Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD¹⁷⁸) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD¹⁷⁸. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype. Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD) and fatal familial insomnia (FFI). The reason for this variability is not known, but assembly of the mutant PrPs into distinct aggregates that spread in the brain by promoting PrP aggregation may contribute to the disease phenotype. We previously generated transgenic mice modeling genetic CJD, clinically identified by dementia and motor abnormalities. We have now generated transgenic mice carrying the PrP mutation associated with FFI, and found that they develop severe sleep abnormalities and other key features of the human disorder. Thus, transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs in FFI and CJD mice are aggregated but unable to promote PrP aggregation. They accumulate in different intracellular compartments and cause distinct morphological abnormalities of transport organelles. These results indicate that mutant PrP has disease-encoding properties that are independent of its ability to self-propagate, and suggest that the phenotypic heterogeneity may be due to different effects of aggregated PrP on intracellular transport. Our study provides new insights into the mechanisms of selective neuronal dysfunction due to protein aggregation.

How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α₂δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α₂δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1^G93Amice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1^G93Amouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENTWe provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1^G93Amouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

Marinesco-Sjögren syndrome (MSS) is a rare, early onset, autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts and myopathy. Most MSS cases are caused by loss-of-function mutations in the gene encoding SIL1, a nucleotide exchange factor for the molecular chaperone BiP which is essential for correct protein folding in the endoplasmic reticulum. Woozy mice carrying a spontaneous Sil1 mutation recapitulate key pathological features of MSS, including cerebellar atrophy with degeneration of Purkinje cells and progressive myopathy. Because the PERK branch of the unfolded protein response is activated in degenerating neurons of woozy mice, and inhibiting PERK-mediated translational attenuation has shown protective effects in protein-misfolding neurodegenerative disease models, we tested the therapeutic efficacy of GSK2606414, a potent inhibitor of PERK. Mice were chronically treated with GSK2606414 starting from a presymptomatic stage, and the effects were evaluated on biochemical, histopathological and clinical readouts. GSK2606414 delayed Purkinje cell degeneration and the onset of motor deficits, prolonging the asymptomatic phase of the disease; it also reduced the skeletal muscle abnormalities and improved motor performance during the symptomatic phase. The protein but not the mRNA level of ORP150, a nucleotide exchange factor which can substitute for SIL1, was increased in the cerebellum of GSK2606414-treated woozy mice, suggesting that translational recovery promoted the synthesis of this alternative BiP co-factor. Targeting PERK signaling may have beneficial disease-modifying effects in carriers of SIL1 mutations.

α-Synuclein oligomers are crucial players in the pathogenesis of Parkinson's disease. Some mechanisms involved in α-synuclein oligomer detrimental effects include membrane damage, neuroinflammation and protein-protein interactions. Recently, the cellular prion protein (PrPC) emerged as an interactor of α-synuclein oligomers, apparently mediating their detrimental activities. Through direct in vivo and in vitro approaches we herein investigated the existence of a direct cross-talk between α-synuclein oligomers and PrPC. In vitro, we assessed α-synuclein oligomer toxicity by comparing the effect in Prnp+/+ versus PrPC knockout (Prnp0/0) hippocampal neurons. Through an in vivo acute mouse model, where α-synuclein oligomers injected intracerebroventricularly induce memory impairment and neuroinflammation, we verified whether these detrimental effects were preserved in Prnp0/0 mice. In addition, PrPC-α-synuclein oligomer direct binding was investigated through surface plasmon resonance. We found that PrPC was not mandatory to mediate α-synuclein oligomer detrimental effects in vitro or in vivo. Indeed, α-synuclein oligomer toxicity was comparable in Prnp+/+ and Prnp0/0 neurons and both Prnp+/+ and Prnp0/0 mice injected with α-synuclein oligomers displayed memory deficit and hippocampal gliosis. Moreover, surface plasmon resonance analyses ruled out PrPC-α-synuclein oligomer binding. Our findings indicate that PrPC neither binds α-synuclein oligomers nor mediates their detrimental actions. Therefore, it is likely that PrPC-dependent and PrPC-independent pathways co-exist in Parkinson's disease.