Efficacy and Safety of Cilostazol in Mild Cognitive Impairment: A Randomized Clinical Trial.

Importance: Recent evidence indicates the efficacy of ß-amyloid immunotherapy for the treatment of Alzheimer disease, highlighting the need to promote ß-amyloid removal from the brain. Cilostazol, a selective type 3 phosphodiesterase inhibitor, promotes such clearance by facilitating intramural periarterial drainage. Objective: To determine the safety and efficacy of cilostazol in mild cognitive impairment. Design, Setting, and Participants: The COMCID trial (A Trial of Cilostazol for Prevention of Conversion from Mild Cognitive Impairment to Dementia) was an investigator-initiated, double-blind, phase 2 randomized clinical trial. Adult participants were registered between May 25, 2015, and March 31, 2018, and received placebo or cilostazol for up to 96 weeks. Participants were treated in the National Cerebral and Cardiovascular Center and 14 other regional core hospitals in Japan. Patients with mild cognitive impairment with Mini-Mental State Examination (MMSE) scores of 22 to 28 points (on a scale of 0 to 30, with lower scores indicating greater cognitive impairment) and Clinical Dementia Rating scores of 0.5 points (on a scale of 0, 0.5, 1, 2, and 3, with higher scores indicating more severe dementia) were enrolled. The data were analyzed from May 1, 2020, to December 1, 2020. Interventions: The participants were treated with placebo, 1 tablet twice daily, or cilostazol, 50 mg twice daily, for up to 96 weeks. Main Outcomes and Measures: The primary end point was the change in the total MMSE score from baseline to the final observation. Safety analyses included all adverse events. Results: The full analysis set included 159 patients (66 [41.5%] male; mean [SD] age, 75.6 [5.2] years) who received placebo or cilostazol at least once. There was no statistically significant difference between the placebo and cilostazol groups for the primary outcome. The least-squares mean (SE) changes in the MMSE scores among patients receiving placebo were -0.1 (0.3) at the 24-week visit, -0.8 (0.3) at 48 weeks, -1.2 (0.4) at 72 weeks, and -1.3 (0.4) at 96 weeks. Among those receiving cilostazol, the least-squares mean (SE) changes in MMSE scores were -0.6 (0.3) at 24 weeks, -1.0 (0.3) at 48 weeks, -1.1 (0.4) at 72 weeks, and -1.8 (0.4) at 96 weeks. Two patients (2.5%) in the placebo group and 3 patients (3.8%) in the cilostazol group withdrew owing to adverse effects. There was 1 case of subdural hematoma in the cilostazol group, which may have been related to the cilostazol treatment; the patient was successfully treated surgically. Conclusions and Relevance: In this randomized clinical trial, cilostazol was well tolerated, although it did not prevent cognitive decline. The efficacy of cilostazol should be tested in future trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02491268.

[A case of neuromyelitis optica spectrum disorders, with slowly progressive bulbar palsy, mimicking a motor neuron disease].

A 52-year old woman first noted dysphagia four months before admission followed by dysarthria two months later. She then developed weakness of all limbs and became unable to walk. All these symptoms, associated with tongue atrophy, slowly progressed, leading to the initial clinical impression of a motor neuron disease, although her nerve conduction study and electromyography showed no abnormalities. Her brain MRI with T2 weighted/diffusion weighted image (DWI)/fluid attenuated inversion recovery (FLAIR) revealed a high signal lesion located at dorsal medulla oblongata. She proved positive for anti-aquaporin 4 antibody, which confirmed the diagnosis of neuromyelitis optica spectrum disorders (NMOSD). We conclude that NMOSD may initially present with progressive bulbar palsy and pyramidal tract disorder over a few months, mimicking a motor neuron disease. Awareness of this atypical presentation helps establish an early diagnosis of this treatable entity.

[A case of limbic encephalitis associated with Sjögren's syndrome mimicking anti N-methyl D-aspartate receptor encephalitis].

A 25-year-old woman in her 37 weeks and 5 days pregnant presented with abnormal behavior and memory impairment following a high fever. Her manifestations were diagnosed as limbic encephalitis, and she delivered a baby by Cesarean section. In the operation, bilateral ovarian tumors were found and resected, though they were revealed as non-teratoma afterward. After operation, she became agitated, and started to present oral dyskinesia, intractable systemic myoclonus, central hypoventilation, and autonomic manifestations such as labile blood pressure, but her symptoms responded well to immunotherapy. Her clinical course was typical for anti-N-methyl D-aspartate (NMDA) receptor encephalitis, but anti-NMDA receptor antibody was not detected in her serum and CSF. On the other hand, anti SS-A antibody was positive in her serum, and the lip biopsy findings confirmed Sjögren's syndrome (SjS). Only several cases of SjS-associated limbic encephalitis have been reported, but none of them mimicked anti-NMDA receptor encephalitis. This patient indicates that SjS should be considered even in a case of limbic encephalitis with a typical clinical spectrum of anti-NMDAR encephalitis.

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS.

A dominant mutation in the gene for copper-zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat-shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1(G93A) transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1(G93A) mice as disease progressed, while levels of expression of two other heat-shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1-containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.

Protective effect of serofendic acid on glutamate-induced neurotoxicity in rat cultured motor neurons.

We have previously reported that a sulfur-containing neuroprotective substance named serofendic acid was purified and isolated from lipophilic extract of fetal calf serum (FCS). In the present study, we investigated the effect of serofendic acid on glutamate neurotoxicity using embryonic rat spinal cord culture. When cultures were exposed to glutamate (20 microM) with a glutamate transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC; 40 microM) for 24 h, motor neurons were injured through both N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole/kainate receptors. This glutamate neurotoxicity was attenuated by nitric oxide (NO) synthase inhibitors. Serofendic acid (0.1-5 microM) prevented glutamate neurotoxicity in a concentration-dependent manner. S-Nitrosocysteine (SNOC; 10 microM), an NO donor, induced motor neuronal death. Serofendic acid (5 microM) also prevented SNOC-induced neurotoxicity. These results indicate that serofendic acid protects cultured motor neurons from glutamate neurotoxicity by reducing the cytotoxic action of NO.

Stimulation of nicotinic acetylcholine receptors protects motor neurons.

The present study demonstrated that administration of nicotine prevented glutamate-induced motor neuronal death in primary cultures of the rat spinal cord. The nicotine-induced neuroprotection was inhibited by either dihydro-beta-erythroidin (DHbetaE) or alpha-bungarotoxin (alphaBT), suggesting that it is mediated through both alpha4beta2 and alpha7 nicotinic acetylcholine receptors (nAChRs). Both alpha4beta2 and alpha7 nAChRs were identified on rat spinal motor neurons by immunohistochemical methods. We also demonstrated that galantamine, an acetylcholinesterase inhibitor with allosteric nAChR-potentiating ligand properties, prevented glutamate-induced motor neuronal death. These results suggest that stimulation of nAChR may be used as a treatment for ALS.

Galantamine modulates nicotinic receptor and blocks Abeta-enhanced glutamate toxicity.

Galantamine is a plant alkaloid that is used in the treatment of Alzheimer's disease. We have studied the effects of galantamine on beta-amyloid-enhanced glutamate toxicity using primary rat cultured cortical neurons. Nicotine and galantamine alone, and in combination, protected neurons against this neurotoxicity. The protection was not blocked by alpha4beta2 nicotinic acetylcholine receptor (nAChR) antagonists, but was partially blocked by alpha7 nAChR antagonists. Galantamine induced phosphorylation of Akt, an effector of phosphatidylinositol 3-kinase (PI3K), while PI3K inhibitors blocked the protective effect and Akt phosphorylation. The antibody FK1, which selectively blocks the allosterically potentiating ligand site on nAChR, significantly reduced the galantamine-induced protection and Akt phosphorylation. Furthermore, suppression of alpha7 nAChR using an RNA interference technique reduced Akt phosphorylation induced by galantamine. Our data suggest that neuroprotection by galantamine is mediated, at least in part, by alpha7 nAChR-PI3K cascade.

Effects of mitochondrial dysfunction on glutamate receptor-mediated neurotoxicity in cultured rat spinal motor neurons.

Glutamate-induced excitotoxicity is implicated as playing a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS), and mitochondrial dysfunction is also found in ALS patients. We investigated the relationship between glutamate excitotoxicity and mitochondrial dysfunction elicited by rotenone (a complex I inhibitor), malonate (a complex II inhibitor), or antimycin (a complex III inhibitor), in primary cultures of the embryonic rat spinal cord. Rotenone and malonate induced relatively selective toxicity against motor neurons as compared to non-motor neurons, whereas antimycin caused non-selective toxicity. The toxicity of rotenone was prevented by a non-N-methyl-D-aspartate (NMDA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by an NMDA receptor antagonist, 5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). The toxicity of malonate was blocked by both CNQX and MK-801. The toxicity of antimycin was affected by neither CNQX nor MK-801. When mitochondrial complex I was mildly inhibited by a sub-lethal concentration of rotenone, AMPA-induced motor neuron death was significantly exacerbated. A sub-lethal concentration of malonate exacerbated both NMDA- and AMPA-induced motor neuron death. These data suggest that mitochondrial dysfunction predisposes motor neurons to ionotropic glutamate receptor-mediated excitotoxicity.

Phosphodiesterase inhibitors are neuroprotective to cultured spinal motor neurons.

We have previously reported that cyclic guanosine-3',5'-monophosphate (cGMP) protects spinal motor neurons against acute reactive oxygen species (ROS)-induced toxicity but not against chronic ROS-induced or glutamate (Glu)-induced toxicity. In this study, we investigated the effects of phosphodiesterase (PDE) inhibitors on the survival of cultured spinal motor neurons. Selective PDE5 inhibitors (dipyridamole, T-1032, and zaprinast) as well as a nonselective PDE inhibitor (aminophylline) protected motor and nonmotor neurons against both acute ROS-induced and chronic Glu-induced neurotoxicity, whereas selective inhibitors of PDE1-4 offered no protection. 8-Bromo-cGMP (8br-cGMP), a cGMP analogue, protected both motor and nonmotor neurons against acute ROS-induced toxicity but protected only nonmotor neurons against chronic Glu-induced toxicity. This neuroprotection was blocked by KT5823, a cGMP-dependent protein kinase (PKG) inhibitor. Immunohistochemical staining confirmed that PDE5 and PKG are located in almost all rat lumbar spinal neurons. Furthermore, semiquantitative analysis of the immunostaining intensity revealed that PDE5 was more abundant in motor neurons than in nonmotor neurons. Our results suggest that this difference in the amount of PDE5 may be responsible for the vulnerability of motor neurons to chronic excitotoxicity. In addition, the results of this study raise the possibility that PDE5 inhibitors might be used as a treatment for amyotrophic lateral sclerosis.

Protective effect of dopamine D2 agonists in cortical neurons via the phosphatidylinositol 3 kinase cascade.

Glutamate, one of the excitatory neurotransmitters, contributes to the neuronal death associated with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, and with ischemia. In Alzheimer's disease brains, there is a decreased number of dopamine D2 receptors, which might cause neuronal dysfunction or death. In the present study, bromocriptine exerted a protective effect against glutamate-induced cytotoxicity in rat cortical neurons. This neuroprotective effect was mediated via D2 receptors, because it was attenuated by domperidone, a D2 dopaminergic receptor antagonist. Another dopamine D2 agonist, quinpirole, also protected cells against glutamate toxicity. D2 agonists protected cells from calcium influx, nitric oxide, and peroxynitrite toxicity, which are thought to be the mediators of glutamate toxicity. The phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) inhibited this neuroprotective effect of bromocriptine, in contrast to the mitogen-activated protein kinase kinase (MAPKK) inhibitor (PD98059), which did not counter the protective effect. Furthermore, Akt protein kinase, which is an effector of PI3K, was activated by bromocriptine, and the antiapoptotic protein Bcl-2 was up-regulated by bromocriptine treatment. These results suggest that D2 dopaminergic receptor activation plays an important role in neuroprotection against glutamate cytotoxicity and that the up-regulation of Bcl-2 expression via the PI3K cascade is, at least partially, involved in this effect.

Estradiol protects dopaminergic neurons in a MPP+Parkinson's disease model.

The prevalence of Parkinson's disease is higher in males than in females. Although the reason for this gender difference is not clear, the level of female steroid hormones or their receptors may be involved in the pathogenesis. The estrogen receptor subtype expressed in the midbrain is limited to the novel beta subtype, whose role in the central nervous system has not been resolved. We demonstrated that ligand-activated estrogen receptor beta suppressed dopaminergic neuronal death in an in vitro Parkinson's disease model which uses 1-methyl-4-phenylpyridinium ions (MPP(+)). MPP(+) treatment caused the upregulation of c-Jun amino-terminal kinase (JNK) and dopaminergic neuronal death, the latter being blocked by curcumin, an inhibitor of the c-Jun/AP-1 cascade. 17alpha- and 17beta-estradiol both protected dopaminergic neurons from MPP(+)-induced neuronal death and this was blocked by a pure antagonist of the estrogen receptor, ICI 182,780, but not by an inhibitor of estrogen receptor dimerization, YP537. These data indicated that the neuroprotection provided by 17alpha-estradiol was via inhibitory transcriptional regulation at the activator protein-1 (AP-1) site mediated by estrogen receptor beta. Thus, 17alpha-estradiol is a suitable candidate for neuroprotective therapy of Parkinson's disease because it is associated with few undesirable feminizing effects.