Novel treatment with neuroprotective and antiviral properties against a neuroinvasive human respiratory virus

Abstract

Human coronaviruses (HCoVs) are recognized respiratory pathogens with neuroinvasive and neurotropic properties in mice and humans. HCoV strain OC43 (HCoV-OC43) can infect and persist in human neural cells and activate neuroinflammatory and neurodegenerative mechanisms, suggesting that it could be involved in neurological disease of unknown etiology in humans. Moreover, we have shown that HCoV-OC43 is neurovirulent in susceptible mice, causing encephalitis, and that a viral mutant with a single point mutation in the viral surface spike (S) protein induces a paralytic disease that involves glutamate excitotoxicity in susceptible mice. Herein, we show that glutamate recycling via the glial transporter 1 protein transporter and glutamine synthetase are central to the dysregulation of glutamate homeostasis and development of motor dysfunctions and paralytic disease in HCoV-OC43-infected mice. Moreover, memantine, an N-methyl-d-aspartate receptor antagonist widely used in the treatment of neurological diseases in humans, improved clinical scores related to paralytic disease and motor disabilities by partially restoring the physiological neurofilament phosphorylation state in virus-infected mice. Interestingly, memantine attenuated mortality rates and body weight loss and reduced HCoV-OC43 replication in the central nervous system in a dose-dependent manner. This novel action of memantine on viral replication strongly suggests that it could be used as an antiviral agent to directly limit viral replication while improving neurological symptoms in various neurological diseases with a viral involvement. Mutations in the surface spike (S) protein of human respiratory coronavirus OC43 appear after persistent infection of human cells of the central nervous system, a possible viral adaptation to this environment. Furthermore, a single amino acid change in the viral S protein modulated virus-induced neuropathology in mice from an encephalitis to a neuropathology characterized by flaccid paralysis, which involves glutamate excitotoxicity. We now show that memantine, a drug that is used for alleviating symptoms associated with neuropathology, such as Alzheimer's disease, can partially restore the physiological state of infected mice by limiting both neurodegeneration and viral replication. This suggests that memantine could be used as an antiviral agent while improving neurological symptoms in various neurological diseases with a viral involvement.

FIG 1

Daily memantine and dizocilpine treatment attenuates the motor dysfunctions and severe paralysis induced following infection of mice by S-mutant human coronavirus OC43. Groups of 10 BALB/c mice infected with rOC/U_S241 were treated daily by intraperitoneal injection of a defined dose of memantine or dizocilpine or of PBS (vehicle). Motor dysfunctions were evaluated and scored according to a scale based on experimental allergic encephalitis (EAE) clinical score (CS) evaluation (0 to 1, normal with no clinical signs; 1.5 to 2, partial hind-limb paralysis with a walk close to ground level; 2.5 to 3.5, complete hind-limb paralysis; 4 to 5, moribund state and death). (A) Daily treatment with 0.5 μg/g dizocilpine attenuated the clinical scores in mice infected with rOC/U_S241. Higher doses of dizocilpine had strong side effects on mice and increased the mortality rate, as illustrated in Fig. 2. (B) Daily memantine treatment attenuated (doses, up to 10 μg/g) and even abolished (dose, 30 μg/g) the clinical scores in a dose-dependent manner. Results are representative of three independent experiments.

FIG 2

Daily memantine treatment attenuates the mortality rates of infected mice compared to those achieved with dizocilpine and vehicle (control) treatment. Groups of 10 BALB/c mice infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) were treated daily with an intraperitoneal injection of a defined dose of memantine or dizocilpine or of PBS (vehicle). (A) Daily memantine treatment attenuated (doses, up to 5 μg/g) and totally suppressed (doses, 10 μg/g and above) the mortality rates for mice infected with either virus in a dose-dependent manner. (B) Daily dizocilpine treatment did not attenuate the mortality rates for mice infected with either virus. A higher dose of dizocilpine (1 μg/g) by itself increased the mortality rate. Results are representative of three independent experiments.

FIG 3

Daily memantine treatment attenuates/abolishes the weight loss of infected mice compared to that achieved with dizocilpine and vehicle (control) treatments. Groups of 10 BALB/c mice infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) were intraperitoneally treated daily with a defined dose of memantine or dizocilpine or with PBS (vehicle). (A) Daily memantine treatment attenuated (doses, up to 5 μg/g) and abolished (dose, 10 μg/g and above) weight loss in mice infected with either virus in a dose-dependent manner. (B) Daily dizocilpine treatment did not attenuate weight loss in mice infected with either virus. *, P < 0.05 (Dukey's test) for comparison with infected mice treated with vehicle for a defined day postinfection; **, P < 0.01 (Dukey's test) for comparison with infected mice treated with vehicle for a defined day postinfection. Results are representative of three independent experiments.

FIG 4

Increase in extracellular glutamate levels and LDH release following infection of primary cultures of mouse CNS. Primary cultures of mouse CNS cells were infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) at different MOIs and treated with memantine (20 μM) or vehicle (medium). (A) Increasing MOIs of both viruses led to an increase in the extracellular glutamate level that was significantly decreased by memantine (MOIs, 0.1 and 1). (B) Increasing MOIs of both viruses also led to an increase in LDH release that was also significantly reduced by memantine (MOIs, 0.1 and 1). #, P < 0.05 (Dukey's test) for comparison to mock-infected cells; ##, P < 0.01 (Dukey's test) for comparison to mock-infected cells; &, P < 0.05 (Dukey's test) for comparison of rOC/ATCC- or rOC/U_S241-infected cells at a defined MOI; *, P < 0.05 (Dukey's test) for comparison of memantine- and vehicle-treated infected cells.

FIG 5

The increase in extracellular glutamate levels is not a result of an increase in expression of phosphate-activated glutaminase. Phosphate-activated kidney-type glutaminase (KGA) or liver-type glutaminase (GLS2) was detected in lumbar spinal cord GM segments in infected mice treated with memantine or vehicle at 10 dpi (black arrows). Primary cultures of murine CNS cells were infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) and treated with memantine (20 μM) or vehicle (medium). Immunofluorescence revealed no changes in KGA (A) or GLS2 (B) activation at 24 h postinfection in lumbar spinal cord GM in primary murine CNS cell cultures following infection by either virus. MAP2, microtubule-associated protein 2. Magnifications, ×400. (C) Semiquantitative Western blot analysis of spinal cord proteins confirmed that activation of KGA and GLS2 was not modulated and that the levels of astrogliosis (a significant increase in GFAP expression) and microglial cell activation (an increase in Mac-2 expression) compared to those in sham-infected mice occurred. Memantine treatment significantly reduced Mac-2 expression in mice infected with rOC/U_S241 compared to that in rOC/ATCC-treated mice. Data are represented as the mean ± SEM (n = 3). ###, P < 0.001 (Dukey's test) for comparison to sham-infected mice; ##, P < 0.01 (Dukey's test) for comparison to sham-infected mice; #, P < 0.05 (Dukey's test) for comparison to sham-infected mice; *, P < 0.05 (Dukey's test) for comparison of groups of memantine treated- or nontreated infected mice. 1, sham infection plus vehicle treatment; 2, rOC/ATCC infection plus vehicle treatment; 3, rOC/ATCC infection plus treatment with 3 μg/g memantine; 4, rOC/U_S241 infection plus vehicle treatment; 5, rOC/U_S241 infection plus treatment with 3 μg/g memantine. Results are representative of those from three independent experiments.

FIG 6

Expression of GS and GLT-1 is downregulated in mice infected with S-mutant human coronavirus OC43 and partially restored following treatment with memantine. GS was evaluated by immunohistochemistry in lumbar spinal cord GM segments of mice infected with rOC/ATCC or rOC/U_S241 and treated with memantine or vehicle at 10 dpi (black arrows) and by immunofluorescence at 24 h postinfection in primary cultures of murine CNS cells infected and treated with memantine (20 μM) or vehicle (medium). (A) GS expression was dramatically downregulated in GM and in primary cultures of murine CNS cells infected with the S-mutant virus (rOC/U_S241), and memantine treatment partially restored GS expression. GFAP, glial fibrillary acidic protein. Magnifications, ×400. (B) Semiquantitative Western blot analysis of spinal cord proteins confirmed the histological findings. Infection of mice with rOC/U_S241 led to a significant downregulation of GS and GLT-1 expression that was significantly restored by memantine. Data are represented as the mean ± SEM (n = 3). ##, P < 0.01 (Dukey's test) for comparison to sham-infected mice; **, P < 0.01 (Dukey's test) for comparison of groups of memantine-treated or nontreated infected mice. 1, sham infection plus vehicle treatment; 2, rOC/ATCC infection plus vehicle treatment; 3, rOC/ATCC infection plus treatment with 3 μg/g memantine; 4, rOC/U_S241 infection plus vehicle treatment; 5, rOC/U_S241 infection plus treatment with 3 μg/g memantine. Results are representative of three independent experiments.

FIG 7

Daily treatment with a low dose of memantine reduces neuronal dysfunctions in infected mice. Groups of three BALB/c mice infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) and treated with vehicle or memantine (3 μg/g) were sacrificed and evaluated by immunohistochemistry for the levels of neurofilament phosphorylation in the lumbar spinal cord GM and WM segments at 10 dpi compared to those in control mice. Following infection, GM showed a loss of normal nonphosphorylated NF immunoreactivity compared to the GM of sham-infected mice. This loss was more pronounced following rOC/U_S241 infection than following rOC/ATCC infection (white arrows). The spinal cord WM of infected mice showed a higher level of abnormal axonal nonphosphorylated NF-H with abnormal axonal swelling, which was more pronounced following infection with rOC/U_S241 than following sham infection (black arrows) and infection with rOC/ATCC. Results are representative of two independent experiments with three mice per group. Magnification, ×400.

FIG 8

Daily treatment with high doses of memantine attenuates viral replication in the CNS of infected mice. Groups of three BALB/c mice infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) were sacrificed and evaluated for viral replication in brains and spinal cords every 2 days. (A) Memantine treatment reduced viral replication in the brains and spinal cords of infected mice in a dose-dependent manner (between 10 μg/g and 30 μg/g). (B) Dizocilpine treatment did not affect viral replication in the brains and spinal cords of infected mice. Data are represented as medians with ranges (n = 3). *, P < 0.05 (Dukey's test) for comparison to control mice (infected mice treated only with vehicle) for a defined day postinfection; **, P < 0.01 (Dukey's test) for comparison to control mice (infected mice treated only with vehicle) for a defined day postinfection; ***, P < 0.001 (Dukey's test) for comparison to control mice (infected mice treated only with vehicle) for a defined day postinfection. Each symbol on the graph represents a single mouse. L.O.D., limit of detection; TCID₅₀, 50% tissue culture infective dose. Results are representative of those from two independent experiments.

FIG 9

Prophylactic transient treatment with memantine attenuates viral replication in the CNS of infected mice. Groups of three BALB/c mice were treated with memantine (5 μg/g) 1 day before viral infection and daily for 4 days postinfection. Mice infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) were sacrificed and evaluated for viral replication in brains and spinal cords every 2 days. The preventive memantine treatment reduced viral replication in the brains and spinal cords of infected mice. Data are represented as medians with ranges (n = 3). *, P < 0.05 (Dukey's test) for comparison to control mice (infected mice treated only with vehicle) for a defined day postinfection. Each symbol on the graph represents a single mouse. L.O.D., limit of detection; TCID₅₀, 50% tissue culture infective dose. Results are representative of those from two independent experiments.

FIG 10

High doses of memantine reduce viral replication in a NMDA-blockade-independent manner. Primary cultures of mouse CNS cells or the HRT-18 cell line were infected with reference wild-type virus (rOC/ATCC) or S-mutant virus (rOC/U_S241) and treated with increasing doses of memantine or dizocilpine. Memantine treatment significantly reduced intra- and extracellular infectious virus titers in primary cultures of mouse CNS cells (A) and in cells of the human epithelial HRT-18 cell line (B) compared to those achieved with dizocilpine and control (medium alone) treatment. h.p.i., hours postinfection. *, P < 0.05 (Dukey's test) for comparison of memantine- or dizocilpine-treated infected cells with PBS-treated infected cells; **, P < 0.01 (Dukey's test) for comparison of memantine- or dizocilpine-treated infected cells with PBS-treated infected cells. (C) Memantine treatment (70 μM) significantly reduced intra- and extracellular infectious virus titers when added after virus adsorption. B, before infection; D, during infection; A, after infection. *, P < 0.05 (Dukey's test) for comparison of memantine-treated infected cells and control (PBS-treated infected) cells. Results are representative of three independent experiments. (D) Expression of the NR1 subunit of the NMDA receptor was confirmed by RT-PCR in primary cultures of mouse CNS cells (lane 1; the 211-bp fragment size is indicated). No expression was detected in the HRT-18 cell line (lane 2). Lane MM, molecular size marker; lane −, negative control for PCR.