“Prions may hold key to stem cell function,” Hutson, New Scientist (2006)


The curative properties of stem cells may rely on prions, a new study suggests, the type of protein made infamous by mad cow disease.

Andrew Steele, Cheng Cheng Zhang and colleagues used radiation to deplete the bone marrow of mice genetically engineered to not produce the prion proteins. The animals’ marrow regenerated quickly at first, but eventually slowed to a stop. The marrow also lost its regenerative abilities when transplanted into normal mice.

“For years we’ve wondered why evolution has preserved this protein, what positive role it could possibly be playing,” says Susan Lindquist, one of the team. “With these findings we have our first answer.”

The question of how prions sustain stem cell activity remains unanswered, but the finding is a first step to understanding the destructive streak of misshapen prion proteins, Steele says. Similar tests on neural and lung stem cells are underway.



“Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal,” Zhang et al. inc. Lindquist, PNAS (2006)


Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells, its physiological function(s) remain unknown, and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations, followed by competitive bone marrow reconstitution assays, shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent, the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore, PrP is a marker for HSCs and supports their self-renewal.


“Cellular prion protein promotes regeneration of adult muscle tissue,” Stella et al., Mol. Cell. Biol. (2010)


It is now well established that the conversion of the cellular prion protein, PrPC, into its anomalous conformer, PrPSc, is central to the onset of prion disease. However, both the mechanism of prion-related neurodegeneration and the physiologic role of PrPC are still unknown. The use of animal and cell models has suggested a number of putative functions for the protein, including cell signaling, adhesion, proliferation, and differentiation. Given that skeletal muscles express significant amounts of PrPC and have been related to PrPC pathophysiology, in the present study, we used skeletal muscles to analyze whether the protein plays a role in adult morphogenesis. We employed an in vivo paradigm that allowed us to compare the regeneration of acutely damaged hind-limb tibialis anterior muscles of mice expressing, or not expressing, PrPC. Using morphometric and biochemical parameters, we provide compelling evidence that the absence of PrPC significantly slows the regeneration process compared to wild-type muscles by attenuating the stress-activated p38 pathway, and the consequent exit from the cell cycle, of myogenic precursor cells. Demonstrating the specificity of this finding, restoring PrPC expression completely rescued the muscle phenotype evidenced in the absence of PrPC.


“Prion protein PrP(c) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis,” Steele et al., inc. Lindquist, PNAS (2005)


The misfolding of the prion protein (PrPc) is a central event in prion diseases, yet the normal function of PrPc remains unknown. PrPc has putative roles in many cellular processes including signaling, survival, adhesion, and differentiation. Given the abundance of PrPc in the developing and mature mammalian CNS, we investigated the role of PrPc in neural development and in adult neurogenesis, which occurs constitutively in the dentate gyrus (DG) of the hippocampus and in the olfactory bulb from precursors in the subventricular zone (SVZ)/rostral migratory stream. In vivo, we find that PrPc is expressed immediately adjacent to the proliferative region of the SVZ but not in mitotic cells. In vivo and in vitro studies further find that PrPc is expressed in multipotent neural precursors and mature neurons but is not detectable in glia. Loss- and gain-of-function experiments demonstrate that PrPc levels correlate with differentiation of multipotent neural precursors into mature neurons in vitro and that PrPc levels positively influence neuronal differentiation in a dose-dependent manner. PrPc also increases cellular proliferation in vivo; in the SVZ, PrPc overexpresser (OE) mice have more proliferating cells compared with wild-type (WT) or knockout (KO) mice; in the DG, PrPc OE and WT mice have more proliferating cells compared with KO mice. Our results demonstrate that PrPc plays an important role in neurogenesis and differentiation. Because the final number of neurons produced in the DG is unchanged by PrPc expression, other factors must control the ultimate fate of new neurons.



“All quiet on the neuronal front: NMDA receptor inhibition by prion protein,” Steele, Journal of Cell Biology (2008)


How do these new findings relate to prion diseases? Clearly, the detection of exaggerated NMDAR activity in prion-diseased samples would be a smoking gun implicating a loss of PrP function in prion disease. Do the familial mutants of PrP fail to effectively silence NMDARs, leading to hyperexcitability and a mechanism of neuronal damage similar to excitotoxicity? The cell death pathways involved in prion disease are far from understood (Steele et al., 2007b), and this new angle of investigation deserves attention, as perhaps NMDAR inhibition will have potential as a prion disease therapeutic strategy. Based on an interaction of PrP with NMDARs, one might speculate that the psychiatric symptoms of prion diseases could relate to defects in glutamatergic neurotransmission brought about either by PrP being titrated away from NR2D subunits or from direct interference by PrP oligomers or aggregates with NMDARs.

How does PrP silence NMDAR? As noted by Khosravani et al. (2008), PrP could block agonist binding, stabilize the closed state of the channel, or indirectly regulate function by interfering with signaling pathways affecting NR2D-containing NMDARs. With respect to NMDAR assembly, very little is known about NR2D subunits other than that they likely require NR1 subunits to reach the cell surface. In wild-type conditions, with ample PrP present on the neuronal cell surface, these channels will not open. What is the molecular logic of building a tonically inhibited NMDAR? Perhaps these channels only respond to extreme stimuli where they need not only a magnesium unblocking event but also a PrP-releasing event to open. The identification and characterization of additional interacting partners of PrP or NMDARs will be a complex and stimulating area of research. These questions aside, it is exciting to see the pieces of the PrP function puzzle start to come together.


“Altered neuron excitability and synaptic plasticity in the cerebellar granular layer of juvenile prion protein knock-out mice with impaired motor control,” Prestori et al., Journal of Neuroscience (2008)


Although the role of abnormal prion protein (PrP) conformation in generating infectious brain diseases (transmissible spongiform encephalopathy) has been recognized, the function of PrP in the normal brain remains mostly unknown. In this investigation, we considered the effect of PrP gene knock-out (PrP0/0) on cerebellar neural circuits and in particular on granule cells, which show intense PrP expression during development and selective affinity for PrP. At the third postnatal week, when PrP expression would normally attain mature levels, PrP0/0 mice showed low performance in the accelerating rotarod and runway tests and the functioning of 40% of granule cells was abnormal. Spikes were slow, nonovershooting, and nonrepetitive in relation with a reduction in transient inward and outward membrane currents, and also the EPSPs and EPSCs had slow kinetics. Overall, these alterations closely resembled an immature phenotype. Moreover, in slow-spiking PrP0/0 granule cells, theta-burst stimulation was unable to induce any long-term potentiation. This profound impairment in synaptic excitation and plasticity was associated with a protracted proliferation of granule cells and disappeared at P40–P50 along with the recovery of normal motor behavior (Büeler et al., 1992). These results suggest that PrP plays an important role in granule cell development eventually regulating cerebellar network formation and motor control.






The function of PrPc

Studies on transgenic mice to deduce the function of PrPc

From all of the evidence put forward above, it seems certain that the production of the aberrant form of the PRNP gene product is responsible in some way for the pathogenesis of the disease. We do know that the PrPc protein is expressed as a glycosylphosphatidyl inositol-anchored glycoprotein found on the outer cell membrane of neurons and to a lesser extent of lymphocytes and other cells, (Stahl et al., 1987, 1990, 1992). However, the role of the normal cellular form of the prion is unclear and unproven.

In 1992, studies on mice with inactive Prn-p genes (homozygously disrupted) by Büeler et al. showed that the mice developed normally. The genes were disrupted by homologous recombination with a 4.8 kilobase DNA fragments in which codons 4 to 187 of the 254-codon open reading frame were replaced by a neomycin phosphotransferase (neo) gene under the control of the herpes simplex virus thymidine kinase promoter. They then obtained mice heterozygous (Prn-p0/+) for the disrupted gene, the presence of which was confirmed by Southern analysis. Heterozygous mice were then mated to other heterozygotes to give a new population. Surprisingly, this population contained 24% Prn-p(0/0)26% Prn-p(+/+) and 50% Prn-p(0/+) mice, surprising because they were all phenotypically the same, and Southern analysis confirmed these results. The Prn-p(0/0) mice were tested for many abnormalities in their behaviour, fitness and fertility and were all confirmed normal for at least 7 months, with no immunological defects. The reason that these results are surprising is that the Prn-p gene is highly conserved through evolution and so it might be thought that the function of this gene is vital to the organism.

In contrast to Büeler et al. (1992), more recent studies have reported that the prion protein is necessary for normal synaptic function (Collinge et al., 1994) and that altered circadian activity rhythms and sleep has been observed in mice also devoid of the prion protein, (Tobler et al., 1996). One might ascertain from PrP knockout mice, that although there appears to be no symptoms, by looking closely at the behaviour of the mice they might be seen to differ phenotypically from the PrP-normal mice. It seems that Büeler et al. in their studies in 1992 may have missed the subtle phenotypic differences observed by Tobler et al. (1996) and Collinge et al. (1994) in later more focused studies.

Collinge et al. (1994) showed that hippocampal slices from PrP null mice have weakened GABAA (g -aminobutyric acid type A) receptor mediated fast inhibition and long term potentiation. Interestingly, this gives a clue as to why Büeler et al (1994) may have missed any differences, as in their studies the PrP-null mice that were used were reported to perform the Morris swim task as well as the controls. This can be seen as surprising, as this behavioural task depends on hippocampal integrity, this was also reported with Collinge et al (1992) but when the mice were extensively studied, the phenotypic differences were exposed. The experiments by Collinge et al. (1994) involved taking slices from the hippocampus in mice expressing normal PrPc and comparing the electrophysiological responses to those of PrP-null mice (PrP0/0). The results were recorded and the extracellular and intracellular responses are shown in Figure 9.

Part (a) shows the extracellular responses evoked by afferent stimulation. The null mice reveal extra population spikes. In diagram part (b) a pair of action potentials (only one observed in the normal) and a lack of a hyperpolarizing fast i.p.s.p. (conductance) which was shown in the normal, can be observed. Despite this, there was no difference in the resting potential. The conductance abnormalities in the PrP-null mice were consistent with GABA having to diffuse further to a significant proportion of receptors with extrasynaptic properties, which could arise if the receptors were not properly localised, or if the synaptic cleft architecture had changed. The abnormalities of synaptic inhibition observed in these studies are relevant to the epileptiform discharges seen in CJD and scrapie-infected mice. So it might be seen that the loss of function of PrPc could bare some relation to neurodegeneration. A later study by Tobler et al. (1996) showed that the null mice had an alteration in both circadian activity rhythms and sleep patterns. Their results were confirmed by producing null mutants in two different ways to achieve the same results; effectively they show that a loss of PrP affects the circadian activity rhythm and sleep.

They suggest that because of the intriguing similarities to rhythm and sleep alterations in fatal familial insomnia, where there is a profound alteration in sleep and the daily rhythms of many hormones, the loss of function PrP may be related to the normal function of the prion protein. Essentially, it can be seen that the probable function of PrPc, being a well-conserved protein present in many mammalian species, may be involved in maintaining sleep continuity or regulating sleep intensity. Another study, this time performed by Sakaguchi et al. (1996), looked at the long-term effects of the disrupted gene on mice. At about 70 weeks of age, their PrP-null mice all began to show progressive symptoms of ataxia. A rotorod test evidently showed up a lack of co-ordination in these mice and upon pathological investigation, there was an extensive loss of Purkinje cells in the vast majority of cerebellar folia. This suggests that PrP plays a role in the long-term survival of Purkinje neurones.

Despite the promising work incorporating the PrP-null idea, it must be mentioned that not all of the PrP gene knockout techniques are the same. They all ultimately result in the loss of gene function, but some are due to small deletions and some are due to large deletions. There is also a chance that during embryogenesis that other proteins or mechanisms may work to compensate the effects of the loss of the PrP-null, so this shows that the mouse PrP-null model is not a perfect model for prion diseases. It is clear from the experiments described, that more studies aimed at the function of PrPand the behavioural effects of PrP-null mice need to be performed. There have been many questions and possibilities raised, including the possibility of glycosylation differences in PrPc/PrPsc affinity, and there seems to be no one-quick-way answer, especially with the differences in the gene knock-out techniques and their corresponding implications and effects. It might then be a good idea to have a kind of standardised way of knocking-out these genes after a full investigation on how the different techniques may affect the mice in different ways.



“Doxycycline control of prion protein transgene expression modulates prion disease in mice,” Tremblay et. al., inc. Prusiner, (1998)

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at ≈50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

On this background, we undertook development of a system where the level of PrP expression could be regulated to modulate the rate of prion formation. We chose the tetracycline-responsive gene system that was developed by using the Escherichia coli tetracycline resistance Tn10 operon (19). It makes use of a transactivator (tTA) obtained by fusing the tetracycline repressor with the transactivation domain of the herpes simplex virus VP16 transcription factor. The tTA binds specifically with high affinity to the tetracycline operator (tetO) and activates transcription from a minimal promoter linked to the target gene. Binding of doxycycline, a tetracycline analog, to tTA prevents the protein from binding to the tetO region, thereby preventing target gene expression. 

The brains from untreated Tg(tTA:PrP)3 mice exhibited extensive neuronal loss in the hippocampal pyramidal cell layer (Fig. 3G) and dentate gyrus and focal loss of Purkinje cells and granular cells in the cerebellum (Fig. 3C). These changes were accompanied by moderate to severe astrocytic gliosis in all regions examined, including the neocortex, hippocampus, entorhinal cortex, thalamus, caudate nucleus, and substantia nigra, as well as cerebellar granular and molecular layers. As noted above, the doxycycline-treated Tg(tTA:PrP)3 mice did not show signs of CNS dysfunction. When these mice were sacrificed ≈200 days after inoculation with prions, their brains showed no signs of neurodegeneration.

Doxycycline administered to adult Tg(tTA:PrP) mice acutely repressed the expression of PrPC but did not produce any recognizable adverse effects in the mice over a 30-day period. Neither the viability nor the neurological status of the mice was compromised, and histological examination of the brains did not reveal any abnormalities. These results indicate that high levels of PrPC are not essential for short-term neuronal survival, as its expression can be repressed over 20-fold without adverse effects. It is noteworthy that adult Tg(tTA:PrP)3 mice were placed on oral doxycycline to repress their PrPC expression and have remained well for >380 days with continual administration of doxycycline (Table 5).

With the production of Tg(tTA:PrP) mice, it is possible to examine the effects of low or intermediate levels of PrPSc in the CNS. We found that low levels of PrPSc did not produce any deleterious clinical or histological effects up to 380 days after inoculation of RML prions in Tg(tTA:PrP)3 mice (Fig. 3C). Studies of Prnp+/0 mice with one functional PrP allele show greatly prolonged incubation times (15) but at a higher accumulation of PrPSc than was anticipated (17). Studies with Tg(tTA:PrP) mice where the levels of PrPC expression are held at different levels throughout the incubation time should help to clarify this issue.

The findings reported here clearly show that repression of PrPC expression in young adult Tg(tTA:PrP) mice is not deleterious, whereas accumulation of PrPSc in the same line of animals is lethal (Table 5). Even though Purkinje cell degeneration in 70-week-old Prnp0/0 mice has been found (9), our data continue to argue that the accumulation of PrPSc and not a loss of PrPC function is responsible for the pathogenesis of prion disease.

Reversing the course of prion diseases by blocking the production of PrPSc through repression of PrPC expression will allow us to measure the removal of PrPSc. Such clearance studies, which were not previously possible, are a prelude to the development of effective therapies where drugs that block PrPSc formation are administered at the earliest onset of symptoms to patients with sporadic Creutzfeldt–Jakob disease. At present, we have no understanding of how much PrPSc can be tolerated by the CNS and how rapidly it will disappear once synthesis of its precursor, PrPC, is repressed.


“In a first, infected mice recover from a prion disease,” Couzin, Science, 2003

Within days after the PrP gene in their neurons shut down, the mice depleted their supply of the normal PrP protein. Remarkably, more than a year later, these nine mice “live a normal life,” says Collinge. Control mice succumbed to prion disease.

Two observations about the recovering animals struck the researchers. The spongiosis long viewed as irreversible disappeared. And non-neuronal brain cells, called glia, which still produced PrP, contained wads of prions.

Apparently, PrP can contort into prions “in the cell right next door, and it’s not hurting the neurons,” says Susan Lindquist, a prion researcher and director of the Whitehead Institute at the Massachusetts Institute of Technology in Cambridge.

The work adds to the growing body of evidence, say Lindquist and others, that the prion form of PrP—at least, when it’s in non-neuronal brain cells—might not be the poison it’s viewed as. Lindquist says the work establishes that attacks on  neurons are key to mouse spongiform disease. But because prions don’t appear toxic elsewhere in the brains of the engineered mice, what harms neurons may be something else entirely. Aguzzi thinks this could be a still-unidentified form of PrP that may fleetingly appear as normal PrP morphs into the scrapie form.


“Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis,” Mallucci et. al., Science (2003)

We found that depleting endogenous neuronal PrPc in mice with established neuroinvasive prion infection reversed early spongiform change and prevented neuronal loss and progression to clinical disease. This occurred despite the accumulation of extraneuronal PrPSc to levels seen in terminally ill wild-type animals. Thus, the propagation of nonneuronal PrPSc is not pathogenic, but arresting the continued conversion of PrPc to PrPSc within neurons during scrapie infection prevents prion neurotoxicity.


“Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration,” Mallucci et. al. out of MRC, EMBO Journal (2002)

Prion protein (PrP) plays a crucial role in prion disease, but its physiological function remains unclear. Mice with gene deletions restricted to the coding region of PrP have only minor phenotypic deficits, but are resistant to prion disease. We generated double transgenic mice using the Cre–loxP system to examine the effects of PrP depletion on neuronal survival and function in adult brain. Cre-mediated ablation of PrP in neurons occurred after 9 weeks. We found that the mice remained healthy without evidence of neurodegeneration or other histopathological changes for up to 15 months post-knockout. However, on neurophysiological evaluation, they showed significant reduction of afterhyperpolarization potentials (AHPs) in hippocampal CA1 cells, suggesting a direct role for PrP in the modulation of neuronal excitability. These data provide new insights into PrP function. Furthermore, they show that acute depletion of PrP does not affect neuronal survival in this model, ruling out loss of PrP function as a pathogenic mechanism in prion disease and validating therapeutic approaches targeting PrP.