2: might work in concert with HC region, as partial deletion of either or the other domain, or ablation of one domain and partial deletion of the other accelerate the pathology in mice. C-terminal: gene KO on H2, H3 or both domains leads to ataxia and neuron disease, BUT FAIL TO REPLICATE PRIONS. No transmission of disease from H2 KO and H3 KO to other animals. H2 and H3 might stabilize the conformation of the protein. C-terminal deletion prevents GPI anchoring of the protein, no development of the disease. Functional domains of the prion protein

Prion Protein: domains and  - and  - helical structures -PrPc contains 208 aminoacid residues and is abundantly expressed in neurons and glial cells -Signal peptide sequence -Octarepeats followed by a short Hydrophobic/toxic structure -The C-terminal portion of the protein is a globular structure that contains 3  -helical domain and 2  -helical domains. This domain folds rapidly and is extremely stable

Amyloid plaques in TSE GSS disease Gerstmann-Straussler-Sheinker disease Kuru disease

Aguzzi et al., Nat Rev Mol Cell Biol Jul;8(7):552-61

Caughey and Byron, 2006 Nature Physiologic role of PrP c

Antiapoptotic function: PrP c KO mice are more susceptible to apoptosis. Following ischemic injury, PrP c KO mice have increased infarct volume and increased caspase 3 activation.

Infarcts are larger in PrP c KO mice after ischemic injury

Levels of activated caspase 3 are increased in PrPc KO mice after ischemic injury

PrP c protects against oxidative stress PrP c Ko mice are more susceptible to damage by H 2 O 2 PrP c KO mice have reduced SOD activity Brain of PrP c KO mice has increased levels of oxidated proteins, lipids, DNA. PrP c also involved in maintaining mitochondrial integrity

Control Prion’s infected Mitochondrial structure is disrupted in Prion’s infected hamsters

PrP c is has a role in maintaining synaptic architecture and function PrP c localizes mainly at the synaptic terminal PrP c KO mice have impaired Glutamatergic and GABAergic transmission, as well as decreased LTP. An early pathologic change in prion’s disease is synaptic loss

PrP stains as a flocculate/amorphous form at a synaptic level

PrP c physiological functions

How does the prion form and how it propagates?

Nature of the prion The prion is the minimum required infectious agent able to convert normal cellular prion protein Prp C into the scrapie PrP sc. Is the prion a virus? NO, the prion is not a virus, as RNA and DNA material are totally absent in its composition, and the minimal molecular weight necessary for infectivity is ~2x10 5 Da, so small to exclude the size of a virus. Is the prion proteinaceous material? YES

The “unfortunate” goal of CDJ disease is to convert normal cellular prion protein PrP C into the scrapie form PrP sc. This will result in 1-reduction of the form with  -sheet conformation, 2-accumulation of the form with  -sheet conformation, which will form aggregates and deposit both at an intracellular and extracellular level.

Sakaguchi Mechanism of replication of PrP Sc from PrP C

Models for conformational conversion of PrP c into PrP sc

Sakaguchi Different biochemical and structural properties between PrP C and PrP Sc

Prion’s disease and PrP c /PrP sc Loss of physiologic function or gain of toxic function?

PrP c KO mice are viable, develop normally and have no severe pathologies observed later in life. PrP c KO do not develop the disease as they do not propagate the formation of PrP sc Gain of toxic function rather than loss of physiological function

The “protein-only” hypothesis -In vitro data suggest that prion infectivity is achieved also de novo in the test tube -Propagation of conformationally changed yeast prions has been achieved -In vivo, prion protein null mice (PrP -/- or PrP KO) are resistant to prion infection, do not propagate prion infectivity after exposure to PrP Sc, suggesting that PrP C is necessary to propagate the infectivity caused by PrP Sc. -Prion protein null mice do not propagate prion infection also when infected with infectious brain tissue. This implies that the protein PrP C ALONE (and not in combination with other cellular or putative viral factors) is sufficient to propagate the prion infectivity. -Brain homogenates spiked with PrP Sc and subjected to sonication and recovery amplify the infectious species which maintains infectivity when “transferred” to new tissue.

Prion Profile: Far left, infectious prion particles extracted from yeast cells. At right is an example of what yeast prions can do when mixed with an isolated yeast protein. These fibrils are essentially long series of prions all linked together after replicating many times in the presence of the protein. Prion Particles Prion Particles+yeast protein Fibrils of prion protein: self propagating mechanism? Nat Med Jul;10 Suppl:S63-7. Review. Soto and Castilla

PMCA Protein Misfolding Cyclic Amplification

PMCA: protein misfolding cyclic amplification: a technique to amplify prions in vitro

Hypothetical model for PrP sc endocytosis

Prion proteins scrapie accumulation and propagation from normal cellular Prion protein (PrP C to PrP sc ) Caughey and Byron, 2006 Nature

Post-translational modifications on Prion protein: Glycosylation and binding to metals Caughey and Byron, 2006 Nature

GPI anchor as a target for the treatment of CJD?

Inhibition of GPI-anchor could improve the internalization AND the degradation of PrP C, that in this way will not be converted into PrP Sc at and intracellular level (PrP Sc intracellular more toxic than extracellular) Caughey and Byron, 2006 Nature

PrP sc is toxic both when extracellular and intracellular MECHANISM?

PrP c and PrP sc form aggregates intracellularly, but only aggregates form PrP sc are toxic UPS impairment as a consequence of PrP sc toxicity

PrP c aggregates accumulate after mild proteasome inhibition…

…but they are not toxic

Prion infection induces apoptosis

Formic acid PrP sc forms large cytosolic aggregates…

…that ARE toxic Colchicine and Nocodazole clear the aggresomes

Aggresomes accumulation is decreased with colchicine and nocodazole

PrP sc accumulates intracellularly in scrapie-infected mouse brain

Prion’s infection causes proteasomal malfunction

Aggregated PrP inhibits the proteolytic activity of 26S Proteasome’s beta subunit

PrP sc Oligomers inhibit the 20S proteasome’s catalytic core

PrP infection inhibits UPS in vivo

How regulation of ER and proteasomes homeostasis may affect PrP sc toxicity? ER-assisted protein degradation (ERAD) is responsible for the degradation of some PrP c Defective transport of substrates to ER promotes proteasomal degradation BUT proteasomal degradation is impaired by PrP sc

Insoluble PrP aggregates accumulate as a consequence of ER stress and proteasomal defect

ER stress reduces PrP c expression, whereas proteasomal inhibition increases it

Transport of PrPc through the secretory pathway is differently affected by ER stress or by proteasomal activity

PrP aggregates accumulating upon proteasomal inhibition localize at the plasma membrane

ER stress and proteasomal dysfunction enhance PrP sc accumulation in prion infected cells

Disrupting ER and proteasomes homeostasis results in more PrP at the cell surface, and consequent increase PrP sc levels in the cell: A mechanism of increased PrP sc propagation

Toxic intracellular aggregates accumulates in prion’s disease Is this the result of defective autophagy? Can autophagy target the degradation of PrP sc aggregates and be used in the therapy of prion’s disease?

Accumulation of degenerating lysosomes and other organelles at the dark synapse in human vCJD brain

Main steps in macroautophagy

Autophagic vacuoles form at the synapse of human vCJD brain

Autophagic vacuoles formed in scrapie-infected hamster brain

Autophagy-inducing drugs can regulate autophagy in scrapie infected cells

Autophagy-inducing drugs can reduce the PK resistant load of PrP sc aggregates

Autophagy as an approach to reduce prion infection

Small molecules used to regulate autophagy in neurodegenerative diseases

Approaches for the treatment of CJD -Use of autophagy-inducing molecules Use of molecules that associate with PrP C and interfere with the capacity of PrP Sc to bind and to propagate the infectious principle. -GPI inhibitors -PrP Sc antibodies: problems in achieving the right level of immunization (prion protein is a self antigen, and immunesystem is involved in the replication of the prion agent and its ultimate access to the CNS). Active immunization with recombinant protein has relatively modest therapeutic effects in mice. Passive immunization effective within a month after exposure to the contaminant. However, too expensive and not effective if performed in advanced stages of the disease, when the animal is symptomatic.