CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3-5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

Delivery of CDNF by AAV-mediated gene transfer protects dopamine neurons and regulates ER stress and inflammation in an acute MPTP mouse model of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) and its close structural relative, mesencephalic astrocyte-derived neurotrophic factor (MANF), are proteins with neurotrophic properties. CDNF protects and restores the function of dopamine (DA) neurons in rodent and non-human primate (NHP) toxin models of Parkinson's disease (PD) and therefore shows promise as a drug candidate for disease-modifying treatment of PD. Moreover, CDNF was found to be safe and to have some therapeutic effects on PD patients in phase 1/2 clinical trials. However, the mechanism underlying the neurotrophic activity of CDNF is unknown. In this study, we delivered human CDNF (hCDNF) to the brain using an adeno-associated viral (AAV) vector and demonstrated the neurotrophic effect of AAV-hCDNF in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. AAV-hCDNF resulted in the expression of hCDNF in the striatum (STR) and substantia nigra (SN), and no toxic effects on the nigrostriatal pathway were observed. Intrastriatal injection of AAV-hCDNF reduced motor impairment and partially alleviated gait dysfunction in the acute MPTP mouse model. In addition, gene therapy with AAV-hCDNF had significant neuroprotective effects on the nigrostriatal pathway and decreased the levels of interleukin 1beta (IL-1ß) and complement 3 (C3) in glial cells in the acute MPTP mouse model. Moreover, AAV-hCDNF reduced C/EBP homologous protein (CHOP) and glucose regulatory protein 78 (GRP78) expression in astroglia. These results suggest that the neuroprotective effects of CDNF may be mediated at least in part through the regulation of neuroinflammation and the UPR pathway in a mouse MPTP model of PD in vivo.

L-DOPA regulates neuroinflammation and Aß pathology through NEP and ADAM17 in a mouse model of AD.

Dopamine plays important roles in cognitive function and inflammation and therefore is involved in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Drugs that increase or maintain dopamine levels in the brain could be a therapeutic strategy for AD. However, the effects of dopamine and its precursor levodopa (L-DOPA) on Aß/tau pathology in vivo and the underlying molecular mechanisms have not been studied in detail. Here, we investigated whether L-DOPA treatment alters neuroinflammation, Aß pathology, and tau phosphorylation in 5xFAD mice, a model of AD. We found that L-DOPA administration significantly reduced microgliosis and astrogliosis in 5xFAD mice. In addition, L-DOPA treatment significantly decreased Aß plaque number by upregulating NEP and ADAM17 levels in 5xFAD mice. However, L-DOPA-treated 5xFAD mice did not exhibit changes in tau hyperphosphorylation or tau kinase levels. These data suggest that L-DOPA alleviates neuroinflammatory responses and Aß pathology but not tau pathology in this mouse model of AD.

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic ß cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.

MANF Is Neuroprotective in Early Stages of EAE, and Elevated in Spinal White Matter by Treatment With Dexamethasone.

Multiple sclerosis (MS) is a progressive autoimmune disease characterized by T-cell mediated demyelination in central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a widely used in vivo disease model of MS. Glucocorticoids such as dexamethasone (dex) function as immunosuppressants and are commonly used to treat acute exacerbations of MS. Dex is also often used as a positive control in EAE studies, as it has been shown to promote motor behavior, inhibit immune cell infiltration into the CNS and regulate the activation of glial cell in EAE. This study further validated the effects of intravenously administrated dex by time-dependent fashion in EAE. Dex postponed clinical signs and motor defects in early stages of EAE. Histological analysis revealed that the degeneration of myelin and axons, as well as the infiltration of peripheral immune cells into the white matter of spinal cord was inhibited by dex in early stages of EAE. Additionally, dex-treatment delayed the neuroinflammatory activation of microglia and astrocytes. Furthermore, this study analyzed the expression of the neurotrophic factor mesencephalic astrocyte-derived neurotrophic factor (MANF) in EAE, and the effect of treatment with dex on MANF-expression. We show that in dex-treated EAE mice expression MANF increased within myelinated areas of spinal cord white matter. We also show that intravenous administration with hMANF in EAE mice improved clinical signs and motor behavior in the early stage of EAE. Our report gives insight to the progression of EAE by providing a time-dependent analysis. Moreover, this study investigates the link between MANF and the EAE model, and shows that MANF is a potential drug candidate for MS.