The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.

IntroductionWe recently identi®ed the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP) (Rieger et al., 1997; for reviews see Rieger et al., 1999;Gauczynski et al., 2001a). Employing a series of neuronal and non-neuronal cells, we proved that the 37-kDa LRP/67-kDa high-af®nity laminin receptor (LR) acts as the receptor for the cellular PrP (Gauczynski et al., 2001b). In the present manuscript we used the yeast twohybrid system and cell-binding studies on neuronal as well as non-neuronal cells involving the Semliki Forest virus (SFV) system (for reviews see Liljestrom and Garoff, 1991;Tubulekas et al., 1997) to identify domains on the PrP and the LRP involved in the PrP±LRP interaction on the cell surface. We identi®ed two binding domains for LRP on PrP termed PrPLRPbd1 and PrPLRPbd2. The ®rst one binds directly to LRP, whereas the second one depends on the presence of heparan sulfate proteoglycans (HSPGs) on the cell surface. The yeast two-hybrid system and cell-binding assays on wild-type and mutant HSPGde®cient Chinese hamster ovary (CHO) cells also identi®ed two binding domains for PrP on LRP.The relationship between 37-kDa LRP and 67-kDa LR is not yet fully understood and has been explained with homodimerization of 37-kDa LRP (Landowski et al., 1995) or an additional factor, such as a polypeptide (Castronovo et al., 1991), which might bind to 37-kDa LRP to form the 67-kDa form of the receptor. The 67-kDa heterodimer might be stabilized by hydrophobic interactions mediated by fatty acids such as palmitate, oleate and stearate bound to 37-kDa LRP and to a galectin-3 (gal-3) cross reacting polypeptide (Landowski et al., 1995;Buto et al., 1998). However, we recently proved that the b-galactoside lectin gal-3 is not present on the surface of neuronal or non-neuronal cells used for PrP-binding/ internalization studies (Gauczynski et al., 2001b) and antigal-3 antibodies failed to compete for the 37-kDa LRP/67-kDa LR-mediated binding and internalization of the cellular PrP (Gauczynski et al., 2001b), suggesting that gal-3 is not a partner of the 37-kDa LRP in this context. In this study we investigated by a yeast two-hybrid system analysis whether gal-3 interacts with 37-kDa LRP and/or the cellular PrP. In addition, we investigated whether 37-kDa LRP interacts with itself in the yeast two-hybrid and analysed the monomer/dimer status of the receptor by sizeexclusion chromatography. Both PrP (Gabizon et al., 1993;Caughey et al., 1994;Chen,S.G. et al., 1995;Brimacombe et al., 1999) and the 37-kDa/67-kDa LR (Guo et al., 1992;Kazmin et al., 2000) bind to heparan sulfates. HSPGs are required for the binding of the ®broblast growth factor (FGF) to its FGFR receptor (Yayon et al., 1991;Spivak et al., 1994;Venkataraman et al., 1999) and act as initial attachment receptors for bacteria (Chen,T. et al., 1995) and viruses including alphaviruses (Byrnes and Grif®n, 1998), human immunode®ciency virus (HIV) type 1 (Mondor et al., 1998) and vaccinia virus (Chung et al., 1998). Heparan sulfates are...

Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system by prions, where neurons and glial cells are regarded as primary targets. Neuronal loss and gliosis, associated with the accumulation of misfolded prion protein (PrP), are hallmarks of prion diseases; yet the mechanisms underlying such disorders remain unclear. Here we introduced a cell system based on primary cerebellar cultures established from transgenic mice expressing ovine PrP and then exposed to sheep scrapie agent. Upon exposure to low doses of infectious agent, such cultures, unlike cultures originating from PrP null mice, were found to accumulate de novo abnormal PrP and infectivity, as assessed by mouse bioassay. Importantly, using astrocyte and neuron͞astrocyte cocultures, both cell types were found capable of sustaining efficient prion propagation independently, leading to the production of proteinase K-resistant PrP of the same electrophoretic profile as in diseased brain. Moreover, contrasting with data obtained in chronically infected cell lines, late-occurring apoptosis was consistently demonstrated in the infected neuronal cultures. Our results provide evidence that primary cultured neural cells, including postmitotic neurons, are permissive to prion replication, thus establishing an approach to study the mechanisms involved in prion-triggered neurodegeneration at a cellular level.T ransmissible spongiform encephalopathies (TSE), which include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep, are fatal neurodegenerative disorders caused by prions, a class of unconventional agents that targets the CNS in mammals. A hallmark of prion diseases is the accumulation of abnormal prion protein (PrP Sc ), a misfolded form of the cellular PrP (PrP c ). Transmissibility is believed to stem from the ability of the prion isoform to promote the conformational transition from PrP c to PrP Sc . Biologically distinct prion strains can propagate in a same host, presumably through the perpetuation of different specific PrP Sc conformers (1-3).Although it seems clear that neuronal dysfunction must lie at the root of the clinical disorders observed in these diseases, it is still obscure what triggers neurodegeneration and what role nonneuronal cells may play in this process. There is ample evidence to support a primary role of the neurons in prion propagation and neuropathogenesis into the CNS. Intra-or perineuronal PrP Sc deposition, spongiform vacuolation involving cell soma and processes, and neuronal loss are typical histopathological changes observed in TSE-affected brain tissues (4, 5). Transgenic mice with PrP expression specifically targeted to neurons have been obtained that turned out to be fully susceptible to prion disease (6). More recently, it was shown that an acute neuron-targeted depletion of PrP in the brain of mice with ongoing infection is able to prevent neuronal loss and progression to disease and even to reverse early spongiform chang...

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of in vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments in vivo, confirm the power and biological relevance of this microfluidic architecture.Electronic supplementary materialThe online version of this article (doi:10.1007/s12640-010-9152-8) contains supplementary material, which is available to authorized users.

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems).The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency E patt was .75% during lengthy in chip culture, with y85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

SummaryReappraisal of neuropathological studies suggests that pathological hallmarks of Alzheimer’s disease and Parkinson’s disease (PD) spread progressively along predictable neuronal pathways in the human brain through unknown mechanisms. Although there is much evidence supporting the prion-like propagation and amplification of α-synuclein (α-Syn) in vitro and in rodent models, whether this scenario occurs in the human brain remains to be substantiated. Here we reconstructed in microfluidic devices corticocortical neuronal networks using human induced pluripotent stem cells derived from a healthy donor. We provide unique experimental evidence that different strains of human α-Syn disseminate in “wild-type” human neuronal networks in a prion-like manner. We show that two distinct α-Syn strains we named fibrils and ribbons are transported, traffic between neurons, and trigger to different extents, in a dose- and structure-dependent manner, the progressive accumulation of PD-like pathological hallmarks. We further demonstrate that seeded aggregation of endogenous soluble α-Syn affects synaptic integrity and mitochondria morphology.

Radial glia cells both generate neurons and physically guide nascent neurons to their target destination in the cortex, and as such they are essential for CNS development. It has been proposed that in the developing cerebellum, neuronal contact induces radial glia formation, however, the mechanisms involved in this process are not well understood. Here we demonstrate that neuronal induction of radial glia formation is the result of sequential signaling through Notch1 and erbB receptors. First, Notch1 activation by neuronal contact induces the glial expression of the brain lipid binding protein (BLBP) and erbB2 genes. Interestingly, two different signaling pathways mediate these effects of Notch1 on transcription, BLBP expression being dependent on Su(H), whereas erbB2 is regulated by a yet unidentified Notch1 pathway. The subsequent increase in erbB2 receptor expression makes the glia more responsive to neuronal NRG, which then induces the morphological transformation into radial glia. Thus, these results unveil some of the mechanisms underlying radial glia formation.