The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations includingPRNP^E200K, trisomy 21 (T21), andLRRK2^G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson’s disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3–4 and 6–10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6–10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with theHC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the amplitudes of gamma oscillations. Such changes were linked with the detection of hypersynchronous events like spike-and-wave discharges. These dysfunctions were associated with altered production and release of neurotransmitters, compromised activity of excitatory ionotropic receptors including receptors of kainate, AMPA, and NMDA, and changed levels and function of excitatory glutamatergic synapses and inhibitory GABAergic synapses. Neuronal properties that modulate GABAergic inhibition including the activity of Na–K-Cl cotransport 1 (NKCC1) in Cl⁻homeostasis and the levels of synaptic and extra-synaptic localization of GABA receptors (GABARs) were altered in theT21COs only. The neurosteroid allopregnanolone, a positive modulator of GABARs, was downregulated in all the dCOs. Treatment with this neurosteroid significantly improved the neuronal communication in the dCOs, possibly through improving the GABAergic inhibition. Overall, without the manifestation of any disease-related pathology, the genetic mutationsPRNP^E200K,T21, andLRRK2^G2019Ssignificantly altered the neuronal network communication in dCOs by disrupting the excitatory-to-inhibitory balance.

Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human cerebral cortex. COs are a promising new system for modelling pathological features of neurological disorders, including prion diseases. COs expressing normal prion protein (PrPC) are susceptible to prion infection when exposed to the disease isoforms of PrP (PrPD). This causes the COs to develop aspects of prion disease pathology considered hallmarks of disease, including the production of detergent-insoluble, protease-resistant misfolded PrPD species capable of seeding the production of more misfolded species. To determine whether COs can model aspects of familial prion diseases, we produced COs from donor fibroblasts carrying the E200K mutation, the most common cause of human familial prion disease. The mature E200K COs were assessed for the hallmarks of prion disease. We found that up to 12 months post-differentiation, E200K COs harbored no PrPD as confirmed by the absence of detergent-insoluble, protease-resistant, and seeding-active PrP species. Our results suggest that the presence of the E200K mutation within the prion gene is insufficient to cause disease in neuronal tissue. Therefore, other factors, such as further genetic modifiers or aging processes, may influence the onset of misfolding.

Anthracyclines are among the most effective chemotherapeutics ever developed, but they produce grueling side effects and serious adverse events, and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular prion protein (PrP ^C ), which blocks their cytotoxic activity. This effect was dose dependent using either cell line–conditioned medium or human serum as a source of PrP ^C . Genetic depletion of PrP ^C or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pretreatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly among doxorubicin-treated patients with residual disease at surgery. These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrP ^C protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by IHC, particularly soluble isoforms released into the extracellular environment by shedding. These findings have important potential clinical implications for frontline regimen decision making. We suggest there is warranted utility for prognostic PrP ^C / PRNP assays to guide chemosensitization strategies that exploit an understanding of PrP ^C -anthracycline-copper ion complexes.

Fatal familial insomnia (FFI) is a rare neurodegenerative disease caused by a dominantly inherited single amino acid substitution (D178N) within the prion protein (PrP). No in vitro human brain tissue model for this disease has previously been available. Consequently, how this mutation exerts its damaging effect on brain cells is still unknown. Using CRISPR-Cas9 engineered induced pluripotent stem cells, we made D178N cerebral organoids and compared these with isotype control organoids. We found that, in the absence of other hallmarks of FFI, the D178N organoids exhibited astrogliosis with cellular oxidative stress. Abnormal post-translational processing of PrP was evident but no tissue deposition or propagation of mis-folded PrP isoforms were observed. Neuronal electrophysiological function was compromised and levels of neurotransmitters, particularly acetylcholine and GABA, altered. Underlying these dysfunctions were changes in cellular energy homeostasis, with substantially increased glycolytic and Krebs cycle intermediates, and greater mitochondrial activity. This increased energy demand in D178N organoids was associated with increased mitophagy and depletion of lipid droplets, in turn resulting in shifts of cellular lipid composition. Using a double mutation (178NN) we could confirm that most changes were caused by the presence of the mutation rather than interaction with PrP molecules lacking the mutation. Our data strongly suggests that shifting biosynthetic intermediates and oxidative stress, caused by an imbalance of energy supply and demand, results in astrogliosis with compromised neuronal activity in FFI organoids. They further support that many of the disease associated changes are due to a corruption of PrP function and do not require propagation of PrP mis-folding.

Mis-folding of the prion protein (PrP) is known to cause neurodegenerative disease, however the native function of this protein remains poorly defined. PrP has been linked with many cellular functions, including cellular proliferation and senescence. It is also known to influence epidermal growth factor receptor (EGFR) signalling, a pathway that is itself linked with both cell growth and senescence. Adult neural stem cells (NSCs) persist at low levels in the brain throughout life and retain the ability to proliferate and differentiate into new neural lineage cells. Knock-out (KO) of PrP has previously been shown to reduce NSC proliferative capacity. We used PrP KO and wild type (WT) NSCs from adult mouse brain to examine the influence of PrP on cellular senescence, EGFR signalling, and the downstream cellular processes. PrP KO NSCs showed decreased cell proliferation and increased senescence in in vitro cultures. Expression of EGFR was decreased in PrP KO NSCs compared with WT NSCs and additional supplementation of EGF was sufficient to reduce senescence. RNAseq analysis confirmed that significant changes were occurring at the mRNA level within the EGFR signalling pathway and these were associated with reduced expression of mitochondrial components and correspondingly reduced mitochondrial function. Metabolomic analysis of cellular energy pathways showed that blockages were occurring at critical sites for production of energy and biomass, including catabolism of pyruvate. We conclude that, in the absence of PrP, NSC growth pathways are downregulated as a consequence of insufficient energy and growth intermediates.

Although considerable evidence supports that misfolded prion protein (PrP^Sc) is the principal component of “prions”, underpinning both transmissibility and neurotoxicity, clear consensus around a number of fundamental aspects of pathogenesis has not been achieved, including the time of appearance of neurotoxic species during disease evolution. Utilizing a recently reported electrophysiology paradigm, we assessed the acute synaptotoxicity of ex vivo PrP^Sc prepared as crude homogenates from brains of M1000 infected wild-type mice (cM1000) harvested at time-points representing 30%, 50%, 70% and 100% of the terminal stage of disease (TSD). Acute synaptotoxicity was assessed by measuring the capacity of cM1000 to impair hippocampal CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in explant slices. Of particular note, cM1000 from 30% of the TSD was able to cause significant impairment of LTP and PTP, with the induced failure of LTP increasing over subsequent time-points while the capacity of cM1000 to induce PTP failure appeared maximal even at this early stage of disease progression. Evidence that the synaptotoxicity directly related to PrP species was demonstrated by the significant rescue of LTP dysfunction at each time-point through immuno-depletion of >50% of total PrP species from cM1000 preparations. Moreover, similar to our previous observations at the terminal stage of M1000 prion disease, size fractionation chromatography revealed that capacity for acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, with the profile appearing maximised by 50% of the TSD. Using enhanced sensitivity western blotting, modestly proteinase K (PK)-resistant PrP^Sc was detectable at very low levels in cM1000 at 30% of the TSD, becoming robustly detectable by 70% of the TSD at which time substantial levels of highly PK-resistant PrP^Sc was also evident. Further illustrating the biochemical evolution of acutely synaptotoxic species the synaptotoxicity of cM1000 from 30%, 50% and 70% of the TSD, but not at 100% TSD, was abolished by digestion of immuno-captured PrP species with mild PK treatment (5μg/ml for an hour at 37°C), demonstrating that the predominant synaptotoxic PrP^Sc species up to and including 70% of the TSD were proteinase-sensitive. Overall, these findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrP^Sc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, albeit with eventual plateauing of transmitting conformers. Although evidence clearly supports that misfolded prion protein (PrP^Sc) is the principal component of “prions”, underpinning both transmissibility and neurotoxicity, consensus is lacking around the time of appearance and biochemical profile of neurotoxic species during disease evolution. Employing an electrophysiology model, measuring the capacity of brain homogenates derived from across the disease time-course to impair CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in hippocampal slices, we observed that synaptotoxic species were present from 30% of the terminal stage of disease (TSD). Evidence that synaptotoxicity directly related to PrP species was demonstrated by significant rescue of LTP dysfunction at each time-point through immuno-depleting >~50% of total PrP species from cM1000 preparations. Moreover, size fractionation chromatography revealed that acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, while additional characterisation of cM1000 demonstrated that the predominant synaptotoxic PrP^Sc species up to and including 70% of the TSD were quite proteinase-sensitive. These findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrP^Sc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, with biochemical transformation of synaptotoxic conformers continuing until late in disease.