α-Synuclein Oligomers Induce Glutamate Release from Astrocytes and Excessive Extrasynaptic NMDAR Activity in Neurons, Thus Contributing to Synapse Loss.

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca2+-dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamate in vivo In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential.SIGNIFICANCE STATEMENT Loss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson's disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD.

Astrocyte Infection Is Required for Retrovirus-Induced Spongiform Neurodegeneration Despite Suppressed Viral Protein Expression.

The ability of retroviruses (RVs) to cause neurodegeneration is critically dependent upon two activities of the envelope protein (Env). First, Env facilitates viral genome delivery to CNS target cells through receptor binding and membrane fusion. Second, Env expression within one or more targets indirectly alters the physiology of certain neurons. Although the major Env expressing CNS cell types have been identified for many neurovirulent RVs, it remains unresolved, which targets play a causal role in neuropathogenesis. Moreover, this issue is complicated by the potential for post-infection virus suppression. To address these questions we explored herein, whether and how cryptic neurotropism differences between ecotropic and amphotropic murine leukemia viruses (MLVs) impacted neurovirulence. Neurotropism was first explored ex vivo using (1) acute primary glial cell cultures and (2) neural progenitor cell (NPC)- neural stem cell (NSC) neural sphere (NPH) chimeras. These experiments indicated that primary astrocytes and NPCs acutely restrict amphotropic but not ecotropic virus entry. CNS tropism was investigated using NSC transplant-based Cre-vector pseudotyping wherein mTmG transgenic fluorescent protein reporter mice revealed both productive and suppressed infection. Cre-pseudotyping with FrCasE, a prototypic neurovirulent ecotropic virus, identified glia and endothelia, but not neurons, as targets. Almost two-thirds (62%) of mGFP+ cells failed to show Env expression, suggesting widespread virus suppression. To circumvent RV superinfection interference confounds, targets were also identified using ecotropic packaging NSCs. These experiments identified known ecotropic targets: microglia, oligodendrocyte progenitor cells (OPCs) and endothelia. Additionally, one third of mGFP+ cells were identified as protoplasmic astrocytes, cells that rarely express virus in vivo. A CNS targeting comparison between isogenic ecotropic (FrCasE) and amphotropic (FrAmE) viruses showed a fourfold higher astrocyte targeting by FrCasE. Since ecotropic Env pseudotyping of amphotropic virus in the CNS dramatically exacerbates neurodegeneration, these results strongly suggest that astrocyte infection is a major disease requirement. Moreover, since viral Env protein expression is largely subdetectable in astrocytes, minimal viral protein expression appears sufficient for affecting neuronal physiology. More broadly, these findings raise the specter that subdetectable astrocyte expression of exogenous or endogenous RVs could play a major role in human and animal neurodegenerative diseases.

Rebound from Inhibition: Self-Correction against Neurodegeneration?

Neural networks play a critical role in establishing constraints on excitability in the central nervous system. Several recent studies have suggested that network dysfunction in the brain and spinal cord are compromised following insult by a neurodegenerative trigger and might precede eventual neuronal loss and neurological impairment. Early intervention of network excitability and plasticity might therefore be critical in resetting hyperexcitability and preventing later neuronal damage. Here, the behavior of neurons that generate burst firing upon recovery from inhibitory input or intrinsic membrane hyperpolarization (rebound neurons) is examined in the context of neural networks that underlie rhythmic activity observed in areas of the brain and spinal cord that are vulnerable to neurodegeneration. In a non-inflammatory rodent model of spongiform neurodegenerative disease triggered by retrovirus infection of glia, rebound neurons are particularly vulnerable to neurodegeneration, likely due to an inherently low calcium buffering capacity. The dysfunction of rebound neurons translates into a dysfunction of rhythmic neural circuits, compromising normal neurological function and leading to eventual morbidity. Understanding how virus infection of glia can mediate dysfunction of rebound neurons, induce hyperexcitability and loss of rhythmic function, pathologic features observed in neurodegenerative disorders ranging from epilepsy to motor neuron disease, might therefore suggest a common pathway for early therapeutic intervention.

Ecotropic Murine Leukemia Virus Infection of Glial Progenitors Interferes with Oligodendrocyte Differentiation: Implications for Neurovirulence.

UNLABELLED: Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved, infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs), we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here, we demonstrate that OPCs, but not OLs, are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival, proliferation, or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that, while MLVs did not affect cellular engraftment or survival, they did inhibit OL differentiation, irrespective of MLV neurovirulence. In addition, in chimeric brains, where FrCasE-infected NPC transplants caused neurodegeneration, the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration, restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. IMPORTANCE: A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifested as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types; however, the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia, whose CNS functions are only now emerging, are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cells (OPCs), we investigated here whether their infection by the neurovirulent MLV FrCasE contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and nonneurovirulent MLVs interfere with oligodendrocyte differentiation. Thus, NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus effects on other glial subtypes.

Postinhibitory rebound neurons and networks are disrupted in retrovirus-induced spongiform neurodegeneration.

Certain retroviruses induce progressive spongiform motor neuron disease with features resembling prion diseases and amyotrophic lateral sclerosis. With the neurovirulent murine leukemia virus (MLV) FrCasE, Env protein expression within glia leads to postsynaptic vacuolation, cellular effacement, and neuronal loss in the absence of neuroinflammation. To understand the physiological changes associated with MLV-induced spongiosis, and its neuronal specificity, we employed patch-clamp recordings and voltage-sensitive dye imaging in brain slices of the mouse inferior colliculus (IC), a midbrain nucleus that undergoes extensive spongiosis. IC neurons characterized by postinhibitory rebound firing (PIR) were selectively affected in FrCasE-infected mice. Coincident with Env expression in microglia and in glia characterized by NG2 proteoglycan expression (NG2 cells), rebound neurons (RNs) lost PIR, became hyperexcitable, and were reduced in number. PIR loss and hyperexcitability were reversed by raising internal calcium buffer concentrations in RNs. PIR-initiated rhythmic circuits were disrupted, and spontaneous synchronized bursting and prolonged depolarizations were widespread. Other IC neuron cell types and circuits within the same degenerative environment were unaffected. Antagonists of NMDA and/or AMPA receptors reduced burst firing in the IC but did not affect prolonged depolarizations. Antagonists of L-type calcium channels abolished both bursts and slow depolarizations. IC infection by the nonneurovirulent isogenic virus Friend 57E (Fr57E), whose Env protein is structurally similar to FrCasE, showed no RN hyperactivity or cell loss; however, PIR latency increased. These findings suggest that spongiform neurodegeneration arises from the unique excitability of RNs, their local regulation by glia, and the disruption of this relationship by glial expression of abnormal protein.

Unique N-linked glycosylation of CasBrE Env influences its stability, processing, and viral infectivity but not its neurotoxicity.

The envelope protein (Env) from the CasBrE murine leukemia virus (MLV) can cause acute spongiform neurodegeneration analogous to that induced by prions. Upon central nervous system (CNS) infection, Env is expressed as multiple isoforms owing to differential asparagine (N)-linked glycosylation. Because N-glycosylation can affect protein folding, stability, and quality control, we explored whether unique CasBrE Env glycosylation features could influence neurovirulence. CasBrE Env possesses 6/8 consensus MLV glycosylation sites (gs) but is missing gs3 and gs5 and contains a putative site (gs*). Twenty-nine mutants were generated by modifying these three sites, individually or in combination, to mimic the amino acid sequence in the nonneurovirulent Friend 57 MLV. Three basic viral phenotypes were observed: replication defective (dead; titer < 1 focus-forming unit [FFU]/ml), replication compromised (RC) (titer = 10(2) to 10(5) FFU/ml); and wild-type-like (WTL) (titer > 10(5) FFU/ml). Env protein was undetectable in dead mutants, while RC and WTL mutants showed variations in Env expression, processing, virus incorporation, virus entry, and virus spread. The newly introduced gs3 and gs5 sites were glycosylated, whereas gs* was not. Six WTL mutants tested in mice showed no clear attenuation in disease onset or severity versus controls. Furthermore, three RC viruses tested by neural stem cell (NSC)-mediated brainstem dissemination also induced acute spongiosis. Thus, while unique N-glycosylation affected structural features of Env involved in protein stability, proteolytic processing, and virus assembly and entry, these changes had minimal impact on CasBrE Env neurotoxicity. These findings suggest that the Env protein domains responsible for spongiogenesis represent highly stable elements upon which the more variable viral functional domains have evolved.

Retrovirus-induced spongiform neurodegeneration is mediated by unique central nervous system viral targeting and expression of env alone.

Certain murine leukemia viruses (MLVs) can induce progressive noninflammatory spongiform neurodegeneration similar to that caused by prions. The primary MLV determinants responsible have been mapped to within the env gene; however, it has remained unclear how env mediates disease, whether non-Env viral components are required, and what central nervous system (CNS) cells constitute the critical CNS targets. To address these questions, we examined the effect of transplanting engraftable C17.2 neural stem cells engineered to pseudotype, disseminate, and trans-complement neurovirulent (CasBrE, CasE, and CasES) or non-neurovirulent (Friend and SFF-FE) env sequences (SU or SU/TM) within the CNS using either the "non-neurovirulent" amphotropic helper virus, 4070A, or pgag-polgpt (a nonpackaged vector encoding Gag-Pol). These studies revealed that acute MLV-induced spongiosis results from two separable activities of Env. First, Env causes neuropathology through unique viral targeting within the CNS, which was efficiently mediated by ecotropic Envs (CasBrE and Friend), but not 4070A amphotropic Env. Second, Env induces spongiosis through a toxin activity that is MLV-receptor independent and does not require the coexpression of other viral structural proteins. CasBrE and 4070A Envs possess the toxin activity, whereas Friend Env does not. Although the identity of the critical viral target cell(s) remains unresolved, our results appear to exclude microglia and oligodendrocyte lineage cells, while implicating viral entry into susceptible neurons. Thus, MLV-induced disease parallels prionopathies in that a single protein, Env, mediates both the CNS targeting and the toxicity of the infectious agent that manifests itself as progressive vacuolar neurodegeneration.

Misfolding of CasBrE SU is reversed by interactions with 4070A Env: implications for gammaretroviral neuropathogenesis.

BACKGROUND: CasBrE is a neurovirulent murine leukemia virus (MLV) capable of inducing paralytic disease with associated spongiform neurodegeneration. The neurovirulence of this virus has been genetically mapped to the surface expressed subunit (SU) of the env gene. However, CasBrE SU synthesized in the absence of the transmembrane subunit (TM) does not retain ecotropic receptor binding activity, indicating that folding of the receptor binding domain (RBD) requires this domain. Using a neural stem cell (NSC) based viral trans complementation approach to examine whether misfolded CasBrE SU retained neurovirulence, we observed CasBrE SU interaction with the "non-neurovirulent" amphotropic helper virus, 4070A which restored functional activity of CasBrE SU. RESULTS: Herein, we show that infection of NSCs expressing CasBrE SU with 4070A (CasES+4070A-NSCs) resulted in the redistribution of CasBrE SU from a strictly secreted product to include retention on the plasma membrane. Cell surface cross-linking analysis suggested that CasBrE SU membrane localization was due to interactions with 4070A Env. Viral particles produced from CasES+4070A-NSCS contained both CasBrE and 4070A gp70 Env proteins. These particles displayed ecotropic receptor-mediated infection, but were still 100-fold less efficient than CasE+4070A-NSC virus. Infectious center analysis showed CasBrE SU ecotropic transduction efficiencies approaching those of NSCs expressing full length CasBrE Env (CasE; SU+TM). In addition, CasBrE SU-4070A Env interactions resulted in robust ecotropic superinfection interference indicating near native intracellular SU interaction with its receptor, mCAT-1. CONCLUSIONS: In this report we provided evidence that 4070A Env and CasBrE SU physically interact within NSCs leading to CasBrE SU retention on the plasma membrane, incorporation into viral particles, restoration of mCAT-1 binding, and capacity for initiation of TM-mediated fusion events. Thus, heterotropic Env-SU interactions facilitates CasBrE SU folding events that restore Env activity. These findings are consistent with the idea that one protein conformation acts as a folding scaffold or nucleus for a second protein of similar primary structure, a process reminiscent of prion formation. The implication is that template-based protein folding may represent an inherent feature of neuropathogenic proteins that extends to retroviral Envs.

Cross-talk between stem cells and the dysfunctional brain is facilitated by manipulating the niche: evidence from an adhesion molecule.

In the injured brain, the behavior of neural stem/progenitor cells (NSCs) is regulated by multiple converging factors encountered in the niche, which is composed of several neural and non-neural cell types. Signals emanating from the host influence the migration, survival, distribution, and fate of transplanted NSCs, which in turn can create host microenvironments that favor a return to homeostasis. We tested the hypothesis that overexpression of key facilitatory molecules that define the injury niche might enhance this bidirectional stem cell-host interaction to therapeutic advantage. As proof of concept, we investigated whether conditioning the niche with the neural cell adhesion molecule L1 might enhance recovery in a prototypical neurodegenerative milieu-the MPTP-induced model of Parkinson's disease in aged mice-where cross-talk between NSCs and imperiled host dopaminergic neurons is known to be pivotal in rescuing the function and connectivity of the latter. In lesioned mice (and in unlesioned controls), we overexpressed L1 in the NSCs to be transplanted into the ventral mesencephalon. Several pairwise experimental combinations were tested based on variations of engrafting L1 overexpressing versus nonoverexpressing NSCs into wild-type (WT) versus L1-overexpressing transgenic mice (specifically L1 transcribed from the GFAP promoter and, hence, overexpressed in host astrocytes). Enrichment for L1-particularly when expressed simultaneously in both donor NSCs and host brain-led to rapid and extensive distribution of exogenous NSCs, which in turn rescued (with an efficacy greater than in nonengineered controls) dysfunctional host dopaminergic nigral neurons, even when grafting was delayed by a month. L1 overexpression by NSCs also enhanced their own differentiation into tyrosine hydroxylase-expressing neurons in both WT and transgenic hosts. Graft-host interactions were thus favored by progressively increasing levels of L1. More broadly, this study supports the view that manipulating components of the niche (such as an adhesion molecule) that facilitate cross-talk between stem cells and the dysfunctional brain may offer new strategies for more efficacious neurotransplantation, particularly when treatment is delayed as in chronic lesions or advanced stages of a neurodegenerative disease.

A plant lignan, 3'-O-methyl-nordihydroguaiaretic acid, suppresses papillomavirus E6 protein function, stabilizes p53 protein, and induces apoptosis in cervical tumor cells.

Persistent infection with oncogenic human papillomaviruses (HPVs) is the most important factor in the induction of uterine cervical cancer, a leading cause of cancer mortality in women worldwide. Upon cell transformation, continual expression of the viral oncogenes is required to maintain the transformed phenotype. The viral E6 protein forms a ternary complex with the cellular E6-AP protein and p53 protein which promotes the rapid degradation of p53. Recent studies have revealed that lignans from the creosote bush (3'-O-methyl-nordihydroguaiaretic acid) can repress the viral promoter responsible for E6 gene expression. Work reported here shows that the lignan can subvert viral oncogene function resulting in stabilized p53 protein within treated HPV-containing tumor cells. The stabilized p53 is transcriptionally active as demonstrated by a luciferase reporter vector and induction of genes for Bax and PUMA proteins. Apoptosis is detected by annexin V binding to treated cells as analyzed by flow cytometry. Programmed cell death is confirmed by the induction of active caspases and TUNEL assay. Initiator caspase-9 is activated first, followed later by the effector caspase-3 enzyme. The stabilization and induced apoptosis are not observed within treated HPV-negative cervical tumor cells. Quantitative real time RT-PCR analysis of endogenous E6 gene transcription from the integrated HPV 16 promoter shows at least a fivefold repression of expression as compared to untreated cells. These results indicate that the loss of E6 protein in treated cells could be, in part, responsible for the stabilization of p53 within the lignan treated cells.

Gene expression profiling of microglia infected by a highly neurovirulent murine leukemia virus: implications for neuropathogenesis.

BACKGROUND: Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform motor neuron disease in susceptible mice upon infection of the central nervous system (CNS). The major CNS parenchymal target of these neurovirulent retroviruses (NVs) are the microglia, whose infection is largely coincident with neuropathological changes. Despite this close association, the role of microglial infection in disease induction is still unknown. In this paper, we investigate the interaction of the highly virulent MLV, FrCasE, with microglia ex vivo to evaluate whether infection induces specific changes that could account for neurodegeneration. Specifically, we compared microglia infected with FrCasE, a related non-neurovirulent virus (NN) F43/Fr57E, or mock-infected, both at a basic virological level, and at the level of cellular gene expression using quantitative real time RT-PCR (qRT-PCR) and Afffymetrix 430A mouse gene chips. RESULTS: Basic virological comparison of NN, NV, and mock-infected microglia in culture did not reveal differences in virus expression that provided insight into neuropathogenesis. Therefore, microglial analysis was extended to ER stress gene induction based on previous experiments demonstrating ER stress induction in NV-infected mouse brains and cultured fibroblasts. Analysis of message levels for the ER stress genes BiP (grp78), CHOP (Gadd153), calreticulin, and grp58 in cultured microglia, and BiP and CHOP in microglia enriched fractions from infected mouse brains, indicated that FrCasE infection did not induce these ER stress genes either in vitro or in vivo. To broadly identify physiological changes resulting from NV infection of microglia in vitro, we undertook a gene array screen of more than 14,000 well-characterized murine genes and expressed sequence tags (ESTs). This analysis revealed only a small set of gene expression changes between infected and uninfected cells (<18). Remarkably, gene array comparison of NN- and NV-infected microglia revealed only 3 apparent gene expression differences. Validation experiments for these genes by Taqman real-time RT-PCR indicated that only single Ig IL-1 receptor related protein (SIGIRR) transcript was consistently altered in culture; however, SIGIRR changes were not observed in enriched microglial fractions from infected brains. CONCLUSION: The results from this study indicate that infection of microglia by the highly neurovirulent virus, FrCasE, does not induce overt physiological changes in this cell type when assessed ex vivo. In particular, NV does not induce microglial ER stress and thus, FrCasE-associated CNS ER stress likely results from NV interactions with another cell type or from neurodegeneration directly. The lack of NV-induced microglial gene expression changes suggests that FrCasE either affects properties unique to microglia in situ, alters the expression of microglial genes not represented in this survey, or affects microglial cellular processes at a post-transcriptional level. Alternatively, NV-infected microglia may simply serve as an unaffected conduit for persistent dissemination of virus to other neural cells where they produce acute neuropathogenic effects.

Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons.

We investigated the hypothesis that neural stem cells (NSCs) possess an intrinsic capacity to "rescue" dysfunctional neurons in the brains of aged mice. The study focused on a neuronal cell type with stereotypical projections that is commonly compromised in the aged brain-the dopaminergic (DA) neuron. Unilateral implantation of murine NSCs into the midbrains of aged mice, in which the presence of stably impaired but nonapoptotic DA neurons was increased by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was associated with bilateral reconstitution of the mesostriatal system. Functional assays paralleled the spatiotemporal recovery of tyrosine hydroxylase (TH) and dopamine transporter (DAT) activity, which, in turn, mirrored the spatiotemporal distribution of donor-derived cells. Although spontaneous conversion of donor NSCs to TH(+) cells contributed to nigral reconstitution in DA-depleted areas, the majority of DA neurons in the mesostriatal system were "rescued" host cells. Undifferentiated donor progenitors spontaneously expressing neuroprotective substances provided a plausible molecular basis for this finding. These observations suggest that host structures may benefit not only from NSC-derived replacement of lost neurons but also from the "chaperone" effect of some NSC-derived progeny.

Reexamination of amphotropic murine leukemia virus neurovirulence: neural stem cell-mediated microglial infection fails to induce acute neurodegeneration.

The 4070A amphotropic murine leukemia virus (A-MuLV) has been variably reported to harbor neurovirulence determinants within its env gene. In this report we reexamined this issue by applying two approaches previously demonstrated to amplify murine leukemia virus neurovirulence. The first approach involved introducing the 4070A env gene into the background of Friend virus clone FB29 to enhance peripheral virus replication kinetics and central nervous system entry. The resulting chimeric virus, FrAmE, exhibited widespread vascular infection throughout the central nervous system (CNS); however, parenchymal infection was quite limited. Neither clinical neurological signs nor spongiform neurological changes accompanied FrAmE CNS infection. To overcome this CNS entry limitation, 4070A and FrAmE were delivered directly into the CNS via transplantation of infected C17.2 neural stem cells (NSCs). Significantly, NSC dissemination of either 4070A or FrAmE resulted in widespread, high-level amphotropic virus expression within the CNS parenchyma, including the infection of microglia, the critical target required for inducing neurodegeneration. Despite the extensive CNS infection, no associated clinical neurological signs or acute neuropathological changes were observed. Interestingly, we observed the frequent appearance of circulating polytropic (MCF) virus in the serum of amphotropic virus-infected animals. However, neither peripheral inoculation of an amphotropic/MCF virus mixture nor transplantation of NSCs expressing both amphotropic and MCF viruses induced acute clinical neurological signs or spongiform neuropathology. Thus, the results generated in this study suggest that the 4070A env gene is not inherently neurovirulent. However, the frequent appearance of endogenous MCF viruses suggests the possibility that the interactions of amphotropic viruses with endogenous retroviral elements could contribute to the development of retrovirus-induced neurodegenerative disease.